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The role of endoplasmic reticulum chaperones in regulating hematopoietic stem cells and hematological malignancies
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The role of endoplasmic reticulum chaperones in regulating hematopoietic stem cells and hematological malignancies
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Content
THE ROLE OF ENDOPLASMIC RETICULUM CHAPERONES IN REGULATING
HEMATOPOIETIC STEM CELLS AND HEMATOLOGICAL MALIGNANCIES
by
Biquan Luo
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
August 2013
Copyright 2013 Biquan Luo
ii
DEDICATION
To my loving parents, Xinglu Luo and Kebin Li, who have inspired
my lifelong interest in science and have given me unconditional love
and support along the way.
iii
ACKNOWLEDGEMENTS
First and foremost, I would like to express my sincere appreciation to my mentor,
Dr. Amy Lee, for her excellent guidance and enthusiastic support throughout my Ph.D.
study. Amy, with her dedication, courage and enthusiasm, presents me a role model of a
successful scientist and a happy woman. She has made the Lee Lab an exceptionally
efficient and friendly working environment for students, postdoctoral fellows and staff.
I owe my deepest gratitude to my wonderful committee members. Dr. Gregor
Adams for his many insightful suggestions, experiment assistance and the tremendous
amount of inspiration and support. Dr. Michael Stallcup for the helpful discussions,
insightful ideas and supportive encouragement. I would also like to thank Dr. Si-yi Chen
for the wonderful collaboration, many inspiring discussions and resource support on my
projects. Dr. Darryl Shibata and Dr. Louis Dubeau for the helpful consultations on
histology and pathology. Dr. Valter Longo and his lab for the use of hematology analyzer.
And finally, Dr. Yvonne Lin and her lab for the helpful discussions.
My Ph.D. study would not have been as colorful without all the former and
current members of the Lee lab. Former members include Shiuan Wey, Risheng Ye,
Miao Wang, Kyle Pfaffenbach, Hui Zhou, Yong Fu, Dezheng Dong, Changhui Mao,
Michelle Pong and Kate Ott. Current members include Chun-Chih Tseng, Wan-Ting
Chen, Genyuan Zhu, Jieli Shen, Yi Zhang, Yuan-Li Tsai, Daisy Flores, Ninad Agashe
iv
and John Johnson. I wish to thank all of them, whom I shared the credit of my work with,
whom I learned scientific skills and techniques from, and whom I shared laugh and tears
with throughout these years.
I am grateful for the helpful discussions and technical support from my former
and current classmates and fellow researchers, including but not limited to: Ben Lam,
Sung-Hyung Lee, Xiaoying Zhou, Dilani Rosa, Lindsey Jones, Min Wei, Chia-Wei
Cheng, Zong Wei, Han Wang and Kaijie He.
This dissertation would not have been possible without the generous technical
support and advice from the USC core facilities and other departments: Lora Barsky and
Dave Stanley from the USC Norris Comprehensive Cancer Center Flow Cytometry Core
Facility, Denis Alexander Trana and Mo-Li Chen from the USC Norris Comprehensive
Cancer Center Translational Pathology Core, Ernesto Barron and Douglas Hauser from
the USC Norris Comprehensive Cancer Center Cell and Tissue Imaging Core, Michelle
Mac Veigh from the Cell and Tissue Imaging Core of the USC Research Center for Liver
Diseases, Grant Dagliyan, Ryan Park and Lindsey Hughes from the USC Molecular
Imaging Center, Jeanne Willoughby and JaMera Landren from the USC Radiation Safety
Department.
I would like to thank my boyfriend, Chun-Yuan Chen, who just became my fiancé
recently, as well as all my former and current roommates including Kaijie He, Wei Liang,
v
Yujiao Sun, Fan Yang, Liying Zhu, Shih-Yi Jan, Wan-Ting Chen, and Che-Wei Lai.
Together, they have taken care of me and have been a wonderful part of each day
throughout my Ph.D. studies.
Finally, I would like to thank my parents and my family members in China. They
have always been there for me and provided me with unconditional love and support
throughout the adventures and experiences of my life.
vi
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES ix
LIST OF FIGURES
LIST OF ABBREVIATIONS
x
xiii
ABSTRACT xvi
Chapter 1: Overview and Introduction 1
1.1 Endoplasmic reticulum
1.1.1 ER stress and UPR
1.1.2 GRP94 and its functions
1.1.3 GRP78 and other ER chaperones
1
1
5
11
1.2 Hematopoietic system
1.2.1 Hematopoiesis and HSCs
1.2.2 HSC homeostasis
1.2.3 Intrinsic and extrinsic regulation of HSCs
15
15
17
19
Chapter 2: GRP94 is required for maintaining HSC interactions with the
adult BM niche
25
2.1 Introduction 25
2.2 Materials and methods 26
2.3 Results 33
2.3.1 Generation of an inducible GRP94 conditional knockout
mouse model in the hematopoietic system (cGrp94
f/f
)
33
2.3.2 cGrp94
f/f
mice displayed increased myeloid
hematopoiesis and impaired lymphogenesis
35
2.3.3 GRP94 deficiency in the BM leads to expanded HSC
pool and increased granulocyte–monocyte progenitors
38
2.3.4 The increased number of GRP94-deficient HSCs was 42
vii
due to loss of quiescence
2.3.5 GRP94-deficient HSCs exhibited increased mobilization
and increased extramedullary hematopoiesis
44
2.3.6 cGrp94
f/f
HSCs displayed impaired interaction with the
BM niche
46
2.3.7 cGrp94
f/f
HSCs failed to reconstitute the hematopoietic
system in the presence of wild type competitors
2.3.8 GRP94 in HSCs, but not niche cells, is necessary for
maintaining HSC homeostasis
2.3.9 cGrp94
f/f
HSCs lacked cell surface integrin α4
expression and exhibited impaired binding to fibronectin
49
51
53
2.4 Discussion 56
Chapter 3: GRP94 deficiency in the hematopoietic system alters
proliferation regulators in HSCs
62
3.1 Introduction 62
3.2 Materials and methods 65
3.3 Results 72
3.3.1 GRP94 deficiency led to loss of quiescence in HSCs
3.3.2 In vitro expansion of GRP94-null LSK cells
72
74
3.3.3 cGrp94
f/f
primitive hematopoietic cells displayed
increased PI3K-AKT activation
76
3.3.4 Microarray analysis identified cell cycle regulator
Ms4a3 downregulated in cGrp94
f/f
primitive hematopoietic
cells
3.3.5 Context-dependent expression of Ms4a3 in LSK cells
79
81
3.3.6 GRP94 depletion reduced cell surface connexin32
plaque number
3.3.7 GRP94 depletion did not affect cell surface CXCR4 and
Tie2 expression
82
87
3.4 Discussion 89
Chapter 4: Targeting GRP94 by lentivirus-mediated shRNA and
investigating the role of GRP94 in multiple myeloma
96
4.1 Introduction 96
4.2 Materials and methods 101
4.3 Results
4.3.1 GRP94 knockdown in the mouse MM cells affected their
survival
115
115
viii
4.3.2 Determining the feasibility of using optical image to
monitor MM cells in vivo
4.3.3 Establishing a syngeneic mouse MM model
4.4 Discussion
123
124
127
Chapter 5: The role of GRP78 in normal and leukemic HSCs homeostasis 135
5.1 Introduction
5.2 Materials and methods
135
137
5.3 Results
5.3.1 GRP78 depletion led to increased apoptosis in normal
HSCs
5.3.2 Intrinsic effect of GRP78 depletion on HSC pool size
5.3.3 GRP78 depletion reversed the loss of quiescence and
increased proliferation in PTEN-null HSC
5.4 Discussion
Chapter 6: Conclusions and Perspectives
BIBLIOGRAPHY
139
139
141
143
146
149
161
ix
LIST OF TABLES
Table 2.1: Primer sequences for mouse genotyping
27
Table 3.1: Top 10 genes downregulated and upregulated in cGrp94
f/f
LSK cells
compared to Grp94
f/f
LSK cells identified by microarray analysis
80
Table 4.1: shRNA and packaging plasmids
Table 4.2 Lentivirus titer calculation (GFP
+
)
Table 4.3 Lentivirus titer calculation (puromycin
+
)
103
106
111
x
LIST OF FIGURES
Figure 1.1: Unfolded protein response and its regulation on cell activities
4
Figure 1.2: The effect of GRP94 depletion on UPR signaling
7
Figure 1.3: Different localizations and functions of major ER chaperones
14
Figure 1.4: Hematopoietic cell ontogeny
17
Figure 2.1: Conditional knockout of Grp94 in the BM
34
Figure 2.2: cGrp94
f/f
mice displayed altered myeloid and lymphoid
differentiation
36
Figure 2.3: cGrp94
f/f
mice displayed loss of viability
37
Figure 2.4: GRP94 deficiency in the BM expanded the primitive cell pool
39
Figure 2.5: GRP94 deficiency led to increased granulocyte–monocyte
progenitors in the BM.
41
Figure 2.6: GRP94-deficient LSK cells displayed increased proliferation and
loss of quiescence
43
Figure 2.7: Increase extramedullary hematopoiesis and increased HSC
mobilization in cGrp94
f/f
mice.
45
Figure 2.8: cGrp94
f/f
HSCs displayed impaired interaction with the niche
48
Figure 2.9: cGrp94
f/f
HSCs failed to reconstitute the hematopoietic system in
the presence of WT competitors
50
Figure 2.10: Effect of GRP94-null microenvironment on HSC maintenance 52
Figure 2.11: Inability of Grp94 knockout HSCs to express surface integrin α4
or bind to fibronectin.
54
Figure 2.12: Changes in hematopoietic stem cell homeostasis and
hematopoiesis upon GRP94 depletion
55
xi
Figure 2.13: Scheme of how GRP94 deficiency affects HSC homeostasis
56
Figure 3.1: GRP94 deficiency in the BM expanded the primitive
hematopoietic cell pool
73
Figure 3.2: In vitro culture of GRP94-null LSK cells
75
Figure 3.3: Increased AKT activation is required for cGrp94
f/f
LSK
proliferation
78
Figure 3.4: Context-dependent expression of Ms4a3 in LSK cells
82
Figure 3.5: Reduction of Cx32 cell surface plaques reduced upon Grp94
deletion
84
Figure 3.6: Effect of Cx32 mimetic peptides on LSK cell proliferation in vitro
86
Figure 3.7: Validation of the ability of
32
Gap27 to block cellular
communication using the scrape-loading and dye transfer assay
87
Figure 3.8: Lack of effect of GRP94 depletion on Tie2 and CXCR4 surface
expression
88
Figure 4.1: Interaction between MM cells with the BM microenvironment
100
Figure 4.2: Restriction enzyme digestion on pGIPZ plasmids
104
Figure 4.3: Lentivirus-mediated knockdown of human GRP94
108
Figure 4.4: Restriction enzyme digestion on packaging and shRNA plasmids
110
Figure 4.5: Lentivirus titering and knockdown of mouse GRP94
113
Figure 4.6: Lentivirus-mediated knockdown of mouse GRP94 in 5TGM1/luc
cells caused increased apoptosis
117
Figure 4.7: GRP94 knockdown in 5TGM1/luc cells were not persistent even
under puromycin selection
119
Figure 4.8: shGrp94 caused moderate increase of apoptosis in MEFs 121
xii
Figure 4.9: shGrp94 caused moderate increase of cell death in U266 cells
122
Figure 4.10: Luciferase activity of 5TGM1/luc cells in vitro
124
Figure 4.11: Pathological characteristics of 5TGM1/luc MM model
126
Figure 5.1: GRP78-deficient LSK cells displayed increased cell death
Figure 5.2: Experiment scheme of BM transplantation
Figure 5.3: Reduction of HSC-enriched population is intrinsic to GRP78
deficiency in BM cells
Figure 5.4: GRP78-deficient LSK cells displayed increased cell death
140
142
143
145
xiii
LIST OF ABBREVIATIONS
17-AAG 17-N-allylamino-17-demethoxygeldanamycin
7-AAD 7-Aminoactinomycin D
ADP adenosine diphosphate
AGM aorta-gonad-mesonephros
AGN angiopoietin
ATF 4 activating transcription factor 4
ATF6 activating transcription factor 6
ATP adenosine triphosphate
BCL-2 B-cell CLL/lymphoma 2
BFU-E burst-forming unit-erythroid
BIK Bcl-2-interacting killer
BM bone marrow
BSA bovine serum albumin
bZIP basic leucine zipper
CaBP4 calcium binding protein 4
CaR calcium-sensing receptor
CDK cyclin-depedent kinase
CFC colony-forming cell
CFSE carboxyfluorescein succinimidyl ester
CFU-G colony-forming unit-granulocyte
CFU-GEMM colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte
CFU-GM colony forming unit-granulocyte/macrophage
CFU-M colony-forming unit-macrophage
CHOP C/EBP homology protein
CLP common lymphoid progenitor
CMP common myeloid progenitor
CRT calreticulin
Cx32 connexin 32
Cx43 connexin 43
CXCR4 chemokine (C-X-C motif) receptor 4
DAPI 4',6-diamidino-2-phenylindole
DC dendritic cell
DMEM Dulbecco's Modified Eagle Medium
DMSO dimethyl sulfoxide
ER endoplasmic reticulum
ERAD ER-associated protein degradation
ESC embryonic stem cell
FBS fetal bovine serum
xiv
FN fibronectin
GMP granulocyte-monocyte progenitor
gp96 96-kDa glycoprotein
GPIb-IX-V platelet glycoprotein Ib-IX-V
GRP78 glucose regulated protein 78
GRP94 glucose regulated protein 94
Gy Gray
HOX homeobox
HSC hematopoietic stem cell
HSP90 heat shock protein 90
IGF I insulin-like growth factor I
IGF II insulin-like growth factor II
IL-3 interleukin-3
IL-6 interleukin-6
IMDM Iscove's Modified Dulbecco's Media
IRE 1α inositol-requiring kinase 1α
KDEL Lys-Asp-Glu-Leu
Lin lineage
LK Lin
-
c-Kit
+
LSK Lin
-
c-Kit
+
Sca-1
+
LT-HSC long-term hematopoietic stem cell
MEF mouse embryonic fibroblasts
MEM minimum essential media
MEP megakaryocyte-erythrocyte progenitor
MHC
microCT
major histocompatibility complex
micro computed tomography
MM multiple myeloma
MNC mononuclear cell
MPL myeloproliferative leukemia virus oncogene
MPP multipotent progenitor
NT non-treated
OPG osteoprotegerin
OPN osteopoietin
P/S penicillin/streptomycin
PBS phosphate buffered saline
PDI protein disulfide isomerase
PERK protein kinase RNA (PKR)-like ER kinase
PI(3,4,5)P3 phosphatidylinositol (3,4,5)-trisphosphate
pI.pC polyinosine-polycytosine
PI3K phosphoinositide 3-kinase
PTEN phosphatase and tensin homolog
xv
RANKL receptor activator of nuclear factor kappa-B ligand
ROS reactive oxygen species
RT-PCR reverse transcription polymerase chain reaction
SCF stem cell factor
SDF
SNARF
stromal cell-derived factor-1
carboxylic acid, acetate, succinimidyl ester
SLAM signaling lymphocytic activating molecule
ST-HSC short-term hematopoietic stem cell
TBS tris-buffered saline
Tg thapsigargin
TLR toll-like receptor
TNF tumor necrosis factor
TPO thrombopoietin
TRA-1 tumor rejection antigen-1
Tu tunicamycin
UPR unfolded protein response
VCAM-1 vascular cell adhesion molecule 1
VEGF vascular endothelial growth factor
VLA-4 very late antigen-4
WT wild type
XBP-1 x-box-binding protein 1
xvi
Abstract
The endoplasmic reticulum (ER) is a cellular organelle essential for protein
folding and transportation, as well as lipid synthesis and Ca
2+
storage. ER chaperones
play critical roles in assisting protein folding and processing. Two most abundant ER
chaperones are GRP94 and GRP78.
Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is
regulated by both intrinsic gene expression products and interactions with extrinsic
factors in the HSC niche. The adult BM niche maintains HSC quiescence via soluble
cues and direct contact. Traditionally regarded as an ER chaperone that assists the folding
and processing of secretory and membrane bound proteins, GRP94 is speculated to play
an important part in regulating HSC homeostasis. To directly elucidate the requirement of
GRP94 in HSC homeostasis, we employed an inducible conditional knockout strategy to
eliminate GRP94 in the hematopoietic system. The mutant mice displayed a 2-fold
increase in HSC pool size resulted from a loss of quiescence and an increase in
proliferation. The expansion of the HSC pool can be attributed to the impaired interaction
of HSCs with the BM niche, evidenced by enhanced HSC mobilization and severely
compromised homing and lodging ability of primitive hematopoietic cells. Transplanting
wild type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a
normal hematology profile and comparable numbers of HSCs as compared to WT
controls, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining
xvii
HSC homeostasis. Furthermore, GRP94-null HSCs exhibited a near complete loss of
integrin α4 expression on the cell surface and showed impaired binding with fibronectin,
which are key molecules known to mediate HSC-niche interactions, providing a possible
explanation for the loss of interaction of GRP94-null HSCs with the adult BM niche.
Further investigation into the underlying mechanisms for this intrinsic
hyperproliferation in GRP94-defiecient BM HSCs discovered an 1.8-fold increase in
AKT activation, corresponding with higher production of PI(3,4,5)P3, indicating elevated
PI3K activation. Treatment of GRP94-null HSCs with AKT inhibitors demonstrated that
this elevated AKT activation is required for the increased proliferation in GRP94-null
HSCs. Microarray analysis on freshly isolated Lin
-
c-Kit
+
Sca-1
+
(LSK) cells
demonstrated a 97% reduction in the expression of the hematopoietic cell cycle regulator
Ms4a3 in the GRP94-null LSK cells. Furthermore, we observed a reduced expression of
cell surface connexin 32 plaques in GRP94-null LSK cells. However, suppression of
connexin 32 hemichannel activity in WT LSK cells through mimetic peptides did not
lead to increased LSK proliferation in vitro. In addition, two other important cell surface
proteins that mediate HSC-niche interactions, specifically Tie2 and CXCR4, were not
impaired by Grp94 deletion.
The requirement of GRP94 for maintaining HSC-niche interaction provides a
therapeutic strategy of targeting GRP94 in the treatment of multiple myeloma (MM), the
progress of which relies closely on the interaction of tumor cells with the BM
microenvironment. We utilized a syngeneic MM mouse model to test whether GRP94
xviii
knockdown in tumor cells suppresses disease progression. Knockdown of GRP94 in
mouse MM cells with lentivirus-mediated shRNA led to increased apoptosis in vitro, and
GRP94 protein expression was gradually restored despite the presence of puromycin
selection. Despite the unsuccessful knockdown of GRP94 in vitro, the syngeneic MM
mouse model was established via tail vein tumor cell inoculation. MM mice displayed
clusters of cells with lymphocyte-like morphology in the BM, as well as lesions of bone
resorption. Further analysis at late stage MM mice demonstrated multiple tumor
formation in the BM, accompanied by significant weight loss and hunched posture.
In parallel with the study on GRP94 in HSC homeostasis, this dissertation also
includes studies on the role of GRP78 in regulating HSC proliferation and apoptosis
under both physiological condition and PTEN-null driven leukemia progression. Unlike
GRP94 whose depletion causes HSC hyperproliferation, GRP78 depletion did not affect
the cell cycle distribution of HSCs. However, in the context of PTEN-null driven
leukemia, partial reduction of GRP78 suppressed the hyperproliferation induced by
PTEN inactivation. Examining the requirement of GRP78 for HSC survival and apoptosis
revealed an increased HSC apoptosis upon complete loss of GRP78, whereas
heterozygous deletion of Grp78 did not lead to increased HSC apoptosis even in the
context of cancer.
In summary, studies in this dissertation revealed the comprehensive roles of ER
chaperones, namely GRP94 and GRP78, in regulating HSC homeostasis and
hematological malignancies.
1
Chapter 1
Overview and Introduction
1.1 Endoplasmic reticulum
1.1.1 ER stress and UPR
The endoplasmic reticulum (ER) is a cellular organelle that forms a network
connecting the nucleus, cytosol, organelles and the plasma membrane. The ER is
essential for the synthesis, folding and modification of secretory and membrane proteins.
It is also the site for lipid synthesis and a major intracellular site for calcium storage.
Physiological and pathological conditions that perturb the ER, such as nutrient
deprivation, hypoxia, ER calcium depletion, impaired glycosylation or disulfide bond
formation, oxidative stress, and viral or bacterial infection, may lead to ER stress. ER
stress occurs when the protein load exceeds the ER capacity to fold or degrade them, and
is manifested by the accumulation of malfolded proteins in the ER. ER stress triggers an
evolutionarily conserved quality control mechanism, the unfolded protein response
(UPR), which aims at restoring ER homeostasis by activating a cascade of signaling
molecules to transiently arrest protein translation, to induce ER molecular chaperones and
enzymes that enhance the protein folding capacity, and to initiate a process to export and
degrade the misfolded ER proteins (Ron and Walter, 2007).
2
There are three canonical branches in the UPR signaling pathway, which are
mediated by three ER stress sensors: protein kinase RNA (PKR)-like ER kinase (PERK),
inositol-requiring kinase 1α (IRE1α), and the activating transcription factor 6 (ATF6). A
major ER chaperone, the 78 kDa glucose regulated protein (GRP78), also referred to as
BiP/HSPA5, acts as a master regulator of UPR through direct interaction with all three
sensors and maintains them in an inactive form in non-stressed situations (Ni and Lee,
2007; Wang et al., 2009). Upon ER stress, GRP78 is titrated away by the accumulated
malfolded proteins, releasing the UPR sensors, which allows the activation and
transduction of UPR signals across the ER membrane to the cytosol and the nucleus.
PERK is an ER transmembrane protein with an ER luminal stress-sensing domain and a
cytosolic kinase domain. One important function of PERK in the UPR is to facilitate the
attenuation of global protein synthesis via the phosphorylation of eIF2α, which
suppresses 80S ribosome assembly. While global translation is suppressed under
conditions of eIF2α phosphorylation, select mRNAs containing regulatory sequences in
the open reading frame in 5’-untranslated regions require the phosphorylation of eIF2α
for translation. The transcription factor ATF4 is one example, and the translational
upregulation of ATF4 can induce the expression of UPR target genes that promote ER
folding capacity and adaptation to stress. IRE1α is a transmembrane Ser/Thr protein
kinase that also has site-specific endoribonuclease (RNase) activity. Upon ER stress,
IRE1α dimerizes and autophosphorylates, and thereby activates its RNase activity to
cleave a 26-base intron from the mRNA encoding X-box-binding protein 1 (XBP1),
3
resulting in a translational frameshift and a translation of a spliced form of XBP1
(XBP1-s) which is a more stable and potent transcription factor of target genes including
DnaJ, p58, ERdj4, EDEM and PDI, all involved in protein folding and ER-associated
degradation (ERAD). ATF6, a basic leucine zipper (bZIP) transcription factor, when
released from GRP78 upon ER stress, translocates from the ER to the Golgi apparatus,
where it is cleaved by S1P and S2P proteases to generate the active nuclear form of ATF6
(p50). Cleaved ATF6 and spliced XBP1 act in parallel to mainly induce the transcription
of genes encoding ER chaperones and enzymes that facilitate protein folding and
maturation. Interestingly, while the PERK/peIF2a/ATF4 pathway is canonically regarded
as the translational controlling arm of the UPR, a recent report suggests that it is also
required for the activation of ATF6 and its target genes, thereby fully integrating the
regulatory networks of the UPR (Teske et al., 2011). While UPR activation leads to
adaptations that may sustain cell survival, under severe and prolonged ER stress
conditions where the cells fail to restore ER homeostasis, the UPR activates pathways
that lead to apoptotic cell death. Such measures protect the organism by eliminating
damaged cells beyond repair (Ron and Walter, 2007; Wu and Kaufman, 2006).
4
Figure 1.1 Unfolded protein response and its regulation on cell activities. Under non-stress condition (left),
GRP78 binds to IRE1α,β, ATF6, PERK and Caspase to quench their activation. When cells are under ER
stress (right), GRP78 is titrated to bind to the malfolded proteins that are accumulated in the ER and release
IRE1α,β, ATF6, PERK and Caspase. IRE1a activates its RNase activity to cleave the mRNA of XBP1,
resulting in a spliced form of XBP1 (XBP1-s). ATF6 translocates from the ER to the Golgi apparatus,
where it is cleaved into the active nuclear form ATF6(c). PERK dimerizes and autophosphorylates, and
thereby phosphorylates its two major substrates eukaryotic translation initiation factor 2α (eIF2α) and
nuclear factor-like 2 (Nrf2). eIF2α phosphorylation attenuates global protein synthesis, and inhibits cyclin
D1 translation through which contributes to cell cycle arrest. The phosphorylation of eIF2α also activates
the transcription of ATF4. XBP1(s), ATF6(c) and ATF4 act in parallel to mainly induce transcription of
genes encoding ER chaperones and enzymes that promote protein folding and maturation. Another function
of phosporylated eIF2α is to activate CHOP, which is a marker for ER stress induced apoptosis. The
phosphorylation of Nrf2 activates the expression of enzymes required for ROS quenching, and thereby
inhibits ROS accumulation. Caspase, upon released from the ER membrane, is cleaved into caspase(c) and
triggers ER stress induced apoptosis.
