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3-Phosphoinositide-dependent kinase-1 (PDK1)-mediated signaling regulates hematopoietic stem cell (HSC) quiescence by governing the
oxidative response
Danielle Erina Matsushima
Submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in the Graduate School of Arts and Sciences
COLUMBIA UNIVERSITY
2016
© 2016
Danielle Erina Matsushima
All rights reserved
ABSTRACT
3-Phosphoinositide-dependent kinase-1 (PDK1)-mediated signaling regulates hematopoietic stem cell (HSC) quiescence by governing the oxidative response
Danielle Erina Matsushima
Regulation of hematopoiesis through the finite control of hematopoietic stem cell (HSC)
quiescence and proliferation is critical to the health of the organism since disturbances in blood
production can lead to clinical malignancies such as anemia or leukemia. Therefore, elucidating the
processes that govern HSCs is vital to advance our understanding of hematological diseases.
Interestingly, HSCs can be regulated through a variety of ways. Extrinsic cues from the niche provide
signals that govern HSC quiescence, proliferation, self-renewal, and differentiation. These external
signals are converted to internal messages through the use of signal transduction pathways that relay
information from the cytoplasm to the nucleus. While many pathways contribute to HSC regulation, the
PI3K/AKT/mTOR-pathway is especially pertinent because it has been implicated in cell survival,
metabolism, proliferation, and death. Many groups have identified key players within PI3K/AKT/mTOR-
signaling that regulate HSC quiescence; however, these studies are hindered by the redundancy of
multiple isoforms and compensatory signaling mechanisms by other members within the pathway. PDK1
is considered to be a master regulator of PI3K-signaling because it is directly activated by PI3Ks and can
govern activity of a variety of substrates within the PI3K-pathway. Because of this, it is an excellent
candidate to fully delineate how PDK1-mediated PI3K-signaling functions to maintain HSC quiescence.
In the current study, conditional deletion of PDK1 in HSCs was achieved through the use of a
hematopoietic specific Vav-Cre line. The loss of PDK1 in HSCs led to reduced numbers and an inability to
provide radioprotection in primary BMTs. Furthermore, PDK1-deficient HSCs exhibit impaired quiescence
and increased cycling. Strikingly, PDK1-mutant HSCs have markedly high levels of reactive oxygen
species (ROS), which led to increased proliferation, DNA damage, and apoptosis in progenitor cells.
Administration of a ROS scavenger, N-acetyl cysteine (NAC) partially rescued the increased proliferation
and differentiation of phenotype Pdk1Hem-KO cells both in vitro and in vivo, suggesting that abnormal HSC
activity in PDK1-deficient cells was in part due to increased ROS. Furthermore, mechanistic studies
identified a remarkable decrease in the levels of nuclear HIF1α in HSCs. Immunoblots and phosflow
studies demonstrated reduced activation of p-p70S6K, a well defined positive regulator, and increased
GSK3β, a key negative regulator of the HIF1α protein. These data suggested that ROS levels are
unrestrained since HIF1α is not present in Pdk1Hem-KO HSCs to activate transcription of genes that
moderate oxidative stress. In addition, Pdk1Hem-KO HSCs also show increased levels of Hif3α and IPAS
mRNA, which are believed to inhibit the transcriptional activation of HIF1α. In essence, the results from
these experiments are the first to implicate PDK1 as a critical regulator of HSC quiescence and as a
moderator of PI3K-signaling to alleviate oxidative stress. In addition, this study is the first to suggest that
HIF3α and IPAS may play a role in HSCs.
i
Table of Contents
List of Figures ...……………………………………………………………………………….v
List of Tables …………………………………………………………………………………vii
Chapter 1: General introduction to PI3K-signaling regulates HSC quiescence ....... 1
The role of HSCs in hematopoiesis ........................................................................................... 1
Differentiation ....................................................................................................................................... 2
Self-renewal .......................................................................................................................................... 4
Quiescence vs. Proliferation ................................................................................................................. 5
Extrinsic vs. intrinsic regulation of HSCs ................................................................................... 6
The hematopoietic niche ...................................................................................................................... 6
Signaling events in HSCs ................................................................................................................... 10
Post-Translational Modifications (PTMs) regulate HSCs ........................................................ 12
Ubiquitination and deubiquitination in HSC quiescence ..................................................................... 13
Phosphorylation events regulate pathways that promote or repress HSC quiescence ...................... 14
PI3K-signaling regulates quiescence in HSCs ........................................................................ 16
PI3K-signaling .................................................................................................................................... 16
Major downstream targets of PI3K-signaling ...................................................................................... 17
PI3K-signaling in HSC quiescence ..................................................................................................... 19
PDK1-signaling in hematopoiesis ............................................................................................ 21
3-phosphoinosidtide-dependent kinase-1 (Pdk1) ............................................................................... 21
PDK1 in hematopoiesis ...................................................................................................................... 23
Abnormal levels of PDK1 result in cancer .......................................................................................... 24
Importance of PDK1-signaling in HSCs .............................................................................................. 24
ii
Chapter 2: Pdk1 is needed for proper hematopoiesis. ............................................. 26
Introduction .............................................................................................................................. 26
HSCs are finely regulated for proper hematopoiesis .......................................................................... 26
HSC identification ............................................................................................................................... 27
PI3K-signaling in HSCs ...................................................................................................................... 28
Results ..................................................................................................................................... 29
Pdk1 is expressed in HSCs ................................................................................................................ 29
PDK1 is important for HSC maintenance in vitro ................................................................................ 30
Pdk1Hem-KO mice have hematopoietic defects ..................................................................................... 30
Loss of PDK1 results in improper hematopoietic lineage differentiation ............................................ 34
Pdk1Hem-KO mice have reduced HSCs in the bone marrow ................................................................. 40
Discussion ............................................................................................................................... 42
PDK1 is necessary for proper lineage differentiation ......................................................................... 43
Loss of PDK1 results in HSPC disturbances and fewer HSCs ........................................................... 44
Chapter 3: The functional ability of Pdk1Hem-KO HSCs is compromised. ................. 47
Introduction .............................................................................................................................. 47
HSC functional assays ....................................................................................................................... 47
Quiescence maintains adult HSCs ..................................................................................................... 48
Results ..................................................................................................................................... 49
Hypotheses for fewer HSCs in the Pdk1Hem-KO mouse ....................................................................... 49
PDK1-deficient HSCs are unable to provide radioprotection .............................................................. 50
Increased HSPCs in the Pdk1Hem-KO spleen ....................................................................................... 53
Loss of PDK1 in HSCs results in increased proliferation and fewer quiescent cells .......................... 55
HSPCs have increased cell death and lineage differentiation in vitro ................................................ 58
Discussion ............................................................................................................................... 59
BMT analysis ...................................................................................................................................... 60
Pdk1Hem-KO HSPCs are homing to the spleen ..................................................................................... 61
iii
Loss of quiescence, hyperproliferation and increased differentiation of PDK1-deficient cells ............ 61
Chapter 4: PDK1-deficient HSPCs exhibit reduced response to oxidative stress . 63
Introduction .............................................................................................................................. 63
Oxidative Stress ................................................................................................................................. 63
Mitochondrial dynamics ...................................................................................................................... 63
ROS and HSC maintenance ............................................................................................................... 64
PI3K-signaling regulates ROS levels .................................................................................................. 65
Results ..................................................................................................................................... 66
Microarray analysis reveals Pdk1Hem-KO HSPCs have reduced response to oxidative stress ............. 66
Deficiency of PDK1 in HSPCs alters the transcription factor networks associated with HSCs and
oxidative stress ................................................................................................................................... 66
PDK1-deficient cells have increased levels of ROS ........................................................................... 71
Treatment with NAC suppresses proliferation and differentiation in PDK1-deficient cells ................. 74
Discussion ............................................................................................................................... 79
Microarray ........................................................................................................................................... 80
Mitochondrial bioenergetics ................................................................................................................ 81
DNA damage/apoptosis assays ......................................................................................................... 84
ROS reversion .................................................................................................................................... 85
Chapter 5: Loss in PDK1 results in reduced Hif1α expression. .............................. 88
Introduction .............................................................................................................................. 88
HIF1α regulation ................................................................................................................................. 88
Hif1α regulates ROS expression ........................................................................................................ 89
PI3K/AKT/mTOR-signaling ................................................................................................................. 90
Results ..................................................................................................................................... 92
Pdk1Hem-KO mice have reduced Hif1α expression ............................................................................... 92
Inhibition of HIF1α in Pdk1Hem-KO cells ................................................................................................ 93
Pharmacological inhibition of PDK1 provides insights into downstream signaling ............................. 98
iv
Reduced AKT-signaling in Pdk1Hem-KO LSK ...................................................................................... 100
Discussion ............................................................................................................................. 103
HIF expression.................................................................................................................................. 103
PI3K/AKT/mTORC-signaling ............................................................................................................ 105
Consequences of reduced HIF1α expression .................................................................................. 106
Chapter 6: Discussion and Future Directions ......................................................... 108
PI3K-signaling in HSCs ......................................................................................................... 108
ROS impacts HSC quiescence .............................................................................................. 113
A novel role for HIF3α in HSC maintenance ......................................................................... 116
Future Directions and perspectives ....................................................................................... 119
Chapter 7: Experimental Procedures ....................................................................... 122
References .................................................................................................................. 129
v
List of Figures
Figure 1: HSCs give rise to all blood cell components. ............................................................................... 4
Figure 3: Schematic of PI3K/PDK1-signaling. ........................................................................................... 19
Figure 4: Inhibitors of PI3K-signaling affect HSPC numbers. .................................................................... 30
Figure 5: Vav-Cre deletes PDK1 in the hematopoietic organs. ................................................................. 32
Figure 6: Pdk1Hem-KO mice show hematopoietic defects. ........................................................................... 34
Figure 7: Lymphoid progenitors are reduced in the Pdk1Hem-KO mice. ....................................................... 36
Figure 8: Pdk1Hem-KO organs show disturbances in differentiation. ............................................................ 37
Figure 9: Pdk1Hem-KO thymus has an increase in DN cells. ........................................................................ 39
Figure 10: The Pdk1Hem-KO BM has fewer HSCs. ....................................................................................... 41
Figure 11: The Pdk1Hem-KO mouse has reduced ST- and LT-HSC numbers. ............................................. 42
Figure 12: Possible explanations for reduced functionality in Pdk1Hem-KO HSCs. ...................................... 50
Figure 13: Pdk1Hem-KO HSCs cannot repopulate the blood cell pool after BMT. ........................................ 51
Figure 14: PDK1-deficient HSCs cannot compete with wild-type HSCs. ................................................... 53
Figure 15: The Pdk1Hem-KO spleen has more HSPCs but HSCs are not significantly increased. ............... 54
Figure 16: Pdk1Hem-KO progenitor cells are less quiescent compared to the control. ................................. 56
Figure 17: Pdk1Hem-KO progenitor cells proliferate more. ............................................................................ 57
Figure 18: In vitro culture reveals that Pdk1Hem-KO LSK cells are apoptotic. .............................................. 59
Figure 19: Pdk1Hem-KO LSK cells exhibit reduced response to oxidative stress. ........................................ 68
Figure 20: PDK1-deficient cells have augmented ROS levels. .................................................................. 72
Figure 21: Pdk1Hem-KO progenitor cells have increased DNA damage. ...................................................... 74
Figure 22: NAC treatment suppresses differentiation and proliferation of Pdk1Hem-KO HSPCs. ................. 75
Figure 23: HIF1α is reduced in Pdk1Hem-KO cells during hypoxic conditions. ............................................. 93
Figure 24: Hif mRNA expression in Pdk1Hem-KO cells. ................................................................................ 95
Figure 25: Pdk1Hem-KO cells have greater IPAS mRNA expression. ........................................................... 97
Figure 26: PI3K-signaling can regulate HIF1α expression. ....................................................................... 99
Figure 27: Drug inhibition of PDK1 provides insight into downstream signaling. ..................................... 100
Figure 28: Loss of PDK1 leads to reduced AKT/mTORC-signaling. ....................................................... 102
vi
Figure 29: PI3K/PDK1-signaling phosphorylates AKT-dependent and AKT-independent AGC kinases. 112
Figure 30: Loss of PI3K-signaling mediated by PDK1 causes HSCs to proliferate more and lose
quiescence. ...................................................................................................................................... 121
vii
List of Tables
Table 1: Haplosufficiency of PDK1 (Pdk1F/+ ; Vav-Cre) is not phenotypically different from Pdk1F/+ or
Pdk1F/F mice. ...................................................................................................................................... 33
Table 2: Up-regulated genes in the Pdk1Hem-KO microarray. ...................................................................... 69
Table 3: Down-regulated genes in the Pdk1Hem-KO LSK microarray. .......................................................... 70
Table 4: Selected transcription factors involved in hematopoiesis or oxidative stress. ............................. 70
Table 5: In vitro NAC treatments limits differentiation of Pdk1Hem-KO lineage negative cells. ..................... 77
Table 6: In vivo NAC treatment impedes proliferation of Pdk1Hem-KO progenitors. ..................................... 78
Table 7: In vivo NAC treatment significantly reduces ROS levels in Pdk1Hem-KO LK. ................................. 79
Table 8: Antibodies used in this study. .................................................................................................... 125
Table 9: Primers for qRT-PCR. ................................................................................................................ 126
viii
ACKNOWLEDGEMENTS
There are so many individuals who have enriched and aided me in my scientific endeavors during
my tenure here at Columbia University. First I would like to thank my advisor, Choz Rathinam, for
accepting me into his lab, lending me his expertise, and allowing me to grow as a scientist. I have truly
enjoyed our conversations regarding this project and I have learned a great deal under his mentorship.
I would also like to thank my committee members, Riccardo Dalla-Favera and Stavroula
Kousteni, for giving me their time and providing critical and helpful feedback during my TRAC meetings
throughout the years. I would especially like to thank Stavroula for kindly agreeing to be the second
reader for my thesis. In addition, I would like to thank Joji Fujisaki and Martin Picard for graciously serving
on my dissertation committee and lending me their expertise from their respective fields. Furthermore,
examiners on my Qualifying Committee, Mimi Shirasu-Hiza, Ginny Papaiannou, and Lori Sussel, provided
initial guidance when I first joined Choz’s lab. Completion of my dissertation work and graduate school
career could not be possible without these professors.
I was lucky to be surrounded by two individuals that enriched my lab experience while at
Columbia. Keyur Thummar aided me in maintaining a mouse colony at Rockefeller as well as kept up
morale with his quiet but incredibly witty comments. I also would not be where I am today without the
guidance and help of Marshall Nakagawa. Marshall is so knowledgeable in the field and helped me
troubleshoot experiments while always putting a smile on my face with his antics.
Furthermore, my scientific colleagues have greatly enriched my experiences during my years at
Columbia. To the Genetics Girls, Lori Khrimian, Junko Shimazu, Angela Pring-Mill Churchill, and Kayla
Hager, I will forever remember discussions in 1609 and late night skit writing. I would also like to thank
Mike Churchill for always attending my student seminars and giving me helpful comments on my project. I
had so much fun envisioning an improved graduate school community and implementing various projects
and events with my fellow Co-President of the Graduate Student Organization, Stephanie Siegmund. We
were able to initiate programs that promoted interactions between graduate students within CUMC as well
increased outreach with the greater New York community. Also, when aspects of this project veered
towards mitochondrial dynamics, discussions with Stephanie were incredibly useful. In addition, thank you
ix
to Lucja Grajkowska, Tulsi Patel, and John Seeley for offering me wisdom that can only be given from
older students. I have truly enjoyed getting to know all of you over the years. Also, many thanks to
Caroline Ng, Leila Ross, and Becca Lewis for keeping me company at lunch as well as for working in a
malaria lab so that I knew who to turn to when I needed advice on hypoxia experiments. Furthermore,
thank you to Thomas Postler, who has been a wonderful sounding board and kept me smiling and well-
fed during these last few months of thesis writing. I could not have accomplished writing this thesis
without your support.
Lastly, I would like to thank the Genetics Department for accepting me into the program and for
providing me with a resource rich environment. Thank you to the faculty, for offering classes that
promoted discussion on a wide range of topics; to members of the Genetics office, who have been
incredibly helpful from the time I interviewed; and to the students for their encouraging words and helpful
advice. This work would not have been completed without funding from the training grant and support
from all of members in the Department of Genetics and Development.
x
DEDICATION
In dedication to my parents, Dr. Glenn and Deborah Matsushima, who have lovingly supported and
encouraged both my personal and academic endeavors, and to my brother Brian, who has patiently
listened to the joys and tribulations of those endeavors.
In addition, to Dr. Larysa Pevny (1965 - 2012), whose mentorship, unwavering encouragement,
professional poise, inquisitiveness, and love for life continues to inspire me to be a better scientist and
person.
1
Chapter 1: General introduction to PI3K-signaling regulates HSC
quiescence
The role of HSCs in hematopoiesis
Throughout the history of mankind, from battle wounds in ancient wars to hemorrhaging during
modern day surgeries, the loss of blood has been associated with the loss of life. Indeed, the blood
system is vital for maintaining homeostasis. Blood delivers oxygen to tissues and fights off foreign
pathogens and without it organisms cannot survive. Understanding the mechanisms that regulate blood
production is vital to scientists and clinicians when treating blood diseases. For instance, a reduction in
blood cells can result in disorders such as anemia and the overabundance of blood cell production can
result in leukemia. Given this, blood equilibrium must be maintained to meet the demands of the
organism.
Blood generation occurs by a process known as hematopoiesis where all the mature cells of the
blood system are produced from a common ancestor cell, known as a hematopoietic stem cell (HSC).
Unlike embryonic stem cells, which are totipotent, HSCs are multipotent and are restricted in terms of
types of cells their progeny can become. The process by which HSCs proliferate and divide into other
more restricted cell types is called differentiation. In order to differentiate, HSCs go from a state of
inactivity, termed quiescence or dormancy, then enter the cell cycle, where they divide, and produce two
daughter cells. Often, one daughter cell is a copy of the HSC, created by a process termed self-renewal,
and the other daughter cell is a more differentiated cell. The stem cell properties of self-renewal,
quiescence, proliferation, and differentiation are vital in maintaining an organism’s blood homeostasis.
These cellular properties of HSCs will be discussed in depth later.
Vertebrate hematopoiesis is thought to occur in successive waves. The first wave, termed
primitive hematopoiesis, occurs around embryonic day (E) 7.5 in the yolk sac and fetal HSCs primarily
function to produce red blood cells, which are necessary for oxygen transport (Dzierzak and Medvinsky,
2
1995; Palis et al., 1999; Pietras et al., 2011). The second wave, or definitive hematopoiesis, provides the
foundation for adult hematopoiesis and is characterized by HSCs migrating to and engrafting into the
bone marrow (BM). It is also at this stage that the output of HSCs switches from the generation of red
blood cells to cells of all blood lineages (Pietras et al., 2011). In definitive hematopoiesis, HSCs first
colonize hematopoietic embryonic tissues around E8.5-E10.5, and then expand to the liver, which is
where the majority of fetal hematopoiesis occurs. Then, from E17.5 until three weeks post-natal, HSCs
migrate to their final niche in the BM. The objectives of fetal and adult hematopoiesis are slightly different.
During fetal hematopoiesis, HSCs primarily serve to: (1) generate the entire blood cell system, the
predominant cell being erythrocytes, which serve to oxygenate growing tissues, and (2) establish the
beginnings of an immune system by generating the first white blood cells. Therefore, almost all fetal
HSCs are active, or cycling, and very few are inactive, or quiescent. In contrast, adult hematopoiesis
functions to maintain balanced blood cell production so the number of actively cycling HSCs is
considerably reduced compared to the fetal stage. However, when there is an immune response, adult
HSCs can activate to supply the immune system with necessary cells and then become dormant when
homeostasis is returned (Orford and Scadden, 2008; Pietras et al., 2011). Taken together, HSCs are able
to respond to hematopoietic needs through dynamic regulation of their opposing hallmark properties:
quiescence and proliferation.
Differentiation
Hematopoiesis occurs based on a hierarchy of cell types (Orkin, 2000), with the most plastic
cells, HSCs, giving rise to the most committed and differentiated cells (Figure 1). During differentiation the
most plastic cells divide and become further restricted in terms of the types they can become. As such, a
terminally differentiated cell cannot become another cell type. As a cell goes from undifferentiated to
terminally differentiated, it progresses through a series of progenitor stages as it commits fully to the
differentiated cell type. In adult hematopoiesis, the long-term (LT)-HSC is the most multipotent and is
characterized by its ability to remain dormant, proliferate, self-renew, and differentiate. LT-HSCs can exit
dormancy and generate short-term (ST)-HSCs, which are more active than LT-HSCs but still retain the
ability to repopulate all blood cell types but only for 3-4 months (Suda et al., 2011; Yang et al., 2005). ST-
3
HSCs can give rise to multipotent progenitors (MPPs), which are distinct in their functional capacity to
give rise to more lineage committed progenitor cell types (Pietras et al., 2011; Wilson et al., 2008). For
instance, Pietras et al. showed that MPP2 is biased towards the megakaryocyte/erythrocyte lineage,
MPP3 towards the granulocyte/monocyte lineage, and MPP4 preferentially differentiates into lymphoid
cells. While MPPs are still able to give rise to more differentiated progeny, they lack the capacity to
engraft, or repopulate the blood cell pool when transplanted. This inability to engraft indicates that they
are in an intermediate differentiation stage. Members of the myeloid and lymphoid lineages are derived
from the MPP stages. Myeloid cells consist predominantly of members of the innate immune system such
as granulocytes, monocytes, macrophages, erythrocytes, megakaryocytes, basophils, eosinophils,
neutrophils, and platelets. B cells, T cells, and natural killer (NK) cells are members of the adaptive
lymphoid immune system. Dendritic cells (DC) arise from progenitors of both the myeloid and lymphoid
lineages. As evidenced by the ability to produce a variety of lineage-specific cells that can fight off
pathogens, HSCs possess a remarkable ability to adapt and respond to urgent requirements of the blood
cell system.
HSCs and progenitor cell types are unable to differentiate without instructive cues to determine
their outcome. Indeed, many transcription factors have been implicated in the differentiation of lineage-
committed cells and these genes were predominantly discovered through genetic knock out studies. For
example, Gata1 controls erythroid differentiation (Kulessa et al., 1995; Orkin, 2000; Pevny et al., 1991),
Notch1 is needed for T cell development (Rothenberg, 2007), and Pax5 is highly important for B cell
differentiation (Nutt et al., 1999; Orkin, 2000). Furthermore, many signals from cells in the niche are
important for governing lineage specification (Morrison and Scadden, 2014). In addition to signals from
the microenvironment, oxidative stress and reactive oxygen species (ROS) levels have been shown to be
necessary during myeloid differentiation (Kohli and Passegue, 2014). Based on the variety of forces that
can influence blood production, it is essential that homeostasis is maintained since a disturbance may
alter HSC output, resulting in an imbalance of immune cell types. This can then result in immune defects,
anemia, or various proliferative disorders that can impact the life of the organism.
4
Figure 1: HSCs give rise to all blood cell components.
A schematic of hematopoietic differentiation. Adult LT-HSCs are able to exit quiescence, proliferate, and differentiate to give rise to all cell types in the blood. LT-HSC: long-term HSC, ST-HSC: short-term HSC, MPP: multipotent progenitor: MEP: megakaryocyte/erythroid, CMP: common myeloid progenitor, CLP: common lymphoid progenitor, and NK: natural killer.
Self-renewal
A defining characteristic of HSCs is that they are able to reconstitute the entire blood supply of a
recipient (Orkin and Zon, 2008). In steady state conditions, HSC division normally yields one HSC and
one differentiated progeny. During regenerative conditions two other possibilities may occur: (1) HSC
expansion, where both daughter cells are HSCs and (2) HSC exhaustion, where both daughter cells are
differentiated progeny (Kohli and Passegue, 2014). One way to test for HSC functional ability is to
perform bone marrow transplantation (BMT) experiments in which donor BM is placed into irradiated
recipients and recipient survival and blood regeneration are monitored. This can only be accomplished if
an HSC is able to both differentiate into various blood cell types as well as actively self-renew so that it
can continue to produce more progeny. HSCs are considered immortal since they have the ability to
successively replicate themselves; however, they do suffer from proliferative stress and show reduced
5
ability to repopulate the blood cell pool after subsequent transplantations. This phenomenon is called
stem cell exhaustion. Interestingly, mouse strains that have different life spans show that the shorter-lived
strains have HSCs that proliferate more than HSCs from longer-lived strains. When HSCs of young
animals from shorter-lived strains were transplanted into recipients, reconstitution capacity was faster
than it was when HSCs from longer-lived strains were transplanted. However, when HSCs of old animals
from shorter-lived strains were transplanted, the cells lost the capacity to quickly reconstitute the blood
cell pool. This suggests that HSCs from shorter-lived strains experience stem cell exhaustion and lose the
ability to self-renewal more quickly than HSCs from longer-lived strains (de Haan et al., 1997; Orford and
Scadden, 2008; Phillips et al., 1992). It is thought that the ability to self-renew is not enough to ensure
HSC survival throughout the lifetime of the organism since the likelihood of stem cell exhaustion
increases with every division. To circumvent this issue, adult HSCs remain in an inactive or quiescent
state for the majority of an organism’s lifetime. This allows the HSC to avoid proliferative activation for
prolonged periods of time, thereby extending the lifespan of the cell (Orford and Scadden, 2008; Suda et
al., 2011).
Quiescence vs. Proliferation
The balance between adult HSC quiescence and proliferation is extremely important to ensure
proper regulation of hematopoiesis as well as to allow HSCs to survive for the lifetime of the organism. In
addition, it is thought that quiescence serves to protect the cell from accumulating damage that can occur
during physiological stress (Kohli and Passegue, 2014; Suda et al., 2011; Wilson et al., 2008). Indeed,
slow cycling HSCs, or LT-HSCs, are more resistant to ultraviolet light, ionizing radiation, and cytotoxic
reagents (Suda et al., 2011). In addition, HSCs and other inactive cell types have highly active ATP-
dependent transport pumps that efflux toxic agents from the cell in order to prevent the accumulation of
hazardous compounds (Challen and Little, 2006; Goodell et al., 1996; Lin and Goodell, 2006). Taken
together, quiescence serves to protect HSCs from deleterious effects that can impact their ability to self-
renew and proliferate when needed. Many studies have investigated numerous biological factors that
regulate HSC quiescence such as signals from the niche, transcriptional factors, and metabolic makeup.
These factors influence HSC quiescence and will be discussed further in the next section.
6
Quiescence in HSCs also serves to promote balanced blood cell production during homeostasis
and after injury. Quiescent murine HSCs divide relatively slowly, once every 145 days or five times
throughout the mouse’s lifetime. Inactive HSCs can be stimulated to initiate hematopoiesis until
homeostasis of the entire blood system is achieved whereupon they return to a quiescent state (Wilson et
al., 2008). This suggests that HSCs are able to dynamically respond to the organism’s immediate
requirements while also ensuring the longevity of the cell.
In summary, HSCs are able to quickly respond to the needs of the organism, such as
replenishment due to blood loss bleeding, battling infections, or defending against environmental trauma.
When necessary, HSCs are able to swiftly activate and produce large quantities of differentiated progeny.
However, to maintain homeostasis, there is a tightly controlled balance between two conflicting forces:
quiescence and proliferation. Without exquisite regulation of these two phenomena, HSCs can be
depleted or expanded, leading to various hematological disorders such as anemia, bone marrow failure,
or leukemia. Therefore, it is critical to have a comprehensive understanding of the mechanisms that
govern quiescence versus proliferation in HSCs.
Extrinsic vs. intrinsic regulation of HSCs
The hematopoietic niche
External cues, including cytokines, chemokines, and growth factors, are vital to maintaining HSC
quiescence in the adult animal. These signaling factors can come from other hematopoietic cell types or
from the cells that compose the HSC niche. Throughout development, the location of the niche changes.
For instance, in the mouse, the first major site of definitive hematopoiesis occurs in the fetal liver while
adult hematopoiesis takes place in the BM. However, in response to hematopoietic stress, the niche can
switch to or be supplemented by other hematopoietic sites such as the spleen or liver (Johns and
Christopher, 2012; Morrison and Scadden, 2014). It is thought that the murine adult BM niche is
composed of two zones: the endosteal niche and the vascular niche. Some evidence suggests that the
location of an HSC, near the endosteal interface or near vasculature, plays a role in its functionality (Orkin
7
and Zon, 2008). For instance, Angiopoeitin-1 is needed to maintain HSC adherence to osteoblasts, which
are located at the endosteal interface. According to this study, close proximity to osteoblasts promotes
HSC quiescence (Arai et al., 2004). This suggests that cell-to-cell contacts may also influence HSC
dynamics (Schofield, 1978). In addition, CXCL12, a chemokine, encourages migration of HSCs to the
vascular niche (Kiel and Morrison, 2006) suggesting that the vascular zone is important for HSC
maintenance. It is important to note that these two regions are very closely situated and that much
controversy surrounds whether or not there are distinct sub-regions within the BM that define HSC
functionality (Orkin and Zon, 2008). For instance, many argue that the vascular niche serves to activate
HSCs while the endosteal niche promotes dormancy (Boulais and Frenette, 2015; Morrison and Scadden,
2014; Orkin and Zon, 2008). In line with the controversy, many genetic studies demonstrate that
osteoblasts are vital for HSC maintenance while recent imaging studies suggest that less than 20% of
HSCs are found within 10uM of the endosteum (Morrison and Scadden, 2014). Despite this discrepancy,
extrinsic signals and the cells that compose the adult BM HSC niche are essential for regulating the
balance between active and quiescent HSCs.
Signaling molecules from the niche are vital to maintain stem cell homeostasis. For instance,
stem cell factor (SCF) and thrombopoietin (TPO) are cytokines produced by immune cells and other cells
in the BM niche. These signaling molecules have been shown to regulate quiescence in adult HSCs
(Wilson et al., 2009; Yamazaki et al., 2006). In addition, the loss of the cytokine receptors Tpo and Kit
(SCF receptor) result in increased HSC cell cycle activity and premature depletion of HSCs (Qian et al.,
2007; Thoren et al., 2008). Interestingly, many of the chemokines and cytokines that are vital for adult
HSC quiescence, are not necessary in fetal hematopoiesis (Wilson et al., 2009), probably because they
are not needed as this is a time when HSCs are highly active. This difference in the requirement of
regulatory molecules at different developmental stages, highlights the importance of identifying
mechanisms that govern the inactive state of adult HSCs.
Many of the signaling molecules that drive quiescence and proliferation in HSCs originate from
cells within the BM niche. Indeed, many of the niche cell types play a positive or negative role in
regulating HSC quiescence and proliferation. It is thought that adipocytes negatively impact HSCs since
HSC and adipocyte numbers are inversely correlated in microenvironments (Naveiras et al., 2009). In
8
contrast, studies demonstrate that osteoblasts, mesenchymal stem cells (MSCs), and endothelial cells
actively contribute to HSC quiescence and cell numbers. For instance, it has been shown that osteoblasts
produce TPO to maintain HSCs in a quiescent state (Yoshihara et al., 2007). Furthermore, co-culturing
osteoblasts with HSCs causes HSC expansion (Lord et al., 1975); however, genetic loss of mature
osteoblasts using target specific Cre-lines have shown that mature osteoblasts may not play a role in
HSC maintenance (Morrison and Scadden, 2014). It is important to note that some of this theory is based
on experiments that deleted SCF from osteoblasts using Col2.3-Cre and showed that HSC frequency was
not changed (Ding and Morrison, 2013). This study is flawed because it assumes that SCF is the only
factor HSCs need; however, other groups have shown that other factors are important for HSC
maintenance including TPO and angiopoietin-1 (Arai et al., 2004; Qian et al., 2007; Yoshihara et al.,
2007). Because the Col2.3-Cre experiments did not evaluate other potential factors released by
osteoblasts, conclusive evidence suggesting osteoblasts do not directly impact HSC quiescence is
lacking.
Despite conflicting evidence regarding whether osteoblasts directly impact HSC maintenance,
there is general agreement that cells of the osteoblastic lineage have a role in progenitor differentiation,
and in particular, development of the B cell lineage (Visnjic et al., 2004; Zhu et al., 2007). Osteoblasts
may not secrete SCF but mesenchymal stem cells (MSCs) and endothelial cells generate this cytokine to
promote HSC maintenance. In addition, these cells are found near sinusoids and other blood vessels,
suggesting that the vascular niche is important for HSC maintenance (Ding et al., 2012). Indeed,
perivascular MSCs are necessary to organize the niche (Sacchetti et al., 2007) and they also express the
chemokine CXCL12 that is needed for HSC retention in the BM (Ding and Morrison, 2013; Greenbaum et
al., 2013). Interestingly, other cell types in the niche produce CXCL12 such as sympathetic nerves
(Katayama et al., 2006). Furthermore, non-myelinating Schwann cells were shown to be responsible for
releasing TGFβ, a cytokine that is necessary for promoting HSC self-renewal (Yamazaki et al., 2011).
Taken together, these experiments demonstrate that different cells in the perivascular and endosteal
zones locate HSCs within the niche and secrete factors that contribute to HSC maintenance and
quiescence (Figure 2).
9
In addition to cell-to-cell contacts and signaling events, the metabolic physiology of the BM can
help promote quiescence in HSCs. The BM niche is highly hypoxic compared to other tissues and
mechanisms controlling the hypoxic response in HSCs are vital for their existence under this low oxygen
condition (Suda et al., 2011). Evidence that HSCs reside in a low oxygen environment comes from
studies that stained HSCs with pimonidazole, a dye that recognizes proteins associated with low oxygen
status, or by staining with Hoescht 33342 and then compared the distance between HSPCs and sites
blood flow. These studies found that HSCs were predominantly located at sites within the BM where the
oxygen gradient was the lowest (Parmar et al., 2007; Winkler et al., 2010). Interestingly, culturing human
CD34+ cells in low oxygen levels or transplanting these cells into the murine BM niche promotes a G0 cell
cycle or quiescent state (Hermitte et al., 2006; Shima et al., 2010). Taken together, these experiments
suggest that HSCs reside in low oxygen conditions, which promote quiescence.
Figure 2: The HSC BM niche.
Adult hematopoiesis occurs in the bone marrow (BM), which is composed of a variety of cell types that can promote or inhibit HSC functions such as by releasing cytokines or chemokines. MSC: mesenchymal stem cell, SNS: sympathetic nervous system.
10
HIf1α is a master regulator of hypoxic stress response in HSCs (Lee et al., 2004; Simsek et al.,
2010; Takubo et al., 2010) since the Hif1α driven metabolic program is critical for maintaining HSCs in a
quiescent state. Indeed, genetic inactivation of Hif1α in vivo has been documented to disrupt HSC
quiescence and functions (Takubo et al., 2010). HIF1α is a bHLH-PAS-type transcriptional regulator that
under normal oxygen conditions is degraded when prolyl hydroxylases hydroxylate its oxygen-dependent
degradation (ODD) domain. The ODD domain is recognized by an E3 ligase known as the von Hippel-
Lindau protein (VHL), which will promote HIF1α degradation in normoxic conditions. In low oxygen
conditions, prolyl hydroxylases are inactivated, allowing the stabilization of HIF1α, which forms a
heterodimer with HIF1β and translocates to the nucleus to activate transcription of downstream targets
such as antioxidants (Dengler et al., 2014; Suda et al., 2011). Interestingly, recent evidence suggests
that the regulation of Hif1α is not only dependent on oxygen levels within the niche but also other
signaling mechanisms, such as the cytokines TPO and SCF, that can stabilize HIF1α to promote
quiescence (Nombela-Arrieta et al., 2013; Pedersen et al., 2008). Taken together, these studies suggest
the hypoxic composition of the niche contributes to quiescence in HSCs.
Signaling events in HSCs
Quiescence in HSCs is also governed by a variety of internal signals including: transcription
factors, cell cycle inhibitors, microRNAs, and post-translational modifications (PTMs). These cell-intrinsic
mechanisms function to promote quiescence, or a G0 state, within HSCs, while allowing for immediate
entry into the cell cycle when prompted. Much work has been done to elucidate the genes and signaling
networks that maintain HSC quiescence and some studies are highlighted below.
Transcriptional regulation of HSCs occurs at all stages of the HSC life cycle. For instance, Runx1
is essential for the formation of HSCs in the embryo and without it hematopoietic clusters do not form
(North et al., 1999; Orkin, 2000). With regard to HSC quiescence, genetic loss of Gfi1 (growth factor
independent 1) resulted in increased HSC cycling and loss of long term repopulating ability (Hock et al.,
2004). Notch1, a major regulator of cell specification, is needed to promote HSC expansion and self-
renewal in mice and humans through the transcription of its target genes (Guruharsha et al., 2012).
Interestingly, deletion of the Notch ligand, Jagged-1 in endothelial cells caused reduced LT-HSC numbers
11
and a loss in quiescence (Poulos et al., 2013). In contrast, loss-of-function studies suggest that the
absence of Notch-signaling does not affect HSC maintenance (Cerdan and Bhatia, 2010). The
discrepancy between the two experiments could be a result of the experimental conditions such as
conditional deletion in the mouse versus studies with hematopoietic cells derived from human pluripotent
stem cells. Another transcription factor that mediates quiescence is Pbx1. Pbx1 promotes proliferation
during development and when it is conditionally deleted, quiescent HSCs are reduced and serial
repopulating ability is lost (Ficara et al., 2008). In all, a variety of transcription factors function to mediate
HSC quiescence.
Proteins involved in cell cycle regulation play a role in HSC quiescence and self-renewal. For
example, when all three Retinoblastoma (Rb) family members are conditionally deleted in adult mice,
severe myeloproliferation occurs presumably as a result of increased HSC numbers and proliferation
(Viatour et al., 2008). Furthermore, the loss of cell cycle inhibitors p21, p27, and p57 has been shown to
be critical for the maintenance of HSCs in an inactive state (Cheng et al., 2000; Matsumoto et al., 2011;
Pietras et al., 2011; Zou et al., 2011). Taken together, these studies show how important regulation of
cell cycle proteins are for HSC quiescence.
Proper microRNA processing is also critical to HSC quiescence since conditional deletion of
Dicer, a component in the microRNA processing machinery, causes a reduction of LSK cells (Guo et al.,
2010). In addition, conditional knockouts of Ago2, a slicer endonuclease responsible for cleaving
microRNAs to match their targets, display compromised HSC pool functions (Lu et al., 2016).
Furthermore, miR-125b increases HSC engraftment and overexpression of this microRNA causes a
dose-dependent myeloproliferative disorder suggesting that it encourages HSC proliferation. The same
study identified other microRNAs that were enriched in HSCs, which have positive or negative effects on
HSC proliferation (O'Connell et al., 2010). Other microRNAs that have been identified to promote HSC
expansion are miR-125a, miR-99b, and let-7e (Guo et al., 2010). These studies demonstrate that
mircoRNAs work to aid in the balance of blood production. In addition to the transcriptional and post-
transcriptional mechanisms already discussed, emerging evidence suggests that PTMs of proteins play
key roles in normal HSC and leukemic HSC regulation (Moran-Crusio et al., 2012; Nakagawa et al., 2015;
Rathinam and Flavell, 2010; Rathinam et al., 2011).
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Post-Translational Modifications (PTMs) regulate HSCs
Post-translational modifications (PTMs) are unique in that they allow the cell to modify signaling
events in order to promote cellular changes that meet its physiological needs. A unique property of PTMs
is that they are site specific and can often be reversed (Jensen, 2006; Prabakaran et al., 2012). This
allows for a dynamic response to a change of status within the cell. There are two types of PTMs that can
dictate the function of a protein: (1) polypeptide modifications and (2) addition of small molecular groups
(Prabakaran et al., 2012). These modifications are initially made on the protein by an activating enzyme
(E1). After this, a second conjugating enzyme (E2) adds additional modifiers. Lastly, E3 ligase is needed
to build the modifying polypeptide that can then be targeted for further ubiquitin-like modifications
(Schwartz and Hochstrasser, 2003; Ulrich and Walden, 2010). One of the most well known polypeptide
modifiers is ubiquitin, which is predominantly responsible for tagging a protein for degradation by the
proteasome. However, ubiquitin and ubiquitin-like proteins can also help maintain homeostasis by
contributing to DNA repair (Ulrich and Walden, 2010). Protein substrates may be either be
monoubiquitinated or polyubiquitinated, which dictates the fate of the protein. It is thought that
monoubiquitination serves to alter protein activity whereas polyubiquitination leads to protein termination
via the proteasome. Furthermore, individual ubiquitin substrates can be removed by deubiquitinating
enzymes (DUBs), which allows for precise regulation of the protein (Moran-Crusio et al., 2012).
The second type of PTMs, addition of small molecular groups, adds material from the cell to a
protein to modulate protein expression. Some of the molecular groups that may be added include:
phosphorylation, acetylation, GlcNAcylation, palmitoylation, and methylation (Prabakaran et al., 2012). Of
particular interest are modifications by phosphorylation because they can be added or removed at site-
specific locations on a given protein. Furthermore, activation or partial activation of the protein can occur
depending on how many sites are phosphorylated. The number of phosphorylation events provides an
extra level of regulation that can have important physiological consequences. In this study, I
demonstrated that phosphorylation cascades are vital for the transduction of signals from the external
13
environment to the nucleus of the cell and that this communication is important for maintaining HSC
homeostasis.
Ubiquitination and deubiquitination in HSC quiescence
In the past 20 years, various studies have provided insight into the role of ubiquitination and its
reversal, deubiquitination, on HSC quiescence. Evidence that E3 ligases contribute to HSC self-renewal
was provided through studies with Casitas B-lineage Lymphoma (c-CBL) knockouts, where it was shown
that c-CBL-deficient HSCs were hyperproliferative and had impaired differentiation capabilities.
Interestingly, increased HSC cycling in the c-CBL knockout was due to increased STAT5 phosphorylation
in response the cytokine TPO (Rathinam et al., 2008). Another E3 ligase, Itch, has been shown to be
responsible for promoting HSC quiescence since knockouts display increased proliferation and
competitive BMT experiments show increased self-renewal capacity. Partial rescue in HSC progenitors
occurred when Notch1 was knocked down through shRNA, suggesting that increased Notch-signaling
promoted quiescence (Rathinam et al., 2011). F-box and WD repeat domain-containing 7 (Fbw-7) is also
an E3 ligase, and its loss in the hematopoietic system results in a highly proliferative LSK fraction that led
to reduced HSC numbers and downregulation of genes associated with HSC quiescence (Matsuoka et
al., 2008; Thompson et al., 2008). Furthermore, the von Hippel-Lindau (VHL) domain can be targeted by
RING finger E3 ligases to tag proteins for degradation. Deleting the VHL protein in HSCs led to HIF1α
stabilization, which caused an increase in LSK cells in the G0 phase of the cell cycle (Takubo et al.,
2010). Another example of a RING finger E3 ligase controlling HSC proliferation is Mouse double minute
2 (MDM-2). MDM-2 targets p53 for degradation and MDM2-deficient HSCs in a p53 hypomorphic
background led to stabilized p53, increased cell cycle arrest, increased levels of ROS, and HSC death
(Abbas et al., 2010). Taken together, these studies suggest that E3 ligases function to maintain HSC
quiescence based on the proteins they target.
Deubiquitinases are responsible for decoupling the ubiquitin molecules that ubiquitinases link
together. It has been shown that the loss of Usp1 in HSCs results in greater numbers of HSPCs that are
not as competent at reconstituting the blood pool (Parmar et al., 2010). Furthermore, A20, a dual-function
ubiquitin-editing enzyme, functions to promote HSC quiescence under steady state conditions since
14
without A20, HSCs hyperproliferate and experience exhaustion (Nakagawa et al., 2015). In essence,
PTMs that use polypeptide modifications are critical for HSC maintenance.
Phosphorylation events regulate pathways that promote or repress HSC quiescence
Phosphorylation is a PTM that allows the status of a protein to be changed quickly since
phosphorylation events often act as an “on/off” switch, meaning that an individual protein substrate or
amino acid can either be phosphorylated or unphosphorylated. Threonine, serine, and tyrosine are the
commonly phosphorylated amino acids and although some sites are constitutively phosphorylated, others
are occupied in a binary fashion that can lead to activation or degradation of a protein (Prabakaran et al.,
2012). Intact phosphorylation cascades are critical to transducing external signals from the niche to the
HSC nucleus so that HSCs can dynamically respond to their environment. To date, many signaling
pathways have been implicated in moderating HSC quiescence and a few are discussed below.
There are controversial reports regarding the role of Wnt-signaling, a major regulator in
embryonic development, in HSC quiescence (Boulais and Frenette, 2015; Jagannathan-Bogdan and Zon,
2013). β-catenin gain-of-function studies showed that increased Wnt-signaling correlates with HSC
expansion in vitro. β-catenin over-activation promoted sustained ability to reconstitute lineage colony
formation (Reya et al., 2003; Willert et al., 2003) but in vivo β-catenin over-activation using Mx-Cre led to
early stem cell exhaustion and impaired multilineage reconstitution (Kirstetter et al., 2006; Scheller et al.,
2006). Loss-of-function studies of Wnt-signaling have shown that deleting the Wnt ligand, Wnt3α, led to
decreased HSC numbers and loss of self-renewal in fetal liver HSCs (Luis et al., 2009). Furthermore,
conditionally deleting β-catenin with Vav-Cre showed reduced reconstitution ability (Cobas et al., 2004);
however, deleting β-catenin using Mx-Cre did not affect self-renewal (Koch et al., 2008). The disparate
results can be explained by differences in methods used in the experiments to delete β-catenin. For
instance deleting in fetal (Vav-Cre) versus adult (Mx-Cre) might contribute to the overall phenotype since
fetal HSCs are highly proliferative (Mendelson and Frenette, 2014).
TGFβ-signaling has been shown to be a negative regulator of HSC proliferation. Previous in vitro
reports have implicated TGFβ-signaling in HSC quiescence (Sitnicka et al., 1996) but a genetic in vivo
study has not recapitulated these results. In vivo deletion of the TGFβ cytokine or total knockouts for the
15
two receptors, TGFβ-RI or TGFβ-RII, results in embryonic lethality (Capron et al., 2010), therefore
inducible Cre lines have been used to assess the role of TGFβ-signaling in HSCs. TGFβ-RI or TGFβ-RII
conditional knockouts with Mx-Cre yielded no major HSC phenotype but the mice developed lethal
inflammatory disorder (Larsson et al., 2005). In contrast, the same group showed that in vivo retroviral
overexpression of Smad7, the negative feedback loop inhibitor of TGFβ-signaling, resulted in increased
stem cell proliferation (Blank et al., 2006). Further adding to the confusion, HSCs lacking SMAD4, a
positive downstream TGFβ -signaling regulator, showed reduced ability to repopulate the hematopoietic
system (Karlsson et al., 2007) while the Smad 2/3 total knockouts did not show a noticeable HSC
phenotype (Blank and Karlsson, 2011). Although TGFβ and its downstream components have been well
studied, clearly there are major discrepancies in the field and further in vivo studies are needed to
conclude how TGFβ-signaling impacts HSC quiescence. Interestingly, cytokines use lipid rafts, areas
where there are large receptor concentrations, to stimulate dormant HSCs to enter cell cycle and start
proliferating (Yamazaki et al., 2006). In vitro studies have shown that TGFβ1 acts to repress lipid raft
formation in HSCs and thus promotes HSC dormancy by repressing the AKT/FOXO-pathway (Yamazaki
et al., 2009). This probably occurs through activation of p57 since it has been shown that TGFβ induces
cell cycle arrest in human hematopoietic cells (Scandura et al., 2004).
The MAPK-signaling family has been shown to be important in converting cytokine signals from
the niche to the HSC nucleus to govern HSC cellular events. MAPKs are a group of serine/threonine
kinases that transduce signals from the cell surface to the nucleus through phosphorylation cascades.
They govern the transcription of many target genes involved in proliferation, differentiation, migration, and
apoptosis (Geest and Coffer, 2009). Furthermore, the MAPKs have been implicated in the maintenance
of HSC quiescence. For instance loss of ATM (ataxia telangiectasia mutated), a cell-cycle checkpoint
regulator, caused increased levels of ROS and constitutively active p38 MAPK phosphorylation, which
reduced HSC self-renewal (Ito et al., 2004). Treatment with an inhibitor of p38 or antioxidants reverted the
phenotype. Deletion of p38 MAPK in HSCs also caused increased levels of ROS and p38-deficient HSCs
have reduced quiescence as well as reduced repopulating activity (Ito et al., 2006). In addition, single
knockouts of either ERK1 or ERK2, needed for ERK MAPK-signaling, did not show a hematopoietic
phenotype probably due to genetic redundancy. However, double knockouts of ERK1 and ERK2 using
16
Mx-Cre showed rapid loss of HSPCs that manifested in leukopenia and anemia (Chan et al., 2013; Staser
et al., 2013). To date, the role of the third MAPK pathway, JNK, has not been established in HSCs. Taken
together, this information indicates that MAPK-signaling is important in maintaining HSC quiescence.
PI3K-signaling regulates quiescence in HSCs
PI3K-signaling
Phosphoinositide 3-kinases (PI3K) are a family of lipid kinases that act as intracellular second
messengers that activate downstream AGC kinases, such as AKT. These kinases regulate many
biological processes including: protein synthesis, insulin metabolism, proliferation, differentiation, and
apoptosis (Buitenhuis and Coffer, 2009). In addition, constitutive activation of PI3K-signaling has been
observed in many leukemias including acute myeloid leukemia (Polak and Buitenhuis, 2012) rendering
the study of this signal transduction pathway clinically relevant. Furthermore, PI3K-signaling is highly
conserved in evolution and the role of insulin-PI3K-AKT can be seen in a range of organisms, the
nematode C. elegans, to mammals (Engelman et al., 2006).
The PI3Ks are heteromeric proteins that contain catalytic and regulatory subunits. The eight
catalytic subunits are divided into three different classes and it is the Class I of PI3Ks that generates
PI(3,4,5)P3 in response to external stimuli and receptor activation. In vertebrates, the PI3K catalytic
subunits are further divided into Class 1A (p110a, p110b, and p110d) and class 1B (110y). The SH2
domain of the regulatory subunits (p85 for Class 1A and p101 for Class 1B) will associate with their
catalytic counterparts and recruit them to the plasma membrane. The PI3Ks are responsible for
converting inositol rings into lipid products that can activate other kinases. The inositol rings have a
glycerol backbone with fatty acids located at positions 1 and 2 and a phosphate group at position 3
(PI(3)). Additional phosphate groups at various points in the inositol ring can be phosphorylated when
there is tyrosine kinase activity and in the case of HSCs, this predominantly occurs when cytokines
stimulate receptor tyrosine kinases (Vanhaesebroeck and Alessi, 2000). The important conversion from
the PI(3,4)P2 (PIP2) substrate to PI(3,4,5)P3 (PIP3) is essential because the triphosphorylated
phosphoinositide (PIP3) is a membrane tether for other proteins that have one or more pleckstrin
17
homology (PH) domains. The phosphate and tensin homolog (PTEN) can inhibit PI3K-signaling by
converting PIP3 back to PIP2 via phosphorylation. In addition, SHIP1 (SH2-containing inositol-5’-
phosphate) and SHIP2 can also perform the PIP3 to PIP2 conversion through hydrolyzation. In PI3K-
signaling, the AGC protein, RAC-alpha serine/threonine-protein kinase (AKT or PKB), is one of the most
heavily studied and it is recruited to the plasma membrane for activation through the interaction of its PH
domain with PIP3. Once activated, AKT can go on to phosphorylate a number of targets that can lead to
transcription of genes involved in a variety of cellular processes (Buitenhuis and Coffer, 2009;
Okkenhaug, 2013; Polak and Buitenhuis, 2012).
Major downstream targets of PI3K-signaling
Many groups have investigated the AKT-pathway because of its ability to regulate the mammalian
target of rapamycin (mTOR) complex and the Forkhead box (FOXOs) transcription factors, two proteins
conserved across evolution (Figure 3). Furthermore, AKT targets are involved in many biological
processes such as cell survival and proliferation (Manning and Cantley, 2007). There are three isoforms
of AKT and they are activated when their PH domain binds to PIP3. This causes a conformational
change, which allows for phosphorylation on Ser473, predominantly by mTORC2, and phosphorylation on
Thr308 by 3-phosphoinosidtide-dependent kinase (PDK1) (Calamito et al., 2010; Okkenhaug, 2013). AKT
is constitutively activated at Thr450, which ensures that the hydrophobic motif is protected so that the
protein is active and stabilized (Pearce et al., 2010). The classical AKT-signaling target is considered to
be mTORC1 although there are multiple substrates that are affected by AKT phosphorylation. AKT can
activate mTORC1 by phosphorylating TSC2, which inhibits the TSC2-TSC1 complex from acting as a
GTPase-activating protein for Rheb. This allows Rheb-GTP to accumulate and then activate mTORC1. In
addition, ERK and RSK, can also inhibit TSC2 and therefore activate mTORC1. Once mTORC1 is
activated, it can go on to phosphorylate p70S6K and 4E-BP1, two proteins involved in protein translation
and synthesis. Once active, p70S6K and 4E-BP1 can phosphorylate IRS1, a scaffolding protein that is
needed to bind and activate PI3K to the insulin receptor. The loss IRS1 can reduce PI3K activation at the
receptor thus providing a negative feedback loop (Manning and Cantley, 2007). AKT can also
phosphorylate FOXOs, causing their inhibition. By blocking FOXO-mediated transcription, AKT can
18
impede apoptosis, cell cycle arrest, and other metabolic properties. Furthermore, AKT can phosphorylate
MDM2, an E3 ubiquitin ligase that can degrade p53, and thus further regulate apoptotic events. In
addition, glycogen synthase kinase-3 (GSK3), a signaling protein often associated with Wnt/β-catenin-
signaling, is inhibited when phosphorylated by AKT. GSK3 can also be inhibited by S6K allowing for the
dephosphorylation of GSK3 substrates, glycogen synthase and eIF2b, which are involved in insulin and
protein synthesis (Cohen and Frame, 2001). Taken together, AKT can act on many substrates to control
the regulation of metabolism, protein synthesis, cell survival, and proliferation.
19
Figure 3: Schematic of PI3K/PDK1-signaling.
PI3K-signaling is activated by external factors such as cytokines and insulin receptors that phosphorylate PI3K. In turn, PI3K converts PIP2 to PIP3 by phosphorylation, which can be removed by the phosphatase, PTEN, or hydrolyzed by SHIP1. Once PIP3 is activated, it can recruit AGC kinases that have a pleckstrin homology (PH) domain to the plasma membrane for activation. PDK1 phosphorylates AKT at residue Thr308. The mTORC2 complex phosphorylates a second phosphorylation site on AKT. Once AKT is fully active, it can negatively regulate the TSC1/TSC2 complex, which will allow enough RHEB-GTP to accumulate to activate the mTROC1 complex. mTORC1 is upstream of two proteins involved in protein synthesis, p70S6K and 4EBP1. The mTORC1 complex is responsible for phosphorylating S6K at Thr389 while PDK1 can phosphorylate S6K at Thr229. AKT can also inhibit GSK3 and FOXOs through phosphorylation, thus promoting cell survival. PDK1 can also activate PI3K/AKT-independent kinases such as RSK and PKC. RSK is also downstream of ERK-signaling while PKC is needed to recruit IKK to lipid rafts when NEMO is ubiquitinated by the CARD11-Bcl10-MALT complex in T cells. This allows IKK to phosphorylate IκB, allowing for NFκB-activation.
PI3K-signaling in HSC quiescence
Many inhibitors of PI3K, such as wortmannin and LY294002, have been used to investigate how
PI3K impacts hematopoiesis, cell survival, and cell proliferation (Recher et al., 2005; Wandzioch et al.,
2004). Mice lacking the p85a and p85b regulatory subunits of PI3K die a few days after birth.
Furthermore, these mice have decreased fetal HSPC numbers and decreased multilineage repopulating
20
ability (Haneline et al., 2006). However, to date no study has deleted PI3K subunits in adult HSCs.
Conditional loss of PTEN, a negative regulator of PI3K-signaling, in adult HSCs resulted in short term
expansion followed by exhaustion as well as myeloproliferative disorder that can be reverted when
treated with the mTORC1 inhibitor, rapamycin (Yilmaz et al., 2006; Zhang et al., 2006). Another group
suggested that PTEN is not needed for fetal hematopoiesis, but is required in adult HSCs to regulate
mTORC2-signaling (Magee et al., 2012). Similarly, the loss of SHIP1 in HSCs, another inhibitor of PI3K-
signaling, also resulted in increased HSC proliferation and reduced long-term repopulation ability
(Desponts et al., 2006). Furthermore, targeted loss of Itpkb (Inositol(1,4,5)trisphosphate 3-kinase-b), a
competitor that blocks AKT activation by PIP3, reduced HSC quiescence and repopulating capacity
(Siegemund et al., 2015). Taken together, these studies suggest that PI3K-signaling is needed to
promote HSC maintenance.
Many other experiments have investigated the mechanism by which downstream signaling
molecules in the PI3K-pathway affect HSC maintenance. For instance, loss of AKT1 and AKT2 in fetal
HSCs results in a prolonged G0 phase and reduced repopulation capacity (Juntilla et al., 2010). In
addition, over-activating AKT in HSCs caused short-term expansion and increased proliferation, which led
to apoptosis (Kharas et al., 2010). In line with these experiments, loss of FOXO1/3a/4 in adult HSCs led
to increased proliferation, a decrease in HSC numbers, loss of reconstitution ability, and increased levels
of ROS (Miyamoto et al., 2007; Tothova and Gilliland, 2007). Inactivation of GSK3, another AKT target,
by pharmacologic inhibition in HSCs caused an increase of HSPCs that had normal repopulating capacity
(Trowbridge et al., 2006); however, in vivo loss of GSK3 showed a transient increase in HSC cycling that
was due to augmented β-catenin expression. This was followed by a decrease in HSC numbers, which
was mTORC1 mediated (Huang et al., 2009). In summary, negative regulation of AKT substrates is
necessary for proper HSC cycling.
Unlike FOXO or GSK3, which are inhibited by AKT phosphorylation, mTORC is activated by AKT.
Conditional loss of TSC1, a negative regulator of mTORC1, led to increased HSC cycling as well as self-
renewal during serial transplantation experiments (Chen et al., 2008; Gan et al., 2008). One of the main
subunits of mTORC1 is Raptor and conditional deletion of Raptor showed non-lethal pancytopenia;
however, complications arose upon transplantation implicating a role for mTORC1-signaling in HSC self-
21
renewal under regenerative conditions (Kalaitzidis et al., 2012). At the same time, another group deleted
Rictor, a critical factor in mTORC2, which is responsible for phosphorylating AKT at residue Ser473. This
study found that the HSC phenotype was similar to the control. However, when Rictor was deleted in a
PTEN-deficient background, leukemogenesis and HSC depletion were prevented suggesting that
mTORC2-signaling plays a role downstream of PTEN/PI3K mediated self-renewal and proliferation
(Magee et al., 2012). Taken together, members of the PI3K-signaling cascade are vital in HSC
maintenance. Furthermore, PI3K-signaling functions to promote proper regulation of HSC quiescence and
proliferation during normal and regenerative hematopoiesis.
PDK1-signaling in hematopoiesis
3-phosphoinosidtide-dependent kinase-1 (Pdk1)
3-phosphoinosidtide-dependent kinase-1 (Pdk1), also known as protein kinase C (PKC), is a 63
kDa serine/threonine kinase that has a N-terminal kinase domain and a C-terminal pleckstrin homology
(PH) domain (Alessi et al., 1997; Vanhaesebroeck and Alessi, 2000). The PH domain is able to interact
with lipids that cause changes in the proteins subcellular localization, conformation, activation state, and
interaction with other proteins. It is through the PH domain that the lipid PIP3 is able to recruit PDK1 to
the cell membrane for activation. PDK1 is constitutively activated since it can trans-autophosphorylate on
residue Ser241 (Pearce et al., 2010). Once PDK1 is activated in the cell it is able to target up to 23 other
AGC kinases for activation. PDK1 is required to phosphorylate the activation loop of a variety of AGC
kinases within the PI3K-signaling pathway including: protein kinase B (PKB)/AKT, mTORC1, GSK3, and
p70 ribosomal S6K (see Figure 3). Therefore, PDK1 is a master regulator of PI3K-signaling. Furthermore,
PDK1 has other well-known AKT-independent substrates such as: PKCθ, serum- and glucocorticoid-
induced protein kinase (SGK), and p90 ribosomal S6K (RSK) (see Figure 29 in Chapter 6). Taken
together, this information demonstrates that PDK1 is an essential signal transduction regulator that plays
a role in a variety of biological pathways.
PDK1 was first identified as the kinase responsible for phosphorylating AKT at Thr308 (Alessi et
al., 1997; Stokoe et al., 1997). In addition, Pdk1 is evolutionarily conserved. Indeed, genetic studies with
22
C. elegans have placed it downstream of AGE-1, the equivalent PI3K catalytic subunit in the worm.
Furthermore, in both mammals and worms, PDK1 has been found to activate AKT, which led to the
activation of mTOR-signaling (Vanhaesebroeck and Alessi, 2000). Some studies suggest that AKT must
first be localized to the plasma membrane by PtdIns(3,4,5)P3 before it can be activated by PDK1 (Mora et
al., 2004) since interaction with PtdIns lead to a conformational change in AKT that promotes
phosphorylation by PDK1. It is important to note that some studies have shown that AKT can still be
activated when a mutated form of PDK1 is unable to act with the plasma membrane, thus suggesting that
co-localization of both proteins may not be needed for AKT activation (Pearce et al., 2010). Without
phosphorylation at Thr308 by PDK1, it is thought that AKT is not fully activated (Vanhaesebroeck and
Alessi, 2000) although phosphorylation at this site alone can cause partial AKT activation. AKT activation
has been implicated in inhibiting GSK3 as well as FOXOs to promote a variety of cellular events such as
proliferation, survival, metabolism, and growth are affected (Manning 2007). Interestingly, the
PDK1/FOXO pathway has been implicated in energy regulation and glucose metabolism (Kawano et al.,
2012).