5
1.1.2 GRP94 and its functions
ER chaperones are located inside the ER lumen and their function is to facilitate
protein folding and transport. One of the most abundant ER chaperones is glucose
regulated protein (GRP94). GRP94 is the ER paralog of HSP90 and the most abundant
glycoprotein in the ER. It is also known as 96-kDa glycoprotein (gp96), calcium binding
protein 4 (CaBP4), tumor rejection antigen-1 (TRA-1), Endoplasmin and ERp99 (Yang
and Li, 2005). Like all members in the HSP90 family, GRP94 exists as homodimers, with
each subunit containing four domains: the N-terminal regulatory and ligand binding
domain, the acidic linker domain, a middle domain and the C-terminal dimerization
domain (Eletto et al., 2010). Unlike HSP90 which has a micromolar binding constant to
ATP/ADP, GRP94 binds to ATP/ADP with weak affinity, and undergoes conformational
changes upon binding (Immormino et al., 2004). ATP binding and hydrolysis is
important for the chaperone function of GRP94, as mutation in the ATP binding pocket
or the amino acids required for ATP hydrolysis renders impaired or complete loss of
chaperone activity in GRP94 (Ostrovsky et al., 2009a). Compounds that inhibit the
function of GRP94 by targeting its ATPase domain include Geldanamycin and 17-AAG,
which, however, also inhibit the activity of HSP90 (Gorska et al., 2012). Recently, a
novel GRP94 inhibitor has been developed and demonstrated to have no effect on cell
viability or HSP90 client proteins at similar concentrations that inhibit GRP94 activity
(Duerfeldt et al., 2012).
6
As part of the ER quality control machinery, GRP94 is ubiquitously expressed in
multicellular eukaryotic metazoan, and can be induced under conditions that trigger ER
stress, such as hypoxia, nutrient deprivation, ER-calcium store depletion, and the
accumulation of malfolded proteins. GRP94 is not required for the viability of the
majority of cells in vitro, but exerts cytoprotective effect under special condition such as
serum deprivation (Ostrovsky et al., 2009b). Our previous study has also shown that
GRP94 was cytoprotective in etoposide-induced apoptosis in hematopoietic cells (Reddy
et al., 1999). Furthermore, complete depletion of GRP94 in mouse embryonic stem cells
(ESCs) results in elevated apoptosis at both basal level and under thapsigargin-induced
ER stress (Mao et al., 2010; Figure 1.2). However, detailed mechanisms underlying this
increased apoptosis caused by GRP94 depletion is not clear. Unlike GRP78, GRP94
deficiency does not cause ER stress or trigger UPR signaling. Grp94
−/−
ESCs displayed
compensatory upregulation of major ER chaperones and reduction of XBP-1 spliced and
unspliced proteins, without affecting the PERK-eIF2α signaling branch and ERAD
pathways (Mao et al., 2010; Figure 1.2).
7
Figure 1.2 (A) Compensatory increased ER chaperones in Grp94
−/−
ESCs. Grp94
+/+
and Grp94
−/−
ESCs
were treated with 300 nM ER stress inducer thapsigargin (Tg) for the indicated time period. (B) Reduction
of the spliced and unspliced form of XBP-1 protein in GRP94 null ESCs. Whole cell lysates of Grp94
+/+
and Grp94
−/−
ESCs treated with 300 nM Tg for the indicated time period. (C) Representative Western blot
results on the effect of GRP94 depletion on UPR targets and ERAD associated proteins. Grp94
+/+
and
Grp94
−/−
ESCs were treated with 300 nM Tg for the indicated time. (D) Representative Western blot
8
analysis on the effect of GRP94 depletion on UPR targets and cytosolic chaperone in Grp94
+/+
and
Grp94
−/−
ESCs treated with 300 nM Tg for the indicated time. (E) Representative Western blot (left) and
quantitation (right) results on effect of GRP94 depletion on cell proliferation and apoptosis. Grp94
+/+
and
Grp94
−/−
ESCs were treated with 300 nM Tg for the indicated time. The cleaved C-7 protein level was
quantitated and normalized against β-actin. The level of cleaved C-7 in Grp94
+/+
ESCs treated with Tg for 4
hr was set as 1. (F) Representative Western blot (left) and quantitation (right) results on effect of GRP94
depletion on cell apoptosis. Grp94
+/+
and Grp94
−/−
ESCs were treated with 1.5 µg/mL tunicamycin (Tu).
The level of cleaved C-3 in Grp94
+/+
ESCs treated with Tu for 4 hr was set as 1.
Unlike GRP78 which is an ER master chaperone for many secreted and
membrane proteins, GRP94 has a short list of client proteins and controls specific
pathways critical for cell growth, differentiation and other functions. Homozygous
deletion of Grp94 in mice causes defected mesoderm development and embryonic
lethality, as GRP94-null mouse ESCs cannot differentiation into muscle cells (Mao et al.,
2010; Wanderling et al., 2007) because of the requirement of GRP94 for the processing
and secretion of IFG-I and IGF-II (Ostrovsky et al., 2009b). In Drosophila, Gp93
(ortholog of GRP94) is required for gut epithelial homeostasis and nutrient
assimilation-coupled growth control, suggesting an essential role in the functional
expression of specific secretory/integral membrane proteins in tissue specialization
(Maynard et al., 2010). GRP94 has been long implicated in innate and adaptive immunity.
As a major ER chaperone, GRP94 is uniquely obligatory for the process of multiple
Toll-like receptors (TLRs) including TLR1, TLR2, TLR4, TLR5, TLR6, TLR7 and
9
TLR9 (Randow and Seed, 2001; Yang et al., 2007). Other than its function in TLR2 and
TLR4 expression in innate immunity, GRP94 is also required for adaptive immune
response. It is shown to be a master chaperone for the expression of a majority of
integrins including integrin α1, α2, α4, αD, αE, αL, αM, αX, αV, β2, β5, β6, β7 and β8 in
the hematopoietic system, and GRP94 deficiency in mouse hematopoietic system leads to
impaired T and B lymphocyte differentiation (Liu and Li, 2008; Staron et al., 2010). In
addition to its requirement for the differentiation of lymphocytes in adaptive immunity,
GRP94 has long been shown to play important roles in antigen presentation. During
antigen presentation, GRP94 elicit the activation of antigen-presenting cells such as
macrophages and dendritic cells, and escort antigenic peptides into cross-presentation
pathways via interacting with antigen-presenting cell receptors such as CD91
(Jockheck-Clark et al., 2010) and scavenger receptor-A (Berwin et al., 2003). Recent
studies with mouse models have expanded the list of proteins whose expression is
dependent on GRP94. For example, GRP94 is an essential chaperone for platelet
glycoprotein Ib-IX-V complex which is responsible for platelet activation and
aggregation, as deletion of Grp94 in mouse hematopoietic system results in
ERAD-mediated reduced expression of GPIb-IX-V and thrombocytopenia (Staron et al.,
2011). Other than directly interacting with its client proteins, GRP94 also regulates
protein expression through interacting with other chaperones. For instance, GRP94 is
required for the surface expression of Wnt co-receptor LRP6 via interacting with its
critical chaperone MesD (Liu et al., 2013). Disrupting the function of GRP94 in vitro also
10
compromised the cell-surface processing of the metalloprotease pro-ADAMTS9 via
direct or indirect mechanisms (Koo and Apte, 2010).
Because of its essential functions in antigen presentation, GRP94-based
anti-tumor vaccination has been under extensive study. Several reports demonstrated that
a vaccination of lethally irradiated cancer cells expressing various non-ER-retainable
autologous GRP94 fusion proteins (devoid of the ER retention/retrieval signal KDEL)
protected mice from primary tumor growth as well as metastasis (Baker-LePain et al.,
2002; Liu et al., 2005; Schreiber et al., 2009). One explanation for this effect could be
that autologous tumor-derived secretory GRP94 stimulates the maturation of
macrophages and DCs, enhances the antigen cross-presentation and amplifies the
inflammatory signals (Podack and Raez, 2007; Strbo and Podack, 2008). As an
alternative to autologous GRP94, a pooled GRP94 vaccine derived from different
multiple myeloma cells was shown to be as effective as the autologous GRP94 vaccine
when tested in mouse models (Qian et al., 2009). Taken together, these studies
demonstrate that the use of GRP94 as an antitumor vaccination can prevent cancer
development. However, GRP94 vaccination in mice appear less promising in the
treatment of established tumors (Schreiber et al., 2009; Pakravan and Hassan, 2011;
Pakravan et al., 2011), and clinical trials across various types of tumors suggest that
autologous tumor-derived GRP94 vaccination may have limited efficacy (Oki et al., 2007;
Testori et al., 2008; Wood et al., 2008; Eton et al., 2010).
11
1.1.3 GRP78 and other ER chaperones
As the most abundant chaperone and the master regulator in the ER, glucose
regulated protein 78 (GRP78) is the most studied ER chaperone. The majority of studies
on GRP78 have been focusing on its function in cancer. GRP78 has been reported to be
upregulated in solid tumors in various organs including breast, liver, gastric, esophagus,
brain, prostate, head and neck and melanoma, correlating with aggressive tumor behavior
and recurrence (Luo and Lee, 2013). Genetic knockout of Grp78 led to a much reduced
embryonic cell proliferation as well as massive apoptotic death of the inner cell mass,
leading to embryonic lethality at day E3.5 (Luo et al., 2006). GRP78 haploinsufficiency,
while not affecting mouse growth rate, organ development, and antibody production,
prolonged the latency period and retards the progression of the oncogene-induced
mammary tumors in the MMTV-PyVT breast adenocarcinoma mouse model (Dong et al.,
2008). Likewise, heterozygous or homozygous deletion of Grp78 specifically in the
mouse prostate epithelium suppressed PTEN-null driven prostate tumorigenesis without
affecting postnatal prostate development and growth (Fu et al., 2008). Similar phenomena
has been observed in PTEN-null driven leukemogenesis (Wey et al., 2012a). GRP78 also
plays a critical role in tumor angiogenesis. WT syngeneic tumor cells injected into Grp78
heterozygous host mice showed suppressed tumor growth and pulmonary metastatic
lesions. Furthermore, creation of a conditional heterozygous deletion of Grp78 in the host
endothelial cells showed severe reduction of tumor angiogenesis and metastatic growth
12
(Dong et al., 2011).
The mechanism of GRP78 facilitating cancer development could be attributed to
its function in cell proliferation and apoptosis. The chaperone function of GRP78 in the
ER may facilitate the secretion of growth factor and/or the maturation of growth factor
receptors, or the potential function of GRP78 as a multifunctional receptor or co-receptor
for growth signaling pathways on the surface of cells, notably cancer cells (Zhang et al.,
2010). Another major function of GRP78 is to protect cells against stress-induced
apoptosis through binding and inactivating pro-apoptotic components such as BIK and
caspase 7 that localize to the ER, as well as suppressing the induction of CHOP, which
mediates the apoptotic arm of the UPR (Fu et al., 2007). Recent studies show that GRP78
and BCL-2 form separate complexes with different domains of BIK, and GRP78 binding
to BIK can compete for BIK binding to BCL-2 and therefore protects cells from
apoptosis (Zhou et al., 2011).
Calreticulin (CRT) is an evolutionarily conserved 46-kDa ER luminal protein
traditionally regarded as a Ca
2+
homeostasis regulator and an ER chaperone. Additionally,
CRT regulates other cellular processes, including cell adhesion, MHC class I molecules
assembly and hormone-sensitive gene expression. While the majority of CRT resides in
the ER, evidence from various studies indicates that CRT is also found on the cell surface
and can be secreted from cells under specific conditions (Johnson et al., 2001). CRT on
the surface (ecto-CRT) of tumor cells that are treated with apoptotic inducing agents has
13
been demonstrated to induce immunogenic tumor cell death (Obeid et al., 2007).
Ecto-CRT functions as an “eat me” signal that is recognized by dendritic cells and elicits
phagocytosis of tumor cells. Phagocytosis leads to increased tumor antigen
cross-presentation, which activates cytotoxic T cells and triggers an anti-tumor immune
response and immunogenic tumor cell death. In human cancer samples, ecto-CRT
expression is enhanced in both solid tumors and hematological malignancies when
compared to the corresponding normal tissues (Chao et al., 2010), which could possibly
explain the increased susceptibility of tumors in immune-based cancer therapy when
compared to normal cells. The presence of ecto-CRT offers a new anti-cancer therapeutic
strategy specifically against the tumor. Such tumor specificity raises the possibility of
personalized tumor vaccination, in which primary tumor cells from a patient could be
treated with apoptosis inducer ex vivo, and subsequently injected back into the patient in
order to both eradicate existing tumor cells and to induce long-term anti-tumor immunity
(Luo and Lee, 2013).
Protein disulfide isomerase (PDI) is a thiol-disulfide oxidoreductase that is
recognized for catalyzing the formation of disulfide bonds in newly synthesized proteins
in the ER lumen. PDI is also found in the cytosol, nucleus, on the cell surface, and can be
secreted (Turano et al., 2002). Similar to other ER chaperones, PDI upregulation in
response to ER stress helps ameliorate misfolded proteins and stress-induced apoptosis
(Uehara et al., 2006). Studies on the role of PDI on cancer have demonstrated that PDI is
14
important for tumor formation and migration, as well as antibody production in humoral
immune response (Lovat et al., 2008; Goplen et al., 2006; Haefliger et al., 2011; Fonseca
et al., 2009).
Due to the interconnectivity of the ER with other cellular compartments, it is
now becoming clear that chaperones traditionally thought to remain in the ER, can
function beyond this compartment and are involved in processes outside of protein
folding and posttranslational modification (Luo and Lee, 2013). The diverse localization
and function of major ER chaperones are summarized in Figure 1.3.
Figure 1.3 Different localizations and functions of major ER chaperones. The circle in the center indicates
ER, where the majority of ER chaperones are located. The squares outside of ER indicate non-traditional
localization of these chaperones and their functions.
15
1.2 Hematopoietic system
1.2.1 Hematopoiesis and HSCs
Hematopoiesis is the formation of different blood cellular components from the
hematopoietic stem cells (HSCs), which are multipotent stem cells residing within bone
marrow (BM) or fetal liver that give rise to all lineages of mature blood cells (Giebel and
Punzel, 2008; Weissman and Shizuru, 2008; Figure 1.4). In mice, hematopoiesis begins
by the appearance of red blood cell-generating primitive progenitors in the yolk sac on
embryonic day E7.5. Starting from E9.5, HSCs start to emerge in the
aorta-gonad-mesonephros (AGM) region of the mouse, and the production of definitive
hematopoietic cells begins in the AGM and placental regions on around E10.5. The fetal
liver is then become the main source of hematopoiesis until E16.5, when HSCs
translocate via the peripheral circulation to the bone cavities to form the BM (Adams and
Scadden, 2006; Medvinsky et al., 2011).
Hematopoiesis is carried out throughout life. Long-term HSCs (LT-HSCs)
maintain self-renewal capacity and give rise to all lineages of blood cells throughout life.
LT-HSCs differentiate into immediate downstream progeny short-term HSCs
(ST-HSCs), which have the capability of self-renewal and differentiation into blood
cells for 8-10 weeks. ST-HSCs differentiate into multipotent progenitors (MPPs), which
further give rise to lineage-restricted progenitors including common myeloid
16
progenitors (CMP), common lymphoid progenitors, (CLP), megakaryocyte-erythrocyte
progenitors (MEP), and granulocyte-monocyte progenitors (GMP) (Weissman and
Shizuru, 2008). The bipotent MEP differentiate into erythrocytes and megakaryocytes.
The main function of the former is carrying O
2
and CO
2
in the blood stream, while the
fragmentation of the latter is responsible for the formation of blood clots. GMP is the
progenitor for the monocytes and granulocytes. Monocytes, which mature from GMP
and monoblasts, further developed into macrophages in local tissues. Distinguished by
their appearance under Wright's stain, Granulocytes can be further categorized into
three populations: eosinophils, basophils and neutrophils. CLP is the progenitor for the
lymphoid lineage, which includes natural killer cells, T-lymphocytes, and
B-lymphocytes. Subpopulations of these lymphocytes can be further distinguished by
their differentiation stage, phenotype and function. For example, plasma cells are
terminally differentiated B cells that secret antibodies and play important roles in
humoral immunity. Unlike other blood cells, dendritic cells contain both myeloid-origin
and lymphoid-origin subgroups. In a healthy adult person, approximately 10
11
-10
12
new
blood cells are produced daily in order to maintain steady state levels in the peripheral
circulation (Surhone et al., 2010). Therefore, the regulation of HSC and hematopoiesis
is critical for blood cell homeostasis. Dysregulation of the hematopoietic system could
result in pathological conditions of insufficient blood cells such as anemia or
thrombocytopenia, or hematological malignancies including leukemia, lymphoma and
myeloma.
17
Figure 1.4 Hematopoietic cell ontogeny. LT-HSCs differentiate into ST-HSCs and further differentiate into
the myeloid lineage (including erythrocytes, thrombocytes, monocytes and granulocytes) and lymphoid
lineage (inducing T cells, B cells and NK cells). Dendritic cells can be either myeloid-origin or lymphoid
origin.
1.2.2 HSC homeostasis
The homeostasis of HSCs is essential for maintaining a normal and functional
hematopoietic system. The physiological features of HSCs include self-renewal,
differentiation, quiescence, apoptosis, mobilization, and engraftment, all of which are
tightly regulated under physiological conditions (Cheng, 2008). The cell fate
18
determination between self-renewal and differentiation is critical for HSC maintenance
and function. HSC self-renewal is achieved through cell division. There are two types of
cell division: one is asymmetrical which generates one daughter stem cell and one
differentiated daughter cell, and the other one is symmetrical which generates two
daughter stem cells or two differentiated daughter cells (Wilson and Trumpp, 2006).
Adjusting the balance between asymmetrical and symmetrical division according to the
environment is crucial for stem cell maintenance, as loss of this balance can result in
HSC exhaustion or expansion (Cheng, 2008). Although in steady state the number of BM
HSCs remains constant, under special circumstances tipping this balance towards one end
can be a regulatory mechanism for HSCs to accommodate to different environments. For
example, HSCs expand through symmetrical division into two daughter stem cells in
response to hematopoietic stress (Wagers et al., 2002). However, uncontrolled
symmetrical division into two differentiated daughter cells can result in depletion of the
HSC pool (Lessard et al., 2004). Consistently, HSCs, like other adult stem cells, divide
infrequently during homeostasis. Studies have shown that a small portion of HSCs with
the highest self-renewal potential displays extremely slow cycling (Wilson et al., 2008).
Other than self-renewal and differentiation, HSC mobilization and engraftment
are indispensible physiology processes for HSC to maintain homeostasis and to carry out
their function of reconstituting the hematopoietic system. Like most of other stem cells,
HSCs resides in a specialized microenvironment named HSC niche (Jones and Wagers,
19
2008; Wang and Wagers, 2011). Under physiological conditions particularly in response
to stress signals, small portions of HSCs migrate out of the BM and enter the circulation,
a process known as mobilization. Trafficking to blood is also an important death pathway
to regulate the steady-state number of HSCs (Lemoli and D’Addio, 2008).
Pharmacologically induced HSC mobilization by G-CSF is a common regimen in clinical
HSC transplantation (Winkler et al., 2012). Circulating HSCs return to the BM through a
process named homing, and lodge back to their niche. Homing and lodgment is the
primary step for transplanted HSCs to engraft in the BM before being able to proliferate
and repopulate the blood cells. Therefore, maintaining HSC homeostasis is critical for
their proper functions to sustain blood supply throughout life, to support local
hematopoiesis in response to stress, and to reconstitute the hematopoietic system after
BM transplantation.
1.2.3 Intrinsic and extrinsic regulation of HSCs
Under physiological condition, HSC homeostasis is strictly regulated by intrinsic
gene expression and extrinsic cues from the microenvironment. Extensive studies have
been performed to identify intrinsic regulators of HSC homeostasis, which can be
categorized as cell cycle regulators, transcription factors, chromatin-associated factors, as
well as CDX and HOX proteins (Zon, 2008). Cell cycle regulators play crucial roles in
regulating HSC self-renewal (Pietras et al., 2011; Matsumoto and Nakayama, 2013). For
example, inactivation of PTEN in the hematopoietic system leads to short-term expansion
20
but long-term decline of HSCs, as well as myeloproliferative disorder which later
develops into leukemia (Zhang et al., 2006; Yilmaz et al., 2006). Similarly, loss of all
three members in the Retinoblastoma family results in intrinsic myeloproliferative
disease, as well as expansion and impaired reconstitution potential in HSCs (Viatour et
al., 2008). Cyclin-dependent kinase (CDK) inhibitors, including p21
cip/waf1
, p16
INK4A
, and
p27
KIP1
, serve as the molecular switch governing the entry of HSCs into
non-self-renewing cell cycle, as inactivating any one of them results in HSC expansion
which eventually leads to the depletion of HSC pool (Cheng et al., 2000; Janzen et al.,
2006; Walkley et al., 2005). In contrast, the deletion of another CDK inhibitor, p18
INK4C
,
results in strikingly increased self-renewing
division and improved long-term engraftment
(Yuan et al., 2004). Other cell cycle regulators such as cyclin D, cyclin A, p53, and p57
have also been demonstrated to regulate HSC number and stemness (Matsumoto and
Nakayama, 2013). Other than cell cycle regulator, transcription factors such as Gfi1,
FOXO, and Mef also play essential parts in the regulation of HSC cell cycle entry as well
as their downstream differentiation (Orford and Scadden, 2008). Modulation of essential
genetic and epigenetic regulators in embryonic development, such as the HOX proteins
and chromatin-associated factors like Bmi1, also affects the self-renewal and normal
function of HSCs (Orford and Scadden, 2008). Interestingly, the functions of these HSC
regulators are interconnected. For example, CDK inhibitors p21, p27 and p57 have been
shown to crosstalk among each other (Matsumoto et al., 2011), whereas p16, p19 and p53
have been demonstrated to play distinct roles under the context of PTEN inactivation
21
(Lee et al., 2010). Therefore, intrinsic signaling regulating HSCs is part of a cellular
regulatory network that involves numerous players and multiple levels of regulation.
Moreover, these multilevel regulations are controlled by signals from the HSC
niche. A stem cell niche can be identified as a spatial structure in which stem cells are
housed and maintained by allowing self-renewal in the absence of differentiation (Wilson
and Trumpp, 2006; Taichman, 2005; Jones and Wagers, 2008). The stem cell niche was
first characterized in D. melanogaster where germline stem cells are maintained and
controlled for differentiation (Xie and Spradling, 2000; Kiger et al., 2000). In higher
organisms, studies on stem cell niche are more challenging because of the uncertainty in
their location. One of the most well-characterized HSC niche is the endosteal niche at the
interface between the bone and the BM. HSCs with low proliferation frequency and high
hematopoietic potential have been identified at the endosteal surface (Arai et al., 2004;
Haylock et al., 2007), and exogenous HSCs infused into nonmyeloablative BM
demonstrated selective accumulation at the endosteal region (Nilsson et al., 2001),
identifying the endosteal region as the harbor for quiescent HSCs. The endosteal HSC
niche is composed of a variety of cellular compartments, including specialized
osteoblasts which are the major compartment, as well as endothelial, cells, reticular cells,
osteoclasts and other BM stromal cells (Kiel and Morrison, 2008; Garrett and Emerson,
2009; Adams and Scadden, 2006). Activation of osteoblasts via constitutive activation of
parathyroid hormone receptor or inactivation of BMP receptor results in increased HSC
22
number and engraftment in the BM (Calvi et al., 2003; Zhang et al., 2003). In addition to
the endosteal niche, HSCs have also been identified located approximate to the sinusoidal
endosteum using SLAM markers, leading to the discovery of the perivascular HSC niche
(Kiel et al., 2005). Main components of this niche include endothelial cells, BM stromal
cells, mesenchymal stem cells, adipocytes, perivascular cells and hematopoietic
progenies of HSCs (Kiel and Morrison, 2008; Wang and Wagers, 2011). The discovery
of the perivascular niche provides an explanation for the maintenance of HSC during
embryonic and extramendullary hematopoiesis, as trabecular bone or osteoblasts are not
components of AGM, fetal liver or spleen. While the endosteal niche is believed to
maintain quiescent HSC and preserve their long-term self-renewal capacity, the
perivascular niche is thought to regulate the proliferation and differentiation of HSCs and
provide them a portal for hematopoiesis reconstitution especially in response to
hematopoietic stress (Yin and Li, 2006; Nakamura-Ishizu and Suda, 2013).
How do these HSC niche components regulate HSCs? There are four major
pathways: 1) Interactions between soluble ligand and their receptors. Local soluble
factors interact with their receptors to regulate the maintenance of HSC pool and their
functions. For instance, the interactions between angiopoietin (AGN)/Tie2 (Arai et al.,
2004), thrombopoietin (TPO)/MPL (Yoshihara et al., 2007; Qian et al., 2007), and
osteopoietin (OPN)/integrins (Nilsson et al., 2005; Stier et al., 2005), SDF-1/CXCR4
(Nie et al., 2008) negatively regulates HSC proliferation and pool size, whereas the
23
binding of Jagged to Notch increases HSC self-renewal and engraftment (Stier, 2002).
The bindings of local growth factors, chemokines and environment factors to their
receptors, such as SCF/KIT (Wilson and Trumpp, 2006), SDF-1/CXCR4 (Wright, 2002),
and Ca
2+
/CaR (Adams et al., 2006), also play important role in regulating HSC
mobilization, homing and engraftment. 2) Direct cell-cell contact. Cell adhesion
molecules such as integrin α4 and α5 are crucial in mediating the homing and retention of
HSCs in the BM via binding to their partners such as VCAM-1 (Jiang et al., 2009; Van
der Loo et al., 1998). Another example is the binding of membrane-bound SCF to KIT,
which has a higher and more sustained activity in regulating the motility, self-renewal
and survival of HSCs than soluble SCF (Driessen et al., 2003). 3) Interactions between
HSCs and extracellular matrix (ECM). ECM components have been identified in the
HSC niche region, such as fibronectin, collagens, and hyaluronic acid. These molecules
play important role in maintaining the structure of the HSC niche, as well as regulating
the homing and retention of HSCs (Adams, 2008). 4) Regulations through intermediate
cells. Interestingly, in addition to local factors in the HSC niche, the sympathetic nervous
system activated by circadian rhythms or stress conditions has been shown to influence
HSC migration and development through secretion of adrenergic hormones. This is
mediated by the increased secretion of SDF-1 from BM stromal cells in response to
adrenergic hormones (Méndez-Ferrer et al., 2008), as well as direct regulation of
adrenergic hormones on osteoblasts and bone turnover (Kollet et al., 2012).
24
However, these secretory ligands as well as surface molecules that are critical for
maintaining HSC homeostasis would not acquire their function without being processed
in the ER. And dysregulation of HSC homeostasis can lead to hematological diseases
such as leukemia or myeloma, in which cancer cells may undergo ER stress and
pathological changes that demands ER chaperones. Therefore, in these studies we are
investigating the function the ER chaperones in the regulation of HSC and hematological
malignancies.
25
Chapter 2
GRP94 is required for maintaining HSC
interactions with the adult BM niche
2.1 Introduction
HSCs reside in a specific anatomic location in the BM known as the stem cell
niche (Taichman, 2005; Li and Li, 2006). Signaling cues from neighboring cells in the
niche are key in dictating the function of the cell to maintain the hematopoietic system of
the individual (Wilson and Trumpp, 2006). At the endosteal surface (Nilsson et al., 2001),
which is the interface between bone and the BM, specialized osteoblasts represent the
main components of HSC niche (Calvi et al., 2003; Zhang et al., 2003). It has been
proposed that this heterogeneous group of cells regulates HSC survival, self-renewal,
migration, differentiation, and quiescence (Cheng, 2008) through several soluble factors
and their receptors, as well as direct contact through ECM and cell surface proteins, such
as integrins. These adhesion molecules, soluble ligands and their receptors also control
HSC engraftment, which involves homing, transmarrow migration, and lodging within
the BM niche (Wilson and Trumpp, 2006).
Secretory and cell membrane-bound proteins are folded, processed, and
transported via the endoplasmic reticulum (ER) under the assistance of ER chaperone
proteins. GRP94 is a major ER chaperone assisting in protein folding, assembly and
26
secretion (Luo and Lee, 2013). Due to its client selectivity and interactions with late
folding intermediates, GRP94 is postulated to perform unique chaperone functions in the
ER, and control specific pathways critical for cell growth and differentiation (Eletto et al.,
2010). For example, GRP94 is required for the expression of a large number of integrins,
as well as Toll-like receptors and selectively regulates innate immunity of macrophages,
and early T and B lymphopoiesis (Liu and Li, 2008; Staron et al., 2010; Yang et al.,
2007). Using a BM chimeric approach, an increase in HSCs and multipotent progenitors
was observed in BM devoid of GRP94 (Staron et al., 2010), raising the interesting issue
of how GRP94 deficiency could lead to HSC expansion. Since all of the secretory and
surface proteins meditating the regulatory cues from the HSC niche to HSCs are
processed in the ER under the assistance of ER chaperones, we hypothesized that loss of
GRP94 impairs this process and thereby results in disruption of HSC homeostasis.
2.2 Materials and methods
Mice
Grp94
f/f
mice in a mixed C57BL/6; 129/Sv background were generated as
previously described (Mao et al., 2010). Grp94
f/f
mice were crossed with the transgenic
Mx-1-Cre mice on a C57BL/6 background (Jackson Laboratory) to generate Grp94
f/f
;
Mx-1-Cre (cGrp94
f/f
) mice. Littermates that were negative for Cre transgene were used
27
as controls. Genotyping was performed by PCR using genomic DNA extracted from
mouse tail biopsies as previously described (Fu et al., 2008). To induce the genomic
deletion, 5.5 to 6.5 week-old male and female mice were injected intraperitoneally with
pI.pC (25 mg/kg mouse body weight) three times a week. Mice were analyzed 11 days
post 7 injections, except with modifications as specifically mentioned in the figure
legends. All protocols for animal use were reviewed and approved by the USC
Institutional Animal Care and Use Committee. The protocol number is 9964.
Genotyping
Primer set PAS1-PAS3 was used to differentiate Grp94 WT and floxed allele,
while niL and niR were used to detect knockout allele (Mao et al., 2010). For Genotyping
of the Cre transgene, primers Cre109 and Cre457 were used. Reactions for Grp94 floxed,
WT, knockout alleles and Cre were run under the following conditions: 94°C for 5 min,
followed by 46 cycles of 94°C for 20 sec, 62°C for 30 sec, and 72°C for 30 sec, ended
with 72°C for 7 min. The primer sequences are listed in table 2.1.
Table 2.1: Primer sequences for mouse genotyping
Gene Primer name Sequence
Grp94 (floxed or +)
PAS1 5’- GCTGTGTCCTGCTGACCTTCG -3’
PAS3 5’-TGATCAGCGATCGCCAAAAGTCCTTAGGGAGG -3’
Grp94 (-)
niL 5’- GCTGTGTCCTGCTGACCTTCG -3’
niR 5’- TACCTCACCGATTGAAAAGC -3’
Cre
Cre109 5’-AAGAACCTGATGGACATGTTCAGGGA-3’
Cre457 5’-ACGAACCTGGTCGAAATCAGTGCGTTC-3’
28
Complete blood count
Peripheral blood was collected via tail bleeding and analyzed using an auto
hematology analyzer BC-2800 vet (Mindray) according to manufacturer’s instructions.
Flow cytometry
BM cells were flushed from long bones (tibias and femurs) with Dulbecco’s
phosphate-buffered saline without calcium and magnesium (PBS), and then filtered
through nylon screen (70 micron, BD Biosciences) to obtain single cell suspension.
Splenocytes were collected following disaggregation of the spleens in PBS, with red
blood cells (RBCs) lysed in lysing buffer (BD Biosciences) for 15 min and then filtered
through nylon screen to obtain single cell suspension. Peripheral blood samples were
collected by tail bleeding, with RBCs lysed in lysing buffer twice for 15 min. Cells from
mouse tissue [BM, spleen and peripheral blood mononuclear cells (MNCs)] were
resuspended in PBS with 0.5% BSA and 0.1% sodium azide for antibody staining. To
study hematopoietic stem and progenitor cells, Lineage (Lin) which consists of B220
(RA3-6B2), TER119 (TER119), CD4 (RM4-5), CD8a (53-6.7), Gr-1 (RB6-8C5), and
Mac-1 (WT.5), c-Kit (2B8), Sca-1 (D7), Flk2 (A2F10.1) CD34 (RAM34), IL7Rα
(SB/199) and FcγRII/III (2.4G2, all antibodies from BD Pharmingen) were used. To
study different lineages of blood cells, Gr-1, F4/80 (Invitrogen), B220, CD4, CD8a and
CD3 (1F4) were used. CD45.1 (A20) and CD45.2 (104, both from BD Pharmingen) were
29
used for BM transplantation analysis. Integrin α4 and α5 were detected using CD49d
(R1-2, eBioscience) and CD49e (5H10-27, BD Pharmingen). 3×10
6
-1×10
7
BM or spleen
cells, or MNCs from 200 µL peripheral blood were incubated with antibodies for 30 min,
washed twice with PBS with 0.5% BSA and 0.1% sodium azide, and then resuspended in
PBS. Dead cells were excluded by 7-AAD staining (BD Pharmingen). Cell population
analysis was performed on BD FACS LSR II.
Fluorescence activated cell sorting
Mouse whole BM cells resuspended in PBS were enriched for c-Kit
+
cells using
CD117 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions. C-Kit
+
cells were stained with anti-mouse c-Kit (Miltenyi Biotec), anti-mouse Sca-1, and Lin
antibody cocktail as described above. Primitive hematopoietic stem and progenitor cells
were purified using a FACSAria flow cytometer (Becton Dickinson) based on established
cell surface phenotypes. LSK cells were sorted for the competitive repopulation and
adhesion assays; LK cells were purified for the in vivo homing and lodgment assay.
Cell cycle analysis
1×10
7
whole BM cells were incubated with 10 µg/ml Hoechst 33342
(Sigma-Aldrich) at 37°C for 45 min, then stained with antibodies against cell surface
markers for primitive hematopoietic cells (Lin, c-Kit and Sca-1) as described above. The
stained cells were resuspended in 10% neutral buffered formalin (BDH Chemicals) and
30
incubated at 4°C overnight. To stain for RNA content, Pyronin Y (Polysciences Inc.) was
added to the cells at a final concentration of 0.75 µg/ml and incubated at 4°C for 30 min.
Cell cycle status was examined using a BD LSR II flow cytometer.
Apoptosis assay
For apoptosis detection, 1×10
6
whole BM cells were stained with primitive
hematopoietic cell antibodies (Lin, c-Kit and Sca-1) as described above. The stained cells
were resuspended in 150 µL 1×Annexin V binding buffer (BD Pharmingen) and
incubated with 5 µL Annexin V and 5 µL 7-AAD (both from BD Pharmingen) for 15 min
at room temperature. Cell apoptosis was examined within an hour using a BD LSR II
flow cytometer.
Colony-forming cell (CFC) assay
Freshly isolated BM cells were mixed thoroughly with 300 µL Iscove’s MDM
with 2% fetal bovine serum (FBS) and 4 mL defined methylcellulose medium M3434
(Stem Cell Technologies). Cells were then plated in triplicate in six-well plates with 1.1
mL volume at a density of 1×10
4
BM cells per well, and cultured at 37°C. The colonies
were then scored at day 10 under an inverted microscope and reported as colony number
in specific lineages according to the morphology of colonies described in the
manufacturer’s instructions.
31
Real-time quantitative RT-PCR
To detect Grp94 knockout efficiency, RNA was extracted from mouse whole BM
cells and reverse-transcription was performed as previously described (Ni et al., 2009).
cDNA samples were analyzed in triplicate with the SYBR Green Supermix (Quanta
Biosciences) according to manufacturer’s instructions. The following primers were used:
Grp94, 5’-TGG GCC TCT GCT GTG TCC TGC-3’ and 5’-GGC TTT TAC CCA GGT
CCT CTT CCA CTG T-3’; 18S RNA, 5’-ACG GCC GGT ACA GTG AAA C-3’ and
5’-GAG GGA GCT CAC CGG G-3’.
Competitive repopulation assay
LSK cells were sorted using a FACSAria flow cytometer and 1000 Grp94
f/f
or
cGrp94
f/f
LSK cells (with cell surface marker CD45.2) were mixed with 250,000 WT BM
MNCs (with cell surface marker CD45.1) which is equivalent to approximately 500 LSK
cells, and injected into the tail vein of B6.SJL mice that were lethally irradiated at 9.5Gy
approximately 24 hr prior to transplantation. Engraftment levels and multilineage
reconstitution were measured in peripheral blood samples obtained from the tail vein of
hosts starting week 4.
In vivo homing
Sorted Grp94
f/f
or cGrp94
f/f
Lin
-
c-Kit
+
(LK) cells were labeled with a green
fluorescent dye, carboxyfluorescein diacetate succinimidyl ester (CFSE), and a red
32
fluorescent dye, SNARF-1 carboxylic acid, acetate, succinimidyl ester (SNARF-1, both
from Invitrogen), respectively, according to manufacturer’s instructions. Labeled cells of
equal number (~5×10
5
to 1×10
6
) were injected into the tail vein of non-irradiated Grp94
f/f
C57BL6; 129/Sv background littermates. Mice were then sacrificed after 16 hr, and the
number of labeled cells in the BM and spleen was measured through the detection of
CFSE
+
and SNARF
+
cells by flow cytometry.
In vivo lodgment
LK cells were labeled with 5 µM CFSE or 5 µM SNARF cell-labeling solutions
and injected into the tail vein of non-irradiated mice, as described above. Tibias and
femurs were dissected from recipient mice 16 hr after injection, decalcified for 3 days in
Immunocal (Decal Chemical Corporation), and embedded in paraffin blocks after
processing. A total of 180 bone sections of 5 µm thickness from three recipients were cut
and mounted with Vectashield containing 4’,6-diamidino-2-phenylindole (DAPI) (Vector
Laboratories). To assess the lodgment of injected cells to the endosteal niche, the
numbers of CFSE
+
(green) or SNARF
+
(red) cells within two cell diameters from the
endosteal surface were counted under fluorescent microscope.
Adhesion assay
5×10
2
LSK cells were added to wells coated with fibronectin (1.5 µg/cm
2
, BD
Biosciences) in cell culture treated 96-well plates and incubated in minimum essential
33
medium (MEM) alpha medium with 10% FBS for 3 hr at 37°C/5% CO
2
in a humidified
atmosphere. Non-adherent cells were washed off with PBS and adherent cells were
visually counted microscopically. To control for nonspecific binding, cells adherent to 1%
bovine serum albumin (Sigma-Aldrich) were quantified.
Statistics
Kaplan-Maier survival curves were constructed using Microsoft Excel. For bar
graphs, the 2-tailed Student’s t-test was used to compute P values, and the error bars
reflect standard error (s.e.) (*, P<0.05, **, P<0.01, ***, P<0.001)
2.3 Results
2.3.1 Generation of an inducible Grp94 conditional knockout mouse model in the
hematopoietic system
To study the in vivo function of GRP94, we created Grp94 mutant mice with the
floxed or knockout allele (Figure 2.1A). Deletion of exon 2 flanked by the loxP-FRT sites
led to an early frameshift mutation resulting in the inactivation of the Grp94 allele. To
determine the role of GRP94 in the hematopoietic system, we bred Grp94
f/f
mice with
transgenic mice bearing the Mx-1-Cre transgene that allows GRP94 to be acutely
eliminated in the hematopoietic system in an inducible manner by administration of pI.pC.
In these studies, Grp94 was deleted in 5.5 to 6.5 week old Grp94
f/f
; Mx-1-Cre (cGrp94
f/f
)
34
mice. Littermates lacking the Cre transgene (Grp94
f/f
), which are phenotypically
equivalent to animals with WT Grp94 alleles, were also injected with pI.pC and served as
controls for any side effects of pI.pC injection. The status of the Grp94 allele deletion
was validated by PCR in isolated BM cells (Figure 2.1B). Real-time quantitative PCR
analysis with mouse BM cells 11 days after 7 shots of pI.pC injection showed that Grp94
transcripts were mostly depleted from the BM cells of the cGrp94
f/f
mice (Figure 2.1C).
Figure 2.1 Conditional knockout of Grp94 in the BM. A) Schematic drawings of the Grp94 wild-type (wt)
allele, the floxed allele and the knockout (ko) allele. The exons are boxed and numbered. The loxP sites
(closed triangle) and the FRT site (open triangle) and expected PCR products for genotyping is indicated. B)
Representative BM PCR genotyping results of mice with indicated genotypes after pI.pC injection. C)
Grp94 mRNA expression measured by quantitative real-time PCR from Grp94
f/f
(n=16) and cGrp94
f/f
(n=18) mouse BM after pI.pC injection. The level of Grp94 mRNA was normalized against the level of
35
internal control 18S RNA. The data are presented as mean ± s.e., ***p<0.001.
2.3.2 cGrp94
f/f
mice displayed increased myeloid hematopoiesis and impaired
lymphogenesis
To determine the consequence of acute depletion of GRP94 following Mx-1-Cre
induction, we examined the hematopoietic phenotypes of cGrp94
f/f
mice compared to
control siblings. Complete blood count analysis demonstrated a general leukocytosis and
severe thrombocytopenia, while red blood cell and hemoglobin counts were normal.
Monocytes and granulocytes in peripheral blood were increased 8-fold and 10-fold,
respectively, in cGrp94
f/f
mice compared to control mice (Figure 2.2A). Analysis of
blood smears further confirmed the leukocytosis in peripheral blood (Figure 2.2B).
Compared with Grp94
f/f
littermates, cGrp94
f/f
mice showed significantly lower thymus
and lymph node cellularities (Figure 2.2C), consistent with smaller thymus and peripheral
lymph nodes (data not shown). The increase in myeloid cells and decrease in lymphoid
cells corresponded with a higher proportion of granulocytes and macrophages and a
lower proportion of T-cells and B-cells in the spleen of the cGrp94
f/f
mice (Figure 2.2D
and 2.2E). The expansion of myeloid cells was also evident in the BM, where Gr-1
+
cells
were increased and B220
+
cells were decreased in cGrp94
f/f
mice. The proportion of
CD4
+
and CD8
+
cells in the BM of cGrp94
f/f
mice were comparable to the control mice
(Figure 2.2F and 2.2G).
36
Figure 2.2 cGrp94
f/f
mice displayed altered myeloid and lymphoid differentiation. (A) Complete blood
count of peripheral blood from Grp94
f/f
(n=31) and cGrp94
f/f
(n=37) mice. (B) Representative
Wright-Giemsa staining of blood smear with tail peripheral blood from Grp94
f/f
and cGrp94
f/f
mice. Scale
bar represents 500 µm. (C) Total thymus cell number (left) and total left and right axillary lymph nodes cell
37
number (right) from Grp94
f/f
and cGrp94
f/f
mice (n=7 for each group). (D) Representative flow cytometric
analysis of splenocytes from Grp94
f/f
and cGrp94
f/f
mice using lineage markers Gr-1 and CD3 (left), F4/80
and B220 (right). (E) Quantitation of flow cytometric analysis of splenocytes using lineage markers Gr-1,
CD3, F4/80 and B220 from Grp94
f/f
(n=4) and cGrp94
f/f
(n=7) mice. (F) Representative flow cytometric
analysis with BM cells using lineage markers Gr-1 and B220 (left) and CD4 and CD8a (right). (G)
Quantitation of flow cytometric analysis with BM cells using lineage markers Gr-1, B220, CD4 and CD8a.
Gr-1 and B220 (n=7 for Grp94
f/f
and n=9 for cGrp94
f/f
mice); CD4 and CD8a (n=7 for each genotype). All
data are presented as mean ± s.e., ***p<0.001.
The cGrp94
f/f
mice demonstrated a loss of viability between 3.5 to 6 weeks after
the first pI.pC injection with multiple pathological parameters including weigh loss,
seizure and bleeding spots in the brain (Figure 2.3A and 2.3B), which were not reported
in previous study in which Grp94 was only deleted in the hematopoietic system (Staron
et al., 2010). This is consistent with the ability of the Mx-1-Cre to delete Grp94 in
multiple organs with time, leading to the eventual morbidity of the cGrp94
f/f
mice.
Figure 2.3 cGrp94
f/f
mice displayed loss of viability. (A) Survival curve of Grp94
f/f
(n=9) and cGrp94
f/f
(n=8) mice after pI.pC injection. The bar graphs were presented as mean ± s.e.,*p<0.05, **p<0.01,
***p<0.001. (B) Representative brain image of Grp94
f/f
and cGrp94
f/f
mice under dissection microscope.
38
Arrows indicate bleeding spots on the brain.
2.3.3 GRP94 deficiency in the BM led to expanded HSC pool and increased
granulocyte–monocyte progenitors
In analyzing the effect of acute Grp94 deletion on primitive hematopoietic cells,
we observed that within 3 weeks of GRP94 depletion there was an expansion of the most
primitive Lin
-
c-Kit
+
Sca-1
+
Flk2
-
CD34
-
(LSKFlk2
-
CD34
-
) LT-HSC-enriched cell
population (2-fold, p<0.01) in the BM. Correlating with this was an expansion of the
ST-HSC-enriched LSKFlk2
-
CD34
+
cells, MPP-enriched LSKFlk2
+
and LSK populations
of cells (p<0.001) (Figure 2.4A and B), indicating that all primitive hematopoietic cells
were significantly increased upon acute hematopoietic Grp94 deletion. These findings,
coupled with the observation that the total BM cell number was not significantly different
in the mutant mice (p=0.373) (Figure 2.4C), reveal that the total number of primitive
hematopoietic cells in cGrp94
f/f
mice BM is higher compared to the Grp94
f/f
controls.
39
Figure 2.4 GRP94 deficiency in the BM expanded the primitive cell pool. (A) Representative flow
cytometric analysis with BM cells using Lin, c-Kit, Sca-1, Flk2 and CD34. (B) Quantitation of flow
cytometric analysis of primitive cell proportions. Left panel shows the percentage of LSKFlk2
-
CD34
-
in
BM and right panel shows LSKFlk2
-
CD34
+
, LSKFlk2
+
(n=5 for Grp94
f/f
, n=8 for cGrp94
f/f
) and total LSK
(n=16 for Grp94
f/f
, n=22 for cGrp94
f/f
) cells in BM using Lin, c-Kit, Sca-1. (C) Total BM cell number from
40
Grp94
f/f
(n=19) and cGrp94
f/f
(n=20) mice (p=0.373). All data are presented as mean ± s.e., **p<0.01,
***p<0.001
We also analyzed the progenitor cell populations in the BM. While the common
myeloid progenitor (CMP)-enriched Lin
-
c-Kit
+
Sca-1
-
IL-7Rα
-
CD34
+
FcγII/IIIR
lo
cells,
the common lymphoid progenitor (CLP)-enriched Lin
-
c-Kit
lo
Sca-1
lo
IL-7Rα
+
cells, and
the megakaryocyte-erythroid progenitor (MEP)-enriched Lin
-
c-Kit
+
Sca-1
-
IL-7Rα
-
CD34
-
FcγII/IIIR
lo
cells were all comparable between the cGrp94
f/f
mice and their
Grp94
f/f
littermates, there were significantly more granulocyte-monocyte progenitor
(GMP)-enriched Lin
-
c-Kit
+
Sca-1
-
IL-7Rα
-
CD34
+
FcγII/IIIR
high
cells in the cGrp94
f/f
mice (Figure 2.5A). This correlates with the increased number of colonies (p<0.01 for
total colony number; p<0.05 for CFU-G and CFU-GM; p=0.07 for CFU-M) formed by
unfractioned cGrp94
f/f
BM cells in CFC assays compared to the Grp94
f/f
controls (Figure
2.5B and 2.5C).
41
Figure 2.5 GRP94 deficiency led to increased granulocyte–monocyte progenitors in the BM. (A)
Quantitation of flow cytometric analysis of myeloid and lymphoid progenitors including common myeloid
42
progenitor (CMP), granulocyte-monocyte progenitor (GMP), megakaryocyte-erythroid progenitor (MEP)
and common lymphoid progenitor (CLP) from Grp94
f/f
(n≥7) and cGrp94
f/f
(n≥10) mice. (B)
Representative morphology of colony derived from different progenitors including (CFU-G, CFU-M,
CFU-GM, CFU-GEMM and BFU-E). (C) Quantitation of colonies formed from unfractioned BM from
Grp94
f/f
(n=4) and cGrp94
f/f
(n=7) mice in methylcellulose medium. All data are presented as mean ± s.e.,
*p<0.05, **p<0.01.