In addition, in mice, PDK1 can phosphorylate AKT on Thr308 as well as a variety of other
proteins within PI3K-signaling such as S6K (Pullen et al., 1998). Thus, PDK1 provides multiple sites of
regulation within PI3K-signaling. While the most well known substrate for PDK1 is AKT, PDK1 has also
been implicated in NFκb-signaling. NFkB-signaling is blocked when it is bound to the IκB kinase (IKK)-
NEMO complex. PDK1 phosphorylates PKCθ, and once activated PKCθ interacts and recruits IKKs to
lipid rafts at the cell membrane allowing for NFκB-activation. In addition, PDK1 also interacts with the
CARD11-Bcl10-MALT complex. This causes the ubiquitination and degradation of NEMO allowing NFκB
to be activated. This mechanism is extremely important in T cell signaling (Lee et al., 2005).
The PI3K-pathway is vital for maintaining proper regulation of cell insulin levels, growth, size, and
metabolism. Indeed, many studies involving various organ systems corroborate a role for PDK1 in these
biological processes. PDK1 total knock out mice were embryonic lethal and PDK1 hypomorphic mice
were small with reduced organ size, which was attributed to smaller cell size (Lawlor et al., 2002). This
study also found that cell size in the hypomorphic mice was regulated independently of insulin signaling
(Lawlor et al., 2002). Furthermore, loss of PDK1 in cardiac muscle led to reduced cell size, heart failure,
23
and death, presumably due to increased cardiomyocyte sensitivity to hypoxia (Mora et al., 2003). In
addition, PDK1 was needed for insulin and glycogen synthase tolerance in the heart (Mora et al., 2005b).
Similarly, PDK1-deficient hepatocytes led to glucose intolerance (Mora et al., 2005a) and loss of PDK1 in
pancreatic β cells induces diabetes because β cell mass was reduced. Interestingly, haplosufficiency of
FOXO1 increased β cell number and glucose levels suggesting that β cell mass is regulated through the
PDK1/AKT/FOXO1-pathway (Hashimoto et al., 2006). Taken together, these studies domonstrate that
PDK1 functions in non-hematopoietic organs to regulate metabolic nutrient uptake as well as cell size.
PDK1 in hematopoiesis
Many studies in the past 10 years have focused on the role of PDK1 in hematopoiesis. From
these experiments, it appears that ablation of PDK1 has detrimental effects on the lymphoid lineage. For
instance, ablating PDK1 in T cells using Lck-Cre, which is expressed in T cell progenitors, caused a block
in DN4 T cell differentiation in the thymus. The same study also reduced PDK1 levels to 10% of normal
and found that with low levels of expression, T cell differentiation remained intact; however, T cell
proliferation capacity was hindered (Hinton et al., 2004). In addition, PDK1 was needed to incorporate
CD28 and T cell receptor signaling to activate NFκB, which is needed for proper T cell proliferation and
ability to mount a proper immune response (Hayashi et al., 2007; Lee et al., 2005; Park et al., 2013). In B
cells, PDK1 was needed to promote B cell survival and differentiation. When PDK1 was ablated using B
cell specific Cre lines or the hematopoietic Vav-Cre, B cell antigen receptor expression was lost as well as
VDJ recombination (Venigalla et al., 2013). In NK cells, loss of PDK1 caused fewer cell numbers as well
as reduced mTOR-signaling, which was important for inducing expression of downstream proteins
needed for proper NK development (Yang et al., 2015). Taken together, deletion of PDK1 in lymphoid
progenitors led to reduced NK, T and B cell numbers and decreased capacity to perform as immune cells.
In contrast, overexpression of Ras in human CD34+ progenitor cells was found to stabilize PDK1 and
promote monocyte colony formation (Pearn et al., 2007). Furthermore, loss of PDK1 using a platelet
specific Cre revealed a slight decrease in numbers leading to mild thrombocytopenia and reduced
activation (Chen et al., 2013). In summary, PDK1 is a vital component in the specification of both the
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lymphoid and myeloid lineages and loss of this kinase leads to reduced survival and loss of immune
functions.
Abnormal levels of PDK1 result in cancer
Many cancers possess mutations in genes associated with the PI3K-pathway. For instance, one
of the most commonly mutated proteins is PTEN. Aberrances in PTEN cause excessive proliferation
through PI3K-signaling (Mora et al., 2004). Furthermore, PDK1 overexpression is seen in a wide variety
of cancers including pancreatic, esophageal, ovarian, and breast cancer (Ahmed et al., 2008; Arsenic,
2014; Eser et al., 2013; Iorns et al., 2009). In the context of hematopoiesis, overexpression of PDK1 is
found in 45% of patients with acute myeloid leukemia and is associated with poorer treatment outcome
(Pearn et al., 2007; Zabkiewicz et al., 2014). Interestingly, many leukemias arise from stem cells that
have become deregulated and proliferate unchecked. There is evidence that targeting PDK1 in cancer
treatment is mechanistically relevant because when PDK1 was ablated in PTEN-deficient glioblastomas it
led to reduced cancer proliferation and survival (Flynn et al., 2000). In addition, many studies have
attempted to target malignant cells with mutations in PDK1 or other PI3K-signaling components through
the use inhibitors (Mora et al., 2004; Najafov et al., 2011) or RNAi against Pdk1 because it is a master
regulator of PI3K-signaling (Iorns et al., 2009; Najafov et al., 2011; Yu et al., 2012; Zabkiewicz et al.,
2014). Based on these studies, it is clear that understanding how PDK1 functions in hematopoiesis,
specifically how it regulates HSC quiescence and proliferation, may provide novel mechanistic insight into
clinical treatments for a wide variety of oncogenic malignancies.
Importance of PDK1-signaling in HSCs
In the current study, I hypothesize that the PI3K-pathway plays a role in HSC quiescence since it
is downstream of many cytokines required for HSC maintenance (Pietras et al., 2011; Warr et al., 2011).
Although some information regarding the role of PI3K and AKT in HSC quiescence is already available,
these studies are hindered by redundancy due to multiple PI3K subunits or multiple isoforms of AKT. In
an attempt to validate my hypothesis, I focus on PDK1 because it is a master kinase of PI3K-signaling
and because PDK1 can function beyond the AKT axis to activate downstream targets in HSCs. Through
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the use of a conditional allele of PDK1 and a hematopoietic specific Vav-Cre line, I can specifically ablate
PDK1 in HSCs in vivo and assess the physiological consequences associated with the loss of PDK1.
While previous studies have looked at the role of PDK1 in the differentiation of myeloid and lymphoid
lineages, this is the first study to assess how PDK1 regulates HSC maintenance. Based on previous
experiments implicating PI3K-signaling in HSC self-renewal and metabolism, I hypothesize that PDK1-
mediated PI3K-signaling is vital for maintaining HSC quiescence and cellular functions.
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Chapter 2: Pdk1 is needed for proper hematopoiesis.
Introduction
HSCs are finely regulated for proper hematopoiesis
The adult blood system is composed of a variety of cell types that range from undifferentiated to
fully lineage-committed circulating cells. These differentiated cells are responsible for performing
functions necessary for the animal to survive such as oxygenating tissues and fighting off pathogens.
Indeed, the work force of the immune system, the cells that are activated upon invasion or injury, can be
found throughout the blood network and are generated from a common ancestor, known as a
hematopoietic stem cell (HSC). In a process called hematopoiesis, all of the blood cell components of the
organism are generated primarily in the bone marrow. Hematopoiesis is structured as a hierarchy where
the least differentiated cell type, HSCs, are able to give rise to progenitors, cells with varying levels of
abilities to differentiate into various types of fully committed and functionally distinct cells. The
differentiation process is controlled intrinsically by transcription factors and also extrinsically by signals
from the cellular niche. HSCs have the potential to divide and become any of the differentiated blood cell
types; however, they may have various levels of developmental potential based on internal and external
factors. While differentiation is important for generating immune cells and maintaining the numbers of
differentiated cell types, balanced control of the HSCs themselves is of the utmost importance.
The murine adult hematopoietic system is tightly controlled by a variety of external and internal
forces that dictate the maintenance and differentiation of HSCs. External regulation is dictated by cues
from the microenvironment and cell-to-cell contacts. Internal regulation of HSCs can occur at the
transcriptional, post-transcriptional, or post-translational levels. Indeed, a variety of transcription factors
and signaling pathways have been implicated in the process of HSC self-renewal and differentiation. In
addition, signal transduction pathways have been shown to regulate HSC quiescence. The control of cell
cycle quiescence is thought to be important for HSC longevity and long-term function. It is vital to the
hematopoietic system and the overall health of the organism that HSC self-renewal, differentiation, and
27
quiescence are tightly controlled to avoid unwanted consequences, such as anemia and leukemia, that
can arise.
HSC identification
The hematopoietic system is one of the most widely studied and well defined with regards to
identifying the different developmental stages of immune cells. This is because cell surface markers can
be used to uniquely identify the most primitive and multipotent cells, the long-term HSCs (LT-HSCs), and
subsequent stages, from the progenitors to the committed lineage cell types (see Figure 1 in Chapter 1).
All HSPCs are found in a population of cells known as LSK cells that are defined based on their
immunophenotype. LSK stands for Lineage-negative, c-kit receptor positive, and Sca1 (stem cell antigen
1) positive (Ikuta and Weissman, 1992). Of note, in this work, Lineage- denotes lack of expression of:
Ter119+, B220+, CD19+, CD11b+, Ly6G+, CD3e+, and CD11c+. The LSK fraction is further divided into LT-
HSCs, short-term (ST)-HSCs and multipotent progenitors (MPPs). A few groups have identified the
various progenitor stages through functional assays (Morita et al., 2010; Oguro et al., 2013; Pietras et al.,
2015; Wilson et al., 2008; Yamamoto et al., 2013) and there is some degree of variability between the
identification schemes. In this study, all populations analyzed will also be designated by their cell surface
expression. In essence, LT-HSCs are the most primitive and are often considered to have the highest
level of multipotency. They mostly remain quiescent but can enter the cell cycle when necessary. ST-
HSCs are considered to be “active” HSCs and these cells go on to differentiate into the progenitor, MPP,
population. The MPPs have reduced engraftment ability in BMT experiments and are not able to self-
renew. According to the most recent study by Pietras et al., the MPP group is divided into three subsets:
(1) MPP2 (LSKFlt3-CD150+CD48+), (2) MPP3 (LSKFlt3-CD150-CD48+), and (3) MPP4 (LSKFlt3+CD150-
CD48+). These three subsets give rise to specific progenitor lineages that guarantee a balanced output of
differentiated blood cell types. For instance, MPP2 is biased towards the megakaryocyte/erythroid
lineage, MPP3 to the granulocyte/monocyte lineage, and MPP4 to the lymphoid lineage. Therefore, blood
production is mediated by HSCs through the production of functionally distinct subsets of progenitor cells
that favor differentiation of certain lineages. Furthermore, specific cell surface markers can distinguish
progenitor and differentiated cell types. For instance, myeloid progenitors are found in the LK fraction
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(Lineage-c-Kit+) and most B-cells can be identified based on CD19+B220+ expression. In this work all
immunophenotyping will be referred to by cell type and antigen identity.
PI3K-signaling in HSCs
The phosphatidylinositol-3 kinase (PI3K) signaling pathway incorporates cell extrinsic signals,
such as cytokines, from the niche, with internal processing events that lead to the transcription of various
genes that control HSC functions. PI3Ks are a family of lipid kinases that act as intracellular messengers
that activate downstream AGC kinases, such as AKT, and regulate many biological processes including:
protein synthesis, insulin metabolism, proliferation, differentiation, and apoptosis (Buitenhuis and Coffer,
2009). In addition, constitutive activation of PI3K-signaling has been observed in many leukemias
including acute myeloid leukemia (Polak and Buitenhuis, 2012) rendering the study of this signal
transduction pathway clinically relevant. Upon receptor activation, PI3Ks convert the PI(4,5)P2 substrate
to PI(3,4,5)P3 (PIP3), which can act as a membrane tether for other proteins that have one or more
pleckstrin homology (PH) domains (Buitenhuis and Coffer, 2009; Okkenhaug, 2013). In PI3K-signaling,
the AGC proteins, such as AKT and PDK1, are recruited to the plasma membrane through the interaction
of their PH domains with PIP3. Many groups have investigated the AKT-pathway because it can activate
mTOR and FOXOs, two proteins conserved across evolution, and it is involved in many biological
processes such as cell survival, proliferation, and HSC quiescence (Manning and Cantley, 2007). In the
current study, I hypothesize that the PI3K-pathway plays a crucial role in HSC quiescence since it is
downstream of many cytokines required for HSC maintenance (Pietras et al., 2011; Warr et al., 2011;
Yamazaki et al., 2009).
PIP3, also activates 3-phosphoinosidtide-dependent kinase (Pdk1), which is required to activate a
variety of other AGC kinases, such as AKT, PKCθ and S6K. Because PDK1 can act on many
downstream targets independent of AKT, it is considered to be a master regulator within PI3K-signaling.
In the hematopoietic system, conditional deletion of PDK1 leads to the loss of differentiation and functions
of B cells, T cells, NK cells, and platelets (Baracho et al., 2014; Chen et al., 2013; Kloo et al., 2011; Lee
et al., 2005; Park et al., 2013; Venigalla et al., 2013; Yang et al., 2015). In addition, over-activation of
PDK1 in human CD34+ cells promotes myeloid differentiation (Pearn et al., 2007). These studies illustrate
29
that PDK1 is important for proper end-stage development and function of the immune system; however,
none of the previous reports investigated the importance of PDK1 in early hematopoiesis, specifically in
HSCs. Since a role for PI3K-signaling in HSC quiescence has been demonstrated with the downstream
kinase AKT (Juntilla et al., 2010), it is a likely hypothesis that PDK1 also impacts the maintenance of HSC
cellular functions by governing quiescence.
Results
Pdk1 is expressed in HSCs
Previous investigation into how PI3K/AKT-signaling impacts hematopoiesis has been hindered by
a variety of factors including genetic redundancy between AKT isoforms and embryonic lethality of PI3K
and AKT total knockouts. In this study, I overcome these difficulties by conditionally deleting Pdk1
specifically in the hematopoietic lineages to determine how PDK1 affects hematopoiesis.
Before generation of an in vivo model, it was necessary to assess if Pdk1 is expressed in HSCs.
To check if Pdk1 expression could be detected in HSCs, wild type bone marrow (BM) was sorted by flow
cytometry into hematopoietic cell fractions: LT-HSCs (Lineage-Sca1+c-Kit+CD150+CD48-), ST-HSCs
(LSKFlt3-CD34+Flt3-), MPP (LSKCD34+Flt3+), LK (Lineage-c-Kit+), and total BM. Furthermore, detection of
Pdk1 mRNA by PCR with primers designed to recognize exons 3 and 4 of the Pdk1 coding sequence
revealed detection of Pdk1 mRNA in all of the isolated hematopoietic cell fractions (Figure 4 A). These
results suggested that HSCs express Pdk1; however, what needed to be determined was whether Pdk1
is required for HSC maintenance and function.
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Figure 4: Inhibitors of PI3K-signaling affect HSPC numbers.
(A) cDNA was extracted sorted total BM from wild type animals, converted to cDNA, and Pdk1 mRNA expression was determined by PCR using primers located in exons 3 & 4 (n = 3). (B) Wild type total BM was MACs sorted for lineage negative cells and then cultured in vitro for 48h in 10%FBS/DMEM + 0.5ng/ml cytokines (SCF, TPO, Flt3l, IL6, & IL3) with no inhibitor (1ul/ml DMSO) or 1uM Pdk1 inhibitor. The LK and LSK cell fractions were assessed by flow cytometry and it appears that blocking PDK1 activity reduces HSPC numbers. (C) Quantification of LK and LSK described in (B). LT-HSC = LSKCD150+CD48-, ST-HSC = LSKCD34+Flt3-, MPP = LSKCD34+Flt3+. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
PDK1 is important for HSC maintenance in vitro
To assess whether Pdk1 has a role in HSC maintenance, I MACS sorted wild type lineage
negative cells and cultured them for 48 hours with 1:1000 DMSO or 1uM PDK1 inhibitor (GSK2334470)
(Najafov et al., 2011) and assessed the relative frequency of the LK and LSK compartments. After 48
hours, the cells treated with the PDK1 inhibitor were significantly reduced in both the LSK (6.8±0.9% with
no inhibitor compared to 2.62±0.7% with PDK1 inhibitor) and LK (28.0±3.2% with no inhibitor compared to
23.6±2.6% with PDK1 inhibitor) compartments compared to the cells that were cultured with DMSO
(Figure 4 B, C). This suggests that in vitro, PDK1 is involved in the maintenance of the hematopoietic
stem and progenitor cells (HSPC); however, the use of a chemical inhibitor may not have accurately
depicted what was happening to HSCs in a biological context so it was important to assess the impact of
deleting Pdk1 in vivo.
Pdk1Hem-KO mice have hematopoietic defects
To address the in vivo function of Pdk1 during hematopoiesis, I conditionally ablated Pdk1 by
breeding Pdk1flox mice (Inoue et al., 2006) (kindly provided by Dr. Sankar Ghosh in the Department of
31
Microbiology and Immunology) to the hematopoietic specific Vav-Cre (Georgiades et al., 2002) to obtain
Pdk1Flox/+, Pdk1Flox/Flox, or Pdk1Flox/+ ; VavCre/+ (referred to as control) and Pdk1Flox/Flox ; VavCre/+ (referred to
as Pdk1Hem-KO) mice (Figure 5 A). Of note, no differences in body weight, cellularity of hematopoietic
organs, and HSC numbers were detected among the control genotypes (Table 1). In the Pdk1Flox mouse,
exons 3 and 4 are flanked by loxP sites so I examined by PCR the expression of Pdk1 mRNA at this
locus in the BM and Spleen and found highly reduced Pdk1 mRNA levels in Pdk1Hem-KO mice when
compared to the controls. This suggested that Pdk1 transcription is abolished in the mutant mouse.
Immunoblots showed loss of PDK1 protein in the BM and spleen of Pdk1Hem-KO mice (Figure 5 B), but not
in the brain, a non-hematopoietic organ (Figure 5 C), suggesting that the deletion is specific to the
hematopoietic lineage. Initial characterization of Pdk1Hem-KO mice showed reduced body weight in
Pdk1Hem-KO mice (Figure 6 A & B; 18.4±1.3g in control compared to 12.1±1.7g in Pdk1Hem-KO). To
determine whether reduced body weight was related to a hematopoietic defect, control and Pdk1Hem-KO
mice were bled and a complete blood count analysis found that the Pdk1Hem-KO peripheral blood (PB) had
reduced white blood cells (WBC) (7.5±0.6 x103/uL in control compared to 1.1±0.2 x103/uL in Pdk1Hem-KO)
and reduced platelets (1062±105.7 x104/uL in control compared to 558±83.6 x104/uL in Pdk1Hem-KO) but
similar numbers of red blood cells (RBC), hemoglobin, and hematocrit (Figure 6). In addition, cell count
analysis of Pdk1 mutants showed ~50% reduction in BM cellularity (34.81.9±x106 cells in control
compared to 16.7±1.7x106 cells in Pdk1Hem-KO), ~78% reduction in the spleen (31.8±3.4x106 cells in
control compared to 7.3±1.7x106 cells in Pdk1Hem-KO), and ~93% reduction in thymus (58.0±4.5x106 cells
in control compared to 3.7±2.2x106 cells in Pdk1Hem-KO; Figure 6 D). Taken together, these results suggest
that the ablation of Pdk1 using Vav-Cre results in hematopoietic abnormalities. Since there was reduced
cellularity in the PDK1-deficient hematopoietic organs, I next investigated whether the loss of Pdk1
caused abnormal proportions of lineage-committed cells or just an overall reduction of cells.
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Figure 5: Vav-Cre deletes PDK1 in the hematopoietic organs.
(A) Breeding scheme to generate Pdk1Hem-KO mice. (B) Pdk1 mRNA, normalized to Hprt and the control, for exons 3-4 is decreased in total BM (n = 4; p <0.0001) and in the spleen (n = 2) of the Pdk1Hem-KO mice. (C) Western Blots for PDK1 reveal reduced protein levels in the Pdk1Hem-KO in the BM and spleen but equal levels in the non-hematopoietic brain (n = 2). For mRNA expression, samples were normalized to Hprt and then to the control. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
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Table 1: Haplosufficiency of PDK1 (Pdk1F/+ ; Vav-Cre) is not phenotypically different from Pdk1F/+ or Pdk1F/F mice.
There are no significant differences between Pdk1F/+, Pdk1F/F, and Pdk1F/+ ; Vav-Cre mice in terms of body weight, BM cellularity, spleen cellularity, thymic cellularity and relative HSC numbers. Data are mean ± SEM and two-tailed unpaired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
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Figure 6: Pdk1Hem-KO mice show hematopoietic defects.
(A) Pdk1Hem-KO mice have reduced body weight when compared to littermate controls (n = 23). (B) Quantification of body weight shown in (A). (C) Complete Blood Count (CBC) analysis shows hematopoietic disturbances (n = 9) which is also reflected in (D) reduced cellularity of the hematopoietic organs BM, spleen, and thymus (n = 22, 19, 15, respectively) in Pdk1Hem-KO (CKO) mice. Mice analyzed were between 4-8 weeks old. Data are mean ± SEM and two-tailed paired Student’s t-test were used to assess significance (*P<0.05, **P<0.01, ***P<0.001, not significant (ns) = P>0.05).
Loss of PDK1 results in improper hematopoietic lineage differentiation
To determine, whether the disturbances in HSPC number affected lineage differentiation in the
BM, I analyzed lymphoid and myeloid progenitors through cell surface immunostaining and flow
cytometry. Analysis of the common lymphoid progenitors (CLP: L-SlowK+, Flt3+, CD127+) showed a
reduction in cells lacking PDK1 (12.2±2.0% in the control compared to 3.1±1.6% in the Pdk1Hem-KO; Figure
7 A & C). Analysis of the myeloid progenitors showed no difference in relative proportions in LK,
granulocyte-monocyte progenitors (Figure 7 B & D; GMP: LK, CD16/32+, CD34+), common myeloid
progenitors (CMP: LK, CD16/32-, CD34+), or in the megakaryocyte-erythroid progenitor population (MEP:
35
LK, CD16/32-, CD34-). However, there were significantly fewer absolute cell numbers of LK (0.65±0.3x106
cells in control compared to 0.46±0.1x106 cells in Pdk1Hem-KO) and GMP (0.28±0.04x106 cells in control
compared to 0.17±0.02x106 cells in the Pdk1Hem-KO) in the Pdk1Hem-KO mice (Figure 2.4B&D). Previous
studies have found that PDK1 plays a role in B cell differentiation. Indeed, my analysis found that B cells
are reduced by 66% in the Pdk1Hem-KO BM (30.5±3.6% in the control compared to 10.0±3.9% in the
Pdk1Hem-KO). Furthermore, the relative frequency of BM Ter119+ erythrocytic lineage cells was the same
between the control and Pdk1Hem-KO, the frequency of granulocytes (CD11b+Ly6G+) was decreased
(58.8±1.3% in the control compared to 26.1±9.5% in the Pdk1Hem-KO), the frequency of monocytes
(CD11b+Ly6G-) was increased (30.0±1.3% in the control compared to 72.4±9.8% in the Pdk1Hem-KO), and
the frequency of CD41+ cells was equivalent (Figure 8 A). Thus, Pdk1 appears to mostly affect the
lymphoid progenitor compartment and partly the absolute numbers of the LK and GMP populations.
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Figure 7: Lymphoid progenitors are reduced in the Pdk1Hem-KO mice.
(A) Gating scheme for isolating the common lymphoid progenitors (CLP) and (B) myeloid progenitors. Relative and absolute cell numbers of the (C) CLP show a decrease in PDK1-deficient cells. (D) Relative and absolute numbers of myeloid progenitors show reduced LK and GMP cells in the Pdk1Hem-KO mice. CKO = Pdk1Hem-KO, GMP: granulocyte/monocyte progenitors, MEP: megakaryocyte/erythrocyte progenitors, CMP: common myeloid progenitors. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
37
Figure 8: Pdk1Hem-KO organs show disturbances in differentiation.
(A) Relative and absolute numbers of lineage committed cells in the BM and (B) spleen show reduced B cell numbers and an increase in monocytes in the Pdk1Hem-KO. (C) Analysis of the spleen for T cells show markedly changes in the Pdk1Hem-KO when compared to the control. (D) Complete blood count analysis of peripheral blood shows a decrease in lymphocytes and an (E) increase in monocytes in the Pdk1Hem-KO. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
38
The proportions of hematopoietic lineage-derived cells were also altered in the Pdk1Hem-KO spleen
(Figure 8 B). The relative frequency of CD19+ B cells in the spleen was decreased (42.0±2.8% in the
control compared to 4.7±1.4% in the Pdk1Hem-KO), Ter119+ erythroid cells was increased (3.8±0.5% in the
control compared to 19.7±2.5% in the Pdk1Hem-KO), CD11b+ myeloid cells was increased (17.0±1.2% in
the control compared to 50.5±4.2% in the Pdk1Hem-KO), granulocytes (CD11b+Ly6G+) was increased
(16.4±1.6% in the control compared to 33±1.5% in the Pdk1Hem-KO), monocytes (CD11b+Ly6G-) was
decreased (83.1±1.7% in the control compared to 64.6±1.6% in the Pdk1Hem-KO), and CD41+ cells was
increased (1.5±0.3% in the control compared to 6.0±1.2% in the Pdk1Hem-KO) in the absence of PDK1. Of
note, the trends seen in the relative numbers may not be depicted in the absolute values since the spleen
cellularity was incredibly low. The lymphoid compartment was also evaluated in the Pdk1Hem-KO mice and
T cell disturbances are also detected in the spleen. The relative numbers of CD3e+, a general marker for
T cells, cells was decreased (40.1±3.7% in the control compared to 29±1.2% in the Pdk1Hem-KO, Figure 8
C). Corroborating this trend, CD4+ and CD8+ T cells were decreased in Pdk1Hem-KO mice (Figure 8 C).
Furthermore, the altered proportions of lineage cells can be seen with complete blood count analysis of
the Pdk1Hem-KO peripheral blood which showed severe decreases in the relative and absolute number of
lymphocytes (Figure 8 D; 83.6±2.0% in the control compared to 60.8±5.2% in the Pdk1Hem-KO) and
monocytes (Figure 8 E 4.4±0.5% in the control compared to 28.0±6.0% in the Pdk1Hem-KO) in the absence
of PDK1. Thus, the alterations found in the absence of PDK1 in HSCs also affect the blood and peripheral
secondary lymphoid compartment such as the spleen.
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Figure 9: Pdk1Hem-KO thymus has an increase in DN cells.
(A) Surface staining for thymic cell populations reveals that the Pdk1Hem-KO has an increase in immature thymocytes (DN: CD4-CD8-) and a decrease in maturing CD4+CD8+ double-positive (DP) thymocytes. (B) Relative and absolute numbers of thymic cell populations. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
HSCs provide lymphoid progenitors that migrate to the thymus, which is critical for the
development of T cells. Lymphoid progenitors are devoid of CD4 and CD8 markers and are labeled
double negative (DN) but then progress to an intermediary stage where the thymocytes express both
markers and become double-positive (DP) thymocytes. Once DP thymocytes express a T cell receptor,
they can be further selected to become single positive for either CD4 or CD8. I analyzed the thymic
population to determine whether the absence of PDK1 in HSCs affects the development of T cells. As
shown in Figure 9 A and quantified in Figure 9 B, there is a dramatic decrease in double-positive
(CD4+CD8+) thymocytes when PDK1 is absent. The lack of DP thymocytes is correlated to a greater
number of DN thymocytes suggesting progression into DP thymocytes is severely inhibited in the
absence of PDK1. Furthermore, the DN thymocytes that lack a functional T cell receptor can be divided
into four categories based on their CD44 and CD25 profiles. Thymocytes from control mice display all
four DN populations (Figure 9 A lower left panel): DN1 (CD44-CD25+), DN2 (CD44+CD25+), DN3
(CD44+CD25-), and DN4 (CD44-CD25-). In contrast, Pdk1Hem-KO mice display reduced DN1, DN2, and
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DN4 thymocytes but have an excess percentage of DN3 thymocytes (Figure 9 A, lower right panel and
Figure 9 B upper panel). Thus, PDK1 has a dramatic effect in regulating maturation of DN thymocytes
from the DN3 stage to the DN4 stage and subsequently to DP thymocytes. Taken together, similar to
decreased B cell differentiation, T cell development in the thymus is also altered by PDK1 depletion in
HSCs.
Pdk1Hem-KO mice have reduced HSCs in the bone marrow
To assess whether the loss of PDK1 impacts HSCs, I dissected BM from femurs and tibias of
control and Pdk1Hem-KO mice, processed the cells to single cell suspension and examined the HSC
compartment through cell surface staining and flow cytometry. Immunophenotyping revealed no
difference in the relative frequency of lineage negative cells (Figure 10 B) but a decrease in the absolute
number (2.6±0.2x106 cells in control compared to 1.6±0.3x106 cells in Pdk1Hem-KO). The relative and
absolute numbers of LSK cells (Figure 10 C) were equivalent in the Pdk1Hem-KO mice compared to the
control group. Similarly, analysis of CD150+CD48-LSK (Wilson et al., 2008) revealed no change in the
relative and absolute numbers (Figure 10 D). Strikingly, HSC analysis, as defined by CD150+CD48-Flt3-
CD34-LSK (Wilson et al., 2008), revealed a large decrease in cell number in the Pdk1Hem-KO (23.9±0.4% in
control compared to 6.8±2.1% in Pdk1Hem-KO; Figure 10 E). Furthermore, MPP analysis revealed a relative
increase in MPP1 (68.4±3.7% in control compared to 86.6±4.7% in the Pdk1Hem-KO) and MPP3
(38.4±4.4% in control compared to 67.5±3.5% in Pdk1Hem-KO) and a decrease in MPP4 (60.4±4.4% in
control compared to 30.5±3.7% in Pdk1Hem-KO; Figure 10 F). Recently, Pietras et al. demonstrated another
strategy to isolate various HSPC fractions based on their functional properties. Using this scheme (Figure
2.8A), HSCs are divided into long-term (Flt3-/lowLSKCD48-CD150+), and short-term fractions (Flt3-
/lowLSKCD48-CD150-), and multipotent progenitors are divided into three subsets: MPP2 (Flt3-
/lowLSKCD48+CD150+), MPP3 (Flt3-/lowLSKCD48+CD150-), and MPP4 (LSKFlt3+). Immunophenotyping
analysis of the Pdk1Hem-KO revealed a striking decrease in LT-HSCs (12.9±1.1% in control and 6.0±1.8%
in Pdk1Hem-KO) and ST-HSCs (25.5±2.6% in control compared to 8.1±2.1% in Pdk1Hem-KO; Figure 11 B).