2.3.4 The increased number of GRP94-deficient HSCs was due to loss of quiescence
To examine the underlying cause for the increase in primitive hematopoietic cells
in the BM of the cGrp94
f/f
mice, we analyzed the cell cycle distribution in LSK cells by
Hoechst/Pyronin Y staining (Figure 2.6A). While approximately 75% of the LSK cells in
both genotypes were in G1 phase, the proportion of cells in G0 decreased from 15% in
the Grp94
f/f
mice to 7.7% in the cGrp94
f/f
mice (p<0.001), whereas the amount of
S+G2+M cells increased from 10% in the Grp94
f/f
mice to 17% for cGrp94
f/f
mice
(p<0.01) (Figure 2.6B). Furthermore, the expansion of the cGrp94
f/f
primitive
hematopoietic cell pool was not a consequence of altered cell survival as analysis of the
level of apoptotic cells using Annexin V did not reveal significant differences between
Grp94
f/f
and cGrp94
f/f
LSK cells (p=0.324) (Figure 2.6C). These findings suggest a loss
of quiescence and increase in proliferation account at least in part for the primitive
hematopoietic cell expansion observed in cGrp94
f/f
mice, similar to what has been shown
using other models of HSC function.
43
Figure 2.6 GRP94-deficient LSK cells displayed increased proliferation and loss of quiescence. (A)
Representative flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. To
examine early effects of GRP94 depletion on HSC proliferation, BM was extracted from Grp94
f/f
and
cGrp94
f/f
mice 3 days after 4 shots of pI.pC injection every other day. (B) Summary of cell cycle
distribution of LSK cells from Grp94
f/f
and cGrp94
f/f
mice (n=7 for each genotype). (C) Summary of flow
cytometric analysis of apoptotic LSK cells using Annexin V and 7-AAD (n=5 for Grp94
f/f
, n=8 for
cGrp94
f/f
) (p=0.324). All data are presented as mean ± s.e., **p<0.01, ***p<0.001.
44
2.3.5 GRP94-deficient HSCs exhibited increased mobilization and increased
extramedullary hematopoiesis
We next examined whether the alteration in the cell cycle status of the primitive
hematopoietic cells was accompanied by any changes in their localization. Histological
analysis showed that the spleen size was significantly enlarged in cGrp94
f/f
mice (Figure
2.7A and 2.7B) with signs of increased extramedullary hematopoiesis as indicated by the
increased hematopoietic cells in the red pulps (Figure 2.7C). Flow cytometric analysis
showed that there was a significant increase (2.5-fold, p<0.001) in the percentage of LSK
cells in the spleen of cGrp94
f/f
mice compared to Grp94
f/f
mice (Figure 2.7D and 2.7E).
The number of circulating LSK cells in the peripheral blood of cGrp94
f/f
mice were also
greatly increased (17-fold, p<0.001) compared to control (Figure 2.7F and 2.7G). We
also observed increased Lin
-
Sca-1
-
c-Kit
+
progenitor cells in the peripheral circulation.
These results suggest that primitive cell mobilization was increased as a consequence of
Grp94 deletion.
45
46
Figure 2.7 Increase extramedullary hematopoiesis and increased HSC mobilization in cGrp94
f/f
mice. (A)
Representative spleen size and morphology of Grp94
f/f
and cGrp94
f/f
mice. (B) Average spleen weights of
Grp94
f/f
(n=19) and cGrp94
f/f
(n=25) mice. The data are presented as mean ± s.e., ***p<0.001. (C)
Representative H&E staining of paraffin sections of spleen from Grp94
f/f
and cGrp94
f/f
mice.
Hematopoietic cells in the red pulp are indicated by the area between the two arrows. Scale bar represents 2
mm. (D) Representative flow cytometric analysis of splenocytes from Grp94
f/f
and cGrp94
f/f
mice using Lin,
c-Kit and Sca-1. (E) Quantitation of flow cytometric analysis of splenocytes from Grp94
f/f
(n=9) and
cGrp94
f/f
(n=9) mice using Lin, c-Kit and Sca-1. In these studies, splenocytes were extracted from Grp94
f/f
and cGrp94
f/f
mice 5 days after 7 injections of pI.pC (to examine HSC mobilization before the spleen was
enlarged). (F) Representative flow cytometric analysis of blood MNCs from Grp94
f/f
and cGrp94
f/f
mice. (G)
Quantitation of flow cytometric analysis of blood MNCs from Grp94
f/f
(n=11) and cGrp94
f/f
(n=10) mice.
All data are presented as mean ± s.e., ***p<0.001.
2.3.6 cGrp94
f/f
HSCs displayed impaired interaction with the BM niche
Since the HSC niche has been proposed to maintain stem cells in the BM in a
quiescent state, we hypothesized that the increase in proliferation of the cGrp94
f/f
HSCs
may have been mediated by a loss of interaction with the niche, which thus also led to
increased HSC mobilization and enlargement of the spleen by extramedullary
hematopoiesis in cGrp94
f/f
mice. Therefore, we directly examined the capability of
cGrp94
f/f
progenitors to home and lodge in the endosteal niche. To do this, we performed
a competitive lodgment assay (Figure 2.8A), in which the lodgment of primitive cells is
dependent upon their functional interaction with the niche in vivo. An equal number of
Grp94
f/f
and cGrp94
f/f
Lin
-
c-Kit
+
(LK) cells were labeled with fluorescent vital dyes
47
CFSE and SNARF respectively, and co-injected into non-irradiated WT littermates. In
these experiments we used LK cells, as in agreement with previous studies, the use of a
purified HSC population would result in too few cells to be reliably detected (Haylock et
al., 2007). We observed that fewer labeled cells from mutant mice homed to BM, while
more of them in the spleen of recipient mice (p<0.05) (Figure 2.8B and 2.8C). We cut a
total of 180 sections of femurs and tibias from a total of three recipient mice in three
independent experiments, and visualized the localization of cells as shown in Figure 2.8D.
The endosteal region is defined as the area within 2 cell diameters from endosteal surface.
Among all the 400-1000 cells that homed to the BM (numbers varied each time according
to the number of cells injected), about 13.5% of Grp94
f/f
LK cells lodged at the endosteal
region, while only 5% of the cGrp94
f/f
cells that homed to the BM were found at the
endosteal surface (p<0.05) (Figure 2.8E). These findings suggest that acute deletion of
Grp94 in primitive hematopoietic cells impairs the ability of HSCs to home to BM and
lodge at the endosteal niche.
48
Figure 2.8 cGrp94
f/f
HSCs displayed impaired interaction with the niche. (A) Scheme of the in vivo
competitive lodgment assay. (B) Representative flow cytometric analysis of CFSE-labeled Grp94
f/f
LK
cells and SNARF-labeled cGrp94
f/f
LK cells with host BM and spleen cells. (C) Summary of LK cells
homed to the BM and spleen (n=4 for BM, n=2 for spleen), with the level of Grp94
f/f
cells in BM and
49
spleen set as 1. (D) Bone section of a recipient femur. Grp94
f/f
LK cells were labeled with CFSE (green);
cGrp94
f/f
LK cells were labeled with SNARF (red); and nuclei were stained with DAPI (blue). Solid arrows
indicate cells lodged in the endosteal region (within two cell diameters from the endosteal surface), while
open arrows indicate cells located in the central marrow. Scale bar represents 1 mm. (E) Summary of the
percentage of labeled LK cells found at the endosteal region among those homed to BM from 3
independent experiments. All data are presented as mean ± s.e., *p<0.05.
2.3.7 cGrp94
f/f
HSCs failed to reconstitute the hematopoietic system in the presence
of WT competitors
As a consequence of impaired homing and retention to the BM niche, in the
competitive repopulation assay (Figure 2.9A) cGrp94
f/f
LSK cells failed to engraft and
reconstitute the hematopoietic system in lethally irradiated recipient mice in the presence
of WT competitors (Figure 2.9B and C).
50
Figure 2.9 cGrp94
f/f
HSCs failed to reconstitute the hematopoietic system in the presence of WT
competitors. (A) Scheme of the in vivo competitive repopulation assay. (B) Representative flow cytometric
analysis with tail blood from recipient mice 4 weeks after BM transplantation. CD45.1
+
cells represent
blood cells from WT competitor, while CD45.2
+
cells represent progenies from Grp94
f/f
or cGrp94
f/f
LSK
cells. Tail blood from recipient mice 8, 12, and 24 weeks after BM transplantation yielded the same results
(data not shown). (C) Summary of the percentage contribution of Grp94
f/f
or cGrp94
f/f
LSK cells to the
peripheral blood MNCs (n=5). The data are presented as mean ± s.e., ***p<0.001.
51
2.3.8 GRP94 in HSCs, but not niche cells, is necessary for maintaining HSC
homeostasis
In the competitive repopulation and competitive lodgment assay, where cGrp94
f/f
cells were transplanted into a WT microenvironment, we observed impairment in the
function of the cells indicating that Grp94 deletion in HSCs at least partially contributes
to the disruption of the HSC-niche interaction. However, in our model system Grp94 was
knocked out in the whole BM, therefore the disruption of HSC-niche interactions could
also be attributed to the niche cells. To further explore a possible extrinsic role of GRP94
null niche cells in HSC homeostasis disturbance in cGrp94
f/f
mice, we created a chimeric
mouse model with Grp94
f/f
hematopoietic cells and a GRP94 null microenvironment.
Grp94
f/f
BM cells were transplanted into lethally irradiated Grp94
f/f
or cGrp94
f/f
recipient
mice. Following 8 weeks to allow for full reconstitution of the hematopoietic system,
Grp94 deletion was induced by pI.pC administration (Figure 2.10A). According to
previous reports, BM stromal cells do not engraft after BM transplantation (Perkins and
Fleischman, 1988), therefore, Grp94 deletion only occurred in the hematopoietic cells .
Chimeras with Grp94
f/f
hematopoietic cells and cGrp94
f/f
microenvironment (WT-cKO
chimera) showed a normal hematology profile and spleen size as the control chimeras
(WT-WT chimera) (Figure 2.10B and 2.10C). Flow cytometric analysis revealed a
similar LSK percentage in BM from WT-cKO chimeras and WT-WT chimeras (Figure
2.10D and 2.10E). These findings provide direct evidence that GRP94 in HSCs is
52
required for their interaction with the niche, and depleting GRP94 in BM
microenvironment itself is not sufficient to affect the regulation of HSCs.
Figure 2.10 Effect of GRP94-null microenvironment on HSC maintenance. (A) Scheme of creating
chimeric mice with Grp94
f/f
hematopoietic cells transplanted into Grp94
f/f
or cGrp94
f/f
microenvironment.
(B) Complete blood count with tail peripheral blood from WT-WT (n=5) and WT-cKO (n=8) chimeric
53
mice. (C) Quantitation of spleen weights from WT-WT (n=4) and WT-cKO (n=5) chimeric mice. (D)
Representative flow cytometric analysis with BM from WT-WT and WT-cKO chimeric mice using Lin,
c-Kit and Sca-1. (E) Summary of percentage of LSK cells in the BM from WT-WT (n=5) and WT-cKO
(n=8) chimeric mice. All data are presented as mean ± s.e..
2.3.9 cGrp94
f/f
HSCs lacked cell surface integrin α4 expression and exhibited
impaired binding to fibronectin
To investigate the potential mechanism by which GRP94 maintains the interaction
between HSCs and the niche, we examined the cell surface expression of integrin α4 and
α5, which are known to be important for the homing and adhesion of HSCs to the
endosteal niche. Flow cytometric analysis with mouse BM LSKFlk2
-
and LSKFlk2
+
populations demonstrated significantly lower expression of cell surface CD49d (integrin
α4/β1) on cGrp94
f/f
than Grp94
f/f
cells, whereas CD49e (integrin α5/β1) expression was
comparable between the two groups (Figure 2.11A). These results establish that GRP94
is required for the expression of integrin α4 on the cell surface of hematopoietic stem and
progenitor cells, which is consistent with the recent finding that loss of GRP94 abrogates
integrin α4 but not α5 expression on BM cells (Staron et al., 2010).
To test specific interactions of the primitive hematopoietic cells with adhesion
molecules known to be present at the endosteal surface and play a role in mediating
HSC-niche interactions, we examined the ability of the cells to bind to fibronectin in vitro,
54
the binding partner of integrin α4, with binding to BSA as a negative control. While
Grp94
f/f
LSK cells were able to bind to fibronectin, we did not detect binding of the
cGrp94
f/f
cells to fibronectin above the negative control level (Figure 2.11B). Collectively,
these findings demonstrate that acute depletion of GRP94 in HSCs disrupts the
interaction of HSCs with niche, possibly through the disruption of adhesion to key ECM
molecules mediated by integrin α4.
Figure 2.11 Inability of Grp94 knockout HSCs to express surface integrin α4 or bind to fibronectin. A)
Representative flow cytometric analysis of CD49d and CD49e with BM LSKFlk2
-
and LSKFlk2
+
cells from
Grp94
f/f
and cGrp94
f/f
mice. Grey-filled histogram represents isotype control staining; dashed green line
represents Grp94
f/f
cells; solid red line indicates cGrp94
f/f
cells. B) The percentage of Grp94
f/f
and cGrp94
f/f
LSK cells bound to fibronectin in vitro. The number of cells binding to BSA was subtracted from that
binding to fibronectin, the results then were normalized against the number of Grp94
f/f
cells bound to BSA.
The experiments were performed twice in duplicate; each replicate contained pooled BM from 2 to 4
55
Grp94
f/f
or cGrp94
f/f
mice. The data are presented as mean ± s.e.
Figure 2.12 Changes in hematopoietic stem cell homeostasis and hematopoiesis upon GRP94 depletion.
LT-HSCs contact with the BM niche at the endosteal region to maintain their quiescence and stemness
through soluble factors and adhesion molecules. LT-HSCs differentiate into ST-HSCs, which give rise to
CMP, CLP, myeloid cells and lymphoid cells. HSCs mobilization from the BM, enter circulation, and
home back to the BM niche. Upon Grp94 deletion, GRP94-null HSCs lose contact with the BM niche,
leading to increased mobilization and impaired homing. Loss of interaction with the BM niche renders loss
of quiescence in GRP94-null HSCs. Moreover, Grp94 deletion in the hematopoietic system perturbs
lymphoid and myeloid differentiation, leading to decreased lymphoid cells and increased myeloid cells in
the BM, spleen and peripheral blood.
56
Figure 2.13 Scheme of how GRP94 deficiency affects HSC homeostasis. Without GRP94 the process of
secretory factors and membrane-bound proteins (such as integrin α4) is impaired, which leads to the
blockage of regulatory cues from HSC niche to maintain their quiescence and retention in the BM.
2.4 Discussion
GRP94 is the most abundant glycoprotein in the ER and is emerging as an
important player in protein processing. Its unique interaction with specific client proteins
may allow it to specifically regulate cellular processes distinct from other major
chaperones in the ER. In particular, recent model studies suggested that GRP94 is
critically required for the functional expression of secretory and/or membrane proteins
that enable the integration of cells into tissues (Maynard et al., 2010). Here we examined
the role of GRP94 in the HSC interactions with the adult BM niche. The model system
57
that we employed is the Grp94; Mx-1-Cre mouse model whereby GRP94 can be acutely
eliminated largely in the hematopoietic system in a temporally controlled manner in adult
mice. Our results showed that within 3 weeks of depletion of GRP94 in the hematopoietic
cells, there was a 2-fold increase in primitive HSCs in the mutant mice. The Grp94 KO
LSK cells are more proliferative, likely as a consequence of loss of niche interaction. The
latter is supported by our observation that the mutant LSK cells are more mobilized to the
spleen and peripheral blood. The mutant LSK cells lack cell surface integrin α4
expression and do not bind to fibronectin as efficiently as the WT LSK cells in vitro, are
less able to lodge in the BM niche in vivo, and as a consequence showed no engraftment
in competitive repopulation assays.
The maintenance of HSCs involves the balance between self-renewal and
differentiation, proliferation and quiescence, mobilization and homing, as well as their
capacity to provide lifelong reconstitution of all blood-cell lineages after transplantation
into lethally irradiated recipients. Our study demonstrates the unique function of GRP94
in regulating the homeostasis of HSCs and their interaction with the stem cell niche.
Currently, the regulation of HSC physiology is mostly attributed to transcription factors,
cell-cycle regulators, chromatin-associated proteins, extracellular signaling molecules
and their receptors (Zon, 2008). Here we explore the notion that an ER chaperone protein
may play a role in the regulation of HSC-niche interactions. The HSC niche is believed to
house HSCs, maintain their quiescence and prevent them from differentiation. Here we
58
determined that Grp94 KO primitive cells are less efficient in contacting with BM niche,
with the consequence that the mutant LSK cells can exit quiescence and enter cell cycle.
While the precise mechanisms by which GRP94 affects this awaits future investigation,
one plausible mechanism may be attributed to the requirement of GRP94 to facilitate
processing of factors such as integrins specifically required for HSC-niche adhesion
(Forsberg and Smith-Berdan, 2009). Secretory factors and cell-surface proteins are
synthesized in the rough ER, and translocated into the ER where they are folded into their
functional conformation by ER chaperone proteins. GRP94 has been reported to be
essential for the proper folding of several integrins, including α4 (Staron et al., 2010).
This has been reported to mediate the interaction of HSCs with niche cells and the ECM,
as loss of integrin α4 results in impaired homing and reduced efficiency of HSCs to lodge
to the endosteal surface (Jiang et al., 2009). While integrin α4 and α5 are both important
for HSC retention in the niche (Jiang et al., 2009; Van der Loo et al., 1998), GRP94 is
specifically required for processing integrin α4 but not α5 in HSCs. The failure of the
Grp94 KO HSCs to express integrin α4 on the cell surface and its inability to bind to
fibronectin are supportive of this notion. It is also possible that GRP94 is required for the
proper folding of soluble ligands mediating the cross talk from HSCs to the niche. Other
mechanisms may include cell cycle regulation by GRP94 and the ability of GRP94 to
regulate ER Ca
2+
efflux and signaling. As GRP94 is also recently localized to the cell
surface and also identified as a transmembrane ER protein, it may also control signaling
pathways initiated from the cell surface and/or the ER and cytosol interphase affecting
59
stem cell proliferation.
Our studies raise the general issue concerning the roles of protein folding
chaperones in hematopoiesis and its relationship to the UPR. Hematopoiesis is tightly
regulated by growth factors, cytokines and their receptors, which are secretory and cell
membrane proteins (Zhu and Emerson, 2002). Secretory factors and cell surface proteins
are synthesized in the rough ER where they are folded into their functional conformation
by ER chaperone proteins. Therefore, ER chaperone proteins are likely to be important
for maintaining hematopoiesis, and their function depends on the role of specific client
proteins in hematopoiesis. Other than synthesizing client proteins that are regulating
hematopoiesis, the ER is also involved in hematopoiesis through UPR signaling pathways.
For example, UPR sensor IRE1 ribonuclease activity for XBP-1 splicing is required for
both the early differentiation of T and B lymphocytes and the terminal differentiation of
activated B cells to immunoglobin-secreted plasma cells (Zhang et al., 2005; Brunsing et
al., 2008). Whether UPR signaling is involved in regulating HSC homeostasis is not
known, and awaits further investigation.
In comparing our Mx-1-Cre-mediated acute GRP94 knockout model with the
chimera model using tamoxifen-mediated inducible Grp94 deletion following BM
transplantation (Staron et al., 2010), similar hematopoietic phenotypes, including general
leukocytosis, increased spleen cellularity, decreased thymus and lymph node cellularities,
increased myeloid cells and decreased lymphoid cells in the BM and spleen were
60
observed in both models. The increase of LSK cells in the BM, spleen and peripheral
blood and loss of integrin α4 expression upon GRP94 depletion are also in agreement
with the previous report (Staron et al., 2010), which focuses on the role of GRP94 in
lymphogenesis, specifically pro-B to pre-B transition in B cell development, as well as T
cell development beyond CD4
-
CD8
-
double negative stage. Our work here investigates
the unique function of GRP94 in the homeostasis of HSCs and their interaction with the
stem cell niche. With our model, we provide new evidence that the expansion of
primitive hematopoietic cells is attributed to the change of cell cycle status upon Grp94
deletion and that the change of HSC cell cycle status and localization is due to loss of
interaction with the niche, revealed by the impaired homing and lodging ability of
GRP94-deficient HSCs in vivo. We further determined that GRP94 in the niche cells is
not required for maintaining the interaction with HSCs.
In summary, we identified GRP94 as a novel regulator of HSCs physiology and
their interaction with the niche. It is tempting to speculate that agents that can alter
GRP94 expression or activity may be explored for therapeutic intervention, such as
increasing the proportion of HSCs in peripheral blood as a source for blood stem cell
therapy, or as an alternative method for non-myeloablative conditioning through
emptying the HSC niche (Czechowicz et al., 2007) and these warrant future investigation.
Furthermore, overexpression of GRP94 has been associated with cellular transformation,
tumorigenicity and decreased sensitivity to anti-cancer treatment (Fu and Lee, 2006).
61
With cancer stem cells implicated as the cancer initiating cells responsible for
tumorigenesis and contribute to cancer resistance, it would be interesting to determine in
future studies whether GRP94 regulates cancer stem cell niche interaction and thus
contributes to tumorigenicity and self renewal of resistant cancer cells.
62
Chapter 3
GRP94 deficiency in the hematopoietic system
alters proliferation regulators in HSCs
3.1 Introduction
The self-renewal of HSCs is tightly regulated by intrinsic determinants and
extrinsic cues from the microenvironment (Zon, 2008). Intrinsic determinants of HSC
self-renewal and differentiation include cell cycle regulators, transcription factors and
chromatin-associated factors (Orford and Scadden, 2008). One critical intrinsic regulator
of HSC self-renewal and differentiation is AKT, a serine/threonine kinase. Activated
growth factor receptors recruit PI3K to the plasma membrane, allowing for the
phosphorylation of phosphoinositides and conversion of PI(4,5)P2 to PI(3,4,5)P3. AKT,
through binding to the PI(3,4,5)P3 lipid products, localizes to the cell membrane and
becomes activated. AKT is a major effector of the PI3K pathway and many of its
substrates regulate cell survival and growth (Kharas and Gritsman, 2010). Deleting PTEN,
which is a negative regulator of PI3K-AKT pathway in the mouse hematopoietic system,
results in HSC hyperproliferation, myeloproliferative disorder and leukemia (Zhang et al.,
2006; Yilmaz et al., 2006). Constitutive activation of AKT signaling causes short-term
expansion of the LSK compartment through increased cycling of LSK cells and
eventually leads to HSC depletion and leukemia (Kharas et al., 2010). While
well-established cell cycle regulators such as p53 and p21
cip1/waf1
are known to modulate
63
HSC cell fate (Pietras et al., 2011), novel hematopoietic cell cycle modulators have also
been identified, including MS4A3 (HTm4) (Donato et al., 2002). MS4A3 is a
transmembrane protein of the MS4A family expressed in hematopoietic cells and other
select cell types and tumors (Kutok et al., 2011). MS4A3 interacts with the cyclin
dependent kinase 2 (CDK2), cyclin A and CDK-associated phosphatase complex, and its
overexpression in hematopoietic cells has been reported to cause cell cycle arrest at the
G0/G1 phase (Chinami et al., 2005). Thus, MS4A3 can potentially regulate HSC
proliferation in vivo.
Extrinsic signals from the microenvironment control the expression of intrinsic
determinants of HSC self-renewal and differentiation. HSCs reside in a specialized
microenvironment known as the HSC niche which composes cellular and humoral
signaling cues that regulate the survival, self-renewal, migration, differentiation, and
quiescence of HSCs (Wilson and Trumpp, 2006; Jones and Wagers, 2008). The first
identified HSC niche was the BM endosteal niche where a specific type of osteoblastic
cells represents the major component. However, vasculature, stromal cells, and
hematopoietic progeny compartments in the BM and other hematopoietic organs have
also now been identified to regulate the physiology of HSCs (Kiel and Morrison, 2008;
Wang and Wagers, 2011; Nakamura-Ishizu and Suda, 2013). Local extrinsic elements
from the niche includes soluble factors which function through interactions with their
receptors, as well as direct contact through ECM and cell surface proteins (Adams and
Scadden, 2006). Gap junction proteins have been shown to play important roles in HSC
64
homeostasis. Connexin 43 (Cx43) in the endosteal niche is a crucial regulator of HSC
homing and migration in an irradiated microenvironment (Gonzalez-Nieto et al., 2012),
while connexin 32 (Cx32) is also required for maintaining hematopoietic progenitors in
the BM. It has been reported that Cx32
-/-
mice showed BM LSK cell expansion and
increased LSK cell proliferation (Hirabayashi et al., 2007).
In Chapter 2 we identified the ER chaperone GRP94 as a novel regulator of HSCs
and their interaction with the adult BM endosteal niche. As a chaperone that assists in the
folding, assembly and secretion of a selective collection of client proteins, GRP94
performs unique functions in the ER, and controls specific pathways critical for cell
growth, differentiation, organ homeostasis, and immune functions (Ni and Lee, 2007;
Marzec et al., 2012; Liu et al., 2013). In Chapter 2 we used an Mx-1-Cre-mediated
inducible knockout mouse model (Grp94
f/f
; Mx-1-Cre, cGrp94
f/f
) identified GRP94 as a
regulator for HSC physiology, as loss of GRP94 in the hematopoietic system leads to an
expansion of the hematopoietic stem and progenitor cell pool through increased
proliferation (Luo et al., 2011). This increased proliferation could in part be attributed to
detachment of HSCs from the BM niche as GRP94-null HSCs show increased
mobilization, and impaired homing and engraftment. Furthermore, this impaired homing
and engraftment is cell autonomous, as transplanting WT hematopoietic cells into a
GRP94-null microenvironment yielded a normal hematopoietic profile with comparable
numbers of HSCs as compared to controls. Thus, these earlier results indicated that loss
of GRP94 function in the hematopoietic stem and progenitor cells activate cell intrinsic
65
mechanisms that lead to the loss of quiescence and cell expansion in the GRP94-null
mouse HSCs. Here we explored these mechanisms in LSK cells isolated from the
cGrp94
f/f
mice.
3.2 Materials and methods
Mice
Grp94
f/f
mice in a mixed C57BL/6; 129/Sv background were generated as
previously described (Mao et al., 2010). Grp94
f/f
mice were crossed with the transgenic
Mx-1-Cre mice on a C57BL/6 background (Jackson Laboratory) to generate Grp94
f/f
;
Mx-1-Cre mice. Littermates that were negative for Cre transgene were used as controls.
Genotyping was performed by PCR using genomic DNA extracted from mouse tail
biopsies as previously described. To induce the genomic deletion, 5.5 to 6.5 week-old
male and female mice were injected intraperitoneally with pI.pC (25 mg/kg mouse body
weight) three times a week. Mice were analyzed 11 days post 7 injections, except with
modifications as specifically mentioned in the figure legends. All protocols for animal
use were reviewed and approved by the USC Institutional Animal Care and Use
Committee. The animal assurance number is A3518-01. The protocol number is 9964.
Genotyping
Primer set PAS1-PAS3 was used to differentiate Grp94 WT and floxed allele,
66
while niL and niR were used to detect knockout allele (Mao et al., 2010). For Genotyping
of the Cre transgene, primers Cre109 and Cre457 were used. Reactions for Grp94 floxed,
WT, knockout alleles and Cre were run under the following conditions: 94°C for 5 min,
followed by 46 cycles of 94°C for 20 sec, 62°C for 30 sec, and 72°C for 30 sec, ended
with 72°C for 7 min. The primer sequences are listed in table 2.1.
Flow cytometry
BM cells were flushed from long bones (tibias and femurs) with Dulbecco’s
phosphate-buffered saline without calcium and magnesium (PBS), and then filtered
through nylon screen (70 micron, BD Biosciences) to obtain single cell suspension. To
study hematopoietic stem and progenitor cells, Lineage [Lin; which consists of B220
(RA3-6B2), TER119 (TER119), CD4 (RM4-5), CD8a (53-6.7), Gr-1 (RB6-8C5), Mac-1
(WT.5) and CD41 (eBioMWReg30 from eBioscience)], c-Kit (2B8), Sca-1 (D7, all from
BD Pharmingen except CD41), CD150 (mShad150), and CD48 (HM48-1) were used.
Antibodies against Tie2 (TEK4, eBioscience) and CXCR4 (12G5, BD Pharmingen) were
used individually to detect cell surface Tie2 and CXCR4. Cell population analysis was
performed on BD FACS LSR II.
Fluorescence activated cell sorting
Hematopoietic stem and progenitor cells were stained with cell surface markers
(Lin
-
Sca-1
+
c-Kit
+
) and purified using a FACSAria flow cytometer (Becton Dickinson).
67
Immunofluorescence and confocal microscopy
Sorted BM LSK cells were washed and resuspended in serum-free DMEM (high
glucose). Droplets of cells were then seeded on Superfrost Plus
®
Gold slides
(FT-4981-GLPLUS, Thermo Scientific) and cultured at 37°C for 1 h. The cells were then
fixed in 4% paraformaldehyde at RT for 10 min then dehydrated by serial ethanol
solutions and allowed to dry. The slides were stored at -80°C for later use. For
immunostaining, the cells were rehydrated in PBS and then quenched with 0.25% (w/v)
ammonium chloride in PBS containing 0.01% saponin (PBS/SAPONIN) at RT for 5 min.
The cells were then briefly permeabilized by 0.1% triton X-100 in PBS/SAPONIN and
quenched again with 0.25% (w/v) ammonium chloride. After briefly washing with
PBS/SAPONIN, the cells were pre-blocked with 1% BSA in PBS/SAPONIN
(PBS/BSA/SAPONIN) at RT for 1 h. For PI(3,4,5)P3 staining, endogenous mouse IgG
was blocked with additional treatment with M.O.M.™ Mouse Ig Blocking Reagent
(BMK-2202, Vector Laboratories) at RT for 1 h. Then, the cells were incubated with
primary antibodies at 37°C for 1 h followed by secondary antibodies at 37°C for 30 min
in 5% PBS/BSA/SAPONIN. For PI(3,4,5)P3 staining, mouse anti-PI(3,4,5)P3 (Z-P345b,
Echelon Biosciences, 20 µg/mL) primary antibody and Alexa-fluor 488 conjugated goat
anti-mouse secondary antibody (Life Technologies, 20 µg/mL) were used. For
phosphorylated AKT (pAKT) staining, rabbit anti-pAKT (Ser473, Cell Signaling
Technology, 4 µg/mL) primary antibody and Alexa-fluor 488 conjugated goat anti-rabbit
68
secondary antibody (Life Technologies, 20 µg/mL) were used. For total AKT staining,
mouse anti-pan-AKT (Cell Signaling Technology, 4 µg/mL) primary antibody and
Alexa-fluor 594 conjugated goat anti-mouse secondary antibody (Life Technologies, 20
µg/mL) were used. For Cx32 staining, rat anti-Cx32 antiserum (Developmental Studies
Hybridoma Bank, 1:1) primary antibody and Alexa-fluor 488 conjugated goat anti-rat
secondary antibody (Life Technologies, 20 µg/mL) were used. The cells were then
washed with PBS/SAPONIN and ddH
2
O and mounted with Vectashield antifade medium
with DAPI (H-1200, Vector Laboratories).
Confocal images were obtained by a Zeiss LSM510 confocal microscope
equipped with a Hamamatsu R6357 photomultiplier and LSM 510 Version 4.2 SP1
acquisition software (Carl Zeiss). Representative pAKT images (Fig. 3A) were taken
with a Plan-Apochromat 100×/1.4 oil DIC objective at 2× zoom and cropped in
Powerpoint software (Microsoft). Images for quantification were taken under 40×/1.30
Oil objective without zoom. Compressed Z-stack PI(3,4,5)P3 images (Fig. 3C) were
taken with an EC Plan-Neofluar 40×/1.30 Oil objective without zoom and cropped in
Photoshop. Same number of sections were compressed for each condition. Representative
Cx32 images (Fig. 5A) were taken with a Plan-Apochromat 100×/1.4 oil DIC objective at
8× zoom.
69
HSCs in vitro culture
Human stromal cells (HS-5) were irradiated at 15Gy to stop proliferation before
being seeded into 24-well plates and cultured overnight. Equal number of sorted LSK
cells were plated in wells with or without HS-5 cells and cultured in α-MEM with 10%
serum and 1% P/S for 48 h in the presence of IL-3 (10 ng/mL), IL-6 (50 ng/mL), SCF
(100 ng/mL) and TPO (100 ng/mL). LSK cells cultured without HS-5 cells were
harvested and enumerated with a hemacytometer. LSK cells co-cultured with HS-5 cells
were harvested with medium whereas HS-5 cells were trypsinized, collected and mixed
with the LSK cells. The mixture of cells was stained with hematopoietic cell marker
CD45 (30-F11, eBioscience) and the number of CD45
+
cells were calculated based on the
proportion of CD45
+
cells in the mixture and the number of HS-5 cells seeded.
Real-time quantitative RT-PCR
RNA was extracted from mouse whole BM cells and reverse-transcription was
performed as previously described (Ni et al., 2009). cDNA samples were analyzed in
triplicate with the SYBR Green Supermix (Quanta Biosciences, Gaithersburg, MD)
according to manufacturer’s instructions. The following primers were used: mouse
Ms4a3, 5’-TAC TGC AAG CCC TCG GGG CCA-3’ and 5’-GGG TTT CTC CCT GCG
GCA ACA-3’; human Ms4a3, 5’-AAG CCT GAA GCC TCC AAG TTC-3’; mouse and
70
human 18S RNA, 5’-CCA CTC CCG ACC CGG GGA GGT AGT GAC GAA-3’and
5’-CTC AGC TAA GAG CAT CGA GGG GGC GCC GAG AGG-3’.
Reagents
The AKT inhibitor MK2206 (Hirai et al., 2010) was purchased from Tocris
Bioscience. MK2206 was dissolved in DMSO and diluted with DMEM before use. The
Cx32 mimetic peptide and the control peptide with scrambled sequence were purchased
from GenScript. The sequences of the peptides are as follows: for
32
Gap27,
SRPTEKTVFT (extracellular loop 2, position 182-191 of Cx32) (Maezawa and Jin,
2010); for scrambled control, TFEPIRISITK (Wang et al., 2012).
Lentivirus production and infection
The second generation packaging plasmids PsPAX and PMD2.g were kindly
provided by Dr. Wange Lu’s lab. Lentiviral shRNA against human Grp94 (shGrp94,
Open Biosystems) or scrambled control sequence (shCtrl) was transfected individually
into 293T cells with packaging plasmids for 6 hours. The lentivirus-containing medium
was collected at 48 hours and 72 hours post transfection, and the lentiviral particles were
concentrated with PEG-it
TM
Virus Precipitation Solution according to manufacturer’s
instruction (System Biosciences). U266 human multiple myeloma cells and HL60 human
promyelocytic leukemia cells were maintained in RPMI 1640 supplemented with 10%
FBS and 1% P/S. Cells for lentiviral infection were resuspended in IMDM or RPMI with
71
polybrene (4 µg/mL, Sigma), mixed thoroughly with lentivirus and centrifuged at 1500g
for 30 mins at room temperature. Cells were then incubated with lentivirus-containing
medium from 24 hours, cultured in regular medium for 5 days, and selected with
puromycin (6µg/mL) for 1 week before harvested for analysis.
Scrape-loading dye transfer assay
Gap junction permeability was determined using a Lucifer yellow scrape loading
technique (el-Fouly et al., 1987). HeLa cells which are deficient of all connexin
expression (Plantard et al., 2003) were transfected with Cx32 overexpression plasmid or
the pcDNA3 empty vector by Bio-T transfection reagent (Bioland Scientific) for 24 hours.
The Cx32 expression plasmid was constructed by subcloning the mouse Cx32 cDNA
amplified via RT-PCR from mouse liver RNA into the pcDNA3 expression vector.
Confluent cells were incubated with scrambled peptide or Cx32 mimetic peptide
32
Gap27
for 30 min, washed with TBS, and loaded with mixture of Lucifer yellow and Rhodamine
dextran followed by scratches using 20 µL peptide tips. After incubation at 37°C for 10
min, the cells were washed 7 times with TBS prior to examination by florescent
microscope. Images were obtained by a Nikon ECLIPSE TE300 lnverted Microscope
equipped with Hamamatsu Orca photomultiplier and MetaMorph acquisition software.
Statistics
Statistical significance was assayed by Student’s t-test.
72
3.3 Results
3.3.1 GRP94 deficiency led to loss of quiescence in HSCs
Using the cGrp94
f/f
mouse model that we previously created in Chapter 2 where
GRP94 is depleted in the hematopoietic system upon administration of pI.pC to 5.5 to 6.5
week old mice (Luo et al., 2011), we determined the effect of GRP94 depletion on
primitive hematopoietic cell pool using the signaling lymphocyte activation molecule
(SLAM) family proteins (CD150, CD48 and CD41). Littermates lacking the Cre
transgene (Grp94
f/f
) which are phenotypically equivalent to animals with WT Grp94
alleles were also injected with pI.pC and served as controls for any side effects of pI.pC
injection. There was a 2-fold expansion of the long-term HSC (LT-HSC)-enriched Lin
-
c-Kit
+
Sca-1
+
CD41
-
CD48
-
CD150
+
(LSKCD41
-
CD48
-
CD150
+
) cell population (Figure
3.1A and B), correlating with a 2-fold increased hematopoietic stem and progenitor
cells-enriched Lin
-
c-Kit
+
Sca-1
+
(LSK) population (Figure 3.1C). Interestingly, using the
SLAM markers, short-term HSC (ST-HSC)-enriched Lin
-
c-Kit
+
Sca-1
+
CD41
-
CD48
-
CD150
-
(LSKCD41
-
CD48
-
CD150
-
) cell population was not significantly altered (Figure
3.1B).
73
Figure. 3.1 GRP94 deficiency in the BM expanded the primitive hematopoietic cell pool. (A)
Representative flow cytometric analysis with BM cells using Lin, c-Kit, Sca-1, CD41, CD48 and CD150.
(B) Quantification of flow cytometric analysis of LSKCD41
-
CD48
-
CD150
+
LT-HSCs and
LSKCD41
-
CD48
-
CD150
-
ST-HSCs (n=12 for Grp94
f/f
, n=8 for cGrp94
f/f
). (C) Quantification of flow
cytometric analysis of total LSK cells in BM (n=12 for Grp94
f/f
, n=8 for cGrp94
f/f
). All data are presented
as mean ± s.e., ***p<0.001.
74
3.3.2 In vitro expansion of GRP94-null HSCs
Consistent with the loss of quiescence and increased proliferation in cGrp94
f/f
BM
LSK cells determined by cell cycle analysis as described in Chapter 2, when equal
number of cGrp94
f/f
and Grp94
f/f
BM LSK cells were seeded and cultured in medium
containing IL-3, IL-6, SCF and TPO for two days (Figure 3.2A, top panel), the number of
cGrp94
f/f
hematopoietic cells generated was twice as many as that from the Grp94
f/f
control (Figure 3.2B). Under co-culture conditions with HS-5 human stromal cells for
two days (Figure 3.2A, lower panel), similar increase in cGrp94
f/f
CD45
+
hematopoietic
cells were also observed following equal seeding of LSK cells from the two genotypes
(Figure 3.2C and D). These results confirm that loss of GRP94 in the primitive
hematopoietic cells leads to cell expansion.
75
Figure. 3.2 In vitro culture of GRP94-null LSK cells. (A) Scheme of culturing Grp94
f/f
and cGrp94
f/f
LSK
cells in vitro. (Upper panel) with growth factors and cytokines only; (Lower panel) with stromal cells,
growth factors and cytokines. CD45 staining was used to determine hematopoietic cells derived from the
LSK cells. (B) Cell numbers from cultures without stromal cells determined by hemocytometer. (C) Flow
cytometric analysis using CD45 as a marker for hematopoietic cells derived from LSK cells co-cultured
with stromal cells. The green line represents Grp94
f/f
cells and the red line indicates cGrp94
f/f
cells. (D)
Quantification of hematopoietic cells under co-culture conditions with stromal cells, based on the number
of stromal cells seeded and the proportion of CD45
+
cells determined by flow cytometric analysis. All data
are presented as mean ± s.e., **p<0.01.
76
3.3.3 cGrp94
f/f
primitive hematopoietic cells displayed increased PI3K-AKT
activation
AKT activation is a major contributing factor for HSC proliferation. To
investigate the underlying mechanisms for cell expansion due to GRP94 deficiency, we
examined the activation of AKT in the primitive hematopoietic compartment using
freshly isolated BM LSK cells from the two genotypes. Representative
immunofluorescence staining images for pAKT and total AKT in the LSKs are shown in
Figure 3.3 A and C, and the level of staining was quantified and compared between the
two genotypes (Figure 3.3B and D). Our results revealed that while Grp94
f/f
control
LSK cells exhibited low levels of pAKT staining, cGrp94
f/f
LSK cells showed
significantly higher (1.8-fold) AKT phosphorylation compared to Grp94
f/f
controls.
Interestingly, total AKT level was about 15% lower in the GRP94-null LSKs compared
to Grp94
f/f
controls.
To determine at which step GRP94 regulates AKT activation in the LSK cells, we
measured the effect of Grp94 knockout on the production of PI(3,4,5)P3, which is an
indication of PI3K activity upstream of AKT activation. PI(3,4,5)P3 production was
measured by confocal microscopy following staining of purified LSK cells with a
monoclonal anti-PI(3,4,5)P3 antibody previously established for sensitive detection of
PI(3,4,5)P3 levels in cells, including hematopoietic cells (Wey et al., 2012a).
Representative immunofluorescent images of the LSK cells from the two genotypes are
77
shown in Figure 3.3E. While the staining pattern and intensity showed some
heterogeneity among the LSK cells, cGrp94
f/f
LSK cells in general exhibited a higher
level of PI(3,4,5)P3 production, corresponding with the pAKT staining pattern. Upon
quantitation, we observed about 80% of cGrp94
f/f
LSK cells showed positive PI(3,4,5)P3
production compared to about 45% for the control Grp94
f/f
LSK cells. Thus, GRP94
deficiency activates AKT in LSK cells at least in part via higher PI(3,4,5)P3 production.
To determine the requirement of AKT activity on cGrp94
f/f
LSK cell proliferation,
we isolated the Grp94
f/f
and cGrp94
f/f
LSK cells and cultured them for 2 days in the
presence or absence of an allosteric AKT inhibitor MK2206 (Hirai et al., 2010). We
recently reported that MK2206 was able to block AKT phosphorylation in various cell
lines cultured in vitro at concentrations as low as 50 nM (Gray et al., 2013). While as
expected cGrp94
f/f
LSK cells nearly tripled in cell number during culture when treated
with DMSO alone, MK2206 treatment compromised the cell expansion in a dose
dependent manner, with an effect readily observable at 50 nM of drug concentration
(Figure 3.3F). Thus, AKT activation contributes at least in part to cGrp94
f/f
LSK cell
hyperproliferation.
78
Figure 3.3 Increased AKT activation is required for cGrp94
f/f
LSK proliferation (A) Fluorescent staining of
phosphorylated-AKT (pAKT, Ser473; in green) on freshly isolated BM LSK cells from the indicted
79
genotypes. The corresponding nuclei were stained by DAPI in blue. Scale bar represents 10 µm. (B)
Quantification of pAKT levels in Grp94
f/f
and cGrp94
f/f
LSK cells with ImageJ (n=41 for Grp94
f/f
, n=54 for
cGrp94
f/f
). ***p<0.001. (C) Fluorescence staining of total AKT (in red) on freshly isolated BM LSK cells.
(D) Quantification of total AKT levels in Grp94
f/f
and cGrp94
f/f
LSK cells with ImageJ (n=45 for Grp94
f/f
,
n=64 for cGrp94
f/f
). *p<0.05, (E) Representative PI(3,4,5)P3 staining on freshly isolated BM LSK cells
from Grp94
f/f
and cGrp94
f/f
mice. The corresponding nuclei were stained by DAPI in blue. Scale bar
represents 5 µm. (F) Fold change in cell numbers from cultured LSK cells which were either treated with
DMSO or MK2206 at the indicated concentrations, with the number of cells seeded set as 1. All data are
presented as mean ± s.e..
3.3.4 Microarray analysis identified cell cycle regulator Ms4a3 downregulated in
cGrp94
f/f
primitive hematopoietic cells
To explore other mechanisms that may contribute to HSC expansion upon Grp94
deletion, we isolated mRNA from cGRP94
f/f
and Grp94
f/f
LSK cells and performed
microarray analysis, which showed that the mRNA level of major cell cycle regulators
known to regulate HSCs such as p18, p19, p21, p27 and p57 in cGrp94
f/f
LSK cells were
all comparable with Grp94
f/f
controls. While we did not detect any proliferation
regulators in the top up-regulated category in the microarray, the expression of a
newly-identified cell cycle regulator, Ms4a3 (Donato et al., 2002), was reduced to about
3% of the control. This was one of the genes that were most drastically downregulated
upon GRP94 depletion detected (Table 3.1). The reduced expression of Ms4a3 in
cGrp94
f/f
LSK cells was confirmed by real-time quantitative PCR as the expression of
80
Ms4a3 in cGrp94
f/f
LSK was 10% of that in Grp94
f/f
LSK cells (Figure 3.4A).
Interestingly, the reduced expression of Ms4a3 mRNA level upon Grp94 deletion was
only observed in LSK cells but not in the whole BM of cGrp94
f/f
mice (Figure 3.4A),
suggesting the regulation of GRP94 on Ms4a3 may be restricted to the primitive
hematopoietic cells.
Table 3.1 Top 10 genes downregulated and upregulated in cGrp94
f/f
LSK cells
compared to Grp94
f/f
LSK cells identified by microarray analysis
Gene
Symbol
RefSeq Expression Fold
Changed
Definition / Major function
Grp94
f/f
cGrp94
f/f
(cGrp94
f/f
/
Grp94
f/f
)
Top 10 downregulated genes
Ear1 NM_007894 3037.36 20.21 0.007 eosinophil-associated, ribonuclease A
family, member 1
Prg2 NM_008920 2688.34 35.82 0.013 proteoglycan 2, bone marrow
Ear1 NM_007894 3822.76 58.37 0.015 eosinophil-associated, ribonuclease A
family, member 1
Ear10 NM_053112 2288.84 35.79 0.016 eosinophil-associated, ribonuclease A
family, member 10
Ear10 NM_053112 1996.70 35.82 0.018 eosinophil-associated, ribonuclease A
family, member 10
Epx NM_007946 2668.11 52.31 0.020 eosinophil peroxidase, natural killer cell
activator, eosinophil granule major basic
protein
Ms4a3 NM_133246 4306.50 118.43 0.028 membrane-spanning 4-domains, subfamily
A, member 3, cell cycle regulator
Ear6 NM_053111 1638.55 49.12 0.030 eosinophil-associated, ribonuclease A
family, member 6
Ear2 NM_007895 1471.94 50.49 0.034 eosinophil-associated, ribonuclease A
family, member 2
Mt2 NM_008630 5026.37 203.78 0.041 metallothionein 2, heavy metal binding
Top 10 upregulated genes
Taf1d BC056964 371.85 2105.58 5.662 TATA box binding protein
(Tbp)-associated factor
Rnu12 NR_004432 662.32 2813.99 4.249 RNA U12
Klf12 NM_010636 70.45 279.88 3.973 Kruppel-like factor 12
ATP6 ENSMUST00000119
235
90.66 355.35 3.919 ATP synthase F0 subunit 6
Igh BC018535 254.96 968.49 3.799 immunoglobulin heavy chain complex
ND6 ENSMUST00000082
419
196.98 736.19 3.737 NADH dehydrogenase subunit 6
Lhcgr NM_013582 128.39 468.32 3.648 luteinizing hormone/choriogonadotropin
receptor
Mn1 NM_001081235 215.27 770.12 3.578 meningioma
Ifi44 NM_133871 328.16 1118.38 3.408 interferon-induced protein 44
Mamdc2 NM_174857 190.67 638.64 3.349 MAM domain containing 2
81
3.3.5 Context-dependent expression of Ms4a3 in LSK cells
To further explore the expression pattern and determinants of Ms4a3, we
compared the level of Ms4a3 mRNA from freshly isolated BM LSK with those from
freshly isolated spleen LSK cells, as well as BM LSK cells cultured in vitro. In the
freshly isolated spleen LSK cells of the control Grp94
f/f
mice, the level of Ms4A3 mRNA
was 5% of the level detected in BM LSK (Figure 3.4A). Similarly low level of Ms4a3
mRNA was detected in the spleen LSK of the cGrp94
f/f
mice. In examination of cultured
BM LSK cells, 5-fold reduced Ms4a3 expression was observed in Grp94
f/f
LSK cells
cultured in vitro when compared to freshly isolated ones from the BM. Thus, the
expression of Ms4a3 is context dependent, and the reduction in its expression upon
GRP94 depletion appears to be specific for freshly isolated BM LSK cells (Figure 3.4A).
Furthermore, lentiviral shRNA-mediated GRP94 knockdown in human promyelocytic
leukemia cells HL60 where MS4A3 is abundantly expressed did not result in reduced
Ms4a3 expression (Figure 3.4B and C). Collectively, these results support the notion of
cell type specificity and BM microenvironment dependency in the regulation of Ms4a3
expression by GRP94.
82
Figure 3.4 Context-dependent expression of Ms4a3 in LSK cells. (A) Ms4a3 mRNA expression measured
by quantitative real-time PCR from BM LSK cells, whole BM cells, spleen LSK cells and BM LSK cells
cultured in vitro for two days. The levels of Ms4a3 mRNA were normalized against 18S RNA. (B) Western
blot analysis of GRP94 and b-actin expression in the human promyelocytic leukemia cells (HL60) infected
with lentiviral shCtrl or shGrp94. (C) Ms4a3 mRNA expression from HL60 cells infected with shCtrl and
shGrp94 normalized against 18S RNA. The experiments were repeated 2 to 4 times. All data are presented
as mean ± s.e., *p<0.05, **p<0.01.