Analysis of the Pdk1Hem-KO progenitor populations showed an increase in MPP2 (13.0±2.2% in the control
compared to 28.0±3.2% in the Pdk1Hem-KO), no change in MPP3, and a decrease in MPP4 (44.0±1.9% in
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the control compared to 17.0±5.0% in the Pdk1Hem-KO; Figure 11 C). The aberrant differences in the
Pdk1Hem-KO HSPC populations suggest that PDK1 is necessary for maintaining a proper balance of the
HSPC pool.
Figure 10: The Pdk1Hem-KO BM has fewer HSCs.
(A) Gating scheme and analysis according to Wilson et al. 2008. (B-F) Relative and absolute cell numbers of the cell population show disturbances in the HSPCS. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001)
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Figure 11: The Pdk1Hem-KO mouse has reduced ST- and LT-HSC numbers.
(A) Gating scheme and analysis according to Pietras et al. 2015. (B&C) Relative and absolute cell numbers of the cell population show alterations in the HSPCS. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
Discussion
While previous studies have used both gain- and loss-of-function approaches to study how PI3K-
signaling affects HSC survival, proliferation, and differentiation, to date no study has looked at how PDK1-
mediated PI3K-signaling influences HSC self-renewal and quiescence. Based on this data, abrogation of
PDK1-signaling through genetic deletion by Vav-Cre leads to hematopoietic disturbances as evidenced
by the reduced cellularity of hematopoietic organs and changes in CBC. Furthermore, cell surface
staining and flow cytometry revealed disturbances in lymphoid lineage differentiation and astonishingly
fewer HSCs in the Pdk1Hem-KO mice. While the results regarding lineage differentiation corroborate findings
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from other groups, the HSC phenotype is striking and provides evidence that PDK1 is essential for the
maintenance of HSC numbers.
PDK1 is necessary for proper lineage differentiation
Other groups have shown a role for PDK1 in hematopoietic differentiation and the findings from
this study corroborate these results. For instance, these experiments show reduced numbers of B cells in
both the BM and spleen of Pdk1Hem-KO animals (Figure 8) and earlier reports have demonstrated that
PDK1 regulates B cell differentiation and homeostasis (Baracho et al., 2014; Park et al., 2013; Venigalla
et al., 2013). This corroborates an earlier investigation that established a need for PI3K-signaling in early
B cell differentiation (Baracho et al., 2014). Interestingly, two of these groups used two different B cell
specific Cre lines, mb1-Cre and CD19-Cre, therefore indicating that PDK1 has an independent role in
determining B cell survival; however, this study as well as the one conducted by Venigalla et. al, make
use of Vav-Cre which deletes in HSCs suggesting that PDK1 is needed further upstream at the progenitor
or even HSC level in order to determine B cell fate. The assumption that PDK1 is needed at the
progenitor stage to promote B cell survival, would corroborate the findings from Pietras et al. that suggest
the progenitors are biased in their lineage output. It is important to note that Venigalla et al. did not focus
on the role PDK1 in HSC maintenance and that this study was already in progress at the time of
publication. Furthermore, although the same deletion system was used in both studies, Vav-Cre is the
only non-inducible Cre line that faithfully deletes in HSCs at E 10.5 to adulthood, rendering it a powerful
tool to study how PDK1 governs HSC quiescence. In addition to supporting the B cell finding, this study
also demonstrates a role for PDK1 in T cell survival (Figure 9). Other groups have used T cell specific Cre
lines to demonstrate that PDK1 is needed to activate NFκB-signaling to promote T cell proliferation during
the adaptive immune response (Lee et al., 2005; Park et al., 2013; Park et al., 2009). An extremely small
thymus, a reduction in thymic cellularity, a reduction in splenic CD3e+ cells, and a relative increase in the
DN population in the Pdk1Hem-KO suggests that PDK1 is needed before either CD4 or CD8 is expressed,
and that this kinase is necessary at the progenitor stage to promote T cell differentiation and survival.
With regards to the myeloid lineage, there is an increase in the relative numbers of granulocytes and an
increase in the relative numbers of splenic CD11b+ cells in the Pdk1Hem-KO mice. This is interesting
44
considering that overexpression of PDK1 in human CD34+ progenitor cells through a myristolated N-
terminus retroviral construct promoted a 3-fold increase in monocyte colony formation (Pearn et al.,
2007). It is possible that PDK1 is needed for active differentiation of the lymphoid lineage and that high
levels of PDK1 are not needed to specify granulocyte/monocyte cell types. However, results of the
Pdk1Hem-KO CBC and of Chen et al. suggest that PDK1 is needed to specify, at least, some myeloid
lineages such as platelets (Chen et al., 2013). Taken together, the results from this study and previous
studies suggest that PDK1 is needed for balanced proportions of myeloid and lymphoid lineage cell types
and that it plays a major role in HSPC differentiation.
Interestingly, I found that relative numbers of lineage-committed cells in the spleen do not reflect
the composition seen in the BM. For instance, the relative numbers of Ter119+ erythroid progenitors and
CD41+ megakaryocytes are increased in the spleen and are equal in the BM. These discrepancies
suggest that the spleen is acting as a secondary site of hematopoiesis in Pdk1Hem-KO mice. Indeed, during
times of trauma or injury extramedullary hematopoiesis, which occurs outside of the BM, can act as a
supplement to normal hematopoiesis (Kim, 2010). Furthermore, during infection or injury, the spleen is
able to support myelopoiesis thus providing a possible explanation for increased relative numbers of
CD11b+ cells in the Pdk1Hem-KO spleen. Although, extramedullary hematopoiesis can supplement normal
adult BM hematopoiesis, it is clear based on the Pdk1Hem-KO organ cellularity and CBC results that it is not
enough to sustain the organism for prolonged periods of time. Furthermore, it is unclear if the Pdk1Hem-KO
spleen can serve as a niche for mutant HSCs (discussed in Chapter 3). In summary, despite the
possibility that extramedullary hematopoiesis happens in the Pdk1Hem-KO mice, it is evident that it is not
enough to account for the hematopoietic differences seen in the BM between the control and the Pdk1Hem-
KO mice.
Loss of PDK1 results in HSPC disturbances and fewer HSCs
While many of the previous studies have focused on the role of PDK1 in the committed myeloid
and lymphoid cell types, this study also assessed changes at the BM progenitor level. I found no major
differences in the number of myeloid progenitors (LK, GMP, CMP, and MEP); however, there was a
reduction in CLP numbers (Figure 7 A). In addition, the Pdk1Hem-KO mouse has a large reduction in the
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lymphoid primed MPP4 population and taken together, this suggests that PDK1 is needed in HSCs to
promote lymphoid progenitor specification. It would be interesting to delete Pdk1 at the progenitor MPP
stage to determine if there was an independent role for PDK1 in lymphoid progenitor lineage specification
from the progression of HSC to MPP4. Indeed, crossing the Pdk1Flox mouse with Flt3-Cre (Benz et al.,
2008), which deletes at the progenitor stage, when the Fms-like tyrosine kinase receptor (Flt3 or CD135)
is expressed, would elucidate if there is an independent role for PDK1 in HSCs (Vav-Cre) versus
progenitors (Flt3-Cre). Of note, the deletion efficiency of Pdk1Flox/Flox ; Flt3-Cre mice might be poor since
there is a large reduction in the Fms-like tyrosine kinase receptor (Flt3 or CD135) expression in the
progenitor fractions of Pdk1Hem-KO mice (Figure 11 and data not shown). Low Flt3 expression in HSPCs
also suggests that PDK1 may play a role in this receptor’s expression. Interestingly, Pdk1Hem-KO mice
have reduced expression of other receptors that are important in HSC maintenance. For instance,
expression of c-kit, the receptor for stem cell factor (SCF), is low in all LSK compartments and c-mpl, the
receptor for thrombopoietin (TPO), is reduced in LT- and ST-HSCs (data not shown). Interestingly, a
previous report showed a role for AKT-signaling downstream of lipid rafts in HSCs (Yamazaki et al., 2009)
and another group has implicated a positive feedback loop for AKT/mTORC in maintaining lipid raft
formation in human melanoma cells (Yamauchi et al., 2011). Lipid rafts are areas that allow for the
concentrated formation of cytokine receptors. Since PDK1 mediates AKT phosphorylation as well as a
few other downstream components of PI3K-signaling, it would be interesting to determine whether lipid
raft formation was compromised in the Pdk1Hem-KO mice. However, assessing this connection, and how
PDK1 may regulate expression of cytokine receptors in HSPCs in general, is beyond the scope of this
study.
A major finding of this study is that HSC numbers are fewer when PDK1 is deleted in the most
primitive cells of the hematopoietic system. Reduced HSC number in the Pdk1Hem-KO mice is unexpected
considering that the AKT 1/2 DKO, as described by Juntilla et al., showed increased quiescence and
therefore increased HSC cell numbers. This discrepancy can be explained by the following possibilities:
(1) The AKT mutant used in their study was a double knockout of AKT isoforms 1 and 2 so the third
isoform could have compensated the activity. This isoform could be contributing to some AKT-signaling
and thus allowing for a quiescence signal to be administered; (2) The AKT1/2 DKO used in their study
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were total knockouts and the mice were embryonic lethal. To circumvent this, Juntilla et al. transplanted
fetal liver HSCs into adult recipient mice. Currently, it is widely accepted that the physiology of fetal liver
HSCs are different from adult HSCs and that fetal liver HSCs are controlled by distinct cellular
mechanisms. It is unclear if transplantation of HSCs from fetal liver into the adult BM niche could
recapitulate the physiology of adult HSCs.
Overall, my studies suggest that the numbers of HSCs are clearly affected in Pdk1Hem-KO mice.
Nevertheless, it is unknown if the functions of PDK1-deficient HSCs are impaired. Therefore, what
remains to be determined is whether or not the mutant HSCs are as functional as control HSCs and for
this, future experiments such as bone marrow transplants (BMTs) and cell cycle experiments need to be
performed. It is also important to note that this study was performed with Vav-Cre, which deletes Pdk1
when HSCs first appear, around embryonic day 10. While this ensures a complete understanding of how
PDK1 functions in HSCs, it does not specifically address the role of PDK1 in maintaining adult HSCs. To
answer this question, similar studies will need to be performed using an inducible Cre, such as Mx-Cre, in
which PdkFlox/Flox ; Mx-Cre and control BM can be transplanted into lethally irradiated recipient mice and
then deletion is induced after transplantation. This would ensure accurate depiction of the role for PDK1
once HSCs reside within the BM. Despite these questions, the results from this study establish a novel
role of PDK1 in maintaining HSC numbers and future experiments will elucidate the mechanistic action.
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Chapter 3: The functional ability of Pdk1Hem-KO HSCs is compromised.
Introduction
HSC functional assays
The hallmark of HSCs is that they are able to both self-renew and produce all the blood cell types
of an organism. Therefore, it follows that a single HSC should be able to populate the entire blood
system since it can replenish itself as well as generate progenitors that can go on to differentiate into
committed cells. Although early studies suggested that 200 HSCs are needed to repopulate the BM of an
entire mouse (Becker et al., 1963; Siminovitch et al., 1963; Till and Mc, 1961; Wu et al., 1968), some
evidence suggests that this can be performed by only one HSC (Eaves, 2015). Although, the minimum
number of HSCs needed to repopulate an organism’s blood system is interesting, the more pertinent
question to this study is the functional ability of PDK1-deficient HSCs.
A variety of assays exist to help determine whether a stem cell can perform its responsibilities
appropriately. One is the colony forming cell (CFC) or colony forming unit (CFU) assay (Orford and
Scadden, 2008). CFUs test the ability of a cell to produce more cells as well as reveal the identity of the
progeny. This is especially important when assessing the overall differentiation potential of HSPCs. Other
assays used to assess HSC function involve transplantation studies that allow for the assessment of HSC
migration, implantation and reconstitution of the BM and blood system. One of the most common
transplantations is bone marrow transplantation (BMT) in which total BM or an isolated BM fraction from a
donor is transplanted into a lethally irradiated recipient. Since lethal irradiation kills and depletes the
host’s rapidly dividing BM hematopoietic cells, donor cells can be used to determine repopulation of the
blood system or the function of the BM microenvironment. This concept is also known as radioprotection
since repopulation occurs after irradiation, thereby preventing the organism from dying of bone marrow
failure. Often, HSCs are able to self-renew and regenerate the entire blood system after the first
transplantation; however, if the first transplantation is adequate, then subsequent transplantations may
test the self-renewal capacity of HSCs and provide an understanding of a stem cell’s exhaustion potential.
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In addition, competitive repopulation assays, where a mixture of syngeneic and allogeneic donor cells are
transplanted, test the ability of an experimental cell population to perform at the level of a wild type cell.
This experiment allows for a direct head-to-head comparison (1:1) or can be done at various dilutions to
gain insight into the level of fitness of the experimental population (Eaves, 2015; Orford and Scadden,
2008).
Based on the immunophenotyping results in Chapter 2, HSCs are reduced in Pdk1Hem-KO mice
when compared to control mice. What remains to be determined is whether the HSCs are fully functional
but reduced in numbers or whether the performance of HSCs is compromised. Functional assays such as
total BMT and competitive BMT will shed light on the ability of PDK1-deficient HSCs to perform their
function as the most multipotent hematopoietic cell.
Quiescence maintains adult HSCs
HSCs are one of the longest living cells within the hematopoietic system since they are required
to produce blood cells throughout the lifetime of the organism. Adult HSCs are able to continually
repopulate the blood cell pool through their ability to self-renew; however, proliferation and self-renewal
increase the likelihood of acquiring deleterious mutations, which can cause improper hematopoiesis and
lead to malignancies such as leukemia and anemia. One mechanism HSCs employ to prevent this is
quiescence because cells in the G0 stage of the cell cycle are less likely to accumulate the harmful
mutations that occur during DNA replication in dividing cells (Suda et al., 2011). Indeed, during a 24 hour
period, 5.3-11.1% of adult ST-HSCs and 0.8-1.8% of adult LT-HSCs are actively cycling. Furthermore,
90-95% of all adult HSCs are quiescent since their primary function is to maintain balanced hematopoietic
cell numbers rather than accommodate the growth of an organism as in the case of fetal HSCs where
closer to 100% are cycling (Pietras et al., 2011). In addition to preventing DNA damage, quiescence has
been shown to protect HSCs from immune response such as the killing effect seen with type I interferon
exposure (Pietras et al., 2014; Pietras et al., 2015). In summary, quiescence is vital to HSC survival and
its loss is detrimental to the overall health of the organism. Given this, I wondered whether quiescence
was lost in the Pdk1Hem-KO HSCs and, if so, whether this loss contributed to their reduced HSC numbers in
this model. In order to determine whether PDK1-deficient HSCs have reduced quiescence, a variety of
49
functional assays can be performed. One such assay is challenging the cell to repopulate the entire blood
pool through BMT. Furthermore, if Pdk1Hem-KO cells have reduced quiescence, then it is important to
determine the physiological consequence of this loss: are they proliferating at the same rate as control
HSCs, or are they experiencing other biological phenomenon such as apoptosis? Given that other loss-
of-function mutants, such as TSC1 knockouts (Chen et al., 2008), in PI3K-signaling experience increased
proliferation, I hypothesized that the Pdk1Hem-KO HSCs have reduced quiescence and proliferate more,
and are thus unable to repopulate the blood cell pool during regenerative conditions. The aim of this
chapter is to investigate why there are fewer HSCs in a mutant model and whether the few remaining
HSCs are functioning similar to their wild type counter parts. Since PDK1 is a master regulator of PI3K-
signaling and this pathway has been implicated in cell survival, it is likely that Pdk1Hem-KO HSCs are
experiencing increased rates of cell proliferation and death.
Results
Hypotheses for fewer HSCs in the Pdk1Hem-KO mouse
Immunophenotyping analysis of the Pdk1Hem-KO mouse revealed a reduction in both the quantity
of HSCs and in the numbers of lineage-committed cells (Chapter 2). Lower BM cellularity and an
imbalance of differentiated cell types could be due to: (1) reduced overall HSC numbers since there are
fewer cells that differentiate, (2) the HSCs themselves are functionally compromised and are therefore
unable to perform their responsibilities as a multipotent cell, or (3) a combination of both, fewer and less
competent HSCs. A trademark of HSCs is that they are able to respond to the needs of the organism in
that they have the ability to self-renew, proliferate, and differentiate into progenitors, which can then go on
to repopulate the blood cell pool. The lower lineage-committed numbers found in Pdk1Hem-KO could
suggest that they are unable to perform this repopulating function. Reduced numbers in the Pdk1Hem-KO
mouse could be explained by a variety of reasons: (1) the HSCs could be migrating out of the BM niche to
another site of hematopoiesis, (2) the HSCs could be dying, and (3) the HSCs could be proliferating and
then differentiating into lineage-restricted progenitor cells and simultaneously not self-renewing therefore
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they are not replenishing their numbers (Figure 12). This phenomenon of reduced self-renewal is called
stem cell exhaustion and testing the functional ability of a stem cell can be accomplished with BMT
experiments (Figure 13 A). Total BMT were performed on with the Pdk1Hem-KO mouse model to determine
why there was a reduced number of HSCs and whether the existing HSCs were fully competent. This
model was chosen because it determines the functional ability of Pdk1Hem-KO HSCs to fully repopulate the
blood cell pool.
Figure 12: Possible explanations for reduced functionality in Pdk1Hem-KO HSCs.
Pdk1Hem-KO HSCs may have fewer cell numbers if HSCs were migrating into the blood stream to secondary sites of hematopoiesis such as the spleen. Another possibility for fewer HSCs in the BM is increased cell death. Furthermore, reduced numbers or a loss of functionality can be explained by increased proliferation without self-renewal. Increased proliferation would not explain an overall reduction in numbers therefore the active HSCs/HSPCs could be dying or differentiating into terminally differentiated cell types.
PDK1-deficient HSCs are unable to provide radioprotection
To assess the functional ability of Pdk1Hem-KO HSPCs, I performed BMT. Wild type CD45.1+
recipient mice were lethally irradiated (10gy), and then injected intravenous (i.v.) with either control or
PDK1-deficient BM (106). By day 18 post-transplantation, all recipient mice receiving control BM remained
viable; however, 12/13 of the Pdk1Hem-KO BM recipients had died suggesting that PDK1-deficient HSPCs
are unable to provide radioprotection (Figure 13 B). Analysis of BM from the one surviving recipient of
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PDK1-deficient BM, revealed presence of donor cells (CD45.2+) in the BM. Homing, therefore, appears to
be in tact. However, there was a high percentage (99%) of CD45.1+ BM present, which suggests that
survival of this mouse might have been due to the escape of wild type HSCs (Figure 13 C).
Figure 13: Pdk1Hem-KO HSCs cannot repopulate the blood cell pool after BMT.
(A) BMT experimental set-up. Control or Pdk1Hem-KO total BM was injected into 10 grey irradiated CD45.1+ mice. (B) Survival rates of recipient mice show 12/13 recipients of Pdk1Hem-KO total BM died before day 20 (n = 13 per genotype). Data points represent one or more deaths logged. (C) Donor cell analysis of peripheral blood (PB), bone marrow (BM), and spleen of recipient mice at day 45 after BMT reveals reduced engraftment of Pdk1Hem-KO cells. CKO = Pdk1Hem-KO. Survival Curve comparison was determined by Log-rank (mantel-Cox) Test (*P<0.05, **P<0.01, ***P<0.001) and are representative of animals from two different experiments. Error bars in C represent mean ± SEM.
To determine whether Pdk1Hem-KO HSPCs could function as well as wild type HSPCs, I performed
competitor BMTs. Donor (CD45.2+) BM from either control or Pdk1Hem-KO mice were mixed 1:1 with wild-
type CD45.1+ BM cells and then injected into lethally irradiated CD45.1+ recipients (10gy) (Figure 14 A).
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Peripheral blood (PB) analysis at 2 weeks, 4 weeks, and 6 weeks revealed that the wild-type, CD45.1+,
BM outcompeted Pdk1Hem-KO cells since PDK1-deficient cells were dramatically reduced in number (Week
2: 16.3±2.5% vs. 2.1±0.6%; Week 4: 28.3±2.6% vs. 1.5±0.4%; Week 6: 25.8±3.0% vs. 0.6±0.1%; control
vs. Pdk1Hem-KO, respectively; Figure 14 B). Flow cytometric multilineage analysis of PB at 4 weeks post-
transplantation showed fewer Pdk1Hem-KO donor derived B cells (23.4±1.7% in control compared to
10±4.8% in Pdk1Hem-KO) and fewer Pdk1Hem-KO donor derived T cells (8.7±3.5% in control compared to
0.9±0.3% in Pdk1Hem-KO) when compared to the control donor derived BM (Figure 14 C & D). Most of the
detectable Pdk1Hem-KO donor derived cells were most likely granulocytes or monocytes since the
proportions of myeloid cells present were similar to those seen in the donor control PB (Figure 14 D). Cell
surface-staining and flow cytometric analysis of hematopoietic organs harvested at 6 weeks post-
transplantation, revealed reduced multilineage contribution of Pdk1Hem-KO cells in the BM (<1%), spleen
(<1%), and the thymus (<1%) (Figure 14 E). Collectively, the results from the BMT experiments
demonstrate that without PDK1, HSPCs are functionally compromised since they are unable to self-renew
and provide radioprotection after irradiation and cannot contribute to multilineage differentiation, two
hallmarks of HSC identity.
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Figure 14: PDK1-deficient HSCs cannot compete with wild-type HSCs.
(A) Schematic of a 1:1 competitor BMT. (B) Analysis of peripheral blood (PB) at 2, 4, and 6 weeks show miniscule numbers of Pdk1Hem-KO donor cells. (C) Flow cytometry of PB at 4 weeks show fewer B cells in Pdk1Hem-KO recipient mice. (D) Quantification of donor cells at 4 weeks shown in (C). (E) Analysis at 6 weeks of the hematopoietic organs show an absence of Pdk1Hem-KO donor cells in recipient BM and spleen. B cells = CD19+, T cells = CD4/8+, granulocytes = CD11b+Ly6G+, and monocytes = CD11b+Ly6G-. Data are of 5 mice per genotype from two independent experiments. CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
Increased HSPCs in the Pdk1Hem-KO spleen
The Pdk1Hem-KO mice had fewer HSCs present in the BM (Chapter 2) than the control group and
one reason for this could be that the HSCs are exiting the BM niche and moving to a secondary site of
hematopoiesis. Extramedullary hematopoiesis happens when the generation of blood components occurs
outside of the BM. In the adult mouse, the most common alternative location is the spleen and to some
extent the liver (Johns and Christopher, 2012; Kim, 2010). Using immunophenotyping by staining single
cell suspensions, control and Pdk1Hem-KO spleens were assessed and while there is a relative increase in
the LK (3.9±1.8% in control compared to 31.0±7.0% in Pdk1Hem-KO) and LSK (0.2±0.1% in control
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compared to 1.1±0.2% in Pdk1Hem-KO) fractions, the relative and absolute numbers of HSCs
(LSKCD150+CD48-) were not altered (Figure 15).
Figure 15: The Pdk1Hem-KO spleen has more HSPCs but HSCs are not significantly increased.
(A) Gating scheme for HSC isolation of LSK populations in the spleen by flow cytometric analysis. (B) Quantification of the relative cell numbers by percentages show an increase in LK and LSK in Pdk1Hem-KO spleens and (C) absolute cell numbers of spleen hematopoietic cell fractions show a decrease in lineage negative cell. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
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Loss of PDK1 in HSCs results in increased proliferation and fewer quiescent cells
To determine whether Pdk1Hem-KO BM was unable to provide radioprotection because the HSPCs
were not present due to cell death, control and Pdk1Hem-KO BM were stained with Annexin V, a protein that
is labeled with a dye to detect the externalization of phosphatidylserine in apoptosing cells. Analysis of
the control and Pdk1Hem-KO BM revealed slightly fewer but statistically insignificant Annexin V+ cells in both
the LSK (13.0±2.5% in control compared to 10.6±1.8% in Pdk1Hem-KO) and CD150+CD48+LSK (6.9±1.0%
control compared to 4.8±1.8% in Pdk1Hem-KO) compartments suggesting that the PDK1-deficient HSCs are
not dying (Figure 16 A & B).
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Figure 16: Pdk1Hem-KO progenitor cells are less quiescent compared to the control.
(A) Representative flow cytometric plots of LSK and HSC (CD150+CD48-LSK) show no difference in Annexvin V+ cells (n = 11). (B) Representative FACS plots of cells stained for the side population reveals fewer quiescent CD150+SK cells in Pdk1Hem-KO BM (n = 4). (C) LSK cells deficient in Pdk1, have fewer cells in the side population (n = 4) and (D) are less likely to be in G0 (n = 6). CKO = Pdk1Hem-KO. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
It is possible that Pdk1Hem-KO HSPCs are not as quiescent as control HSPCs and are thus unable
to provide radioprotection due to exhaustion and active proliferation. To determine whether the HSPCs
lacking PDK1 were less quiescent, I performed a Hoescht 33342 dye labeling experiment to identify the
“side population”, or the quiescent cells. According to Goodell et al. 1996, quiescent cells have highly
active ABC transport pumps and therefore, cells that are active retain the dye longer than those that are
quiescent. In the CD150+SK fraction, there was a reduction in the relative percentage of side population
cells (8.5±0.2% in control compared to 3.8±0.9% in Pdk1Hem-KO; Figure 16 C), indicating that these cells
were quiescent and therefore not active. This assay also corroborates that there are reduced HSC
numbers in the Pdk1Hem-KO mice. Pyronin Y is an intercalating dye that is used to measure the separation
between G0 and G1 phases in the cell cycle. Analysis of the Pdk1Hem-KO LSK revealed a reduction in both
the relative and absolute cell numbers in G0 when compared to the control group (14.4±0.9% in control
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compared to 8.6±1.5% in Pdk1Hem-KO; Figure 16 D). Taken together, it appears that fewer Pdk1Hem-KO
HSCs are inactive suggesting that they are proliferating more.
Figure 17: Pdk1Hem-KO progenitor cells proliferate more.
(A) Representative flow cytometric plots of LSK and HSC (CD150+CD48-LSK) show increased levels of cell cycle protein, Ki67 (n = 6). (B) Mice pulsed for 16h with a DNA intercalating agent, 5-bromouridine (BrdU), revealed increased incorporation in Pdk1Hem-KO LSK and HSCs (CD150+CD48-LSK) (n = 6). Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
In order to repopulate the blood cell pool, HSCs need to proliferate and differentiate to meet the
demands of the organism. To determine whether the Pdk1Hem-KO BM had proliferation defects, 5’-bromo-
uridine (BrdU) was administered for a pulse of 16 hours. BrdU is a synthetic thymidine analog that that
intercalates into replicating DNA. Flow cytometric analysis for BrdUhigh cells revealed a significant
increase in the LSK (36.8±4.5% in control compared to 55.9±5.6%in the Pdk1Hem-KO) and CD150+CD48-
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LSK (15.5±2.0% in control compared to 40.9±8.1% in the Pdk1Hem-KO; Figure 17 A) fractions. To
corroborate that the Pdk1Hem-KO HSPCs were cycling more, I next analyzed expression of Ki67, a cell
cycle protein that is present in active phases of the cell cycle. Immuno-staining showed that there were
increased levels of Ki67 in the Pdk1Hem-KO LSK (51.2±3.0% in control compared to 65.1±10% in Pdk1Hem-
KO) and CD150+CD48-LSK (39.5±8.1% in control compared to 66.3±2.8% in Pdk1Hem-KO) fractions (Figure
17 B). Taken together, the increased levels of Ki67 and BrdUhigh cells in Pdk1Hem-KO HSPCs suggested
that these cells were hyperproliferating.
HSPCs have increased cell death and lineage differentiation in vitro
Since Pdk1Hem-KO HPSCs cells were proliferating more, I wanted to determine whether this
resulted in increased differentiation or apoptosis. To determine this, control and Pdk1Hem-KO LSK cells
were stained with a proliferation dye, cultured and assayed for proliferation, cell death, and differentiation
at 24, 48, and 72h. CellTrace is a fluorescent dye that is initially taken up by the cell cytoplasm (Figure 18
A). As the cell divides, the concentration of dye in each cell decreases as it is shared by the daughter
cells. In this way, the amount of dye present in the cell indicates the amount of proliferation cells are
undergoing, i.e. cells with increased proliferation exhibit weaker florescence. At all time points analyzed,
the PDK1-deficient cells showed high mean florescence indicating reduced proliferation, when compared
to the control cells (Figure 18 B & C; 24h: 15876±444 in control compared to 23844±2052 in Pdk1Hem-KO,
48h: 8424±493 in control compared to 14204±382 in Pdk1Hem-KO, 72h: 6572±1266 in control compared to
11288±1909 in Pdk1Hem-KO). Interestingly, cells stained for expression of AnnexinV+7AAD- showed
increased cell death in the Pdk1Hem-KO LSK at all time points (Figure 18 D; 24h: 10.9±1.4% in control and
27±2.2% in Pdk1Hem-KO, 48h: 15.1±5.1% in control and 28.0±2.6% in Pdk1Hem-KO, 72h: 22.1±4.9% in
control compared to 50.6±4.8% in Pdk1Hem-KO). Lastly, Pdk1Hem-KO LSK cells had more cells that were
lineage positive (CD11b+, B220+, CD19+, Ly6G+, Ter119+, CD3e+) at 72h (Figure 18 E; 26.2±9.5% in
control compared to 34.4±7.2% in Pdk1Hem-KO) suggesting these cells differentiated more. In summary,
unlike the in vivo phenotype, the cultured HSPC results suggest that when isolated, PDK1-deficient
HSPCs have increased cell death, do not hyperproliferate, and are more likely to terminally differentiate.
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Figure 18: In vitro culture reveals that Pdk1Hem-KO LSK cells are apoptotic.
(A) Cartoon depicting LSK isolation scheme. LSK cells were cultured at 37C with stem cell cytokines and analyzed for CellTrace expression at 24h, 48h, and 72h. (B) Representative flow cytometric plots (C) and geometric mean florescence intensity (GMI) show increased CellTrace expression meaning cultured Pdk1Hem-KO LSK cells are less proliferative. (D) Cell death analysis at 24h, 48h, and 72h reveals increased Annexin V expression in PDK1-deficient cells. (E) Isolated Pdk1Hem-KO LSK cells significantly differentiate more into lineage-positive cells at 72h. Data are of 6 wells from two independent experiments. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
Discussion
Lineage disturbances in the Pdk1Hem-KO blood could be due to a variety of reasons including
fewer, less functionally competent HSCs. The results from this chapter shed light on the functional ability
of Pdk1Hem-KO HSCs. Since Pdk1Hem-KO cells could be detected in recipient BM and total BMT was unable
to reconstitute the blood cell pool after transplantation, it suggested that the PDK1-deficient cells were
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able to engraft but are not able to overcome the trauma of transplantation and thus fulfill their role as
donor HSCs. Furthermore, competitive BMTs showed that wild type cells outcompeted mutant cells to
reconstitute the blood supply in recipient mice, further providing evidence that Pdk1Hem-KO HSCs could not
appropriately proliferate, differentiate, and self-renew when stressed. Further experiments showed that
loss of reconstitution ability maybe a result of reduced quiescence and hyperproliferation of Pdk1Hem-KO
cells. Taken together, these results suggest that PDK1 is needed for the maintenance of quiescence and
self-renewal properties of HSCs.
BMT analysis
While the results from the total and competitive BMTs conclusively show that Pdk1Hem-KO BM
cannot provide radioprotection to lethally irradiated recipients, there are a few things to consider when
trying to determine the cause. First, although 1 x 106 total BM cells of control and mutant were injected
into each recipient, the composition of the two types of cells differed so fewer Pdk1Hem-KO HSCs could
have been transplanted per recipient compared to control recipients. This difference, however, seems
negligible since theoretically, only one HSC is needed to repopulate the blood system. However, further
homing experiments are needed to properly determine this. Very severe engraftment defects following the
total and competitive transplantation of Pdk1Hem-KO BM cells can also be explained by increased sensitivity
of HSCs to radiation stress. To address this possibility, further studies including a transplantation assay
following sub-lethal irradiation are needed. Detection of higher chimerism of donor cells after
transplantation, would suggest that Pdk1Hem-KO cells are unable to repopulate the entire blood system but
are able to contribute to partial repopulation of the blood. In essence, Pdk1Hem-KO HSCs require a boost
from the host wild type cells since they cannot fully perform their duties in a stressed environment. This is
logical in the context of survivability of the Pdk1Hem-KO animals since they survive well into adulthood (8+
weeks), after the transition from fetal to adult BM has occurred. Injection of PDK1-deficient BM into
partially irradiated donors seems excessive given that competitive BMTs more or less delineate the same
answer. Furthermore, it would be interesting to see if the Pdk1Hem-KO niche was compromised since a
study (Schepers et al., 2013) reports that hyperproliferative myeloid cells can impact cells of the
endosteal niche and the PDK1-deficient BM has increased numbers of monocytes. To assess this, wild
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type donor BM could be injected into sub-lethally irradiated Pdk1Hem-KO recipients and hematopoietic
output could be assessed. HSCs receive many quiescent and self-renewing signals from the niche so
irradiation would make it necessary for the host and donor cells to repopulate the BM. However, if the
external signals coming from the niche are altered, the wild type HSCs may not be able to provide
radioprotection. While this would be interesting, it is beyond the scope of this paper, which focuses on the
intrinsic role of PDK1 in HSCs.
Pdk1Hem-KO HSPCs are homing to the spleen
Interestingly, immunophentoyping of the spleen suggests that Pdk1Hem-KO HSCs are exiting the
niche and homing to a secondary site of hematopoiesis. This is common when an organism is
experiencing trauma or injury or the BM is in capable of facilitating hematopoiesis. Although, the LK and
LSK fractions are increased in Pdk1Hem-KO mice, the relative and absolute numbers of CD150+CD48-LSK
are not increased, suggesting that the HSCs found in the spleen are unable to compensate for the lack of
HSCs in the BM. Indeed, the hematopoietic output and number of differentiated lymphoid or myeloid cell
types are still skewed in Pdk1Hem-KO mice, which indicates that the HSPCs in the spleen are ineffective.
Loss of quiescence, hyperproliferation and increased differentiation of PDK1-deficient cells
Through multiple assays, it is highly evident that Pdk1Hem-KO HSCs, loose quiescence and self-
renewal ability, so that when they hyperproliferate they are unable to maintain their cell numbers.
Interestingly, the results of the in vivo and in vitro apoptosis and proliferation assays yield opposing
results. Detection of increased apoptosis in vitro but not in vivo can be explained by the fact that there are
clearing mechanisms within the host that could be phagocytosing the dead cells. I tried to stain ex vivo
BM with TUNEL, an apoptosis marker, to corroborate levels of cell death. Preliminary experiments failed
due to lack of detection of BM fractions and it is possible that an enzyme in the assay is cleaving the
conjugated antibodies used to identify different cell populations. Future experiments with sorted cells
using TUNEL or Caspase-3 staining via IHC will be helpful to verify that HSCs are not dying more in vivo.
Furthermore, an artifact of in vitro culture is increased cell death. In addition, cultured HSPCs no longer
receive signals from the in vivo niche, which could include cell survival signals as well as proliferation
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signals. Lack of these signals from the niche, could be a reason why the in vitro CellTrace experiments
suggested reduced LSK proliferation. Regardless of the discrepancies, it is clear based on the BrdU and
Ki67 studies that in vivo HSPCs are cycling more.
The in vitro culture experiments shed light into the ramifications of hyperproliferation. It seems
that after 72 hours, Pdk1Hem-KO LSK cells are more likely to have expression for lineage markers, which
are indicative of specific differentiated cell types. In order to determine the full differentiation capacity of
PDK1-deficient HSCs, the cells could be sorted and differentiated in vitro in either liquid culture or on
semi-solid medium. While an elegant differentiation study would be thorough, the results from this study,
as well as others with lineage specific Cre lines have shown that PDK1 is needed for proper
differentiation of the lymphoid lineage.
The results from this study provide a rationale for reduced HSC numbers in the Pdk1Hem-KO
mouse but the reason for cellular status is affected, loss of quiescence and increased proliferation, is
something that remains to be determined. Exploration into the mechanisms that govern HSC quiescence
and cycling will elucidate what is happening in the Pdk1Hem-KO HSCs at a molecular level.
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Chapter 4: PDK1-deficient HSPCs exhibit reduced response to
oxidative stress
Introduction
Oxidative Stress
When the body is subjected to physical and metabolic stressors, an imbalance of free radicals
accumulates within cells and organs and can cause oxidative stress. The generation of free radicals is
fueled by events outside the cell particularly from environmental insults such as infection and disease that
activate immune responses, exposure to heat or cold that trigger heat shock proteins, oxygen deprivation
or hypoxia possibly from trauma or exercise, metabolic deprivation such as from starvation, and ionizing
radiation from natural and man-made sources (Mendelson and Frenette, 2014). Exposure to these events
can cause the cell to generate excessive amounts of reactive oxygen species (ROS), particularly from the
mitochondria, which can lead to signaling cascades and by-products that can cause DNA damage and
cell death (Bigarella et al., 2014; Wilson et al., 2008). High levels of ROS can interact with proteins
causing amino acid oxidation therefore impacting protein signaling and degradation (Bigarella et al., 2014;
Jensen, 2006). Similarly, undue ER stress induced by unfolded proteins or metabolic perturbations can
also generate ROS and then lead to increased autophagy and increased cell death (Zhang, 2010) (Figure
19 C). Taken together, it is clear that metabolic homeostasis and the regulation of ROS within a cell, such
as HSCs, must be maintained or there will be drastic consequences to HSC viability and production of
lineage-specific cells.
Mitochondrial dynamics
One of the largest contributors to oxidative stress within a cell is the mitochondrion. Indeed,
roughly 2% of the oxygen consumed by mitochondria is estimated to reduce to ROS (Finkel, 2012; Handy
and Loscalzo, 2012). Large amounts of ROS are generated by the mitochondrion when the oxidative
phosphorylation pathway (OXPHOS) is used for energy production. In brief, energy production begins
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with glycolysis, which is when glucose is converted to pyruvate. The intermediate of glucose, glucose 6-
phosphate, can be bypassed to the pentose phosphate pathway which can regenerate co-factors needed
during glycolysis as well as the antioxidant glutathione which can repress ROS. Once pyruvate is formed,
it can be directed towards the tricarboxylic acid cycle, which powers the electron transport chain to
produce adenosine triphosphate (ATP). Most intracellular ROS is generated from the electron transport
chain (Bigarella et al., 2014; Ito and Suda, 2014; Suda et al., 2011). Interestingly, progenitors and
differentiated cells predominantly use OXPHOS for energy production while stem cells use glycolysis (Ito
and Suda, 2014; Simsek et al., 2010; Zhang and Sadek, 2014). In addition, it has been shown that
quiescent HSCs have fewer mitochondria when compared to differentiated cells (Lewandowski et al.,
2010), since differentiated cells need mitochondria for OXPHOS ATP production. Taken together, this
suggests that in order to maintain homeostasis, HSCs have metabolic mechanisms that encourage low
levels of ROS production. Many dyes exist to detect intracellular ROS, as well as mitochondria mass, and
mitochondria membrane potential. Furthermore, metabolomics can be used to assess energy output
associated with either glycolysis or OXHPOS. For instance, cellular oxygen consumption, lactate
production, ATP production, NADH levels, and drug inhibition to measure basal and maximal cellular
respiration can provide insight into the energetic output of a cell. These assays can provide insight into
the mitochondrial metabolic properties of Pdk1Hem-KO HSCs.
ROS and HSC maintenance
Many avenues of evidence implicate ROS as a regulator in stem cell activation and progenitor
differentiation. ROS can interact with amino acids in proteins through oxidative modification to influence
many of the signaling cascades that are necessary to activate the transcription of differentiation programs
(Bigarella et al., 2014). Furthermore, work by other groups has suggested that ROS regulates the
epigenetic landscape, a vital component in controlling stem cell fate (Bigarella et al., 2014; Challen et al.,
2012). In addition, major sources of ROS generation can impact stem cell properties. For instance, ROS
due to high levels of irradiation can alter HSC repopulating abilities (Cao et al., 2011; Ito et al., 2004; Ito
et al., 2006). In addition, OXPHOS produces ROS, and overactivation of OXPHOS led to loss of stem cell
dynamics and inhibition of OXPHOS by HIF activation improved proliferation and maintenance of
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embryonic stem cells (Bigarella et al., 2014). Reduced OXPHOS is also seen in HSCs since they have
decreased mitochondrial respiration and are highly dependent on glycolysis (Norddahl et al., 2011;
Unwin, 2006).
Recent work by a number of groups has provided evidence that suggests low ROS levels are vital
to maintain HSCs in an undifferentiated state. Work by Takubo et al. showed that HSCs are highly
positive for pimonidazole, a hypoxic label, and hypoxia has been correlated with lowering levels of ROS
by initiating energy production to glycolysis (Ito and Suda, 2014; Zhang and Sadek, 2014). Furthermore
Jang and Sharkis separated ROShigh and ROSlow HSCs and found that unlike ROSlow HSCs, ROShigh
HSCs undergo stem cell exhaustion much more quickly when serial transplanted. The ROShigh and
ROSlow HSCs were isolated by the same immunophenotype scheme but had different oxidative stress
levels so taken together, this suggests that there is a physiological significance of low levels of ROS in
the maintenance of stem cell self-renewal. For HSCs, the most quiescent and multipotent cell within the
hematopoietic system, a minimal amount of oxidative stress is vital therefore the HSC must remain in an
inactive cycling state that protects the cell from acquiring undue mutations and potential cell death (Naka
and Hirao, 2011; Takubo et al., 2010).
PI3K-signaling regulates ROS levels
Interestingly, genetic knockouts of members in the PI3K/AKT/mTOR-pathway that have
disturbances in HSC quiescence often have an imbalance in oxidative stress. For instance AKT1/2 DKO
HSCs show increased quiescence and decreased levels of ROS (Juntilla et al., 2010). Mutants for TSC,
which is inhibited by AKT activation and negatively regulates mTORC1, have high levels of ROS and
reduced quiescence (Chen et al., 2009). In addition, mice lacking FoxO1/3a/4 show an increase in LSK
cells, which correlates with an increase in ROS and a decrease in quiescence (Miyamoto et al., 2007;
Rimmele et al., 2015; Tothova and Gilliland, 2007). The Pdk1Hem-KO HSCs are proliferating more and are
less quiescent. Therefore, I hypothesize that the loss of a regulator, PDK1, upstream of the AKT/mTOR-
pathway, changes the oxidative stress in HSCs.
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Results
Microarray analysis reveals Pdk1Hem-KO HSPCs have reduced response to oxidative stress
The results in Chapter 3 suggest that HSCs lacking PDK1 loose quiescence, have impaired self-
renewal, and are hyperproliferative. To gain an understanding of why Pdk1Hem-KO HSCs elicit this aberrant
phenotype, I wanted to know which transcriptional targets were deregulated in PDK1-deficient cells. This
would provide insight into the molecular mechanisms that were governing PDK1-mediated HSC
quiescence. To determine the transcriptional regulation differences between control and Pdk1Hem-KO
HSCs, RNA was extracted from sorted LSK cells, converted to cDNA, and then processed for microarray
analysis. Microarray data are useful since they provide a broad overview of gene expression data within a
sample. Using Gene Set Enrichment Analysis (GSEA), I first checked to see whether genes associated
with the HSC profile were affected. GSEA compares the gene clusters associated with our data with data
sets that are publically available. As expected, there was a loss of genes associated with HSC identity in
Pdk1Hem-KO cells (Figure 19 A). In addition, the GSEA indicated that PDK1-deficient HSPCs have reduced
ability to respond to oxidative stress (Figure 19 B). This intrigued me considering other mutants of the
AKT/mTOR pathway experience differences in ROS expression while also seeing differences in HSC
quiescence. Taken together, the microarray data indicate that the loss of PDK1-signaling does result in
different transcriptional programs compared to normal HSCs, and that Pdk1Hem-KO HSPCs may have
changes in metabolic activity.
Deficiency of PDK1 in HSPCs alters the transcription factor networks associated with HSCs and
oxidative stress
The results from the GSEA analysis suggested that the transcription networks between the
Pdk1Hem-KO mice were altered in HSC signature and in the machinery to oxidative stress when compared
to the control mice. To understand the molecular basis for decreased HSC quiescence in PDK1-deficient
HSCs I selected transcription factors (TFs) that were differentially up- or down-regulated in the conditional
knockout. I focused on TFs since their activity is required to promote HSC quiescence, self-renewal, and
differentiation (Orkin, 2000). After I selected any gene higher than a 1.5 expression ratio (Pdk1Hem-KO
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expression/control expression) for up-regulated genes and any gene lower than 0.5 (Pdk1Hem-KO
expression/control expression) for down-regulated genes, there was a list of 422 up-regulated TFs and
128 down-regulated TFs (Table 2 and Table 3). I further narrowed this list by selecting for genes known to
be involved in hematopoiesis, the hypoxic response, or oxidative stress (Table 4). Interestingly, many of
the differentially regulated genes correlated with the Pdk1Hem-KO BM phenotype (Agrawal et al., 2008;
Buscarlet et al., 2014; Carreras et al., 2010; Chan et al., 2001; Elvert et al., 2003; Feng et al., 2010;
Finlay, 2014; Fox et al., 2004; Gross et al., 2008; Jin et al., 2008; Kawanami et al., 2009; Kuure et al.,
2010; Leiherer et al., 2014; Lukin et al., 2010; McKinney-Freeman et al., 2012; Morosetti et al., 1997;
Passegue and Ernst, 2009; Pevny et al., 1991; Schuettpelz et al., 2012; So et al., 2004; Tian et al., 2012;
Ubeda et al., 1999; van der Veer et al., 2014; Wolfler et al., 2010; Xu et al., 2005; Yamamoto et al., 2011;
Yap et al., 2005; Zhou et al., 2015). For example, Klf7, whose overexpression suppresses HSPC
functions (Schuettpelz et al., 2012), is up-regulated, This corroborates with the loss of expression of HSC
maintenance and differentiation genes: Hopx, Fos, Fosb Klf5, and Tal1 (McKinney-Freeman et al., 2012;
Zhou et al., 2015). In addition, Cebpa and Cebpe, two genes that direct myeloid differentiation (Morosetti
et al., 1997; Wolfler et al., 2010), were increased. The data also suggested that inhibitors of HIF1a were
increased, Cited2 and HoxA7 (Agrawal et al., 2008; So et al., 2004), while a downstream target of Hif1,
Fosl2 (Leiherer et al., 2014), was decreased. Furthermore, genes involved in oxidative metabolism, such
as Ncor1 and Nfe2l2 (Chan et al., 2001; Yamamoto et al., 2011), were deregulated. Taken together, this
data suggests that many of the differentially regulated TFs in the Pdk1Hem-KO LSK may explain why PDK1-
deficient HSCs lose quiescence and are unable to respond to oxidative stress. However, further qRT-
PCR experiments are needed to validate the results described in the microarray.
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Figure 19: Pdk1Hem-KO LSK cells exhibit reduced response to oxidative stress.
(A) Microarray GSEA analysis of control and Pdk1Hem-KO LSK cells indicate that the HSC signature is lost and (B) that there is reduced ability to respond to oxidative stress in Pdk1-deficient cells. (C) Schematic representing how oxidative stress can affect an HSC cell.
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Table 2: Up-regulated genes in the Pdk1Hem-KO microarray.
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Table 3: Down-regulated genes in the Pdk1Hem-KO LSK microarray.
Table 4: Selected transcription factors involved in hematopoiesis or oxidative stress.
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PDK1-deficient cells have increased levels of ROS
One of the hallmarks of oxidative stress is the presence of reactive oxygen species (ROS; Figure
19 C). To determine whether the Pdk1Hem-KO HSCs exhibited increased ROS-related oxygen stress, I
stained freshly isolated BM from control and Pdk1Hem-KO mice with a dye that measures acetate
intracellular esterases and oxidation, CM-H2DCFDA. I found increased levels of ROShigh expressing cells
in all of the blood cell populations analyzed (Figure 20 A & B) including the CD150+CD48-LSK HSC
fraction (4.2±0.9% in control compared to 10.4±2.4% in Pdk1Hem-KO), the progenitor LSK fraction
(8.2±1.2% in control compared to 28.8±6.5% in Pdk1Hem-KO), the progenitor LK fraction (35.4±3.0% in
control compared to 49.1±6.3% in Pdk1Hem-KO) and the lineage negative fraction (27.7±4.2% in control
compared to 40.5±4.6% in Pdk1Hem-KO). CM-H2DCFDA is considered to be a general oxidative stress
indicator; however, other specific forms of oxidative stress can be found within the cell. To assay for
oxidative stress due to mitochondria specific superoxide, I stained control and Pdk1Hem-KO BM with
MitoSOX Red. Although I did not detect significant increases in superoxide levels in the HSPC fractions, I
did detect significant increases in oxidative stress due to superoxide in the PDK1-deficient lineage
negative fraction (Figure 20 C & D; 7.6±2.3% in control compared to 17.6±4.4% in Pdk1Hem-KO).
Interestingly, previous reports have shown that high levels of ROS can activate the p38 MAPK pathway,
which then promotes HSC exhaustion and loss of HSC quiescence (Ito et al., 2006; Jang and Sharkis,
2007). To determine whether this pathway was activated in the absence of PDK1, protein was extracted
from wild-type total BM that was either treated with or without 1uM PDK1 inhibitor. Immunoblots showed
an increase of phosphorylated p38 in the absence of PDK1-signaling (Figure 20 E). To corroborate this
with the in vivo model, I next looked at the p38 GSEA from the microarray and indeed, there was
increased transcription of p38 targets in Pdk1Hem-KO LSK cells (Figure 20 F). These experiments
suggested that Pdk1-deficient cells have high levels of ROS that trigger aberrant signaling in associated
pathways. Consequently, a high level of oxidative stress can have detrimental effects on the cell such as
increased proliferation, providing opportunities for DNA damage, apoptosis, and eventually HSC
exhaustion (Figure 19 C).
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Figure 20: PDK1-deficient cells have augmented ROS levels.
(A) Representative flow cytometry plots of cells labeled with CM-H2DCFDA, a dye that measures reactive oxygen species. (B) Analysis of CM-H2DCFDA high cells showed increased ROS in the Pdk1Hem-KO BM (n = 13). (C&D) Cells labeled for mitoSOX, which measures ROS due to mitochondrial superoxide levels, showed an increase in the lineage negative fraction in the mutant (n = 5). (E) Immunoblots of total BM treated with and without 1uM PDK1 inhibitor showed increased p-p38 expression when Pdk1 activity is blocked (n = 2). (F) Microarray analysis revealed that p38 transcriptional targets were up in the Pdk1Hem-KO LSK cells (pool of 5 mice). Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
To determine the functional consequences of increased ROS levels, I first analyzed the
progenitor populations for proliferation. Ki67 is expressed during active phases of the cell cycle and the
Pdk1Hem-KO LK (51.2±2.9% in control compared to 70.8±4.6% in Pdk1Hem-KO) and lineage-negative
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(47.3±4.3% in control compared to 64.5±3.9% in Pdk1Hem-KO) fractions have more Ki67high cells (Figure
4.3A). These results followed the trend I saw with the BrdU and Ki67 analysis of the LSK and
CD150+CD48-LSK in Figure 17. Increased cell cycling exposes the cells’ DNA to mutagens and other
harmful events that can cause damage. To assess whether increased levels of ROS resulted in increased
levels of DNA damage, we assayed for double stranded breaks. Double stranded breaks are
phosphorylated upon cleavage; therefore, control and Pdk1Hem-KO BM were stained with antibody against
phospho-H2A.X. Although there was a trending increase in cells with high levels of DNA damage in all
BM populations analyzed, only the lineage negative fraction was at significant levels (Figure 21 B & C;
13.6±3.6% in control compared to 32.4±4.4% in Pdk1Hem-KO). Cell death analysis of the LSK and
CD150+CD48-LSK showed no significant difference in apoptosis in vivo (Figure 16 A & B); however,
analyzing the progenitor populations revealed that the PDK1-deficient LK (2.4±0.3% in control compared
to 3.6±0.3% in Pdk1Hem-KO) and lineage-negative (6.4±0.7% in control compared to 8.0±0.7% in Pdk1Hem-
KO) fractions have more AnnexinV+7AAD- cells (Figure 21 D). Taken together, perturbations in PDK1-
mediated signaling lead to higher levels of oxidative stress that results in increased cell proliferation, DNA
damage, and cell death.
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Figure 21: Pdk1Hem-KO progenitor cells have increased DNA damage.
(A) Quantification of Ki67 expression shows that Pdk1Hem-KO LK and Lineage negative populations are proliferating more (n = 6). (B) Representative flow cytometry plots and (C) quantification of cells stained for phosphorylation of double stranded breaks, p-H2A.X, shows increased DNA damage in the Pdk1Hem-KO (n = 5 except for CD150+CD48-LSK: n = 3). (D) Quantification of AnnexinV+7AAD- expression shows increased cell death in the Pdk1Hem-KO LK and Lineage negative cells (n = 11). Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
Treatment with NAC suppresses proliferation and differentiation in PDK1-deficient cells
High levels of oxidative stress can be mediated by antioxidants, which terminate the oxidative
chain reactions that lead to ROS generation. N-acetyl cysteine (NAC) is a free radical scavenger that has
been shown to decrease ROS levels and revert hematopoietic physiologic pathologies due to oxidative
stress (Tothova et al., 2007). To determine whether NAC can rescue hematopoietic disturbances in
PDK1-deficient cells, lineage depleted control and Pdk1Hem-KO total BM were cultured with or without NAC.
After 24 hours the cells were harvested and immunophenotyped. Pdk1Hem-KO cells treated with NAC had
decreased levels of lineage positive cells (Figure 22 B; 81.5±18.3% in Pdk1Hem-KO compared to 67.2±3.3%
in Pdk1Hem-KO with NAC) and increased numbers of lineage negative cells (Figure 22 C; 18.0±1.8% in
Pdk1Hem-KO compared to 30.9±3.1% in Pdk1Hem-KO with NAC) when compared to the untreated mutant
cells. These results suggest that treatment with NAC was able to block lineage negative cells from
differentiating into lineage positive cells (Table 5).
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Figure 22: NAC treatment suppresses differentiation and proliferation of Pdk1Hem-KO HSPCs.
(A) Representative flow cytometry plots of lineage negative cells cultured for 24h with PBS or NAC. (B) Culturing Pdk1Hem-KO lineage negative cells with NAC in vitro for 24h resulted in reduced lineage positive cells (C) and increased lineage negative cells when compared to Pdk1Hem-KO cells cultured with PBS. (D) Representative flow cytometry blots of HSCs (CD150+CD48-LSK) from mice treated with PBS or NAC. (E) Pdk1Hem-KO mice treated with NAC retained lineage negative cells although not to significance. (F) Pdk1Hem-KO mice treated with NAC have increased LK cells, (G) fewer LSK cells, and (H) increased HSCs (CD150+CD48-LSK) when compared to Pdk1Hem-KO mice treated with PBS. In vitro data are representative triplicate wells from two independent experiments. In vivo data are a merge of three independent experiments and are represented as mean ± SEM. Control: n = 4; control + NAC: n = 6; Pdk1Hem-KO: n = 5; Pdk1Hem-KO + NAC: n = 6. Two-tailed paired (in vitro) and unpaired (in vivo) Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
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The in vitro NAC results were promising but it still did not provide evidence whether this
mechanism worked in vivo as well. To assess whether BM lineage defects could be rescued in vivo, I
injected control and Pdk1Hem-KO 2-week-old mice with 100mg/kg NAC or PBS daily for 4 weeks. After
treatment, BM was harvested and immunophenotyped for HSPCs. Compared to untreated Pdk1Hem-KO
mice, mutant mice treated with NAC had increased numbers of lineage negative cells, increased numbers
of LK cells, and reduced LSK cells (Figure 22 E – G, Table 6). Most importantly, treating Pdk1Hem-KO mice
with NAC increased the numbers of CD150+CD48-LSK (Figure 22 D & H, Table 6). This suggests that
NAC is able to prevent hyperproliferation and increased differentiation of Pdk1Hem-KO cells. Since NAC is
an antioxidant, I wanted to know whether the rescue of HSPC numbers was due to ROS scavenging by
the drug. To determine whether retention of CD150+CD48-LSK numbers in Pdk1Hem-KO treated with NAC
were due to reduced ROS levels, BM cells were stained with CM-H2DCFDA, that detects ROS as a
reflection of oxidative stress. The results revealed that while the mean florescence values of both control
and Pdk1Hem-KO cells were reduced compared to the untreated mice in all BM fractions, only ROShigh cells
in LK were decreased at significant levels (Table 7). This implies that NAC treatment is able to partially
rescue the detrimental ROS phenotype. Therefore, ROS appears to increase in Pdk1Hem-KO HSCs and
accounts for the loss in quiescence and subsequent increase in proliferate.
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Table 5: In vitro NAC treatments limits differentiation of Pdk1Hem-KO lineage negative cells.
Sorted control and Pdk1Hem-KO lineage negative cells were cultured with PBS or NAC for 24h and then stained for cell-surface markers to assess hematopoietic fractions. Data are representative of triplicate wells from two independent experiments. std = standard deviation, SEM = standard error margin. Paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
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Table 6: In vivo NAC treatment impedes proliferation of Pdk1Hem-KO progenitors.
Control and Pdk1Hem-KO mice were either treated with PBS or 100mg/kg NAC for 4 weeks and then dissected and BM was stained for cell-surface markers to assess hematopoietic fractions. Data are representative of 3 independent groups (control: n = 4, control + NAC: n = 6, Pdk1Hem-KO: n = 5, Pdk1Hem-KO + NAC: n = 6). std = standard deviation, SEM = standard error margin. Unpaired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
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Table 7: In vivo NAC treatment significantly reduces ROS levels in Pdk1Hem-KO LK.
Control and Pdk1Hem-KO mice were either treated with PBS or 100mg/kg NAC for 4 weeks. Values represent the mean percentage of gated ROShigh cells. Data are representative of 3 independent groups (control: n = 4, control + NAC: n = 6, Pdk1Hem-KO: n = 5, Pdk1Hem-KO + NAC: n = 6). std = standard deviation, SEM = standard error margin. Unpaired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
Discussion
In summary, microarray data of Pdk1Hem-KO HSPCs suggest that they are unable to respond to
oxidative stress and indeed, there is accumulation of ROS in these cells. Furthermore, progenitor cells
are more proliferative, have higher levels of DNA damage, and increased cell death in the absence of
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PDK1. Interestingly, in vivo NAC treatment aimed to inhibit ROS is only able to partially rescue the ROS
phenotype; however, NAC treatment increases retention of HSPC populations both in vitro and in vivo.
Microarray
While the microarray led to exciting insight on the physiological status of PDK1-deficient cells,
regrettably, the results of the microarray are not from a pure HSC population. The LSK fraction is
composed of both HSCs and MPPs so the gene expression data is representative of numerous cell
populations rather than purified ones. This could allow for misinterpretation of which gene sets are
actually differential in the Pdk1Hem-KO mouse. For instance, a set of genes could be increased in the HSCs
but severely decreased in MPPs and the microarray would not be able to discriminate separate profiles
because my samples contained both populations. Unfortunately, due to the number of available mice and
extremely low HSC count in the Pdk1Hem-KO mouse, I was unable to obtain enough RNA when I isolated
only HSCs (LSKF-). Indeed, the results from this one microarray are a pool of enriched LSK that were
sorted from 5 animals in each group. Currently, a more accurate method for determining broad scale
gene expression levels as well as other components of the cellular transcriptome is through RNA-
sequencing. Performing this assay would be contingent on obtaining enough material; however, with the
latest technologies in single cell RNA-seq, RNA-seq on LSKF- or even LSKF-CD34-CD150+CD48- cells
would provide a more accurate representation of Pdk1Hem-KO HSC gene expression which could then be
validated at the protein and signaling levels. Despite the limitations, the microarray did serve to provide
an answer regarding why Pdk1Hem-KO mice were loosing quiescence: low response to oxidative stress.
This was corroborated by subsequent experiments measuring ROS and superoxide ROS levels.
PDK1 is an AGC kinase that is capable of activating a variety of downstream signaling pathways
to govern cellular processes. Earlier studies have shown that PDK1 is important for NFkB activation in T
cells (Lee et al., 2005). In addition, loss of the NFkB subunit p65 (RelA) causes increased HSPC cycling,
loss of HSPC differentiation ability (Stein and Baldwin, 2013). Microarray analysis of NFkB targets in
Pdk1Hem-KO LSK cells yielded no significant changes compared to the control (data not shown). Based on
this, it is possible that in HSCs, PDK1 is indispensible for NFkB activation; however, more extensive
studies assessing NFkB-signaling in the Pdk1Hem-KO BM need to be performed in order to determine this.
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Furthermore, other groups as well as this study (Chapter 5) have shown that the loss of PDK1 also leads
to a reduction in AKT-signaling. In addition, loss of the AKT/FOXO-signaling pathway in HSCs has been
shown to cause a decrease in HSPCs due to increased cycling and apoptosis. Furthermore,
FOXO1/3a/4-deficient HSCs were found to have increased ROS levels that could be reverted with the
addition of an antioxidant drug (Miyamoto et al., 2007; Tothova and Gilliland, 2007). Given that many of
the stem cell physiological properties are altered in both Pdk1Hem-KO HSCs and FOXO-deficient HSCs and
that Pdk1Hem-KO HSCs have reduced AKT-signaling, I also looked at FOXO targets with the microarray
and did not see any differences between control and Pdk1Hem-KO LSK. Although it is believed that FOXO is
highly regulated by AKT, there are other non-AKT-mediated PTMs that can regulate FOXO activity in the
nucleus and it is possible that FOXO is being regulated by a PDK1/AKT-independent mechanism (Obsil
and Obsilova, 2008). Thus would make sense that a loss of AKT-signaling should lead to an increase in
FOXO activity; however, I do not see an increase in FOXO targets. While the microarray provides
insights, further experiments immunoblotting for FOXO with Pdk1Hem-KO cells or phosflow of HSCs would
be useful to determine a comprehensive understanding of the downstream consequences of the loss of
PDK1.