3.3.6 GRP94 depletion reduced cell surface connexin32 plaque number
We then further investigated the loss of interaction of GRP94-null HSCs with the
BM microenvironment. Since GRP94-null HSCs have impaired engraftment upon
83
transplantation, whereas transplanting Grp94
f/f
hematopoietic cells into a GRP94-null
microenvironment yielded a normal hematology profile and HSC pools (Luo et al., 2011),
the extrinsic molecules through which GRP94 regulates HSCs are expressed on HSCs but
not niche cells. Therefore, we examined the changes of potential extrinsic proteins that
are maintaining the HSC-niche interaction and the loss of which could affect HSC
proliferation.
Gap junction protein Cx32 has been shown to be specifically expressed on the
membrane of LSK cells and the knockout of Cx32 leads to increased LSK cells in the
BM through increased proliferation (Hirabayashi et al., 2007). Freshly isolated
GRP94-null LSK cells demonstrated decreased Cx32 plaque number on the cell surface
compared to the Grp94
f/f
control, as revealed by confocal microscopy (Figure 3.5A).
Quantitation of the images showed that about 50% of the Grp94
f/f
LSK cells had 5 or 6
plaques on the cell surface, with some cells showing up to 11 plaques (Figure 3.5B). In
contrast, 40% GRP94-null LSK cells had no Cx32 plaque observed on the surface and
only very low number of these cells showed 5 or more plagues (Figure 3.5B). This
indicates that the formation of cell surface Cx32 plaque was disrupted upon GRP94
ablation in LSK cells.
84
Figure 3.5 Reduction of Cx32 cell surface plaques upon Grp94 deletion. (A) Immunofluorescent Cx32
staining on Grp94
f/f
and cGrp94
f/f
BM LSK cells. White arrows indicate Cx32 plaques on the cell surface.
Scale bar represents 2 µm. (B) The number of Cx32 plaques was counted from Z-stack images for whole
cells, and the percentages of Grp94
f/f
(left) and cGrp94
f/f
(right) BM LSK cells with different numbers of
Cx32 plaques on the cell surface are presented.
85
To determine whether the loss of cell surface Cx32 stimulates LSK cell
proliferation, we utilized
32
Gap27, a Cx32 mimetic peptide designed to specifically block
the Cx32 gap junction channel function via targeting its extracellular loop (Maezawa and
Jin, 2010). First, using a well-established scrape-loading and dye transfer assay in HeLa
cells devoid of Cx32, we established that
32
Gap27, at a commonly used dosage (0.25
µg/mL; Maezawa and Jin, 2010; Takeuchi et al., 2006; De Vuyst et al., 2006), was able to
inhibit Cx32-mediated gap junction function in HeLa cells when Cx32 was ectopically
expressed (Figure 3.6A and Figure 3.7). Specifically, cells were scraped and loaded with
the mixture of Lucifer yellow (green) and Rhodamine dextran (red), in which Lucifer
yellow is specifically permeable for intercellular gap junctions, whereas Rhodamine
dextran with a 10,000 kDa molecular weight cannot pass intact cell membrane. Cx32
overexpression in HeLa cells led to formation of functional Cx32 gap junctions and the
transfer of Lucifer yellow to neighboring cells, and incubation with
32
Gap27 reduced the
Lucifer yellow transfer in Cx32-overexpression HeLa cells (Figure 3.7).
To test the effect on LSK cells, we isolated LSKs from Grp94
f/f
mice, which were
equivalent to WT LSKs due to the absence of the Cre-transgene, and the cells were either
non-treated, or treated with scrambled peptides or
32
Gap27 at either 0.25 µg/mL or ten
times higher dose for two days prior to cell counting (Figure 3.6B). We observed an
increase in LSK cell number for non-treated and scrambled peptide-treated samples as
expected, however, the number of cells from the
32
Gap27 groups were similar to the
86
control groups at both peptide dosages (Figure 3.6C). These data indicate that the loss of
hemichannel function of cell surface Cx32 has no effect on LSK proliferation in vitro.
Figure 3.6 Effect of Cx32 mimetic peptides on LSK cell proliferation in vitro. (A) Validation of the ability
of
32
Gap27 in blocking gap-junctional intercellular communication (GJIC) by scrape-loading and dye
transfer assay. HeLa cells transfected with empty vector or Cx32 expression vector were scraped and
loaded with the mixture of Lucifer yellow (green) and Rhodamine dextran (red) dyes. The level of
intracellular communication, as indicated by the ratio between the cells receiving Lucifer yellow from
neighboring cells and the cells initially labeled under various experimental conditions, is shown. (B)
Scheme of experimental design for treatment and assay with mimetic (
32
Gap27) or scrambled peptides. (C)
Cell numbers from the cultures subjected to the different treatment conditions as indicated below. The
concentrations of the peptides in µg/mL are shown. NT: not treated. All data are presented as mean ± s.e.
87
Figure 3.7 Validation of the ability of
32
Gap27 to block cellular communication using the scrape-loading/
dye transfer assay. The solid lines indicate the edge of scratch, and the dashed lines represent the edge of
cells transferred with Lucifer yellow. The colors of cells reflecting different phases of dye transfer are
denoted below. Scale bar represents 120 µm.
3.3.7 GRP94 depletion did not affect cell surface CXCR4 and Tie2 expression
In contrast, two other important cell surface proteins maintaining HSC quiescence
were not altered by GRP94 depletion. Despite the depletion of GRP94 in cGrp94
f/f
BM
cells as confirmed by Western blot (Figure 3.8A), cell surface expression of angiopoietin
receptor Tie2, which is required for maintaining the quiescence of HSCs (Arai et al.,
88
2004), was not affected on GRP94-null LSK cells (Figure 3.8B). CXCR4 is another cell
surface receptor that mediates the retention of HSC to the BM niche, the knockout of
which leads to increased HSC proliferation (Nie et al., 2008). Because the expression of
mouse CXCR4 on LSK cells are too low to detect possibly due to rapid turnover rate
(Smith-Berdan et al., 2011), we utilized the U266 human multiple myeloma cells as a
model where cell surface CXCR4 is abundant and detectable. In this model, knockdown
of GRP94 expression mediated by shRNA (Figure 3.8C) did not affect cell surface
expression of CXCR4 (Figure 3.8D).
Figure 3.8 Lack of effect of GRP94 depletion on Tie2 and CXCR4 surface expression. (A) Western blot
analysis of GRP94 expression in the Grp94
f/f
and cGrp94
f/f
BM, with b-actin serving as loading control. (B)
Cell surface Tie2 expression on Grp94
f/f
and cGrp94
f/f
BM LSK cells determined by flow cytometric
89
analysis. The green line represents Grp94
f/f
cells and the red line indicates cGrp94
f/f
cells. (C) GRP94
expression in the U266 multiple myeloma cells infected with lentiviral shCtrl or shGrp94 determined by
Western blot. (D) Representative flow cytometric analysis on cell surface CXCR4 expression on the U266
cells. The grey area represents isotype control staining; the blue line indicates non-treated (NT) cells; the
green line represents U266 cells infected with shCtrl; and the red line indicates U266 cells infected with
shGrp94.
3.4 Discussion
GRP94, with its unique chaperone function, is emerging as an important player
not only in protein processing but also specific cellular processes distinct from other
major chaperones in the ER. In particular, recent studies suggested that GRP94 is
critically required for the functional expression of secretory and/or membrane proteins
that enable the integration of cells into tissues (Maynard et al., 2010). We previously
reported the role of GRP94 in maintaining the interaction between HSC and the adult BM
niche using the Grp94
f/f
; Mx-1-Cre mouse model. Inducible conditional loss of GRP94 in
the hematopoietic system led to impaired HSC homing and engraftment, and an increase
in proliferation that leads to an expansion of the GRP94-null HSC pool. In this report, we
utilized additional criteria to establish the novel observation that GRP94 depletion has
major impact on HSC proliferation regulators, leading to the discovery of new
mechanisms whereby the lack of GRP94 can lead to loss of quiescence and LSK cell
expansion in the BM.
90
We previously noted that using LSK markers in combination with Flk2 and CD34,
both LT-HSC-enriched and ST-HSC-enriched populations were increased in the BM of
the cGrp94
f/f
mice (Luo et al., 2011). Interestingly, in this study using the SLAM markers,
while a 2-fold increase of LSK cells and LT-HSC-enriched LSKCD41
-
CD48
-
CD150
+
populations were observed in agreement with the earlier study, the ST-HSC-enriched
LSKCD41
-
CD48
-
CD150
-
cells remained comparable with that in the Grp94
f/f
control. As
inconsistency of changes in the LSKSLAM marker populations and LSKFlk2CD34
populations were also observed in other studies (Kaplan et al., 2011), the differences
could be due to different coverage of cell populations identified by the two sets of
markers. Through culture of the isolated LSK cells in vitro either in the presence or
absence of stromal cells, we validated that loss of GRP94 in BM LSK leads to a
significant increased cell expansion. This increase in cell expansion could be a cell
intrinsic property of GRP94-null HSCs. However, it is also possible that the deficiency of
niche retention of the GRP94-null HSCs in vivo alters LSK physiology and leads to cell
expansion both in vivo and in vitro, and this awaits future investigation.
How might this increased HSC expansion be achieved? Here we discovered that
the loss of quiescence in the cGrp94
f/f
HSCs in the BM could be at least partially
attributed to the increased activation of AKT. Examination of freshly isolated LSK cells
immunostained with antibodies against pAKT and total AKT followed by confocal
microscopy imaging and analysis demonstrated an increase in pAKT level and
PI(3,4,5)P3 formation in the GRP94-null LSK cells. Furthermore, an allosteric AKT
91
Inhibitor MK2206 compromised cell expansion of GRP94-null LSKs. The increased
activation of AKT in GRP94-null LSK cells could be intrinsically restricted to primitive
hematopoietic cells or a consequence of HSCs detaching from the BM endosteal niche
and thereby escaping from the extrinsic cues maintaining their quiescence. Interestingly,
the effect of ER chaperone deficiency on the activation of AKT may not only be
context-dependent but also chaperone-specific. Deficiency of another major ER
chaperone, GRP78, impairs the activation of PI3K-AKT pathway in vitro as well as in
PTEN-null prostate cancer and leukemia mouse models (Fu et al., 2008; Wey et al.,
2012a). This reflects the distinct function of individual ER chaperones besides their
common capacity of assisting protein folding and this could be attributed to their unique
localization, characteristics of client proteins, and other possible functions such as
mediating signal transduction. While the mechanisms whereby GRP94 deficiency
promotes PI(3,4,5)P3 production and AKT activation remains to be determined, it is
noted that as in the case of several other ER chaperones, GRP94 can also be localized on
the cell surface of specific cell types (Luo and Lee, 2013). It is tempting to speculate that
cell surface GRP94 could act as a suppressor for PI3K signaling and its ablation will
relieve the inhibition resulting in increase of PI(3,4,5)P3 production and AKT activation.
Through microarray analysis, we explored how GRP94 depletion alters the gene
expression profile of LSK cells. Whereas the well-established cell cycle regulators do not
appear to be affected at the gene expression level in the GRP94-null LSK cells, one
striking gene expression change involves the dramatic down-regulation of Ms4a3, a
92
negative cell cycle regulator in the context of hematopoietic cells. Human MS4A3 is
reported to bind to cyclin-dependent kinase-associated phosphatase-CDK2 (KAP-CDK2)
complexes, stimulates the phosphatase activity of KAP and thereby regulates cell cycle
progression (Chinami et al., 2005). Unfortunately, since no antibody against mouse
MS4A3 is available, our studies have been restricted to measurement of its transcript
level. However, its expression pattern in freshly isolated LSK cells from BM and spleen
and following LSK cell culture in vivo reveal new information. Compared to WT BM
LSK cells, Ms4a3 expression is dramatically reduced in spleen LSK as well as BM LSK
cells subjected to in vitro culture for two days. As a negative regulator of the cell cycle,
the much lower Ms4a3 expression in the spleen LSK cells in comparison with the BM
LSK cells is also consistent with their proliferative status, as spleen HSCs cycle twice as
frequently as do BM HSCs (Morita et al., 2011). While the Ms4a3 expression is similarly
low in Grp94
f/f
and cGrp94
f/f
spleen LSK cells, the number of LSK cells actually
increased by 2.5-fold in the cGrp94
f/
spleen when compared to the control, likely
resulting from increased HSC mobilization upon GRP94 depletion (Luo et al., 2011).
Supporting this notion was the loss of integrin α4 in cGrp94
f/f
LSK cells and a 17-fold
increase in circulating cGrp94
f/f
LSK cells in the peripheral blood (Luo et al., 2011). The
low expression of Ms4a3 in the spleen LSK cells and cultured BM LSK cells supports the
notion of a possible role of BM microenvironment in regulating the expression of Ms4a3.
Thus, loss of GRP94 in the LSK cells and the subsequent loss of niche attachment could
trigger the downregulation of Ms4a3, contributing to loss of quiescence and cell
93
expansion. Unfortunately, we are unable to test the functional contribution of MS4A3
in GRP94-null mediated LSK expansion in vitro, since the differential levels of Ms4a3
between the two genotypes only occurred in vivo and the expression of Ms4a3 was lost
when LSK cells were cultured in vitro.
Our previous study established that, while GRP94-null HSCs do not engraft upon
transplantation, transplanting WT HSC into GRP94-null microenvironment yields normal
HSC profile. This indicates that this loss of interaction between HSC and the BM is cell
autonomous for HSCs (Luo et al., 2011). One defect could be due to the loss of
integrin α4 expression in the GRP94-null LSKs, as GRP94 is known to be required for
the processing of select integrins and Toll-like receptors (Staron et al., 2010), and
integrins play an important role in niche attachment (Forsberg and Smith-Berdan, 2009).
Here our studies revealed that the formation of cell surface Cx32 plaque is impaired on
GRP94-null HSCs likely due to defective processing in the ER. It has been reported that
Cx32-knockout mice showed loss of quiescence and hyperproliferation of HSCs
(Hirabayashi et al., 2007). While it will not be possible to rescue cell surface Cx32
expression by ectopic expression in GRP94-null LSKs with intrinsic protein processing
defects, we were able to test whether Cx32 gap junction function affects LSK
proliferation in vitro. Our studies blocking Cx32 hemichannel function with a Cx32
mimetic peptide did not result in increased proliferation. There could be several
explanations for this. First, the increased Cx32-null LSK proliferation in the mouse
model could be due to the complete depletion of Cx32 by genetic knockout, whereas
94
blocking the function of membrane Cx32 may only partially suppress its activity such
that it is not sufficient to alter LSK proliferation. Second, the regulation of HSC
proliferation by surface Cx32 plaques may be independent from the function of Cx32 as a
gap junction protein, and therefore targeting the hemichannel function of Cx32 with
mimetic peptides will not be effective. Third, it is also possible that the reduced surface
Cx32 plaques upon GRP94 depletion is not a cause but a consequence of increased
proliferative signals in cGrp94
f/f
LSK cells, such as elevated AKT phosphorylation. In
support of this, constitutively activated AKT has previously been shown to suppress
Cx32 mRNA and protein levels (Plante et al., 2006). We also examined the expression of
Cx43 and Cx26 by immunofluorescent staining, however, their levels were below
detection limit in the LSK cells in our experimental system (data not shown).
Interestingly, two other major cell surface receptors linked to stem cell quiescence, Tie2
and CXCR4 expression, were not impaired upon GRP94 depletion in hematopoietic cells.
In summary, our studies provide the first evidence that GRP94 deficiency leads to
AKT activation, suppresses Ms4a3 expression and impairs Cx32 plague formation in BM
LSK cells, factors that have been linked to stem cell proliferation. Studies with inhibitors
of AKT and Cx32 also provide mechanistic insights on their requirement in mediating the
hyperproliferation resulting from GRP94 depletion in the HSCs. Our studies demonstrate
that GRP94, which is traditionally regarded as an ER chaperone protein with a primary
function of protein folding and assembly, has novel roles in regulating HSC proliferation,
growth signaling and cell cycle regulators. The diverse role of ER chaperones reflects the
95
complex nature by which chaperone proteins regulating cellular processes in different
contexts, both intrinsically and in interacting with the microenvironment, warrant future
investigation.
96
Chapter 4
Targeting GRP94 by lentivirus-mediated shRNA and
investigate the role of GRP94 in multiple myeloma
4.1 Introduction
Multiple myeloma (MM) is a B-cell malignancy characterized by the preferential
accumulation of plasma cells, which are differentiated B-cells that secrete monoclonal
immunoglobulins in the BM. MM is often associated with end-organ damage including
osteolytic bone lesions, anemia, immunodeficiency, and renal disease (Borrello, 2012).
The American Cancer Society estimates that in 2013 about 22,350 new cases will be
diagnosed and about 10,710 deaths are expected to occur for multiple myeloma in the
United States. The five-year survival rate of people with multiple myeloma is about 43%.
Treatment methods for MM include conventional high-dose chemotherapy and
autologous BM transplantation, as well as novel therapeutic agents such as thalidomide,
lenalidomide, and bortezomib. Despite improvements in disease management, MM
remains incurable. The course of MM treatment is characterized by a pattern of remission
and relapse, which eventually results in refractory disease (Borrello, 2012).
Cancer cells are under chronic ER stress due to external environment stress such
as hypoxia, low pH, and nutrients deprivation, as well as internal excessive protein loads
for their uncontrolled proliferation. On top of these factors, the highly hypoxic BM
97
environment and the production of a large amount of immunoglobulins create greater ER
stress for MM cells. Therefore, UPR plays important role in the survival of MM cells.
Among the three UPR branches, the IRE1α-XBP1 pathway has been implicated in the
proliferation and survival of MM cells (Ri et al., 2012). Treatment with a novel
small-molecule inhibitor of IRE1 showed significant antimyeloma activity in a mouse
model with human MM xenografts (Papandreou et al., 2011). Proteosome inhibitor
treatment, which further enhances the accumulation of misfolded proteins, sensitizes MM
cells to apoptosis and significantly improves the survival times for MM patients
(Edwards et al., 2009). Therefore, targeting players in the UPR can be a potential
therapeutic strategy for MM treatment.
Myeloma growth in vivo is supported by the BM microenvironment. There is
increasing evidence that the interaction between tumor cells and their BM
microenvironment plays a pivotal role in the development, maintenance, progression and
drug resistance of MM (Manier et al., 2012; De Raeve and Vanderkerken, 2005).
Components of the BM microenvironment refers not only to the cellular components
such as BM stromal cells, osteoclasts, osteoblasts, endothelial cells and immune cells, but
also to the non-cellular components including ECM, cytokines and growth factors (Kuehl
and Bergsagel, 2012; Figure 4.1). The BM microenvironment plays an important role in
supporting HSCs and normal hematopoiesis, and is hijacked by the tumor cells during the
onset and progression of MM. The interaction between MM cells and the BM
98
microenvironment include direct contact of MM cells with BM stromal cells or ECM
protein, and indirect influence of cytokines and growth factors produced by MM cells or
BM stromal cells. This interaction leads to activation of proliferative and anti-apoptotic
signals in MM cells, as well as changes of BM environment such as angiogenesis and
osteolytic bone lesions.
Interactions between MM cells with the BM stromal cells through cell adhesion
molecules such as CD44, CD56, CD49 and seletin have been shown to play crucial roles
in the homing and retention of MM cells in the BM. Among these adhesion molecules,
CD49 family protein CD49d (also known as integrin α4β1, VLA-4) is particularly
important. Myeloma cells express CD49d while stromal cells express its binding partner
vascular cell adhesion molecule-1 (VCAM-1). It is shown that the binding of VLA-4 to
VCAM-1 promotes MM cell colonization in the BM, and treatment with neutralizing
monoclonal antibody to integrin α4 suppresses the development of MM and associated
osteolytic diseases (Mori et al., 2004). Fibronetin is another binding partner of VLA-4 in
the BM, and the adherence of VLA-4 to fibronetin induces drug resistance in the tumor
cells through upregulation of p27 (St Croix et al., 1996).
It is clear that there are complex autocrine and paracrine interactions between
MM cells and BM microenvironment. Adhesion of MM cells to the BM stromal cells
prominently upregulates the production of IL-6, IGF-1 and APRIL, which are major
survival and proliferative factors for MM cells (Borrello, 2012). MM associated bone
99
destruction is mainly mediated by increased osteoclast activity. Receptor activator of
NF-κB ligand (RANKL) is part of the tumor necrosis factor (TNF) gene family and is a
major osteoclastogenic factor involved in MM bone disease through promoting the
differentiation of osteoclasts. Osteoprotegerin (OPG) is a soluble decoy for RANKL that
is produced by BM stromal cells to counterbalance the action of RANKL (Sezer et al.,
2003). Binding the MM cells to the BM stromal cells leads to increased secretion of
RANKL and decreased secretion of OPG, which results in further osteoclast activation
and enhanced bone destruction (Roodman, 2010). The adhesion of MM cells to the
cellular component of the BM microenvironment enhances vascular endothelial growth
factor (VEGF) production in tumor and surrounding cells, which stimulates proliferation
and chemotaxis in both endothelial cells and stromal cells, and promotes stromal cells to
secretion IL-6 for tumor growth (Dankbar et al., 2000).
100
Figure 4.1 Interaction between MM cells with the BM microenvironment [adapted from (Kuehl and
Bergsagel, 2012)]. MM cells adhere to BM stromal cells through VLA-4/VCAM-1 and ECM through
VLA-4/FN, upregulating factors that promote tumor growth and bone destruction. Various cell types in the
BM secrete IL-6 and IGF-1 to promote tumor survival and proliferation. The ratio of RANKL and OPG
determines the activity of osteoclasts, and the adhesion of MM cells upregulates RANKL while
downregulating OPG. VEGF produced by tumor and supporting cells promotes angiogenesis and tumor
growth.
As above discussed, on one hand, UPR signaling especially the IRE1α-XBP1
branch plays important role in the survival of MM cells. On the other hand, interactions
101
between MM cells with the BM microenvironment mediated by cell adhesion molecules,
soluble ligands and receptors are crucial for the survival and proliferation of MM cells.
As discovered in Chapter 2, complete loss of GRP94 in the mouse ESCs leads to
significant decrease in the level of unspliced and ER-stress induced spliced form of
XBP-1 protein (Mao et al., 2010), which implicates the potential therapeutic effect of
depleting GRP94 in MM cells. Moreover, GRP94 depletion in the HSCs leads to
increased HSC mobilization, impaired HSC homing and engraftment to the BM niche,
which can be at least partially attributed to disrupting the expression of secreted and cell
membrane proteins such as integrin α4β1 (Luo et al., 2011). Since these cell adhesion
molecules, ligands and receptors are also shared by MM in their interaction with the BM
microenvironment, we hypothesized that GRP94 deficiency in the MM cells will disrupt
their interactions with the BM microenvironment and compromise their survival and
clonogenic ability in vivo.
4.2 Materials and methods
Cell culture
Mouse MM cell line 5TGM1/luc was kindly provided by Dr. Oyajobi in the
Department of Cellular and Structural Biology at the University of Texas Health Science
Center. These 5TGM1/luc cells were grown in Isocove Modified Dulbecco Media
102
(IMDM, Life Technologies) supplemented with 10% FBS and 1% P/S and passaged
twice a week. WT mouse embryonic fibroblasts (MEFs) were maintained in high glucose
Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% P/S.
U266 human MM cells were grown in RPMI supplemented with 10% FBS and 1% P/S
and passaged twice a week.
Plasmid DNA miniprep and maxiprep
For miniprep, plasmid-containing bacterial glycerol stocks were thawed on ice,
inoculated into 3 mL LB broth in the presence of selection antibiotics (ampicillin or
carbenicillin, 100 µg/mL) and cultured at 37 °C for 12-16 hours. Miniprep was performed
with the Plasmid miniprep kit (Bioland Scientific) according to manufacturer’s
instruction. For maxiprep, bacteria from overnight culturing was inoculated into 3 mL LB
broth and shook vigorously at 37 °C for 6-8 hours to prime as starter culture. The starter
culture were then inoculated into 200 mL LB broth and cultured at 37 °C for 12-16 hours
with vigorous shaking. Maxiprep was performed with the Plasmid maxiprep kit (Bioland
Scientific) according to manufacturer’s instruction. DNA quality and content were
quantified by Nano drop (Thermo Scientific).