Mitochondrial bioenergetics
The results from labeling HSCs with CM-H2DCFDA provided a physiological explanation for the
Pdk1Hem-KO phenotype. Using CM-H2DCFDA, I was able to assess that there are more cells that are
considered to be expressing high levels of ROS. Interestingly, various dyes exist that target organelle
specific ROS as well as the different chemical components responsible for ROS, H2O2 and NO. It is
important to note that there are genetic methods to alter cells so that they emit florescence (Woolley et
al., 2013); however, in the HSC field, the most commonly used method to detect ROS levels is staining
cells with the florescent CM-H2DCFDA dye which is activated upon reduction and it is detected via flow
cytometry (Chen et al., 2008; Ito et al., 2004; Ito et al., 2006; Jang and Sharkis, 2007; Juntilla et al., 2010;
Kocabas et al., 2012; Lee et al., 2010; Miyamoto et al., 2007; Nakada et al., 2010; Qian et al., 2016;
Rimmele et al., 2015; Tothova and Gilliland, 2007; Warr et al., 2013; Yu et al., 2013). Employing this
method makes use of the fact that an isolated population of cells can be identified either by cell surface
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markers or by purification via cell sorting and the overall florescence within a population can be measured
by mean florescence intensity. This provides a quantifiable measure of the number of cells expressing a
certain intensity, based on the florescence of a particular dye, which in this case is a readout of ROS. In
addition, unlike many other cell types, hematopoietic cells are free floating allowing them to easily be
studied by flow cytometry rather than adhering, fixing, and then staining the cells in preparation for
analysis by another methods such as confocal analysis of immunohistochemistry, which is more time
consuming, more labor intensive, and more expensive. Furthermore, using flow cytometry analysis, high
ROS expressing cells can be distinguished from low ROS expressing cells based on the level of
florescence intensity. This is particularly useful given that some studies have shown that the level of HSC
quiescence is based on the amount of ROS within the niche (Jang and Sharkis, 2007; Ludin et al., 2014).
Furthermore, since ROS has been shown to cause a variety of cellular events, such as protein
modification and DNA damage, I verified this in the Pdk1Hem-KO cells. The purpose of this study was to
determine why PDK1-deficient HSCs are no longer quiescent with a focus on what the cellular
consequences are for increased ROS, and not the exact location where ROS generation occurs.
However, it should be noted that there are reports that mitochondrial ROS can be measured by staining
with MitoTracker and performing confocal microscopy (Li et al., 2013).
Since CM-H2DCFDA measures general ROS levels, I also additionally assayed for ROS
generated by superoxide. Although the trend seen with mitoSOX staining was similar to the CM-
H2DCFDA staining, it was only significant in the lineage-negative population. It is possible that assaying a
greater number of animals would provide a more accurate assessment and may eventually lead to
significant differences. Conversely, there may actually be less superoxide ROS in the HSPC fractions.
Interestingly, ROS and superoxide-specific ROS have been shown to promote differentiation (Sauer et
al., 2001; St Clair et al., 1994; Zhang et al., 2013); therefore, significant expression in the lineage-
negative fraction could be indicative of increased differentiation. Furthermore, it has been shown that
myeloid cells need higher levels of ROS in order to differentiate properly (Tothova and Gilliland, 2007;
Zhang et al., 2013). Thus, it is plausible that detection of superoxide ROS is highest in the lineage-
negative fraction because Pdk1Hem-KO BM is predominately composed of myeloid progenitors that are
known to readily generate ROS during differentiation (Chapter 2). In this study, I did not assay for free
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radical production due to H2O2. However, when I incubated control and Pdk1Hem-KO BM with H2O2 as a
positive control and then stained with CM-H2DCFDA, I saw significantly increased ROShigh cells in the
control and a slight shift in the Pdk1Hem-KO BM (data not shown). Firstly, this suggests that the Pdk1Hem-KO
cells were already producing almost maximum ROS levels. Secondly, CM-H2DCFDA detects free radicals
produced by H2O2 so it is not necessary to measure what percentage of ROS is due specifically to H2O2
especially since the mitoSOX staining is in line with the CM-H2DCFDA staining. Taken together, detecting
ROS levels with CM-H2DCFDA provided novel insight into why Pdk1Hem-KO HSCs were cycling more.
Within a cell, one of the primary sites of ROS generation is in the mitochondria. ROS is typically
generated when there is mitochondrial distress. Analysis of Pdk1Hem-KO mitochondrial biogenesis would be
interesting. Preliminary results suggest that Pdk1Hem-KO BM populations have greater mitochondrial mass
when assayed via flow cytometry with a fluorescent dye, MitotrackerGreen. Although this method can
determine the relative mass per cell, it cannot distinguish between the number mitochondrial species
within a cell. Nonetheless, the amount of mitochondrial mass could be explanation for increased
metabolic differences between the control and Pdk1Hem-KO mice and can be used in combination with
other assays to determine the energetic consequences in the mutant cells. A greater number or larger
mitochondria would provide an explanation for increased ROS levels or delineate that the Pdk1Hem-KO
mitochondria are able to generate higher levels ROS per equivalent amount of mitochondria.
Interestingly, treating cells with an electron transport chain inhibitor, Antimycin A, reverted the
mitochondria mass levels back to the control. I did not see a correlated reduction in ROS in this
experiment but that could be due to Antimycin A that is known to cause high levels of H2O2, which can
lead to more ROS. I have only performed this experiment once so further replicates are needed for
verification and statistics. An alternative to assess for mitochondrial mass would be to look for the amount
of mtDNA per cell or to assess for TAM20 or TFAM by protein expression. Similar to the signaling studies,
protein expression by immunoblots is difficult given the number of HSCs per mouse. Furthermore,
assaying for mitochondrial membrane potential through the use of available dyes such as MitotrackerRed
would provide insights into the ability for ATP generation and OXPHOS activity, a major producer of ROS
(Bigarella et al., 2014; Suda et al., 2011). In addition, stress tests can be performed using drugs to target
the oxidation chain to measure cellular respiration or promote and inhibit glycolysis to determine the
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glycolytic output of a cell. These studies would provide an in depth understanding of how loss of PDK1
impacts mitochondrial processes.
Another readout of oxidative stress is autophagy. Autophagy is a part of the lysosome pathway
whereby components within the cell are degraded to prevent cellular damage. Interestingly, loss of ATG7,
a component of the autophagosome causes loss of reconstitution ability of HSCs in transplantation, loss
of lymphoid and myeloid production, increased ROS, and increased mitochondrial mass (Mortensen et
al., 2011). A specific form of autophagy, known as mitophagy, is instrumental in ensuring that aberrantly
active mitochondrial are inactivated. If Pdk1Hem-KO mitochondria are malfunctioning and the machinery to
maintain mitochondrial quality control is affected, then this would provide an explanation for the buildup of
ROS levels. Taken together, further experiments into mitochondria bioenergetics and mitophagy may
provide a rationale for why ROS is elevated in Pdk1Hem-KO cells and may further provide insights for
PDK1-regulation in metabolism.
DNA damage/apoptosis assays
While the results of the p-H2A.X staining showed increased phosphorylation of double stranded
breaks in the lineage negative fraction, the trend is not significant in the HPSC fractions. One reason may
be due to the lower numbers of cells. Only three animals were useable to assess for CD150+CD48-LSK
cells while these cells were not detected in two additional animals. Although PDK1 ablation showed
dramatic effects in the BM, unfortunately the Pdk1Hem-KO mice have very few HSCs, thus limiting the
number of cells we can work with. I assessed p-H2A.X levels via flow cytometry and an alternative
method would be to perform IHC and count the number of p-H2A.X foci per cell (Flach et al., 2014). This
may provide visual representation of p-H2A.X foci in each HSC and this can be counted by a researcher
for statistical numbers; however, since HSCs are hematopoietic cells and are thus free floating, it is just
as easy to stain and asses by flow cytometry in order to generate a readout of the number of cells that
express a certain level of florescence associated with positive stain. Furthermore, since cell surface
markers can be used to distinguish subpopulations, data can be quickly and simultaneously obtained
regarding a variety of cell types proving this method cost and time effective. Since p-H2A.X stain was not
statistically significant in the LT-HSC population, a possible explanation is that DNA damage is not
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increased in PDK1-deficient HSCs. This would suggest that Pdk1Hem-KO HSCs instead proliferate into
progenitors where consequences manifest themselves, such as greater numbers of DSBs (lineage
negative) and apoptosis (LK and lineage negative).
ROS reversion
Intriguingly, I saw an increase of phosphorylated p38 expression by immunoblots from treated
with PDK1 inhibitor and an increase in p38 transcriptional targets in Pdk1Hem-KO LSK from the microarray
analysis. Interestingly, a pilot study where I cultured sorted LK and LSK from control and Pdk1Hem-KO mice
with 10uM p38 inhibitor (data not shown, SB203590, Selleckchem). This treatment did not lower ROS
levels as shown in other studies (Jang and Sharkis, 2007). This suggests that the increase of p38 activity
is a readout of ROS rather than a cause of ROS. This also suggests that there is another target that is
responsible for reducing ROS levels in the PDK1-pathway, which I will expand upon in Chapter 5.
While the results of the in vitro and in vivo NAC treatment studies show that antioxidant exposure
results in retention of HSPC numbers, it was not able to significantly decreases ROS levels as seen in
other studies (Tothova and Gilliland, 2007). Interestingly, other groups have used an inducible deletion
model (Mx-Cre) so that treatment of NAC was administered the day after deletion. In this study, I used
Vav-Cre, which is a temporal and spatially specific hematopoietic marker where vav expression occurs as
early as embryonic day 10.5. The mice in this study were treated with NAC on post-natal day 14 when
there was a large time period in which the Pdk1Hem-KO mice were not treated and this could explain the
partial rescue phenotype. To circumvent this, pregnant mothers could be treated as well as administered
NAC in their drinking water. In addition, pups can be treated from birth so that oxidative stress can be
suppressed during critical periods of lymphoid development. Furthermore, crossing the Pdk1Flox mouse
with Mx-Cre would allow me to treat animals with NAC from the onset of deletion.
It is important to note that the cell has a variety of mechanisms to counteract the accumulation of
ROS. One mechanism is that cell has antioxidants and there are many that have been studied such as:
vitamins, inorganic antioxidants, synthetic antioxidants, and plant-derived polyphenols. One is the
enzyme superoxide dismutase (SOD), which catalyzes superoxide anions into H2O2 and oxygen.
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Catalase is also a ROS scavenger that is found in the cell (Li et al., 2013). In addition, a group of
enzymes, gluthathione peroxidase, degrades H2O2 and peroxides into alcohols. Vitamin C and E have
also been suggested to limit the consequences of an abundance of ROS (Gorrini et al., 2013). Since
some studies need ROS scavengers to cross the blood brain barrier and some biological compounds
cannot cross the mitochondrial membrane, many synthetic forms of antioxidants have been created to
directly target mitochondria. For instance MitoVit-E (Vitamin E) has been shown to decrease ROS
production and limit apoptosis in endothelial cells and it was created to selective inhibit oxidative damage
due to mitochondria (Sheu et al., 2006). Another drug, MitoQ, makes use of the fact that ubiquitnone and
its derivatives have known antioxidant effects. MitoQ is specifically taken up by energized mitochondria
and specifically blocks H2O2 activation of apoptosis (Li et al., 2013; Sheu et al., 2006). Furthermore,
MitoPBN specifically targets ROS due to the uncoupling of proteins within the electron transport chain.
PBN does not react with superoxide so when used in combination with MitoVitE and MitoQ, which do
block superoxide, inferences can occur about what types of ROS are being generated from the
mitochondria (Sheu et al., 2006). Another drug, MitoPeroxidase, when used in combination with the
glutathione peroxidase-like ebselen, can inhibit mitochondrial respiration, decrease the mitochondrial
membrane potential, leading to reduced ROS (Sheu et al., 2006). Since, in this study, it is unclear where
the source of ROS is, either from mitochondria or other sites within the cell, I chose to use a general ROS
scavenger drug, NAC, to revert the effects of ROS in HSCs. It is important to note that some studies
show that NAC was created to specifically target mitochondria (Sheu et al., 2006) while it has also been
reported to be a general scavenger (Tothova et al., 2007). In HSCs, NAC has commonly been used to
rescue detrimental effects of ROS in a variety of different studies (Chen et al., 2008; Gurumurthy et al.,
2010; Ito et al., 2004; Ito et al., 2006; Kocabas et al., 2012; Lee et al., 2010; Miyamoto et al., 2007; Qian
et al., 2016; Rimmele et al., 2015; Tothova and Gilliland, 2007), many of which find that the loss of certain
genes associated with various pathways including PI3K/mTOR have increased ROS levels due to
mitochondrial dysfunction. Further experiments using drugs to directly inhibit mitochondrial ROS in
Pdk1Hem-KO cells are needed to attribute elevated ROS levels solely to mitochondrial causes. Taken
together, the results from these experiments show that Pdk1Hem-KO HSCs have increased oxidative stress
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which can be partially reverted through NAC treatment and thus provide a cellular explanation for why
PDK1-deficent HSCs loose quiescence.
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Chapter 5: Loss in PDK1 results in reduced Hif1α expression.
Introduction
HIF1α regulation
The bone marrow niche is naturally a hypoxic environment; therefore, HSCs must have
intracellular machinery that allows for their survival in low oxygenic conditions. A group of DNA-binding
proteins, known as Hypoxia Inducible Factors (HIFs), orchestrates the expression of several genes that
allow the cell to cope with a hypoxic environment such as controlling energy production, antioxidant
transcription, angiogenesis, oxygen supply, proliferation, and apoptosis (Dengler et al., 2014). These
mechanisms provide the basis for quiescence as a means for HSCs to survive throughout the lifetime of
mammals.
HIFs are transcription factors that heterodimerize with each other in order to regulate transcription
of target genes. They are basic helix-loop-helix DNA-binding proteins and in mammals, they are
separated into alpha and beta subunits. In mice, there are three genes that encode the alpha subunits:
Hif1α, Hif2α (EPAS), and Hif3α. The beta subunit, HIF1β or aryl hydrocarbon receptor 1 (ARNT), is
constitutively expressed and can bind to any of the alpha subunits with varying effects on nuclear
translocation and transcription. Heterodimerization only occurs if the alpha subunit is stabilized, which
occurs during hypoxic conditions. In normoxia, the alpha subunits are hydroxylated at specific proline and
asparagine residues by prolyl hydroxylases, inhibiting their transactivation functions and targeting them
for degradation. When oxygen is low, hypoxia inhibits prolyl hydroxylases allowing HIFαs to accumulate
and dimerize with HIF1β to initiate transcription (Dengler et al., 2014; Lee et al., 2004). HIF1α is
expressed in all cell types whereas HIF2α and HIF3α appear to be tissue-specific. HIF1α and HIF2α are
structurally similar and appear to activate similar targets (Dengler et al., 2014). Not much is known about
HIF3α, but evidence suggests that it exists in many variant isoforms, some of which act to inhibit HIF1α
and HIF2α (Kaelin and Ratcliffe, 2008). The most well known negative regulator of HIF1α is inhibitory
PAS domain (IPAS) domain protein and it is thought that HIF1α can regulate IPAS transcription, which
IPAS can then, in turn, generate a negative feedback loop by inhibiting HIF1α (Makino et al., 2001;
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Makino et al., 2007). Taken together, the HIF family works in both in concert and opposition to each other
to respond to low oxygen conditions.
HIF1α activity can be regulated through various mechanisms that act to ensure HSC survival in
the niche. First, other transcription factors, such as Meis1, can alter the transcription of HIF1α (Kocabas
et al., 2012). Second, translation of HIF1α protein could be affected. Third, oxygen levels can impact
HIF1a stabilization (Dengler et al., 2014; Suda et al., 2011). In addition, modifications of redox conditions
can activate HIF1α expression (Fukuda et al., 2007; Suda et al., 2011). Also, interactions with co-
activators, such as ARNT or HIF3α, can affect HIF1α translocation into the nucleus (Dengler et al., 2014;
Makino et al., 2007). Furthermore, other transcription factors, such as Cited2, can compete with HIF1α for
the p300 subunit to prevent DNA binding (Bakker et al., 2007; Kranc et al., 2009; Majmundar et al., 2010).
Lastly, HIF1α expression can be regulated by phosphorylation from other signaling molecules, such as
members of the PI3K/AKT-pathway (Flugel et al., 2007; Mottet et al., 2003). Thus, the regulation of HIFα
occurs from the transcriptional level with feedback loops to the protein interactions and signal
transduction pathways within the HSC.
Hif1α regulates ROS expression
Regulation of metabolic activity of HSCs is vital for the health of the cell because it is required to
maintain self-renewal and multipotent differentiation abilities, two disparate functions. In order to protect
HSCs from a variety of physiological stresses, including oxidative stress, there are cellular regulators that
help maintain homeostasis. One defense mechanism is that the cell can remain in quiescence as it
protects a cell from accumulating mutations that can occur during division. It has been shown that
hypoxia induces quiescence in cultured HSCs (Hermitte et al., 2006; Shima et al., 2010) and it has been
suggested that most LT-HSCs reside within the hypoxic endosteal BM niche (Arai et al., 2004; Calvi et al.,
2003; Zhang et al., 2003). Taken together, it appears that HSCs are able to use hypoxia as a mechanism
to maintain quiescence.
Response to oxygen levels and hypoxic conditions within a cell is predominantly governed by the
hypoxia inducible factor-1 (HIF1) transcription factor. In low oxygenic conditions, the HIF1α isoform is
stabilized through its VHL domain and can then form a heterodimer with HIF1β and then translocate to
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the nucleus to initiate transcription of hypoxic response genes (Kaelin and Ratcliffe, 2008; Semenza,
2010). HIF1α is highly expressed in HSCs and conditional knockouts of HIF1α in HSCs have reduced
quiescence (Simsek et al., 2010; Takubo et al., 2010). Furthermore, deletion of ARNT caused increased
HSC apoptosis and loss of long-term reconstitution ability and double knockouts of HIF1α and HIF2α
phenocopied the ARNT phenotype (Krock et al., 2015). However, excessive levels of HIF1α led to HSC
exhaustion suggesting that a fine balance must be maintained (Suda et al., 2011). Nonetheless, these
experiments demonstrated that the role of HIF1α in HSCs is essential because it is needed to reprogram
energy output from oxidative to glycolytic pathways (Lou et al., 2011; Semenza, 2009). This is interesting
because oxidative phosphorylation by mitochondria tend to generate high levels of ROS, which mandates
intracellular responses that can cause destabilization of the cell. Indeed, loss of HIF1α in mouse
embryonic fibroblasts (MEFs) resulted in increased ROS production in hypoxic conditions (Fukuda et al.,
2007; Suda et al., 2011). Furthermore, it has been shown that ROSlow HSCs retain self-renewal properties
while ROShigh HSCs experience exhaustion (Jang and Sharkis, 2007). In addition, ROS expression has
been shown to inactivate the destabilization enzymes that tag HIF1α for degradation therefore, causing
HIF1α transcriptional activation, which can then regulate ROS levels (Bonello et al., 2007; Kaelin, 2005;
Klimova and Chandel, 2008). Given the link between hypoxia, quiescence and ROS, I speculated that
defects in HIF1α may affect the PDK1-signaling pathway and this may explain the high levels of ROS and
loss of quiescence found in the Pdk1Hem-KO HSCs.
PI3K/AKT/mTOR-signaling
Extrinsic cues from the BM niche are transduced primarily from membrane cytokine receptors to
the nucleus through intermediary signaling components. The PI3K/AKT-signaling pathway is a major
pathway involved in the regulation of cell growth, survival, and proliferation. This pathway is activated
when cytokines or other stimuli, such as growth factors, bind to receptor tyrosine kinases or when
intracellular scaffolding proteins, such as IRS1, that are bound to the receptor cytosolic tails help recruit
and activate PI3K. PI3K can then phosphorylate PIP2 to PIP3, and thus allowing for either PDK1 or AKT1
recruitment to the plasma membrane. Of note, PTEN, a phosphatase, can revert PIP3 to PIP2 therefore
acting as a negative regulator of the pathway. Although AKT can be directly activated by PIP3, one of the
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prevailing dogmas is that PDK1 is needed to phosphorylate the activation loop of AKT at Thr308. For full
AKT activation, the second phosphorylation site, Ser473, must be phosphorylated, which is predominantly
accomplished by the mTOR2 complex. Once AKT is phosphorylated, it can lead to activation of the
mTOR1 complex by inhibition of TSC1/2 that then releases Rheb from inhibiting mTOR1, and thereby
leading to the activation of downstream S6K by phosphorylation at Thr389. Interestingly, PDK1 is also
responsible for phosphorylating S6K at Thr229 indicating that PDK1 regulates PI3K-signaling at multiple
levels and can regulate targets independently from AKT (Pullen et al., 1998). Furthermore, it is well
established that AKT phosphorylates glycogen synthase kinase-3 (GSK3), causing the inhibition of GSK3
that then regulates several pathways including limiting apoptosis (Cross et al., 1995). Interestingly, GSK3
has been shown to phosphorylate the VHL domain on HIF1α, thus causing its degradation (Flugel et al.,
2007; Mottet et al., 2003). To prevent over-activation of the PI3K/AKT-pathway, active mTORC1 and S6K
can phosphorylate the scaffolding protein IRS1, impeding PI3K-signaling (Manning and Cantley, 2007).
While many of the signaling events in this cascade have been well established, I wanted to determine
whether the loss of PDK1 was perturbing AKT-signaling in the Pdk1Hem-KO mice. This would allow me to
gain insight into the molecular mechanisms that cause reduced quiescence and increased proliferation in
the mutant HSCs.
Members of the PI3K/AKT/mTOR-pathway can regulate HIF1α expression, which can support
HSC quiescence. For instance, the inhibition or loss of GSK3 induced HIF1α expression and the over-
activation of GSK3 caused HIF1α depletion in HepG2 cells (Flugel et al., 2007). Furthermore, a role for
mTORC1 and its downstream substrate, S6K has been linked to increased HIF1α expression by
regulating its transcription and translation (Duvel et al., 2010; Hudson et al., 2002; Laplante and Sabatini,
2012). Interestingly, both GSK3 and mTORC1 are separately activated by the AGC kinase AKT therefore,
the loss of AKT could lead to decreased HIF1α expression. Given that PDK1 is responsible for
phosphorylating the activation loop on AKT, I hypothesized that PDK1 may mediate HIF1α expression by
modulating the downstream signaling events in the PI3K-pathway. My approach to determine the role of
PDK1 in HIF1α expression will use in vitro and in vivo approaches taking advantage of PDK1-deficient
mice.
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Results
Pdk1Hem-KO mice have reduced Hif1α expression
While partial rescue of HSC cell numbers through NAC treatment was encouraging (Chapter 4),
it did not explain why NAC-treated animals still maintained high levels of ROS (Table 7). During steady-
state conditions, ROS is predominantly produced by oxidative phosphorylation (OXPHOS) during
mitochondrial energy production. When there is excessive oxidative stress, negative feedback
mechanisms activate HIF1α, a transcription factor that responds to hypoxic stress, to reduce ROS by: (1)
activating transcription of antioxidants and (2) shunting the primary mode of energy production from
OXPHOS to glycolysis, which produces less ROS. To determine whether this feedback system was
abrogated in the Pdk1Hem-KO mouse, I first assessed whether PDK1-deficient cells had abnormal levels of
HIF1α that translocated to the nucleus. Sorted control or Pdk1Hem-KO HSCs (LSKF-) and progenitor (LK)
cells were pipetted onto poly-D-lysine coated plates and cultured for 4 hours in hypoxic conditions
(5%O2/5%CO2/90%N2). HIF1α nuclear expression is associated with transcriptional activation, therefore
HIF1α was evaluated by immunohistochemistry and captured images showed the mean florescence
intensity (MFI) levels were reduced in Pdk1Hem-KO HSCs (Figure 23 A & C; 2.4±0.3 in control compared to
1.2±0.1 in Pdk1Hem-KO) and the progenitor cells (Figure 23 B & C: 3.0±0.2 in control compared to 0.8±0.03
in Pdk1Hem-KO). These results suggest that under hypoxic conditions, PDK1-deficient cells have reduced
levels of HIF1α in the nucleus and are thus unable to initiate the transcriptional machinery that would lead
to reduced levels of ROS expression.
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Figure 23: HIF1α is reduced in Pdk1Hem-KO cells during hypoxic conditions.
(A) Representative confocal images of IHC for HIF1α expression in HSCs (LSKF-) and (B) myeloid progenitors (LK) that have been incubated in 5% O2 for 4h show reduced HIF1α in Pdk1Hem-KO cells. (C) Quantification of mean florescencent intensity (MFI) of HIF1α reveals reduced expression in Pdk1Hem-KO LSKF- (control: n = 18, Pdk1Hem-KO: n = 19) and LK (control: n = 45, Pdk1Hem-KO: n = 70) fractions. Scale bars indicate 10um. Data are a merge of two independent experiments and are represented as mean ± SEM. Two-tailed unpaired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
Inhibition of HIF1α in Pdk1Hem-KO cells
The reduced HIF1α expression in the Pdk1Hem-KO cells was intriguing but the consequence of
limited nuclear expression of HIF1α was not clear. There are a few possible explanations for reduced
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nuclear HIF1α expression in PDK1-deficient HSCs: (1) there is less transcription of the Hif1α gene, (2)
Hif1α is transcribed but not translated, and (3) HIF1α is sequestered in the cytoplasm for degradation and
therefore cannot activate its downstream promoter targets in the nucleus.
To investigate why HIF1α was decreased in the mutant, I first quantified Hif1α mRNA in the BM.
In total BM, qRT-PCR revealed that Pdk1Hem-KO Hif1a mRNA is present at low levels similar to the control,
whereas expression of Arnt is slightly, yet significantly, reduced, and that Hif2α mRNA is increased
(Figure 24 B). Because Pdk1Hem-KO BM is composed of an abnormal proportion of cell types, the low
abundance of Hif1α mRNA may be due to the limited cells that express Hifs. I next examined Hifs in
sorted LSK and LK fractions and I found increased levels of Hif1α mRNA (Figure 24 C & D). These
results suggest that transcription of Hif1α is unaffected or even increased in the LSK and LK fractions
suggesting that there is a possible disturbance in translation of the transcript or post-translational
problems. It is possible that there is reduced nuclear HIF1α in the Pdk1Hem-KO mouse because HIF1α
protein is not being translated. Of note, some of the translational machinery must be in tact since there
translation of HIF1α is occurring because HIF1α expression is detectable in the IHC confocal images in
HSCs (LSKF-) and progenitors (LK) (Figure 23 and data not shown). Because detecting HIF1α protein
levels by IHC implies that there must be translation of mRNA protein, I speculated that HIF1α is present in
the cytosol but it is being hindered from entering the nucleus. In assessing other HIFs in cell fractions of
the BM, analysis of Hif3α mRNA in LSK and LK showed large increases compared to the control (Figure
24 B & C). In contrast, Hif2α did not reveal a large difference and there was no difference between control
and Pdk1Hem-KO mRNA. This result was fascinating because in other systems, such as eye
vascularization, HIF3α has been shown to inhibit HIF1α expression (Makino et al. 2001, 2007)
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Figure 24: Hif mRNA expression in Pdk1Hem-KO cells.
(A) Schematic of HIF1α activation by ROS. Oxidative stress causes increased HIF1α protein, promoting binding to ARNT. The HIF1α/ARNT heterodimer translocates to the nucleus were it transcriptionally activates hypoxic response genes. (B) qRT-PCR showed reduced Arnt, no change in Hif1α, and increased Hif2α mRNA in Pdk1Hem-KO total BM. (C) qRT-CR of Hif1α, Hif2α, and Hif3α in LSK and (D) LK revealed increased Hif3α mRNA expression. qRT-PCR expression was normalized to Hprt and control. Total BM: n = 3, LSK: n = 2, LK: n = 2. Data are representative of two-wells from each experiment. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
This finding led me to consider three possibilities that could explain the results (Figure 25 A). The
first is that HIF1α is able to bind to its coactivator, ARNT, heterodimerize and translocate to the nucleus to
activate Hif transcriptional targets. This scenario may explain what is occruing in the control BM. The
second scenario is that HIF3α is directly competing with HIF1α for binding with ARNT (Dengler et al.
2014). If this happens, then HIF3α/ARNT can translocate to the nucleus altering Hif targets resulting in
low transcription. Based on reduced Arnt mRNA in total BM (Figure 24 B) this seems unlikely, although
Arnt mRNA levels and ARNT protein expression must be verified in Pdk1Hem-KO HSCs. The third possibility
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is that HIF3α, specifically the inhibitory pas domain (IPAS) isoform, is directly binding to HIF1α and
sequestering it in the cytosol for degradation (Makino et al. 2001). The later scenario, is an attractive
hypothesis because it explains why nuclear HIF1α is lower in PDK1-deficient cells and as well, implies
that there are downstream consequences to the loss of HIF1α-signaling, such as reduced transcription of
antioxidant genes.
The previous experiment reveals that the levels of Hif3α mRNA is up in Pdk1Hem-KO LSK and LK;
however, these results were using primers that did not distinguish between IPAS and other isoforms of
Hif3α. To resolve which mechanism inhibits Hif1α function in PDK1-deficient cells, qRT-PCR was
performed with total BM using primers specifically designed to recognize the IPAS isoform, forward primer
in exon 4a and reverse primer in exon 6 (Figure 25 B), and the full length Hif3α (Hif3αFL) isoform, forward
primer in exon 11 and reverse primer in exon 13. Independent experiments from three different pairs of
mice show increased Hif3αFL and IPAS (Figure 25 C) mRNA. To confirm that the primers were amplifying
Hif3αFL and IPAS, sequencing of PCR amplification product was performed, with the aforementioned
primers, on both control and Pdk1Hem-KO BM. Amplicons were then gel-purified and the extracted DNA
was sent for sequencing (Figure 25 D). NCBI BLAST of sequencing results revealed that both the control
and Pdk1Hem-KO samples align with known sequences of Hif3αFL (data not shown) and Hif3α isoform1/2 or
IPAS (Figure 25 E). This data show that transcription of Hif3αFL and IPAS are increased and therefore, if
translated appropriately, IPAS could be acting to inhibit HIF1α. Future experiments to elucidate whether
HIF3α and IPAS protein expression are increased in Pdk1Hem-KO cells, and if either HIF3α or IPAS are
hindering HIF1α expression, will elucidate which scenario is occurring.