Production of lentivirus mediated shRNA against human GRP94
The second-generation packaging plasmids PsPAX and PMD2.g were kindly
provided by Dr. Wange Lu at USC. The pGIPZ plasmids containing lentiviral shRNA of
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scrambled control sequence (shCtrl) and against human Grp94 (shGrp94, Open
Biosystems) (Table 4.1) from maxiprep were first confirmed with restriction enzyme
digestion (Figure 4.2). Restriction enzyme digestion was designed based on the
manufacturer’s instruction and the sequence of plasmids. Mixtures containing plasmids,
restriction endonuclease and corresponding 10X NEBuffer (New England Biolabs) were
incubated at 37 °C for 2 hours and then subjected to electrophoresis. To further exclude
potential mutations on the shRNA sequence, the pGIPZ plasmids containing shGrp94
were sequenced and aligned with the designed sequence using sequencing primer 5’-
GCATTAAAGCAGCGTATC -3’.
Table 4.1 shRNA and packaging plasmids
Generation Package
Plasmids
Vector shRNA Selection
markers
Human 2
psPAX
pGIPZ
shGrp94
V3LHS_371778
Puromycin
&
GFP
V3LHS_371779
V3LHS_371780
pMD2.g
V3LHS_371781
GFP
V3LHS_371536
scrambled SHC002
Mouse 3
pLP1
pLKO.1
shGrp94 NM_011631.1-252s1c1
Puromycin
pLP2
pLP/VSVG scrambled RHS4346
104
Figure 4.2 Restriction enzyme digestion on pGIPZ plasmids. (A) Lane 1: 100 bp DNA ladder; Lane 2: 1kb
DNA ladder; Lane 3-7: shCtrl pGIPZ plasmid from maxiprep (lane 3) and digested with Kpn I (lane 4), Sac
105
II (lane 5), Sal I (lane 6), Xho I and Not I (lane 7); Lane 8-12: clone 778 pGIPZ plasmid from maxiprep
(lane 8) and digested with Kpn I (lane 9), Sac II (lane 10), Sal I (lane 11), Xho I and Not I (lane 12). (B)
Lane 1: 100 bp DNA ladder; Lane 2: 1kb DNA ladder; Lane 3-7: clone 779 pGIPZ plasmid from maxiprep
(lane 3) and digested with Kpn I (lane 4), Sac II (lane 5), Sal I (lane 6), Xho I and Not I (lane 7); Lane 8-12:
clone 780 pGIPZ plasmid from maxiprep (lane 8) and digested with Kpn I (lane 9), Sac II (lane 10), Sal I
(lane 11), Xho I and Not I (lane 12). (C) Lane 1: 100 bp DNA ladder; Lane 2: 1kb DNA ladder; Lane 3-7:
clone 781 pGIPZ plasmid from maxiprep (lane 3) and digested with Kpn I (lane 4), Sac II (lane 5), Sal I
(lane 6), Xho I and Not I (lane 7); Lane 8-12: clone 536 pGIPZ plasmid from maxiprep (lane 8) and
digested with Kpn I (lane 9), Sac II (lane 10), Sal I (lane 11), Xho I and Not I (lane 12).
To produce lentivirus mediated shRNA against human GRP94, the confirmed
plasmids were transfected into 293T cells at a ratio of shRNA: psPAX: pMD2.g = 3:5:2.
After transfection, the lentivirus-containing medium was collected at 48 hours and 72
hours post transfection, and the lentiviral particles were concentrated with PEG-it
TM
Virus Precipitation Solution according to manufacturer’s instruction (System
Biosciences). These 293T cells expressed high level of GFP when examined under the
fluorescent microscope (data not shown), which results from both the initial transfection
of GFP-containing shRNA plasmids and the transduction of GFP-containing lentivirus in
the medium.
To determine the titer, all clones of lentivirus were first serial-diluted in a 96-well
plate from 1:5 to 1:5
8
, among which 1:5
2
to 1:5
7
were used for tittering (Figure 4.3A).
293T cells were seeded on a 24-well plate and treated with polybrene before infection. To
106
titer the virus, lentivirus of selected dilutions was incubated with 293T cells for 4 hours
(Figure 4.3B), and the percentage of GFP+ cells were detected two days post-infection by
flow cytometry (Figure 4.3C). The titer of lentivirus was calculated using the following
formula and representative titer is shown in table 4.2:
𝑡𝑖𝑡𝑒𝑟 (/𝑚𝐿)=
𝐺𝐹𝑃 𝑐𝑒𝑙𝑙𝑠 %×𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑠𝑒𝑒𝑑𝑒𝑑×𝑙𝑒𝑛𝑡𝑖𝑣𝑖𝑟𝑢𝑠 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑙𝑒𝑛𝑡𝑖𝑣𝑖𝑟𝑢𝑠 𝑎𝑑𝑑𝑒𝑑
Table 4.2 Lentivirus titer calculation (GFP
+
)
Because of their transduction susceptibility, 293T cells were infected with
lentivirus to determine the activity and knockdown efficiency of different clones of
lentivirus. As determined by flow cytometry, around 90% of 293T cells expressed GFP,
while non-infected cells had only basal level autofluorescent signal (Figure 4.3D). To
determine the appropriate MOI for GRP94 knockdown, lentivirus from the same
sequence were mixed with 293T cells at different MOIs for 2 days, and the level of
GRP94 was detected by Western blot, showing decent knockdown efficiency at MOI of 5
(Figure 4.3E). To determine the time period for optimal knockdown efficiency, 293T
GFP+ % GFP+ Dilution Cells Volume Titer (TU/mL)
CTRL 2.1 0.021 625 50000 0.025 2.63E+07
778 3.2 0.032 625 50000 0.025 4.00E+07
779 3.9 0.039 625 50000 0.025 4.88E+07
780 4.4 0.044 625 50000 0.025 5.50E+07
781 2 0.02 625 50000 0.025 2.50E+07
536 3.9 0.039 625 50000 0.025 4.88E+07
107
cells were transduced with the same lentivirus at MOI of 5 and harvested on various days
post transduction. Western blot on the level of GRP94 demonstrated that the knockdown
efficiency started to reach maximal at day 5 post transduction (Figure 4.3F). To
determine the knockdown efficiency of different shGrp94 sequences, 293T cells were
infected with 5 individual sequences of lentiviral shGrp94 at MOI of 5 for 5 days, and the
level of GRP94 was determined by Western blot, which shows near complete knockdown
of GRP94 with all the 5 sequences (Figure 4.3G).
108
109
Figure 4.3 Lentivirus-mediated knockdown of human GRP94. (A) Scheme of lentivirus serial dilution in
96-well plate for tittering. (B) Scheme of 293T cell transduction by different dilutions of lentivirus in
24-well plate for tittering. (C) Representative flow cytometry analysis of lentivirus-transduced 293T cells
for tittering, among which transduced cells expressed GFP. (D) Representative flow cytometry analysis of
non-infected 293T cells (left) and lentivirus-infected 293T cells (right), among which transduced cells
expressed GFP. (E) Western blot analysis of GRP94 and GRP78 level in 293T cells transduced with
lentiviral shRNA against human Grp94 at different MOIs. (F) Western blot analysis of GRP94 and GRP78
level in 293T cells transduced with lentiviral shRNA against human Grp94 for different time periods. (G)
Western blot analysis of GRP94 in 293T cells transduced with lentiviral shRNA against human Grp94 at
MOI of 5 for 5 days.
Production of lentivirus mediated shRNA against mouse GRP94
The pLKO.1 plasmids containing lentiviral shRNA of scrambled control sequence
(shCtrl) and against mouse Grp94 (shGrp94, Sigma) as well as the third-generation
packaging plasmids pLP1, pLP2, pLP/VSVG were kindly provided by Dr. Davide Eletto
in the lab of Dr. Yair Argon at the University of Pennsylvania (Table 4.1). The pLKO.1
plasmids from maxiprep were first confirmed with restriction enzyme digestion (Figure
4.4). To further exclude potential mutations on the shRNA sequence, the pLKO.1
plasmids containing shGrp94 were sequenced and aligned with the designed sequence
using sequencing primer: 5’-AAACCCAGGGCTGCCTTGGAAAAG-3’
110
Figure 4.4 Restriction enzyme digestion on packaging plasmids and shRNA plasmids. (A) Restriction
enzyme digestion on packaging plasmids. Lane 1: 100 bp DNA ladder; Lane 2: 1 kb DNA ladder; Lane 3-6:
pLP1 plasmid from maxiprep (lane 3) and digested with Kpn I (lane 4), Nco 1 (lane 5), and Pvu II (lane 6);
Lane 7-10: pLP2 plasmid from maxiprep (lane 7) and digested with Pvu I (lane 8), Pvu II (lane 9), Bgl II
and EcoR I (lane 10); Lane 11-15: pLP/VSVG plasmid from maxiprep (lane 11) and digested with Bgl II
(lane 12), EcoR I (lane 13), Nco I (lane 14) and Nde I (lane 15). (B) Restriction enzyme digestion on
pLKO.1. Lane 1: 100 bp DNA ladder; Lane 2: 1 kb DNA ladder; Lane 3-5: pLKO.1 shCtrl plasmid from
maxiprep (lane 3) and digested with BamH I and Nde I (lane 4) and (lane 5); Lane 6-8: pLKO.1 shGrp94
plasmid from maxiprep (lane 6) and digested with BamH I and Nde I (lane 7) and (lane 8).
To produce lentivirus-mediated shRNA against mouse GRP94, The confirmed
plasmids were transfected into 293T cells at a ratio of shRNA: pLP1: pLP2: pLP/VSVG
= 1:1:1:1 for 6 to 8 hours. After transfection, the lentivirus-containing medium was
collected at 48 hours and 72 hours post transfection, and the lentiviral particles were
concentrated with PEG-it
TM
Virus Precipitation Solution according to manufacturer’s
111
instruction (System Biosciences). However, the plasmid transfection efficiency or
lentivirus titer cannot be determined using fluorescent microscope or flow cytometry, as
GFP selection marker is not available on pLKO.1.
To titer the lentivirus, HeLa cells seeded on a six-well plate were first incubated
with polybrene in serum-free medium then transduced with lentivirus at serial dilutions
from 10
-2
to 10
-7
for 4 hours (Figure 4.5A and B). Starting three days after transduction,
the cells were subjected to puromycin selection for a week, during which the
lentivirus-transduced cells survived and grew into visible clones (Figure 4.4B). The
survival clones were stained with crystal violet and enumerated under the inverted
microscope (Figure 4.5C). Only wells in which colonies are determinable were used to
calculate the titer. For example, in the plate in Figure 4.5C, only the colonies in the wells
with lentivuris at 10
-4
, 10
-5
, and 10
-6
dilution were enumerated (Table 4.3). The titer of
lentivirus was calculated as the average of the number of colonies (C) times dilution
factor (D) as following:
𝑇𝑖𝑡𝑒𝑟= (𝐶1×𝐷1+𝐶2×𝐷2+⋯+𝐶𝑛×𝐷𝑛) 𝑛
Table 4.3 Lentivirus titer calculation (puromycin
+
)
Lentivirus Dilutions Titer (TU/mL)
10
4
10
5
10
6
Number of
colonies
shCtrl 136 19 3 2.09E+06
shGrp94 123 15 1 1.24E+06
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Similar as utilizing 293T for testing the lentivirus against human Grp94, MEF
cells were infected with lentivirus to determine the activity and knockdown efficiency of
the lentivirus against mouse Grp94. Since GFP selection marker is not available on
pLKO.1, puromycin selection was applied to ensure the expression of GRP94 comes only
from infected cells. As determined by Western blot, GRP94 was near completely
depleted in the MEFs infected with shGrp94 lentivirus, while non-treated cells as well as
cells infected with shCtrl lentivirus expressed abundant GRP94 (Figure 4.5D).
113
Figure 4.5 Lentivirus titering and knockdown of mouse GRP94. (A) Scheme of lentivirus serial dilution in
96-well plate for tittering. (B) Scheme of MEFs transduction by different dilutions of lentivirus in 6-well
plate for tittering. (C) Representative plate showing colonies formed after transduction with different
dilutions of lentivirus and 12 days of puromycin selection. (D) Western blot analysis of GRP94 level in
114
MEFs transduced with lentiviral shRNA against mouse Grp94 followed by puromycin selection for
different time periods.
Lentivirus infection
5TGM1/luc or U266 cells for lentiviral infection were resuspended in IMDM or
RPMI individually with polybrene (4 µg/mL, Sigma), mixed thoroughly with lentivirus
and centrifuged at 1500g for 30 min at room temperature. MEFs for lentiviral infection
were incubated in DMEM with polybrene (4 µg/mL, Sigma) and mixed with lentivirus.
Cells were then incubated with lentivirus-containing medium from 24 hours, cultured in
regular medium for 5 days, and selected with puromycin (6µg/mL) for 1 week before
harvested for analysis.
Apoptosis assay
For apoptosis detection, 1×10
5
5TGM1/luc cells or trypsinized MEFs were
resuspended in 150 µL 1×Annexin V binding buffer (BD Pharmingen) and incubated
with 5 µL Annexin V and 5 µL 7-AAD (both from BD Pharmingen) for 15 min at room
temperature. Cell apoptosis was examined within an hour using a BD LSR II flow
cytometer.
115
5TGM1 MM model
WT C57BL/KaLwRijHsd mice were kindly provided by Dr. Oyajobi in the
Department of Cellular and Structural Biology at the University of Texas Health Science
Center. 5TGM1/luc cells were suspended in PBS at a density of 1×10
7
cells/mL. 1×10
6
cells were inoculated into C57BL/KaLwRijHsd recipient mouse through tail vein
injection. 4-6 weeks following tumor inoculation, mice were sacrificed and femur and
tibia were dissected and subjected to histological analysis. All protocols for animal use
were reviewed and approved by the USC Institutional Animal Care and Use Committee.
The animal assurance number is A3518-01. The protocol number is 9964.
4.3 Results
4.3.1 GRP94 knockdown in the mouse MM cells affects their survival
To investigate the role of GRP94 in MM progression, we knocked down GRP94
in mouse MM cell line 5TGM1/luc with lentivirus-mediated shRNA. In order to increase
the transduction efficiency, 5TGM1/luc cells resuspended in IMDM supplemented with
polybrene were mixed thoroughly with lentivirus and followed by the protocol of
spinoculation. Starting two days after transduction, the cells were selected with
puromycin for five days. Western blot showed that after five days of puromycin selection
GRP94 in 5TGM1/luc cells was knocked down by around 60% (Figure 4.6A and B).
116
Under normal culture condition, 5TGM1/luc cells were round shape with smooth
membrane, homogeneous in size and aggregating together when multiplying. While
shCtrl-infected 5TGM1/luc cells were growing like healthy uninfected cells, the GRP94
knocked down cells demonstrated fewer cell number and less aggregation, as well as
signs of cell death such as irregular cell shape, rough cell surface, and shrinkage in cell
size (Figure 4.6C). Apoptosis analysis with Annexin V and 7-AAD demonstrated a
significant increase of early and late apoptosis from the 17.8% in the shCtrl-infected
5TGM1/luc cells to the 34.8% in the shGrp94-infected cells under normal culture
condition (Figure 4.6D and E). The shGrp94-infected cells also demonstrated increased
early and late apoptosis in the presence of different concentrations of etoposide when
compared to the shCtrl-infected cells (Figure 4.6D and E).
117
118
Figure 4.6 Lentivirus-mediated knockdown of mouse GRP94 in 5TGM1/luc cells caused increased
apoptosis. (A) Western blot analysis on GRP94 level in 5TGM1/luc cells infected with lentiviral shRNA.
(B) Quantitation of Western blot in A. (C) Morphology of non-infected 5TGM1/luc cells and 5TGM1/luc
cells infected with shCtrl or shGrp94 lentivirus. Scale bar represents 50 µm. (D) Flow cytometry analysis
on non-infected 5TGM1/luc cells and 5TGM1/luc cells infected with shCtrl or shGrp94 lentivirus in the
presence and absence of etoposide. (E) Quantitation of apoptotic 5TGM1/luc cells determined by flow
cytometry analysis.
Interestingly, the shGrp94 infected 5TGM1/luc cells gradually restored the
morphology of healthy cells along the process of puromycin selection (data not shown),
however, the GRP94 expression level was also gradually restored despite the presence of
puromycin selection, reaching the level of that in untreated and shCtrl-infected cells after
17 days of puromycin selection, and this is possibly due to the survival selection pressure
against GRP94-depleted 5TGM1/luc cells in culture (Figure 4.7A and B).
119
Figure 4.7 GRP94 knockdown in 5TGM1/luc cells were not persistent even under puromycin selection. (A)
Western blot analysis on GRP94 level in 5TGM1/luc cells subjected to different time periods of puromycin
selection. (B) Quantitation of GRP94 level in 5TGM1/luc cells after normalizing against β-actin.
To determine whether the cell death caused by GRP94 knockdown is cell-type
specific, same shRNA was used to infect MEFs. While shCtrl-infected MEFs exhibit
similar morphology as non-treated cells, GRP94 knockdown MEFs displayed
morphology of early and late apoptosis, including blebs and shrinkage (Figure 4.8A).
While non-infected MEFs and shCtrl-infected MEFs displayed 5.54% and 3.98% of
apoptosis (early and late combined), respectively, 14.99% of shGrp94-infected MEFs
demonstrated early or late apoptosis (Figure 4.8B and C). However, despite increased
120
apoptosis in GRP94 knockdown MEFs, the majority of cells survived without GRP94 for
up to 14 days of puromycin selection (Figure 4.8D), which is different from 5TGM1/luc
cells with GRP94 knockdown.
Similarly, U266 human MM cells were also infected with lentviral shCtrl or
shGrp94 (Figure 4.9A). Cells infected with shGrp94 did not display significantly
reduction in cell number or altered morphology from the non-infected group or shCtrl-
infected group (Figure 4.9B). Cell death of these U266 cells determined by 7AAD
staining demonstrated a significant increased of cell death upon GRP94 knockdown, from
the 15% in the shCtrl group to the 22.6% in the shGrp94 group (Figure 4.9C and D).
These data indicate the cytoprotective function of GRP94 in the MEFs, and a special
requirement of GRP94 in MM cells, among which the mouse 5TGM1/luc MM cells have
higher dependency than the U266 human MM cells.
121
Figure 4.8 shGrp94 caused moderate increase of apoptosis in MEFs. (A) Morphology of shRNA-infected
MEFs in cultured after with 7 days of puromycin selection. Non-infected cells were not subjected to
122
puromycin selection. Black arrows indicate apoptotic cells based on their morphology under the
microscope. (B) Apoptotic analysis on MEFs cells using 7-AAD and Annexin V, among which 7-AAD
-
Annexin V
+
cells indicate early apoptotic cells while 7-AAD
+
Annexin V
+
represent late apoptotic cells. (C)
Quantitation of apoptotic MEFs analyzed with 7-AAD and Annexin V. (D) Western blot analysis on
GRP94 level in MEFs subjected to different time periods of puromycin selection.
Figure 4.9 shGrp94 caused increase of cell death in U266 cells. (A) The protein level of GRP94 and
β-actin in U266 cells determined by Western blot. (B) Morphology of non-infected or shRNA-infected
U266 cells in cultured after with 5 days of puromycin selection. Non-infected cells were not subjected to
puromycin selection. (C) Analysis on cell death of U266 cells using 7-AAD. (D) Quantitation of 7AAD
+
U266 cells analyzed by flow cytometry.
123
4.3.2 Determining the feasibility of using optical image to monitor MM cells in vivo
To determine the effect of GRP94 depletion in the homing and growth of multiple
myeloma cells in the BM, we planned to use the well-characterized 5TGM1 mouse MM
model in which the 5TGM1/luc MM cells are inoculated into the recipient
C57BL6/KaLwRijHsd mice through intravenous tail vein inoculation (Asosingh et al.,
2000; Oyajobi et al., 2003). However, GRP94 is required for the in vitro survival of
5TGM1/luc cells, making it impossible to deplete GRP94 in the 5TGM1/luc cells before
inoculating into the recipient mice; moreover, persistent-depletion of GRP94 in these
cells was not achievable as the level of GRP94 was restore even in the presence of
puromycin selection, making it impossible to monitor the effect of GRP94 depletion in
the progression of MM throughout the time.
Therefore, we switched gear to establishing this 5TGM1 mouse MM model in the
lab, preparing for testing other treatments for MM in the future. Since 5TGM1/luc cells
express firefly luciferase gene, tumor growth in vivo could be monitored by
bioluminescence imaging. Before performing live animal whole body bioluminescence
optical imaging, the luciferase activity of the 5TGM1/luc cells was evaluated to make
sure the bioluminescence signal is strong enough to penetrate the tissue during in vivo
imaging (Figure 4.10A). As a result, at 10s of exposure 5TGM1/luc cells did not emit
bioluminescence signal until reaching the density of 1×10
6
/100 uL in the 96-well plate,
moreover, at 2 mins of exposure, only the 5TGM1/luc cells at the density of 1×10
5
/100
124
uL and1×10
6
/100 uL in the 96-well plate emitted detectable bioluminescence signal
(Figure 4.10B). In contrast, luciferase-expressing cells that produce good quality in vivo
imaging emits strong signal at the density of 1×10
3
/100 uL at 1s of exposure. Therefore,
the tumor progression of these 5TGM1/luc cells in vivo could not be monitored by mouse
whole body bioluminescence imaging optical imaging unless re-introduced with the
luciferase gene.
Figure 4.10 Luciferase activity of 5TGM1/luc cells in vitro. (A) Different numbers of 5TGM1/luc cells
were seeded as indicated. (B) Bioluminescence optical imaging of 5TGM1/luc cells incubated with
luciferin for 5 mins and exposed for indicative time period.
4.3.3 Establishing a syngeneic MM mouse model
To establish the MM mouse model and monitor the tumor progression,
5TGM1/luc were inoculated into the recipient C57BL6/KaLwRijHsd mice through
125
intravenous tail vein injection and allowed to grow for 4-6 weeks. Examination with
H&E staining on the femur of PBS-injected control mice demonstrated hematopoietic
cell type heterogeneity as well as the presence of adipocytes and sinusoids, whereas
tumor cell-injected mice femur exhibited gradual accumulation of uni-morphology
clusters of MM cells, as well as a gradual loss of trabecular bones at 4 to 6 weeks post
inoculation, indicating the progression of MM in C57BL6/KaLwRijHsd mice inoculated
with 5TGM1/luc cells along the time (Figure 4.11A). At six weeks post inoculation, MM
mice exhibit severe symptoms including significant weight loss, hunched posture, and
visible tumors on lower back (Figure 4.11B). H&E staining on the femur of
tumor-inoculated mice demonstrated lytic bone lesions (Figure 4.11C), which is another
signature of the MM disease resulting from increased osteoclast activity. Complete blood
count analysis demonstrated significant white blood cell reduction in the peripheral blood
of 5TGM1/luc cell-inoculated mice starting from 3-4 weeks post inoculation (data not
shown). So far all of the 7 mice injected with 5TGM1/luc cells showed characteristics of
MM disease, indicating the establishment of this MM model with the penetrance of
100%.
126
Figure 4.11 Pathological characteristics of 5TGM1/luc MM model. (A) H&E staining of femurs from
C57BL6/KaLwRijHsd mice inoculated with PBS or 5TGM1/luc after various time periods as indicated. (B)
Gross morphology of 5TGM1/luc MM model 6 weeks post inoculation, including hunched posture (upper)
and tumor on lower back (lower). White arrow indicates tumor on lower back. (C) H&E staining of femurs
127
from C57BL6/KaLwRijHsd mice 6 weeks after inoculated with PBS or 5TGM1/luc. Black arrow indicates
an osteoclast.
4.4 Discussion
The role of microenvironment in the progression of MM, including the survival,
proliferation and clonogenic capacity of tumor cells, has been a major focus in studies to
identify therapeutic strategy for MM. The adhesion of MM cells to the stromal cells in
the BM has been suggested to be not only crucial for their colonization in the BM, but
also important for protecting the tumor cells from proteasome inhibitor induced apoptosis.
The interaction between MM cells and BM stromal cells is mediated by direct contact or
soluble cues, including the interaction of integrin α4β1 with VCAM-1. Since GRP94
ablation impairs the expression of cell adhesion molecules including integrin α4β1, we
speculate that GRP94 deficiency in MM cells will compromise their interaction with the
BM stromal cells and therefore suppress the disease progression.
MM disease models could be classified as xenogeneic and syngeneic. Currently
established xenogeneic MM models involve the inoculation of human MM cells such as
U266, RPMI8226 and NCI949 into immunocompromised mice through tail vein,
subcutaneous, or direct BM inoculation (Campbell and Berenson, 2008). Since immune
cells are part of the BM microenvironment for MM cells, syngeneic mouse model with a
competent immune system represents an ideal model for studying the interaction of MM
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cells with the BM, only second to xenograft MM model in humanized immune system.
So far the only well-established syngeneic MM model is the 5TGM1 model, in which
5TGM1 cells are inoculated into immunocompromised mice or a specialized type of
immunocompetent recipient mice C57BL6/KaLwRijHsd through intravenous inoculation.