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Figure 25: Pdk1Hem-KO cells have greater IPAS mRNA expression.
(A) Schematic demonstrating how HIF3α can inhibit HIF1α. (B) Schematic of IPAS and HIF3α CDS and primer design. (C) Full length Hif3α (Hif3αFL) and IPAS mRNA transcription is increased in the Pdk1Hem-KO BM (n = 3). (D) PCR amplification using primers for Hif3αFL and IPAS of control and Pdk1Hem-KO total BM cDNA show increased expression in Pdk1Hem-KO cells. (E) BLAST of sequencing results of samples from (D) show that the primers are amplifying Hif3α isoforms 1 and 2 (n = 1). qRT-PCR expression was normalized to Hprt and control. Data are of two-wells from three independent experiments. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
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Pharmacological inhibition of PDK1 provides insights into downstream signaling
While the hypothesis that IPAS may bind to and inhibit the function of HIF1α is an attractive
model that explains a plausible regulatory mechanism, I wanted to determine whether there were other
post-translational events that could be disrupting HIF1α expression. The AKT/mTOR-pathway has been
implicated in the regulation of HIF1α expression (Figure 26). Indeed, PDK1 is considered to be a master
regulator of PI3K-signaling and it is thought that the primary role of PDK1 is to activate AKT via
phosphorylation. To determine whether AKT-signaling was reduced in the absence of PDK1-signaling, I
decided to treat cells with a PDK1-specific inhibitor and perform immunoblot analysis. Other groups have
demonstrated that pharmacological inhibition of PDK1 can be used to recapitulate the downstream-
signaling events (Park et al., 2013). Furthermore, due to the small number of HSCs within a mouse it is
difficult to perform immunoblots on such few cells. In addition, the Pdk1Hem-KO has reduced B cells so
protein extraction from total BM would be representative of a different composition of cell types. To
circumvent these issues and to delineate which signaling events were abrogated in Pdk1Hem-KO mice, I
used a highly specific small molecule inhibitor of PDK1, GSK2334470 (Najafov et al. 2011) in my studies.
I chose this inhibitor because GSK2334470 does not block activity of 93 other AGC kinases while also
inhibiting T-loop activation in AKT, the classical target of PDK1. Total BM was treated with or without 1uM
PDK1 inhibitor, starved for 5.5 hours or starved for 5 hours and then stimulated with 10%FBS/DMEM and
0.5ug cytokines (IL6, IL3, SCF, TPO, Flt3l) for 30 minutes. Protein cell lysates were extracted and
immunoblotting for components of the AKT/mTOR-signaling pathway were assessed for phosphorylation.
Similar to the Pdk1Hem-KO total BM immunoblot, there was reduced AKT phosphorylation at residues
Thr308 and Ser473 as well as reduced downstream p70S6K phosphorylation (Figure 27 A). Furthermore,
immunoblots of protein from wild type total BM treated with and without PDK1 inhibitor show loss of
phosphorylation of GSK3b at residue Ser9 (Figure 27 A). Overall, as expected, PI3K/AKT-signaling is
reduced in the absence of PDK1 activity.
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Figure 26: PI3K-signaling can regulate HIF1α expression.
Schematic shows that PI3K is activated when cytokines or growth factors bind to receptors. PI3K then phosphorylates PIP2 to become PIP3. PIP3 recruits PDK1and AKT to the membrane through their PH domains allowing PDK1 to phosphorylate AKT at Thr308. mTORC2 (not pictured) is needed to phosphorylate AKT at Ser473. AKT signals through mTORC1 by inhibiting TSC1/TSC2 (not pictured). mTORC1 activates p70S6K (S6K) through phosphorylation at Thr389. S6K initiates synthesis of a variety proteins needed for cell survival, such as HIF1α. PDK1 phosphorylates p70S6K at Thr229 independently of AKT activation. In addition to mTORC1 activation, AKT can inhibit GSK3 and FOXO through phosphorylation. FOXO acts to suppress reactive oxygen species (ROS) levels. Elevated ROS levels activate p38, which initiate proliferation and cell death signaling cascades that may cause DNA damage. GSK is able to inhibit HIF1α by phosphorylating the HIF1α VHL domain. During hypoxic conditions, or in response to oxidative stress, HIF1α bind with ARNT to form a heterodimer that will translocate to the nucleus to transcriptonally activate a variety of HIF1α target genes including antioxidants and glycolytic genes. The HIF3α isoform, IPAS can block the HIF1α/ARNT heterodimer by binding to HIF1a and sequestering in the nucleus for degradation.
PDK1 is an intermediate signaling kinase, which means that it converts signals from upstream
receptors to activate other downstream-signaling cascades. To determine whether PDK1 inhibition had
an effect on other pathways known to be activated by stem cell cytokine receptors, I examined the MAPK
pathways, which are known for integrating external signals to transcriptional programs and play roles in
proliferation, differentiation, and apoptosis (Geest et al. 2009). I found that there was very little difference
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in p-ERK and p-JNK levels in immunoblots of cells treated with and without PDK1 inhibitor (Figure 27 B).
Taken together, pharmacological inhibition of PDK1 recapitulates the loss of AKT/mTOR-signaling
reported in the literature and shows that the ERK- and JNK-signaling pathways in HSCs remain in tact.
Figure 27: Drug inhibition of PDK1 provides insight into downstream signaling.
(A) Total BM with or without 1uM PDK1 inhibitor (GSK2334470) was serum starved for 5.5h or serum starved for 5h and then stimulated for 30 min with 10% FBS and 0.5ug cytokines (IL6, IL3, Flt3l, TPO, SCF). Components of the PI3K/AKT/mTOR-signaling cascade were checked for phosphorylation and there was reduced AKT, p70S6K, and GSK3β phosphorylation indicating reduced signaling via this pathway. (B) Immunoblots of other known signaling pathways downstream of cytokine receptors showed no major differences in the ERK or JNK pathways. Data are representative of two or three independent experiments.
Reduced AKT-signaling in Pdk1Hem-KO LSK
To corroborate the findings using PDK1 inhibitor studies that PDK1-signaling affects
AKT/mTORC-signaling in vivo, control and Pdk1Hem-KO total BM were depleted of B cells to ensure similar
cell composition between the two groups and protein was immunoblotted for phosphorylated AKT at
residues Thr308 and Ser473. In both the starved and stimulated conditions, there is reduced
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phosphorylation of AKT at both residues whereas total AKT protein levels appear to be the same (Figure
28 A). Total BM has many differentiated cell types, so to ensure that the progenitor populations also had
reduced AKT activity, phosflow on sorted LK and LSK cells for phospho-AKT at T308 revealed reduced
phosphorylation in the Pdk1Hem-KO cells (Figure 28 B & C; LSK: 77.8±10.1% in control and in Pdk1Hem-KO,
LK: 75.8±18.6% in control compared to 34.9±4.8% in Pdk1Hem-KO). In the PI3K/AKT/mTOR-signaling
cascade, mTOR is activated by AKT and in turn phosphorylates p70S6K. To corroborate that there is
reduced PI3K-signaling in the Pdk1Hem-KO cells, I sorted LSK and LK and found decreased phosho-
p70S6K in the Pdk1Hem-KO cells (Figure 28 D & E; LSK: 67.3±2.4% in control and in 28.2±4.7% Pdk1Hem-
KO, LK: 62.6±4.1% in control compared to 21.8±6.1% in Pdk1Hem-KO). Taken together, loss of PDK1 results
in reduced AKT/mTOR-signaling in the myeloid progenitor fraction, LK, and the HSPC fraction, LSK.
Interestingly, loss of mTOC1 and p70S6K activation can lead to reduced transcription and translation of
HIF1α (Duvel et al., 2010; Hudson et al., 2002; Laplante and Sabatini, 2012). In light of this data, PDK1
appears to be regulating HIF1α through PI3K/AKT/mTORC1-signaling.
Previous reports have shown that activated GSK3β negatively regulates HIF1α (Chan et al.,
2001; Flugel et al., 2007; Mottet et al., 2003). AKT has been shown to inactivate GSK3 by
phosphorylating residues Ser21, GSK3α, and Ser9, GSK3β (Cohen and Frame 2001). Taken together,
this led us to hypothesize that loss of PDK1 leads to reduced AKT-signaling, allowing for the activation of
GSK3, which causes HIF1α protein levels to be downregulated. Phosflow analysis of sorted LSK
(86.9±5.7% in control compared to 40.3±7.8% in Pdk1Hem-KO) and LK (71.2±12.0% in control compared to
34.4±3.5% in Pdk1Hem-KO) for p-GSK3β(Ser9) showed less phosphorylation in Pdk1Hem-KO cells (Figure 28
B & C). These results suggest that GSK3β is activated in PDK1-deficient cells. Taken together, it appears
that HIF1α expression is reduced in Pdk1Hem-KO mice since a positive regulator, p-p70S6K, is reduced,
and the negative regulator, GSK3β, is increased. These experiments provide mechanistic insights into
Pdk1Hem-KO HSCs by showing that increased GSK3β activity and lack of p-p70S6K lead to reduced HIF1α
expression. With HIF1α expression reduced, that ROS levels remain high that causes reduced HSC
numbers because of loss of HSC quiescence and increased HSC exhaustion.
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Figure 28: Loss of PDK1 leads to reduced AKT/mTORC-signaling.
(A) Immunoblot of control and Pdk1Hem-KO (CKO) total BM either starved or starved then stimulated with 10%FBS + cytokines (IL6, SCF, TPO, IL3, Flt3l) for 30 min showed reduced phosphorylation at residues Thr308 and Ser473 on AKT in cells without PDK1 (n = 2). (B) Representative flow cytometry plots and (C) analysis of phosflow of LSK (n = 4) and LK (n = 3; p = 0.0994) BM fractions showed reduced p-AKT at T308 in cells lacking PDK1. (D) Representative flow cytometry plots and (E) quantification showed loss of PDK1 led to reduced phosphorylation of p70S6K at Thr389, a downstream signaling molecule of AKT and mTORC1 (n = 3). (F) Representative flow cytometry plots and (G) quantification of sorted LSK (n = 3) and LK (n = 4) cells showed reduced p-GSKβ(Ser9) in the Pdk1Hem-KO cells. Secondary lines represent secondary antibody controls. Data are mean ± SEM and two-tailed paired Student’s t-tests were used to assess significance (*P<0.05, **P<0.01, ***P<0.001).
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Discussion
In Chapter 4, experimental evidence suggested that Pdk1Hem-KO HSCs have higher levels of ROS
that leads to a loss of quiescence (Chapter 3) as well as increased progenitor proliferation and cell death.
Based on the most recent data, it appears that high levels of ROS is due to loss of HIF1α expression
which can act as a negative feedback loop. Further investigation into the loss of HIF1α expression in
Pdk1Hem-KO cells, suggested that HIF1α might be mediated by the inhibitory isoform IPAS and that loss of
PDK1-mediated AKT/mTOR and AKT/GSK-signaling might cause decreased Hif1α transcription and
translation. Taken together, the results from this study suggest that PDK1 regulates the PI3K-signaling
pathway to provide cellular balance in HSCs through the regulation of ROS and HIF1α. Without PDK1,
HSCs lose quiescence and proliferate more, resulting in the loss of HSC numbers and cellular functions.
HIF expression
Since ROS levels were elevated in the Pdk1Hem-KO mouse, I wanted to know whether there was a
defect in HIF1α expression, a negative regulator of ROS. The results from IHC for the detection of HIF1α
in HSCs showed that indeed, HIF1α expression is reduced in Pdk1Hem-KO cells. As mentioned before,
there are a number of explanations for reduced HIF1α expression: (1) there is less transcription of Hif1α,
(2) there is normal transcription but reduced translation of HIF1α, and (3) there is post-translational
inhibition of HIF1α. The first possibility seems unlikely since RT-PCR of total BM led to the conclusion that
Hif1α is being transcribed at normal levels and preliminary data show a dramatic increase in Hif1α
expression in the LSK and LK fractions. Of note, the LSK experiment was only performed once and the
LK experiment only twice, so it is necessary to further replicate this data. To address the second
possibility, HIF1α protein is detected in LSKF- and LK as evidenced in the IHC results (Figure 23). This
suggests that translation is occurring and thus, perturbations in transcription nor translation cannot
account for diminished HIF1α expression.
With regards to the third explanation for reduced HIF1α, there are many instances where HIF1α
can be affected post-translationally: (3a) ARNT, which can bind with HIF1α, is reduced therefore the
HIF1α/ARNT heterodimer may not be formed and HIF1α targets are not transcribed, (3b) the other HIFs
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may be competing for ARNT binding, (3c) IPAS is inhibiting HIF1α, (3d) other signaling molecules are
inhibiting HIF1α expression. With regards to scenario 3a, if ARNT is decreased in HSCs, then it is
possible that HIF1α is being translated properly but the co-factor ARNT is not at sufficient levels to
translocate the heterodimer into the nucleus. Further experiments to determine Arnt transcriptional levels
in HSCs needs to be performed. To address scenario 3b, Hif2α mRNA is increased in total BM, so it is
plausible that HIF2α competes with HIF1α for ARNT binding; however, this may not be the case in the
LSK fraction because the role for Hif2α may be different in HSCs. Further replicates of Hif2α mRNA in
HSCs need to be addressed. The same could be true for Hif3α. To determine whether IPAS is inhibiting
HIF1α (scenario 3c), mRNA levels of IPAS in HSCs require further investigation. In addition, Hif3αFL
mRNA levels in HSCs should be checked as well because there are multiple isoforms of Hif3α that could
have different functions. Increased IPAS mRNA does not necessarily suggest increased protein
expression so HIF3α/IPAS IHC with LSKF- cells or Western blots with total BM protein should be
performed. In these experiments, I would predict higher HIF3α/IPAS in the Pdk1Hem-KO cells than in the
control. Ideally, IHC colocalization of HIF1α and HIF3α would suggest a direct inhibition of HIF1α by
HIF3α/IPAS. One caveat would be that the HIF3α antibody may not strongly recognize the IPAS isoform.
Alternatively, future experiments could include retroviral shRNA knock down of HIF3α and IPAS in HSCs
to rescue HIF1α expression and this may point to a suppression mechanism by certain isoforms of HIF3α.
In addition, overexpression of HIF1α can be accomplished in Pdk1Hem-KO cells using similar molecular
approaches to determine whether circumventing PDK1-deficiency could decrease high ROS levels when
PDK1 is ablated. Lastly, it would be interesting to determine whether overexpression of IPAS in normal
HSCs would recapitulate the Pdk1Hem-KO phenotype.
It is possible that transcription factors associated with other pathways could also be regulating
HIF1α expression (scenario 3d). For instance, Cited2 can compete with HIF1α for binding at with the
histone acetyltransferase p300/CBP (Bakker et al., 2007; Kranc et al., 2009); however, RT-PCR analysis
of total BM (n = 2, data not shown) does not show an increase in Cited2 levels. In addition, loss of Meis1
can lead to reduced Hif1α and Hif2α transcription (Kocabas et al., 2012). Interestingly, RT-PCR of Meis1
in total BM shows the same or slightly increased levels and we know that transcription of Hif1α and Hif2α
are normal or increased in total BM so Meis1 is not impacting Hif1α transcriptional activity. Taken
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together, these preliminary results further provide evidence that loss of HIF1α expression is due to
aberrant in PDK1-signaling (see next section), direct inhibition by IPAS, or a combination of both.
PI3K/AKT/mTORC-signaling
In addition to the scenarios mentioned in the previous section regarding HIF1α regulation,
evidence from this study suggests that PDK1/AKT/mTORC-signaling can regulate HIF1α (scenario 3d).
The results from the PDK1 inhibitor immunoblots, the B cell depleted Pdk1Hem-KO total BM Western blots,
and phosflow conclusively show that AKT phosphorylation is decreased in the Pdk1Hem-KO mouse. In
addition, loss of mTORC1-signaling is corroborated by loss of p70S6K phosphorylation. To provide a
comprehensive understanding of how PDK1 impacts AKT/mTORC1-signaling, immunoblots of 4EBP,
another downstream target of mTORC1 involved in protein translation, can be performed with the PDK1
inhibitor. Furthermore, loss of AKT-signaling leads to activated FOXO-signaling. FOXOs have been
implicated in HSC quiescence and conditional knockouts show increased levels of ROS (Tothova and
Gilliland, 2007). Theoretically, FOXO expression should be increased in the absence of PDK1, and
preliminary immunoblots showed reduced p-FOXO3a (data not shown). AKT phosphorylates FOXO for
degradation, so this is in line with the current model; however, further experimentation using immunoblots
or IHC for 4EBP or FOXO needs to be duplicated to corroborate that p-4EBP is down and that FOXO
expression is increased.
AKT is also responsible for inhibiting GSK3 via phosphorylation. Results from these experiments
show that GSK3β phosphorylation is down-regulated but there is an alpha subunit of GSK that can also
be phosphorylated. Investigation into p-GSK3α(Ser21) levels need to be assessed although it is thought
that the two subunits do not have compensatory roles since knocking out either one results in embryonic
lethality (reviewed in McCubrey et al., 2014). Furthermore, overexpression of GSK3β in HepG2 cells led
to reduced HIF1α (Flugel et al., 2007; Mottet et al., 2003). This would suggest that loss of GSK3β
phosphorylation might be enough to actively inhibit HIF1α. It is important to note that GSK3 is also
downstream of Wnt/β-catenin signaling; however, the role for Wnt-signaling in HSCs is controversial with
some studies showing that it impacts HSC quiescence and others demonstrating that Wnt-signaling is
negligible for HSC maintenance (Mendelson and Frenette, 2014). Lastly, it has been shown that NFkB
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can activate HIF1α through functional binding to a promoter site in Hif1α (Bonello et al., 2007), therefore
suggesting that NFkB could promote HIF1α expression. However, it has been shown that PDK1 can
activate NFkB-signaling through PKCθ in T cells (Lee et al., 2005). In our model, PDK1 is ablated
suggesting that there is reduced NFkB-signaling and therefore reduced Hif1α transcription. Although, the
microarray showed that NFkB targets were similar in the control and Pdk1Hem-KO groups, further
experiments to verify that NFkB-signaling is decreased, such as IHC to look for p65, the downstream
target of NFkB, are needed to demonstrate that activated GSK3 and reduced p70S6K phosphorylation
are coordinately working to down-regulate HIF1α expression.
Consequences of reduced HIF1α expression
Many studies have suggested that the primary function of HIF1α in HSCs is to shunt energy
production from the high ATP producing but also high ROS producing oxidative phosphorylation
(OXPHOS) pathway in mitochondria to the lower ATP producing and lower ROS generating glycolysis
method in the cytoplasm. In addition, it has been shown that stem cells primarily use glycolysis as their
mode of energy production since their metabolic need is different from progenitor and differentiated cell
types as well as to help prevent some of the deleterious effects that can occur with high amounts of ROS.
Indeed, glycolysis has been implicated in HSC survival in the hypoxic niche as a means to promote
quiescence (Suda et al., 2011). The Pdk1Hem-KO mouse has reduced quiescence and increased ROS
levels; therefore, it would be interesting to determine whether glycolysis was reduced in Pdk1Hem-KO HSCs
and as a consequence OXPHOS was increased. Preliminary results show a reduction in Glut1 mRNA
expression, the major receptor for glucose transport into cell; however, further investigation into glucose
uptake, through either Seahorse assays or with a florescent glucose dye, in Pdk1Hem-KO cells need to be
performed. In addition, RT-PCR of genes associated with these pathways can shed light on the
consequences of reduced HIF1a expression in Pdk1Hem-KO HSCs.
In conclusion, I have determined that PDK1 controls ROS activity by regulating HIF1α expression
and this ultimately has consequences for HSC quiescence and viability. PDK1 appears critical for HSCs
to favor anaerobic glycolysis and maintain low ROS by regulating HIF1α expression. The expression of
HIF1α and low ROS serves to limit HSC cell cycling, to avoid unnecessary mutations, and to promote
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longevity of the HSC pool. Here, I have examined a number of potential mechanisms by which PDK1 may
regulate HIF1α and my data suggests that PDK1 may regulate HIF1α expression by a number of
plausible mechanisms including direct PI3K/AKT/mTORC-signal transduction that results in HIF1α
expression and by suppressing potential binding partners such as IPAS or HIF3α that may limit HIF1α
dimerization with ARNT. Such competitive binding partners that inhibit HIF1α dimerizing with ARNT may
prevent translocation of HIF1α to the nucleus and inhibit transcription of antioxidation genes. Thus, PDK1
promotes HIF1α expression that ultimately serves in HSC maintenance and homeostasis.
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Chapter 6: Discussion and Future Directions
PI3K-signaling in HSCs
The precise balance between HSC quiescence and proliferation is essential for adequate blood
production within an organism. Through instructive external niche signals, such as cytokines, and internal
signals that govern the activation of cellular programs, adult HSCs are able to exit quiescence, and then
self-renew and differentiate to maintain hematopoietic homeostasis. Many signal transduction pathways,
such as MAPKs, TGFβ, NFκB, and Wnt have been implicated in the maintenance of HSCs; however, the
PI3K/AKT/mTORC-signaling cascade is one of the major pathways involved in HSC cell survival,
proliferation, and metabolism. The PI3K/AKT/mTORC-signaling pathway lies downstream of a variety of
growth factors and cytokines that are needed to maintain HSCs during homeostasis as well as under
regenerative conditions. Indeed, TPO has been shown to signal through mTOR in stress conditions (Qian
et al., 2007) and SCF promotes lipid raft clustering and AKT-activation to promote HSC cell cycle entry
(Yamazaki et al., 2006). In the current study, I was interested in unraveling the role of PI3K-signaling in
HSC biology. For this purpose, I decided to focus on PDK1 as this protein has been considered the
“master kinase” of the PI3K-signaling pathway. PDK1 is directly activated by PI3Ks and can initiate the
activity of many other PI3K-signaling members by phosphorylating their T-activation loop such as AKT
and S6K. Furthermore, PDK1 is needed for proper lineage differentiation but a role for PDK1 in HSC
maintenance has not been established. In this study, I show that PDK1 unequivocally functions as a
master regulator of PI3K-signaling to promote HSC quiescence.
While previous groups have explored how PDK1 impacts lymphoid and myeloid lineage
differentiation, this is the first study to investigate how PDK1 governs HSC maintenance. Through the use
of a hematopoietic specific Cre line, I was able to ablate PDK1 in the most multipotent hematopoietic
cells, which unsurprisingly led to aberrant defects in lymphoid differentiation as reported by previous
studies (Baracho et al., 2014; Hinton et al., 2004; Lee et al., 2005; Park et al., 2013; Park et al., 2009;
Yang et al., 2015). Interestingly, myeloid progenitor numbers were not significantly affected; however, the
relative ratio of monocyte and granulocyte levels was changed. Furthermore, CBC analysis of the
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Pdk1Hem-KO BM recapitulated the thrombocytopenia defect reported earlier (Chen et al., 2013). This
suggests that PDK1 may be dispensable for myeloid progenitor specification but necessary for lineage
differentiation of specific myeloid cell types. Consistently, Pearn et al. suggested that PDK1
overexpression promotes monocyte formation. In the Pdk1Hem-KO mice, I see a decrease of monocytes in
the BM and an increase in the spleen. Thus, these studies highlight the importance of PDK1-signaling in
monocyte differentiation. Overall, it appears that the loss of PDK1 expression in Pdk1Hem-KO mice had
detrimental effects on the lymphoid lineage and influenced the differentiation of specific cell types within
the myeloid lineage.
Through the use of genetic gain- and loss-of function studies, many groups have demonstrated
that signaling through the PI3K-pathway is essential for HSC survival, proliferation, and differentiation
during normal and regenerative hematopoiesis (Chen et al., 2008; Juntilla et al., 2010; Kalaitzidis et al.,
2012; Kharas et al., 2010; Magee et al., 2012; Miyamoto et al., 2007; Siegemund et al., 2015; Tothova
and Gilliland, 2007; Yilmaz et al., 2006; Zhang et al., 2006). In this study, immunophenotyping of the
Pdk1Hem-KO BM revealed a striking reduction in the number of HSCs and that PDK1-deficient cells were
unable to provide radioprotection in primary BMTs. Furthermore, Pdk1Hem-KO HSCs had normal levels of
viability staining, fewer cells in the G0 phase, and increased cycling. In Pdk1Hem-KO mice, there was
reduced phosphorylation of AKT at Thr308 and Ser473 as well as reduced phosphorylation of p70S6K at
Thr389, a downstream target of mTORC1-signaling. Thus, these results clearly demonstrate that key
downstream targets of the PI3K-pathway are not activated in the absence of PDK1. It should be noted
that although PDK1 and AKT act in concert to activate mTORC1-signaling, activation of the downstream
targets of PI3K-signaling is not strictly dependent on AKT. This is because PDK1 has been shown to
directly phosphorylate the targets of PI3K, such as RSK and PKC, independently of AKT (Figure 29)
(Najafov et al., 2011; Vanhaesebroeck and Alessi, 2000; Vasudevan et al., 2009). Of note, it is thought
that phosphorylation of AKT at Thr308 is predominantly the responsibility of PDK1 whereas
phosphorylation of AKT at Ser473 is executed via mTORC2 (Vivanco et al., 2002; Sarbassov et al.,
2005). Without proper activation of AKT at both of these phosphorylation sites, there is less activated AKT
to facilitate cell cycle progression (Gao et al., 2014). Interestingly, in the Pdk1Hem-KO BM, I see a decrease
in AKT phosphorylation at both residues rather than just at Thr308. This could be explained by the fact
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that loss of phosphorylation at Thr308 leads to reduced AKT activity, which then subsequently results in
reduced TSC activity. Reduction of TSC activity would cause a decrease in both mTORC1 and mTORC2
activity since TSC is responsible for releasing RHEB from the mTOR complexes. Therefore, a reduction
in mTORC2 activity could cause further reduction of AKT activity by diminishing phosphorylation at
Ser473.
In contrast to the Pdk1Hem-KO results described in this study, genetic knockouts of AKT1 and 2, a
key effector kinase in PI3K-signaling, revealed reduced expansion of transplanted fetal HSCs due to
increased levels of quiescence. AKT1/2 mutant HSCs competed poorly against wild-type HSCs during
competitive repopulation experiments suggesting that they had reduced differentiation capacity (Juntilla et
al., 2010). When AKT is activated, it can sequester FOXOs in the cytoplasm to prevent their transcription
of a variety of genes associated with the cell cycle as well as oxidative stress. Conditional deletion of
FOXO1/3a/4 using Mx-Cre, caused reduced HSC numbers and reduced ability to repopulate the blood
cell pool during BMT (Tothova and Gilliland, 2007). Interestingly, targeted mutants for FoxO3a alone had
HSCs that cycled normally and had proper differentiation capacity. Intriguingly, FOX3a-deficient HSCs
had reduced ability to compete during competitive transplantation experiments (Miyamoto et al., 2007).
The discrepancies could be due to functional redundancy among the different isoforms of FOXO in the
second study as well as the type of deletion system used: Mx-Cre versus targeted deletion, respectively.
Nonetheless, both studies reveal that loss of FoxO transcription can impact HSC functions.
Another target of AKT is the mTOR1 complex. AKT negatively regulates the TSC1/TSC2
complex, which in turn negatively regulates mTORC1 by interacting with RHEB, which is needed for
mTORC1 activation. Conditional loss of TSC1 led to decreased HSC quiescence and increased
proliferation. TSC1-deficient HSCs lost self-renewal capacity and reduced hematopoietic reconstitution
ability (Chen et al., 2008). The TSC1 mutant phenotype is interesting for three reasons: (1) PDK1 is
upstream of AKT/mTORC1-signaling, (2) PDK1 can directly impact mTORC1-mediated protein synthesis
by phosphorylating S6K, and (3) the TSC1 mutant HSC phenotype is similar to the Pdk1Hem-KO HSC
phenotype. Taken together, this suggests that the Pdk1Hem-KO phenotype is partially due to loss of
mTORC1-signaling and indeed, there is reduced phos-p70S6K, a readout of mTORC1-signaling, in
Pdk1Hem-KO LSK and LK (Figure 28). Furthermore, loss of Raptor, a key component of the mTOR1
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complex, resulted in pancytopenia and splenomegaly under normal hematopoiesis and after BMT, or
stress conditions, HSCs were unable self-renew (Kalaitzidis et al., 2012). Another study deleted Rictor, a
key component in the mTOR2 complex, and found very few changes in HSC functions during steady
state conditions. However, loss of Rictor, or mTORC2-signaling, in a PTEN-null background prevented
HSC depletion and leukemogensis that can occur when PI3K-signaling is over-activated (Magee et al.,
2012). Pdk1Hem-KO mice might have a different phenotype from these studies because PI3K/PDK1/AKT-
signaling is not altered in the Raptor or Rictor mutants since these kinases act earlier in the signaling
cascade. Furthermore, the normal HSC phenotype seen in both the Raptor and Rictor mutants during
steady state conditions suggests that there could be compensatory mechanisms that maintain mTORC-
signaling. For instance, PDK1 can phosphorylate S6K at Thr229 independently of mTORC1, which
primarily phosphorylates S6K at Thr389 (Figure 29) (Pullen et al., 1998). Thus, the downstream target of
mTORC1, S6K, can still be activated or at least partially activated. Collectively, these experiments show
that loss of PI3K-signal transduction through PDK1/AKT/mTORC is necessary to maintain HSC
quiescence and repopulating capacity both during normal and regenerative hematopoiesis.
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Figure 29: PI3K/PDK1-signaling phosphorylates AKT-dependent and AKT-independent AGC kinases.
In addition to the ‘classical’ PI3K/AKT/mTORC-pathway, PI3K/PDK1-signaling can activate a variety of other AGC kinases including: SGK, RSK, and PKC. These other kinases need to be phosphorylated on different residues by other signaling components for full activation.
Previous studies have shown that increased PI3K-signaling through the loss of PTEN, results in
myeloproliferative disease since HSCs proliferate more. This causes HSC depletion and inability to
repopulate the blood cell pool. Treatment with rapamycin restored HSC function, which suggests that the
loss of quiescence was due to reduced mTORC1-signaling (Yilmaz et al., 2006; Zhang et al., 2006). The
loss of SHIP1 should also lead to increased ATK/mTOR-signaling. However, unlike the PTEN mutant,
SHIP1 deficient mice have increased HSC numbers and fewer apoptotic cells. In addition, SHIP1-
deficient HSCs have reduced reconstitution ability due to decreased expression of CXCR4 and VCAM,
which are needed for engraftment (Desponts et al., 2006). Both PTEN and SHIP1 function to convert
PIP3 to PIP2, thus negatively regulating PI3K-signaling; therefore, it is odd that the HSC phenotype
reported in the PTEN and SHIP1 mutants are different. One possible explanation is that the mechanistic
action is different between the two proteins: PTEN dephosphorylates PIP3 while SHIP1 hydrolyzes PIP3.
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Depending on the receptor or signaling cascade, PTEN or SHIP might be preferentially regulated. In
addition, another explanation for different phenotypes is that the PTEN mutants were conditionally
ablated using Mx-Cre in adult HSCs and the SHIP1 mice were total knockouts. As discussed earlier, the
mechanisms that govern fetal versus adult quiescence are different so the experimental conditions
employed might account for the discrepancy. Another group over-activated PI3K-signaling by performing
BMT with an overexpression allele of myristylated Akt (myr-Akt). The data revealed that HSPCs
transiently expanded and had increased cycling that hindered engraftment. These mice also exhibited
myeloproliferative disease and rapamycin treatment was able to elongate their lifespan by limiting
mTORC1 expression (Kharas et al., 2010). In addition, loss of Itpkb, a competitor of AKT for PIP3
activation, should also result in greater PI3K-signaling. Indeed, Itpkb mutants displayed fewer HSC
numbers, loss of HSC quiescence, increased proliferation, and reduced HSC reconstitution ability. Itpkb-
deficient HSCs had augmented mTOR-signaling as evidenced by S6K phosphorylation and rapamycin
treatment reduced HSC hyperactivity (Siegemund et al., 2015). The HSC phenotypes of the myr-Akt and
Itpkb mutants were similar to the PTEN mutant data reported by Yilmaz et al., suggesting that excessive
PI3K-signaling lead reduced HSC self-renewal. Collectively, these studies demonstrate that increased
AKT/mTORC-activity leads to reduced quiescence and loss of reconstitution ability of the blood cell pool.
Taken together, through the use of genetic knockouts the PI3K/AKT/mTORC-signaling pathway
can either be amplified or diminished in HSCs. Based on the previous experiments, regardless of the
level of PI3K-signaling, HSC quiescence and hematopoietic repopulating ability is compromised
suggesting that this pathway must be precisely regulated to maintain proper HSC numbers and
physiology. The results from the current study also suggest that precise regulation of PI3K-signaling
through PDK1 must be maintained to promote HSC quiescence and long-term repopulating ability to
ensure a balanced hematopoietic output.
ROS impacts HSC quiescence
Through recent studies, ROS has been implicated as a major intracellular signal mediator that
causes the destabilization of HSCs by promoting proliferation and differentiation as well as increased
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DNA damage that can lead to apoptosis. Many studies have implicated various cellular pathways in
maintaining oxidative balance such as the p38 MAPK pathway and hypoxia inducible factors (Hifs);
however, the precise mechanisms that regulate oxidative stress in HSCs are still being elucidated.
Previous experiments show that aberrant ROS signaling results from perturbed PI3K-signaling. Indeed
AKT1/2 DKO fetal liver HSCs have reduced levels of ROS and normal expression can be restored by in
vivo treatment with buthionine sulfoximine (BSO) (Juntilla et al., 2010). In line with this observation, the
loss of FOXO1/3a/4 transcription factors caused increased ROS levels that were reduced upon in vivo
NAC treatment (Miyamoto et al., 2007; Tothova and Gilliland, 2007). Interestingly, conditional deletion of
TSC1 results in elevated HSC ROS levels and mitochondrial biogenesis presumably due to the increase
in mTOR-signaling. Treating the TSC1 mutant with NAC restored ROS levels to normal and rescued BM
cellularity (Chen et al., 2008). Collectively, these results suggest that PI3K-signaling is a positive
regulator of the ROS pathway. In sharp contrast, this study posits that PDK1-mediated PI3K-signaling
functions to limit excessive ROS levels.
In the current study, although it was evident from BMT and cell cycle experiments that PDK1-
deficient HSCs loose quiescence and proliferate more, it was unclear why this was occurring. To
investigate possible causes of HSC instability, microarray analysis was performed on control and
Pdk1Hem-KO LSK cells, and GSEA analysis revealed that PDK1-deficient cells had lost the HSC signature
as well as were compromised in their response to oxidative stress conditions. To verify this, I checked for
increased ROS expression in these mice and found that indeed, ROS was augmented in Pdk1Hem-KO
HSCs. Further experiments suggested that increased ROS expression led to more proliferation, DNA
damage, and apoptosis in progenitor cells. NAC administration partially rescued HSC hyperproliferation
and differentiation of Pdk1Hem-KO cells both in vitro and in vivo. This suggested that abnormal HSC activity
in PDK1-deficient cells was largely due to elevated levels of ROS. These data indicate that incredibly
elevated levels of ROS are toxic for the cells. It is important to note that since PI3K-signaling has
pleiotropic effects, it is unlikely that ROS is the only mediator acting in Pdk1Hem-KO HSCs. Further
experiments investigating other mechanistic modes of action, such as the role of cell cycle regulators, are
needed to provide a comprehensive understanding of PDK1-mediated HSC quiescence. Despite this,
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ROS is responsible for a portion of the Pdk1Hem-KO HSC phenotype since NAC treatment was able to
partially rescue the aberrant HSC changes.
In contrast to the previously reported studies that utilized AKT1/2, FOXO1/3a/4, or TSC1 mutants,
Pdk1Hem-KO mice have reduced AKT/mTORC1-signaling and increased levels of ROS, which, superficially,
does not fit into the paradigm. However, previous studies have demonstrated that mTORC1 mutants,
through ablation of Raptor, that ROS levels are normal (Kalaitzidis et al., 2012). There could be a few
explanations for this: (1) The deletion systems of the knockouts are different and therefore, the mode of
deletion as well as the time of deletion, fetal versus adult, could impact the physiological significance
attributed to the deleted gene. This study ablated PDK1 during fetal hematopoiesis (Vav-Cre) and
measured disturbances in adult HSCs. In contrast, the AKT1/2 DKO study used total knockout fetal HSCs
(Juntilla et al., 2010) and assessed fetal HSC physiological differences in the context of adult BMT, while
the TSC1 and FOXO study used Mx-Cre to ablate in adult HSCs (Chen et al., 2008; Tothova and
Gilliland, 2007). Furthermore, Pdk1Hem-KO ROS levels were only partially rescued with NAC treatment
while treating the TSC1 and FOXO1/3a/4 mutants with NAC completely reverted ROS levels as well as
rescued HSC numbers. The mode of deletion as well as the time of NAC administration after deletion
could influence the level of rescue seen in each mutant. Therefore, further experiments utilizing
Pdk1Flox/Flox ; Mx-Cre mice are needed to determine whether loss of PDK1 during adult hematopoiesis has
the same consequences for HSCs and if complete rescue by NAC treatment is possible. In addition,
extensive studies using proper controls, such as performing knockout studies of all these genes in parallel
with the same Cre line, are required to comprehensively explain the discrepancies in these models. (2)
Another rationale for opposite levels of ROS between Pdk1Hem-KO mice and the TSC1 or AKT1/2 or
FOXO1/3a/4 mutants could be that PDK1-signaling is normal in the other three mutant mice. The FOXOs
are transcription factors so loss of transcriptional activation of a variety of cell survival genes could impact
levels of ROS. The other three proteins, PDK1, AKT, and TSC1, are signaling molecules in the “classical”
PI3K-pathway so I will focus on these proteins for the remainder of the discussion. Unlike AKT or TSC1,
PDK1 can phosphorylate other members in the PI3K/mTORC-pathway, such as S6K, independently of
AKT/mTORC-activation and in addition, activate AKT-independent targets. Therefore, it is tempting to
speculate that PDK1 may be playing a compensatory role and there may be a residual mTORC1-
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signaling or signaling through other AKT-independent PDK1-dependent substrates resulting in ROS
production or repression depending on the context. Overall, the data from this study, as well as results
from others, demonstrate that: 1) the PI3K-signal transduction pathway needs to be tightly controlled to
moderate ROS levels in HSCs, and (2) our studies demonstrate that each individual signaling member of
the PI3K-pathway has a distinct role in activating or restricting ROS levels in HSCs. While elevated ROS
levels provided an explanation for increased cycling in Pdk1Hem-KO HSCs, I next wanted to explore the
mechanistic rationale for augmented ROS expression in these cells.
A novel role for HIF3α in HSC maintenance
Earlier studies depicting a critical role for cell cycle regulation by HIF1α to promote HSC
quiescence have highlighted the importance of maintaining proper metabolic homeostasis within the HSC
(Majmundar et al., 2010; Takubo et al., 2010). Furthermore, HSCs reside in a hypoxic BM niche and their
metabolic makeup is distinctly different from progenitor and differentiated cells. Recent evidence
suggested that HSCs use low-oxygen metabolism and a high rate of glycolysis to promote HSC
quiescence by limiting the generation of ROS (Ito and Suda, 2014; Kohli and Passegue, 2014). As
discussed earlier, high levels of ROS can have detrimental effects on the cell leading to increased
proliferation, DNA damage, and cell death. Given that the Pdk1Hem-KO mouse had elevated levels of ROS,
I wanted to ascertain whether this was caused by a defect in PDK1-mediated HIF1α expression. Confocal
images of control and Pdk1Hem-KO HSCs showed reduced nuclear HIF1α expression in PDK1-deficient
cells. Analysis of downstream PI3K-signaling through phosflow assays found that AKT phosphorylation
was reduced and as a consequence, activity of a positive regulator of HIF1α, S6K, was decreased and a
negative regulator of HIF1α, GSK3, was increased. Diminished p-70S6K activity suggests that HIF1α
protein synthesis is less (Koh et al., 2008), which can explain why Pdk1Hem-KO HSCs have reduced
nuclear HIF1α expression. Interestingly, in CD8+ T cells, the PDK1-mTORC-HIF1α pathway is AKT-
independent therefore suggesting that PDK1 can directly regulate S6K-HIF1α (Finlay, 2014); however, it
is unclear if this is happening in Pdk1Hem-KO HSCs since I also see a reduction in AKT phosphorylation. In
addition to loss of HIF1α protein translation by S6K, active GSK3β can interact with the VHL domain on
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HIF1a to promote degradation (Flugel et al., 2007; Mottet et al., 2003), thus suggesting that there are two
mechanisms in Pdk1Hem-KO HSCs that promote the loss of HIF1α activity. HIF1α is vital to maintain normal
levels of ROS in HSCs to initiate transcription of antioxidants as well as direct metabolic processes to
methods that are low oxidative stress generators (Jang and Sharkis, 2007; Suda et al., 2011). Taken
together, these experiments suggested that ROS levels are unrestrained since HIF1α is not present in
Pdk1Hem-KO cells to moderate oxidative stress. Although, loss of AKT-signaling, and therefore the
simultaneous loss of S6K and gain of GSK activity, explains a post-translational rationale for reduced
HIF1α expression, another explanation could be that Hif1α transcription was reduced in Pdk1Hem-KO mice.
It is important to note that the role for HIF1α in HSC maintenance may be different in fetal and
adult HSCs. A recent study deleted Hif1α with Mx-Cre and found that HSCs were able to self-renew and
provide long-term reconstitution during serial transplantation experiments, which contradict the results
described by Takubo et al. (Vukovic et al., 2016). Furthermore, this study did not report on the metabolic
state of the HSCs so it is unclear if ROS levels are normal in these mice. Given that it is accepted that
HIF1α controls energy output towards glycolysis to reduce ROS production and that mTORC-signaling is
a major regulator of metabolism, it is possible that HIF1α is dispensable as long as mTORC-signaling is
intact. However, in this study, mTORC-signaling is abrogated in the Pdk1Hem-KO HSCs and in addition,
HIF1α activity appears to be reduced therefore two metabolic mechanisms are affected, which may
explain the elevated ROS levels in PDK1-deficient cell.
Interestingly, my investigation into the transcriptional regulation of Hif1α in the Pdk1Hem-KO
revealed some fascinating insights regarding Hif transcription in PDK1-deficient cells. In Pdk1Hem-KO total
BM, there was a decrease in Arnt mRNA, normal levels of Hif1α mRNA, an increase in Hif2α mRNA, and
remarkable increases of Hif3αFL and IPAS mRNA expression. Increased Hif3αFL and IPAS mRNA
expression was corroborated by sequencing and BLAST analysis of total BM. In addition, preliminary RT-
PCR analysis of Pdk1Hem-KO LSK and LK cells showed increased Hif1α mRNA as well as increased Hif3α
mRNA but whether Hif3α mRNA was predominantly Hif3αFL or IPAS needs to be validated. Taken
together, RT-PCR analysis of the Hifs revealed a potential novel explanation for reduced HIF1α protein
expression in the Pdk1Hem-KO HSCs: (1) HIF3α is competing against HIF1α for binding to ARNT and/or (2)
increased IPAS is binding to HIF1α and sequestering it in the cytoplasm. Although, before coming to any
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conclusions, it is first important to first ascertain if HIF3α/IPAS protein expression is increased in Pdk1Hem-
KO HSCs (See Chapter 5 Discussion), I can discuss the likelihood and ramifications of each scenario.
The prevailing dogma is that HIF1α binding to ARNT or HIF2α/ARNT are transcriptional activators
and HIF3α primarily serves as an inhibitor of HIF1/2 (Dengler et al., 2014); however, emerging evidence
in the zebrafish suggests that HIF3α might act as a transcriptional activator and have its own set of
distinct target genes (Zhang et al., 2014). Zhang et al. showed, through transcriptomics and chromatin
immunoprecipitation assays, that HIF1α and HIF3α can activate the same set of genes as well as
distinctly separate genes in response to hypoxia. Zhang et al. corroborated these results in human cells
by overexpressing zebrafish Hif3α, containing the HRE-dependent transactivation activity, and detected
HIF3α in the nucleus. Stabilized HIF3α resulted in increased expression of HIF3α endogenous target
genes whose expression was unchanged when HIF1α was stabilized. Given that HIF3α can
transcriptionally activate a set of genes unique from HIF1α in zebrafish, HIF3α in murine HSCs might
have an additional role besides inhibiting the activity of HIF1/2α. To date, no study has reported HIF3α as
a transcriptional activator in murine hematopoiesis therefore, experiments to further elucidate HIF3α
target genes could provide novel insight into HSC quiescence mediated by hypoxia or even provide
unexplored regulators of HSC maintenance.
While the necessity for HIF3α transcriptional activation is one plausible explanation for increased
HIF3α in Pdk1Hem-KO BM, I also saw upregulation of a specific isoform of Hif3α, IPAS, which is known to
directly inhibit HIF1α expression (Makino et al., 2001). This suggested that the second scenario, where
IPAS could sequester HIF1α in the cytoplasm, may also be occurring to inhibit HIF1α expression and
transcription in Pdk1Hem-KO HSCs. Before making any assumptions, the levels of IPAS protein expression
in the Pdk1Hem-KO HSCs must be verified. However, IPAS inhibition of HIF1α provides an additional
explanation for why HIF1α expression is decreased in PDK1-deficient HSCs besides the loss of
PDK1/AKT/mTORC1-signaling and an increase in GSK3 activity.
Interestingly, there are other Hif3α splice variants besides IPAS. Indeed, there are up to six
reported isoforms of Hif3α (Lee et al., 2004) and so far a comprehensive understanding of whether or not
these isoforms are expressed in HSCs and if they serve a function in HSCs is lacking. For example,
knockouts for NEPAS (neonatal embryonic PAS), another Hif3α isoform, were found have defects in
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embryonic heart and lung development. It was shown that NEPAS primarily functioned to block HIF1α
function (Yamashita et al., 2008). Whether NEPAS is expressed in hematopoietic cells and functions to
inhibit HIF1α remains to be determined. Further analysis regarding which Hif3α isoforms are present in
Pdk1Hem-KO HSCs may shed light into which isoforms have roles in hematopoiesis. In addition, it would be
interesting to ascertain why Hif3α and IPAS are increased in the Pdk1Hem-KO BM? Furthermore, does
PDK1 regulate the transcription of Hifs and control Hif3α isoform splicing? These are questions that still
remain to be answered. Regardless, this study is the first to suggest that Hif3α may have a functional role
in HSC quiescence. Further investigation into the Hif mechanism will broaden our understanding of how
hypoxia and PDK1-mediated PI3K-signaling regulate quiescence.
Future Directions and perspectives
In the past 10 years, studies elucidating the metabolic regulation of HSCs have provided insight
into the mechanisms that govern quiescence. It is thought that quiescence serves to limit the amount of
cellular damage from cytotoxic agents and mitochondria respiration (Ito and Suda, 2014; Kohli and
Passegue, 2014; Takubo et al., 2010). In addition, it appears that quiescence has an anaerobic bias since
HSCs reside in a hypoxic niche and their primary mode of energy production is glycolysis, which
predominantly occurs under low oxygenic conditions and produces less oxidative stress. Given that the
Pdk1Hem-KO HSCs have increased levels of ROS and reduced HIF1α expression, future experiments to
assess the metabolic profile of Pdk1Hem-KO HSCs to gain a comprehensive understanding of how PDK1-
deficiency impacts HSC quiescence are needed. Based on the current data and what is reported in the
literature, I expect to see decreased glycolysis in Pdk1Hem-KO HSCs, which can be assessed via RT-PCR
of glycolytic and oxidative phosphorylation genes are needed to assess if this is true.
Many hematological malignancies show mutations in the PI3K-pathway. Indeed, many cancers
have mutations in PTEN and augmented PDK1 expression is seen in a variety of other cancer types
(Ahmed et al., 2008; Arsenic, 2014; Eser et al., 2013; Iorns et al., 2009). Furthermore, 40% of acute
myeloid leukemia (AML) patients have mutations that overexpress PDK1 (Mora et al., 2004; Pearn et al.,
2007; Zabkiewicz et al., 2014) and it has been suggested chronic or acute myeloid leukemias develop
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from transformed HSCs that exhibit leukemia-initiating stem cell properties (Passegue et al., 2003).
Thus, understanding the biology behind PDK1-mediated signaling in HSCs to maintain quiescence as
well as promote balanced proliferation is highly relevant to our understanding of clinical diseases. This
study, along with other studies of genetic mutants in the PI3K-pathway, suggests a link between HSC
metabolism and the onset of leukemic-like properties in HSCs. Interestingly, cancer cells use glycolysis
even in the presence of oxygen in what is known as the ‘Warburg effect’ (Kohli and Passegue, 2014).
Therefore, elucidating how mitochondrial biogenesis and energy production are affected in Pdk1Hem-KO
could provide novel insight into cancer therapies.
The results from this study are the first to suggest that PDK1 is responsible for maintaining HSC
quiescence (Figure 30). Without proper PDK1-signaling, HSCs hyperproliferate, loose self-renewal
capacity, and are unable to provide radioprotection presumably because of augmented ROS expression.
This caused increased differentiation and apoptosis in the progenitor cells. NAC treatment partially
rescued HSC hyperproliferation, differentiation, and increased ROS levels in the Pdk1Hem-KO mice. Further
experiments suggested that elevated ROS levels were due to decreased HIF1α protein expression. There
are two possible explanations for decreased HIF1α: (1) A coordinated loss of PDK1/AKT/mTOR1-
signaling and increased GSK3 activity, which results in less HIF1α translation and promotes HIF1α
degradation, respectively; and (2) high levels of Hif3α and IPAS mRNA expression in total BM could
indicate inhibition of HIF1α at the protein level. While further experiments are needed, taken together, the
results from this body of work suggest that PI3K-signlaing mediated by PDK1 is essential for promoting
HSC quiescence.
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Figure 30: Loss of PI3K-signaling mediated by PDK1 causes HSCs to proliferate more and lose quiescence.
Schematic shows that reduced PI3K-signaling can cause reduced S6K activity and increased GSK3 activity. Thus, a positive and negative regulator of HIF1α expression and a negative regulator of HIF1α expression.
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Chapter 7: Experimental Procedures
Mouse Generation
Pdk1Flox mice have previously described (Inoue et al., 2006) and were crossed to Vav-Cre (Georgiades et
al., 2002) mice. Pdk1Flox/+ ; VavCre/+ mice were then crossed to Pdk1Flox/+ or Pdk1Flox/Flox to generate control
(Pdk1Flox/+, Pdk1Flox/Flox, or Pdk1Flox/+ ; VavCre/+) or Pdk1Hem-KO (Pdk1Flox/Flox ; VavCre/+) mice. For a schematic
of the breeding scheme, see Figure 5. Sex matched littermate 4-8 week old mice were used for all
experiments.
Cell Preparation
Single cell suspension of total bone marrow (BM) was created after flushing the tibias and femurs from
mice ages 4-8 weeks and lysing RBCs with ammonium chloride (STEMCELL Technologies) for two
minutes at RT, washing with PBS and then straining through 100um mesh. Live cell counts were negative
for Trypan Blue staining (Amresco). Single cell suspensions were cell surface stained for 10 min on ice,
washed with PBS, spun at 1200rpm for 5 minutes at 4C, reconstituted in PBS, and then acquired by flow
cytometry. Lineage positive markers were CD11b, CD19, B220, CD3e, CD11c, Ter119, and Ly6G and
were obtained from BD biosciences.
Bone Marrow Transplantation Experiments
For radioprotection assessment, 106 total BM cells were injected into lethally irradiated (10 Gy) congenic
wild-type (CD45.1+) recipient mice. For competitive repopulation experiments, 0.5 x 106 total BM from
either control of Pdk1Hem-KO mice were mixed 1:1 with WT (CD45.1+) total BM cells and injected into
lethally irradiated congenic wild-type (CD45.1+) recipients.
Apoptosis Assay
Apoptotic cells were detected with the Annexin V PE Apoptosis Detection Kit (BD559763). Briefly, 2.5-3 x
106 total BM cells were stained for cell surface markers on ice for 10 minutes, washed with PBS, and spun
at 1200rpm for 5 minutes at 4C. For each sample, 5ul of Annexin V was added to 100ul of 1X buffer and
123
incubated at room temperature for 15 minutes. 200-400ul of 1X buffer was added to dilute out the stain
and 1ul of 7AAD was added to denote live cells. Cells were acquired by flow cytometry immediately.
Cell Proliferation/Cell Cycle Measurements
For BrdU incorporation experiments, 6-week old mice were i.p. injected with 3.33 mg of BrdU (Sigma
B5002) and drinking water was treated with 0.8mg/ml of BrdU. At 16 hours after injection, mice were
sacrificed and BM cells were stained for cell surface markers for 10 minutes on ice, washed with PBS,
and spun down at 1200rpm for 5 minutes at 4C. Cells were then prepped using BD fix/perm Buffer kit by
adding 100ul of fix/perm buffer and incubating at 4C for 20 minutes. Cells were washed with 1ml of 1X
perm/wash buffer and then digested with DNaseI for 1 hour at 37C. Cells were washed with PBS and
then stained with anti-BrdU-FITC (BD347583) for 80 minutes at room temperature (RT) before detection
by flow cytometry. For Ki67 analysis, BM cells were stained for cell surface markers, fixed, and
permeabilized with BD Fix/Perm kit. Cells were stained with anti-Ki67—APC (BD558615) for 30 minutes
on ice and analyzed by flow cytometry. For Pyronin Y analysis, cells were surface stained, incubated with
5ug/ml of Hoechst 33342 at 37C for 45 minutes and then 1ug/ml Pyronin Y (Sigma) was added for an
additional 45 minutes at 37C before acquisition by flow cytometry.
Side Population Analysis
BM cells were stained for cell surface markers, incubated with 5ug/ml of Hoechst 33342 (Life
Technologies) in DMEM (+ 2% FBS + 1% HEPES) for 90 minutes at 37C and washed twice with HBSS (+
2% FBS + 1% HEPES). 1ul of 7AAD was added to each sample before flow cytometry acquisition.
Measuring ROS levels
BM cells were stained with cell surface markers and then incubated with 2mM CM-H2DCFDA (Life
Technologies C6827) in pre-warmed HBSS at 37C for 15 minutes. The cells were then pelleted and
resuspended in PBS before acquisition. For positive controls, cells were incubated with 100uM H2O2
while staining with CM-H2DCFDA.
124
mitoSOX staining
BM cells were surface stained and then incubated with 5uM MitoSOX Red (Life Technologies M36008) in
HBSS for 30 minutes at 37C. Cells were then pelleted and resuspended in PBS before FACS acquisition.
DNA damage assessment
BM cells were surface stained and processed according to the BD cyto fix/perm kit instructions 554714.
Cells were stained with rabbit anti-phos-H2A.X (Cell Signaling, 2718) diluted 1:300 in perm/wash buffer
for 1 hour at RT. Cells were washed in perm/wash buffer and then stained with Alexa 488 anti-rabbit (Life
Technologies, 0.2ul/100ul of perm/wash buffer) for 1 hour at RT. Cells were washed and then
resuspended in PBS before acquisition. For positive controls, BM cells were either irradiated at 2 grey or
incubated with 200uM H2O2 in HBSS for 3 hours at 37C.
Western Blot Analysis
For immunoblot analysis, control or Pdk1HemKO BM cells were MACS B cell depleted (CD19, B220),
starved for 5.5 hours in 1%BSA/DMEM or starved for 5 hours and then stimulated for 30 minutes. with
10%FBS/DMEM with 50ng/ml stem cell cytokines (Stemgent: IL6, IL3, TPO, SCF, Flt3l) before being
lysed with 1X Cell Lysis Buffer (Cell Signaling) with 1:25 protease inhibitor cocktail (Complete; Roche)
and 1mM PMSF (Santa Cruz). Cell lysates were incubated at 95C for 5 minutes with sample buffer
(NuPAGE; Life Technologies) containing 1% 2-Mercaptoethanol (Sigma). For inhibitor WB, wild type total
BM was incubated with either no inhibitor or 1uM PDK1 inhibitor (Sigma GSK2334470) while being
starved for 5.5 hours in 1%BSA/DMEM or starved for 5 hours and then stimulated with 10%FBS/DMEM
with stem cell cytokines for 30 minutes. before being lysed. Proteins were run on an 8-10% SDS-PAGE
and transferred to PVDF membrane (BioRad Laboratories). The membranes were blocked with 5% BSA
(Fisher) and then treated with primary and secondary antibodies. The blots were visualized using
ProtoGlow ECL (National Diagnostics) or SuperSignal West Pico Chemiluminescent Substrate (Life
Technologies, 34080) and detected on Image Station 440 (Kodak). Antibodies used can be found in
Table 8.
125
Phosflow
BM cells were starved in 1% BSA/DMEM for 2 hours, stained for cell surface markers, and then
stimulated for 30 min with 10%FBS/DMEM and 50 ng/ml stem cell cytokines (Stemgent: Flt3l, SCF, TPO,
IL3, IL6). Cells were then fixed (BD558049) for 10 min at 37C and incubated with primary antibodies in
permeabilization buffer (BD558050) for 1 hour on ice and then with secondary antibodies for 30 minutes
on ice. Cells stained only with secondary were used as a control. Antibodies used can be found in Table
8.
Table 8: Antibodies used in this study.
126
RNA Extraction and Microarray Analysis
Total RNA was isolated with RNeasy Mini kit (Qiagen). cDNA was synthesized with Oligo(dT) primer and
Maxima reverse transcription (Thermo Fisher). For transcriptional profiling studies, HSPCs (Lin-Sca1+c-
kit+) cells were sorted using BD FACS Aria from both control and Pdk1Hem-KO mice (n = 5 and 10,
respectively) before RNA extraction. RNA samples taken from three independent sorts were pooled for
microarray studies. Expression profiling was performed suing Illumina’s MouseWG-6 v2.0 Expression
BeadChip at Yale Center for Genome Analysis with max probes by CollapseDataset modeule in
Genepattern (Reich et al., 2006). These genes were preranked for log2 fold change and analyzed for
gene enrichment set analysis (GSEA) (Mootha et al., 2003; Subramanian et al., 2005). The list of
transcription factors described in this study was selected using GO0003700 (sequence-specific DNA
binding transcription factor activity) from Quickgo using its default behavior (is_a, part_of and occurs_in
relationships only) (Binns et al., 2009; Huang da et al., 2009a, b).
Table 9: Primers for qRT-PCR.
127
In vitro culture
Lineage depleted BM cells were placed in culture with 10%FBS/DMEM + 50ng/ml stem cell cytokines
(Flt3l, SCF, TPO, IL3, IL6; STEMGENT). Cells were cultured in the presence of no drug or 1uM PDK1
inhibitor (GSK2334470; Sigma). Cells were harvested and stained for LSK or lineage positive markers
(CD11b, CD19, B220, CD3e, CD11c, Ter119, Ly6G) and acquired by flow cytometry.
Cell Trace staining
In vitro cells were stained for proliferation with 5uM Cell Trace CFSE (Life Technologies C34554) or 5uM
Cell Trace Violet (C34557) in PBS for 30 minutes at 37C. Cells were washed twice and proliferation was
assessed at 24, 48, or 72 hours.
In vivo NAC treatment
Two-week-old mice were i.p. injected with PBS (vehicle) or with 100mg/kg N-acetyl cysteine (Sigma)
diluted in PBS and pH adjusted to 7.4 everyday for 4 weeks.
In vitro NAC treatment
Lineage depleted BM cells from either control of Pdk1Hem-KO mice were placed in culture with
10%FBS/DMEM + 50ng/ml stem cell cytokines (Flt3l, SCF, TPO, IL3, IL6; STEMGENT). After 24 hours,
cells were harvested and stained for LSK.
In vitro rescue with p38 Inhibitor and NAC
Control and Pdk1Hem-KO BM was sorted by FACS Aria for LSK. 50,000 LSK cells were plated per condition
in 10%FBS/DMEM + 50ng/ml stem cell cytokines (IL3, SCF, TPO, Flt3l, IL6) with vehicle, 100uM NAC
(Sigma A7250), or 10uM 38 inhibitor (SB203580; Selleckchem). Cells were harvested at 24 and 48 hours,
stained for cell surface markers and assessed for ROS levels using CM-H2DCFDA.
128
IHC staining and Microscopy
Freshly sorted cells, typically 500-2000, were plated onto poly-D-lysine (Sigma) coated chamber slides
and incubated at 37C in 10%FBS/DMEM + 50ng/ml stem cell cytokines (IL3, SCF, TPO, Flt3l, IL6) for 2h
before fixing for 10 minutes with 4%PFA at RT. Cells were permeabilized in 0.15% Triton-X100 for 2
minutes at RT and then blocked overnight in 1%BSA/PBS at 4C. Cells were incubated with primary in
blocking solution for 2 hours at 37C and then with secondary for 1 hour at 37C. For nuclear stain, 5ug/ml
Hoescht/PBS was added for 6 minutes at RT and then slides were mounted with VectaShield and imaged
on a Zeiss 710 Confocal using a 100x objective. Antibodies used can be found in Table 8. Florescence
quantification was performed by ImageJ (NIH) analysis similar to McCloy et al.
(http://www.tandfonline.com/doi/abs/10.4161/cc.28401). In summary, Mean Florescence Intensity (MFI)
was calculated by determining the corrected total cellular fluorescence (CTCF).
CTCF = integrated density – (area of selected cell X mean florescence of background)
For these experiments, the background is an average of three measurements taken from the surrounding
cell. For statistical analysis, two-tailed unpaired Student’s t-test were performed using Graphpad Prism
v4.
Statistical analysis
Statistics were calculated using Graphpad Prism v4. Two-tailed, paired Student’s t-tests were performed,
unless otherwise specified, to determine how different the means of the control and Pdk1Hem-KO groups
were with a one star significant p-value set at less than 0.05. Two stars is equivalent to p <0.01 and three
stars p<0.001.
129
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