The 5TGM1 model and tail vein inoculation was chosen in our study because in this
model the interaction between MM cells and the stromal cells in the BM can be preserved
with minimal invasion during tumor inoculation. GRP94 can be knocked down in the
MM cells either before inoculation through traditional lentivirus-mediated knockdown, or
after the establishment of the disease by the inducible expression of lentiviral shRNA.
Because one of our goals was to study whether GRP94 depletion affects the homing of
MM cells to the BM upon inoculated into the peripheral blood circulation, we chose the
traditional lentivirus-mediated knockdown of GRP94 before tumor inoculation.
However, knocking down GRP94 in the 5TGM1/luc cells with the lentiviral
shGrp94 caused severe cell death in the presence of puromycin selection. This cell death
could be attributed to several possible reasons. 1) Loss of GRP94 in these cells. MM cells
are featured by excessive production of antibodies and therefore have a high demand on
ER chaperone activities. The cell death, if caused by loss of GRP94, could reflect the
general requirement of ER chaperone proteins, or particularly GRP94 in this process.
Supporting this notion is our previous study in which GRP94 protected hematopoietic
cells in etoposide-induced apoptosis (Reddy et al., 1999). The cytoprotective effect of
129
GRP94 was also observed in our previous studies with GRP94-null ESCs (Mao et al.,
2010). 2) Silence of the shGrp94 plasmids containing puromycin-resistant gene. During
puromycin selection, uninfected cells will be eradicated within the first three days of
puromycin selection at the concentration determined by the puromycin kill-curve,
whereas the transduced cells remain alive throughout the selection if the
puromycin-resistant gene is persistently expressed. However, in our study, shGrp94
transduced 5TGM1/luc cells displayed heterogeneity in the survival capacity throughout
the selection until GRP94 level was restored after 17 days of selection, indicating that
cell populations with stably expressed puromycin-resistant gene were never established
until late stage. Considering these two possibilities, a probable scenario would be: loss of
GRP94 in 5TGM1/luc cells caused such a huge selection pressure against their survival
that the cells had to shut down the expression of this shRNA, and shutting it down caused
cell death in the presence of puromycin selection. Supporting this notion is the fully
restored GRP94 protein level in the transduced 5TGM1/luc cells after 17 days of
purimycin selection. This could result from acquired mutations that silenced Grp94
shRNA while keeping the expression of the puromycin-resistant gene. 3) Off-target effect
of the shGrp94 sequence. As much as all the evidence supporting our hypothsis, we
cannot rule out the possibility of the off-target effect by shGrp94 treatment. Since this
shRNA was a kind gift from our collaborator, other shGrp94 sequences are not available
for testing. One way to examine this hypothesis is to overexpress GRP94 in the cells
treated with shGrp94, for instance in MEFs since 5TGM1 cells die after shGrp94
130
treatment and therefore are not suitable for GRP94 transduction, and see if the
overexpression could rescue these phenotypes. A piece of evidence that may shed light
on excluding the possibility of shGrp94 off-target effect is the data from our collaborator
Dr. Davide Eletto in the lab of Dr. Yair Argon, who has worked with four other lentiviral
shGrp94 sequences. In his experience, efficient deletion of GRP94 leads to a profound
reduction in cell proliferation which could at least partially attributed to increased p21
and decreased cyclin D1. Moreover, these GRP94 depleted cells were observed undergo a
crisis stage after around four days post transduction, which can be terminal in some cells.
In our case, knocking down GRP94 in 5TGM1/luc cells could not be achieved before
inoculation, leaving inducible system the only option for knocking down GRP94 and
investigate the in vivo progression of MM. However, we decided not to pursue this
research goal.
Interestingly, the dependency of GRP94 is different in 5TGM1/luc cells, U266
cells and MEFs. Despite the three types of cells displayed different basal levels of
apoptosis/cell death (20.87% apoptosis for 5TGM1/luc cells as determined by Annexin V
and 7-AAD, 12.3% cell death for U266 cells as determined by 7AAD, and 5.54%
apoptosis for MEFs as determined by Annexin V and 7-AAD), all of them exhibited
increased apoptosis/cell death upon GRP94 knockdown (34.83% for 5TGM1/luc cells,
22.6% for U266 cells, and 14.99% for MEFs), which is consistent with our previous
reports on GRP94 knockdown in mouse ESCs and Jukat cells (Mao et al., 2010; Reddy et
131
al., 1999). Moreover, the fold changes of apoptosis/cell death in shGrp94-infected cells
over shCtrl-infected ones are similar in 5TGM1/luc cells (1.67) and U266 cells (1.83),
indicating the important role of GRP94 in MM cells. However, these three types of cells
exhibit much difference in the number of viable cells upon GRP94 knockdown. The
number of viable cells was reduced dramatically in the 5TGM1/luc cells upon GRP94
knockdown (around 75%). Similar reduction was also observed in MEFs but to a less
extend (around 60%), whereas the number of viable cells in U266 cells upon GRP94
knockdown remains not much changed (around 10%) when compared to non-infected or
shCtrl-infected cells. This reduction in cell number may be attributed to three possible
causes. One is reduced proliferation upon GRP94 knockdown, and a second is increased
necrosis upon GRP94 knockdown. A third possibility is that, the long-dead cells were
dismantled after GRP94 knockdown, leaving only live cells and the cells undergoing
apoptosis available for flow cytometry analysis. The different dependence of GRP94 in
these three cells is also reflected in their levels of GRP94 in the surviving populations, as
MEFs cells and U266 cells can survive with no or low level of GRP94, whereas
5TGM1/luc cells need to restore the level of GRP94 before their survival and
repopulation can be achieved. We have known that loss of viability contributes to the cell
number reduction, but so far it is not clear that whether GRP94 knockdown affects
necrosis or cell proliferation. A BrdU labeling experiment can be performed to determine
cell proliferation.
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One thing accomplished in this study was the establishment of lentivirus
packaging and infection system for knocking down GRP94 in human and mouse cell
lines. Depending on the packaging plasmids and selection markers on the shRNA vectors,
the lentiviral shRNA against human and mouse GRP94 were produced following two
different protocols. For safety concern, replication-deficient lentivirus vector is designed
to leave out genes for self-replication and only keep the essential components for virus
packaging, named gag, pol and rev. VSV-G, which is the pseudotype envelope of the
vesicular stomatitis virus (VSV), is not necessary for virus packaging but can
significantly increase viral particle stability, broaden target cell range and facilitate high
transduction efficiency in vitro and in vivo. In the second-generation packaging system,
PsPAX encodes gag, pol and rev while PMD2.g encodes VSV-G pseudotype. In the three
plasmid packaging system, gag, pol and rev are separated onto two different plasmids,
pLP1 (encodes gag, pol) and pLP2 (encodes rev), which has minimal relation to the WT
human HIV virus and therefore provides enhanced biosafety to the system.
The GFP selection marker on the pGIPZ plasmid enables monitoring transduced
cells by fluorescent microscopy or flow cytometry. The protocol of titering lentivirus
with pLKO.1 vector could be adapted to titer lentivirus without GFP selection marker, in
which the puromycin selection could be substitute with any other selection markers.
There are factors affecting the “real” viral titer, such as the size of the gene of interest,
the age of lentiviral stock, the number of freeze/thaw cycles and improper storage of the
133
lentviral stock, all of which affect virus activity. However, there are factors affecting the
“readout” of viral titer using the puromycin selection protocol. The first factor is the
characteristics of the cell line used for tittering. In general, they should be mammalian,
adherent, non-migratory cell lines that exhibit a doubling time in the range of 18-25 hours.
Since titer is calculated based on the number of single cell-derived colonies, using cells
with poor adherence such as 293T cells will significantly underestimate the titer. A
second factor is the timing of selection. Puromycin selection should be added at least
three days post transduction, to allow the integration and expression of
puromycin-resistant gene. Adding puromycin selection too early will result in
underestimation of virus titer.
Spinoculation, or centrifugal inoculation, has been widely used in virology
research since 1950s to enhance viral infection on suspension cells. In our study using
lentiviral shRNA against 5TGM1/luc cells, the transduction efficiency was increased
from around 5-10% by regular “virus-cell-mixing procedure” to 50% by spinoculation.
Although the efficiency of virus infection can be substantially improved by spinoculation,
the underlying mechanism of this enhancement has not been well defined. Some study
have showed that centrifugation increases the binding of virus to cells through increased
viral deposition (O’Doherty and Swiggard, 2002); however, the speed for spinoculation
(1500 g, around 3000 rpm) is much lower than the ultracentrifuge speed (20000 rpm) for
virus concentration. A recent study have demonstrated that spin-induced cytoskeletal
134
dynamics upregulates CD4 and CXCR4, promotes viral entry and post-entry processes,
as actin inhibitor diminished spin-mediated transduction enhancement (Guo et al., 2011).
Spinoculation can be used on not only suspension cells but also adherent cells to increase
transduction efficiency if necessary (Miyauchi et al., 2009).
Despite the failed attempt of studying the role of GRP94 in MM progression via
lentivirus-mediated depletion of GRP94, this study established the 5TGM1/luc syngeneic
MM mouse model, which could be of great use to investigate future potential therapeutics
targeting GRP94 or other ER chaperones against MM.
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Chapter 5
The role of GRP78 in normal and leukemic HSCs homeostasis
5.1 Introduction
The last three chapters are centered around GRP94 and its role in HSC
homeostasis and hematological cancer. Other than GRP94, GRP78 is another major ER
chaperone protein that plays important roles in various cellular processes and disease
development (Ni and Lee, 2007; Luo and Lee, 2013). Genetic knockout of Grp78 leads to
a much reduced embryonic cell proliferation as well as massive apoptotic death of the
inner cell mass, leading to embryonic lethality at day E3.5. ER stress occurs when cells
are under conditions such as hypoxia, low pH, or glucose deprivation (Luo and Lee,
2013). As the master regulator of the UPR, GRP78 binds to UPR sensors PERK, IRE1
and ATF6 on the ER membrane and prevent them from being activated. GRP78 depletion
releases these sensors from the ER membrane and triggers the activation of UPR
signaling (Pfaffenbach and Lee, 2010). HSCs reside in the endosteal niche, which is
characterized highly hypoxic due to lack of blood vessel access (Eliasson and Jönsson,
2010). Therefore, GRP78 may play important roles in regulating HSCs in the BM. It has
been reported that cell surface GRP78 interacting with Cripto plays an important role for
maintains HSCs in hypoxia (Miharada et al., 2011). To further study the role of GRP78 in
regulating HSC and hematopoiesis, our former lab member Shiuan Wey has created a
mouse model in which Grp78 is deleted by Mx-1-Cre driven conditional knockout (Wey
136
et al., 2012b). The first half of this chapter is to evaluate the functional consequence of
GRP78 depletion in HSC homeostasis.
The majority of studies on GRP78 have been focusing on its function in cancer.
GRP78 has been reported to be upregulated in solid tumors in various organs including
breast, liver, gastric, esophagus, brain, prostate, head and neck and melanoma, correlating
with aggressive tumor behavior and recurrence. Recent studies from our lab have
demonstrated interaction between GRP78 and the oncogenic PI3K-AKT signaling
pathway, as GRP78 knockdown reduces AKT phosphorylation (Fu et al., 2008).
Activation of PI3K-AKT signaling pathway has been shown in a variety of human
cancers. For example, inactivation of PTEN in the hematopoietic system leads to
short-term expansion but long-term decline of HSCs, as well as myeloproliferative
disorder which later developed into leukemia (Zhang et al., 2006; Yilmaz et al., 2006).
Based on previous study in our lab in which heterozygous or homozygous deletion of
Grp78 specifically in the mouse prostate epithelium suppresses PTEN-null driven
prostate tumorigenesis, we wanted to study if similar phenomena happens in PTEN-null
driven leukemogenesis. Our former lab member Shiuan Wey has created a mouse model
containing genetically modified Pten and Grp78 (Wey et al., 2012a). The second half of
this chapter is to evaluate whether GRP78 depletion rescues the HSC hyperproliferation
induced by PTEN inactivation.
137
5.2 Materials and methods
Flow cytometry
BM cells were flushed from long bones (tibias and femurs) with Dulbecco’s
phosphate-buffered saline without calcium and magnesium (PBS), and then filtered
through nylon screen (70 micron, BD Biosciences) to obtain single cell suspension. To
study hematopoietic stem and progenitor cells, Lineage [Lin; which consists of B220
(RA3-6B2), TER119 (TER119), CD4 (RM4-5), CD8a (53-6.7), Gr-1 (RB6-8C5), and
Mac-1 (WT.5)], c-Kit (2B8), and Sca-1 (D7, all from BD Pharmingen) were used. Cell
population analysis was performed on BD FACS LSR II.
Fluorescence activated cell sorting
Primitive hematopoietic stem and progenitor cells were stained with cell surface
markers (Lin
-
Sca-1
+
c-Kit
+
) and purified using a FACSAria flow cytometer (Becton
Dickinson).
Cell cycle analysis
1×10
7
whole BM cells were incubated with 10 µg/ml Hoechst 33342
(Sigma-Aldrich) at 37°C for 45 min, then stained with antibodies against cell surface
markers for primitive hematopoietic cells (Lin, c-Kit and Sca-1) as described above. The
stained cells were resuspended in 10% neutral buffered formalin (BDH Chemicals) and
138
incubated at 4°C overnight. To stain for RNA content, Pyronin Y (Polysciences Inc.) was
added to the cells at a final concentration of 0.75 µg/ml and incubated at 4°C for 30 min.
Cell cycle status was examined using a BD LSR II flow cytometer.
Apoptosis assay
For apoptosis detection, 1×10
6
whole BM cells were stained with primitive
hematopoietic cell antibodies (Lin, c-Kit and Sca-1) as described above. The stained cells
were resuspended in 150 µL 1×Annexin V binding buffer (BD Pharmingen) and
incubated with 5 µL Annexin V and 5 µL 7-AAD (both from BD Pharmingen) for 15 min
at room temperature. Cell apoptosis was examined within an hour using a BD LSR II
flow cytometer.
BM transplantation
1×10
6
Grp78
f/f
or cGrp78
f/f
whole BM cells were injected into the tail vein of
Grp78
f/f
mice that were lethally irradiated at 9.5Gy approximately 24 hr prior to
transplantation and allowed 8 weeks for reconstitution before pI.pC administration. In the
reciprocal transplantation, 1×10
6
Grp78
f/f
whole BM cells were injected into the tail vein
of irradiated Grp78
f/f
or cGrp78
f/f
mice, and allowed 8 weeks for reconstitution before
pI.pC administration. LSK cell number and hematopoiesis were measured with BM cells
and tail vein peripheral blood samples 6 days post 7 times of pI.pC injection.
139
5.3 Results
5.3.1 GRP78 depletion led to increased apoptosis in normal HSCs
To investigate the role of GRP78 in the hematopoietic system, our former lab
member Shiuan Wey has created a mouse model by crossing Grp78
f/f
mice with
Mx-1-Cre transgenic mouse line that allows Grp78 to be acutely deleted in the
hematopoietic system. In these studies, Grp78 was deleted in 5.5 to 6.5 week old Grp78
f/f
;
Mx-1-Cre (cGrp78
f/f
) mice. Littermates lacking the Cre transgene (Grp78
f/f
), which are
phenotypically equivalent to animals with WT Grp78 alleles, were also injected with
pI.pC and served as controls for any side effects of pI.pC injection. cGrp78
f/f
mice
displayed a statistically significant 30% reduction in the HSC-enriched Lin
-
c-Kit
+
Sca-1
+
(LSK) cell population in the BM (Wey et al., 2012b). Upon further analysis, the reduction
in LSK population in the BM stems from decrease in both the Lin
-
c-Kit
+
Sca-1
+
CD34
-
LT-HSC and Lin
-
c-Kit
+
Sca-1
+
CD34
+
ST-HSC (Wey et al., 2012b).
To determine whether the decrease of the primitive hematopoietic cells in the BM
of the cGrp78
f/f
mice was due to increased cell death or decreased proliferation, we
analyzed the apoptotic and cell cycle profile in LSK cells. Flow cytometric analysis of
Annexin V and 7-AAD staining on LSK cells showed that there was a significant
increase in the percentage of apoptotic LSK cells in cGrp78
f/f
mice compared to Grp78
f/f
mice (Figure 5.1A and B). Whereas the cell cycle distribution analyzed by
140
Hoechst/Pyronin Y staining showed that there was no significant difference between
cGrp78
f/f
and Grp78
f/f
mice (Figure 5.1C and D). This suggests that GRP78 is essential in
the maintenance and survival of adult hematopoietic stem cells, as GRP78 deficiency
results in decrease in the primitive hematopoietic cells at least in part through enhanced
cell death.
Figure 5.1 GRP78-deficient LSK cells displayed increased cell death. (A) Representative flow cytometric
analysis of apoptotic LSK cells using Annexin V and 7-AAD. (B) Summary of flow cytometric analysis of
apoptotic LSK cells using Annexin V and 7-AAD (n=5 for Grp78
f/f
, n=6 for cGrp78
f/f
). (C) Representative
flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. (D) Summary of cell
cycle distribution of LSK cells from Grp78
f/f
(n=4) and cGrp78
f/f
(n=4) mice. All data are presented as
141
mean ± s.e (*P<0.05, Student’s t-test).
5.3.2 Intrinsic effect of GRP78 depletion on HSC pool size
To examine whether the requirement of GRP78 in maintaining the HSC
populations is HSC-intrinsic or extrinsic, reciprocal BM transplant was performed and
primitive hematopoietic populations were examined. As shown in Figure 5.2A, total BM
cells of cGrp78
f/f
mice were transplanted into lethally irradiated Grp78
f/f
siblings
(cKO-WT). As control, BM cells of Grp78
f/f
mice were also transplanted into lethally
irradiated Grp78
f/f
littermates (WT-WT). Two months following transplantation to allow
full BM reconstitution, recipient mice were intraperitoneally injected with pI.pC (25
mg/kg body weight) every other day for a total of 7 injections to induce Mx-1-Cre
mediated deletion. WT-WT mice were also administrated with pI.pC as control. The mice
were analyzed 6 days after the completion of the pI.pC induction interval. A reverse BM
transplant was performed (Figure 5.2B), in which total BM cells of Grp78
f/f
mice were
transplanted into lethally irradiated cGrp78
f/f
siblings (WT-cKO). As control, BM cells of
Grp78
f/f
mice were also transplanted into lethally irradiated Grp78
f/f
littermates
(WT-WT).
142
Figure 5.2 Experiment scheme of BM transplantation. (A) Transplanting cGrp78
f/f
or Grp78
f/f
BM into
Grp78
f/f
siblings. (B) Transplanting Grp78
f/f
BM into cGrp78
f/f
or Grp78
f/f
siblings.
While the total BM cellularity was the same, we observed a significant reduction
of HSC-enriched LSK cells in the BM upon pI.pC induction in cKO-WT mice (Figure
5.3A and B). Furthermore, the level of LSK reduction was similar to that in the cGrp78
f/f
mice, suggesting GRP78 to be essential in maintaining normal HSC homeostasis and this
property is intrinsic to the cell. The intrinsic requirement of GRP78 in maintaining BM
HSCs is supported by a reciprocal transplantation, in which the LSK cell number in
WT-cKO mice were comparable with that in WT-WT mice. These data demonstrated that
the reduction of HSC-enriched population is intrinsic to GRP78 deficiency in BM cells.
143
Figure 5.3 Reduction of the LSK cells is intrinsic to GRP78 deficiency in BM cells. (A) Quantitation of
flow cytometric analysis of LSK percentage in BM after transplanting cGrp78
f/f
or Grp78
f/f
BM into
Grp78
f/f
recipients. (B) Quantitation of total BM cell number after transplanting cGrp78
f/f
or Grp78
f/f
BM
into Grp78
f/f
recipients. (C) Quantitation of flow cytometric analysis of LSK percentage in BM after
transplanting Grp78
f/f
BM into cGrp78
f/f
or Grp78
f/f
recipients. (D) Quantitation of total BM cell number
after transplanting Grp78
f/f
BM into cGrp78
f/f
or Grp78
f/f
recipients.
5.3.3 GRP78 depletion reversed the loss of quiescence and increased proliferation in
PTEN-null HSC
To test the role of GRP78 in leukemogenesis, our former lab member Shiuan Wey
has created a new mouse model containing the Pten floxed allele, the Grp78 floxed allele
and the Mx-1-Cre transgene. Pten and Grp78 were deleted in the hematopoietic system
from 6-8 week old cPten
f/f
Grp78
f/+
mice by administrating pI.pC every other day for a
total of 7 doses to induce Cre expression. The mice were analyzed 6 days post pI.pC
injection. Littermates without the Cre transgene (Pten
f/f
Grp78
+/+
and Pten
f/f
Grp78
f/+
) are
144
phenotypically equivalent to animals with WT Pten and WT Grp78 alleles; they served
as WT normal controls and were also injected with pI.pC. Since mice with homozygous
deletion of both Pten and Grp78 (cPten
f/f
Grp78
f/f
) died during the pI.pC administration
interval, Pten null, Grp78 heterozygous (cPten
f/f
Grp78
f/+
) mice were used in studying the
role of GRP78 in PTEN-null leukemogenesis.
Grp78 heterozygosity rescued the leukemic phenotypes resulted from Pten
deletion, including reduction of BM cells and BM LSK cells (Wey et al., 2012a). To
investigate the mechanism for this rescue, we performed flow cytometric analysis on the
cell cycle distribution and apoptosis status on the LSK cells. The cell cycle distribution
determined that the increased proliferation in cPten
f/f
HSC was suppressed by Grp78
heterozygosity (Figure 5.4A and B), while Annexin V and 7-AAD analysis demonstrated
that apoptosis was comparable among the three groups (Figure 5.4C and D).
145
Figure 5.4 GRP78-deficient LSK cells displayed increased cell death. (A) Representative flow cytometric
analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. (B) Summary of cell cycle
146
distribution of LSK cells from WT (n=3), cPten
f/f
(n=5) and cPten
f/f
Grp78
f/+
(n=5) mice. (C) Representative
flow cytometric analysis of apoptotic LSK cells using Annexin V and 7-AAD. (D) Summary of flow
cytometric analysis of apoptotic LSK cells using Annexin V and 7-AAD from WT (n=3), cPten
f/f
(n=5) and
cPten
f/f
Grp78
f/+
(n=5). All data are presented as mean ± s.e (*P<0.05, Student’s t-test).
5.4 Discussion
Utilizing a loss-of-function approach, here we demonstrate the roles of GRP78 in
regulating the homeostasis of HSC under normal and PTEN-null driven pathological
conditions. We employed flow cytometric analysis to examine the LSK cell number as
well as their proliferation and apoptosis status upon GRP78 depletion in the
hematopoietic system.
Under physiological condition, GRP78 depletion does not result in altered HSC
proliferation, whereas during leukemia progression GRP78 depletion suppresses the HSC
hyperproliferation induced by PTEN inactivation. This reflects different requirements for
GRP78 under physiological and malignant conditions, despite GRP78 is expressed
ubiquitously in eukaryotic cells. Cancer cells are in need of increased protein synthesis
due to their uncontrolled rapid proliferation, and enhanced ER activity is required to
facilitate the folding, assembly and transportation of membrane and secretory proteins for
their proliferation. Moreover, solid tumors are under ER stress because of their
surrounding hypoxic and acidic environment, and the upregulation of GRP78 in solid
147
tumors alleviates the ER stress and facilitate tumor progression. Similar to solid tumors,
leukemia cells also reside in the hypoxic BM environment, with demand for synthesizing
a large amount of proteins. Therefore, the proper function of GRP78 is more important in
the context of cancer. The chaperone function of GRP78 in the ER may facilitate the
secretion of growth factor and/or the maturation of growth factor receptors, or the
potential chaperone function of GRP78 helps stabilize the receptor or co-receptor for
growth signaling pathways on the surface of cancer cells. In contrast, under physiological
condition HSCs divide in low frequencies, which deemphasizes the requirement for
GRP78 and its chaperone function. Another possible explanation is through the potential
interaction of GRP78 with the PI3K-AKT signaling pathway for cell survival and
proliferation, as studies have shown that loss of GRP78 suppresses AKT phosphorylation
in vitro and in vivo (Fu et al., 2008; Wey et al., 2012a). AKT is activated at a low basal
level in normal BM, but highly activated in the PTEN-null driven leukemic BM.
Therefore, GRP78 is more required in PTEN-null driven leukemia progression. Similar to
this study, in PTEN-null driven prostate cancer, loss of GRP78 suppresses the
progression of prostate cancer while not affecting the development and homeostasis of
normal prostate epithelial cells (Fu et al., 2008).
Studies in this chapter demonstrate the requirement of GRP78 for maintaining
HSCs under physiological condition, and loss of GRP78 results in increased HSC
apoptosis. This can be attributed to the constitutive UPR activation upon GRP78
148
depletion (Wey et al., 2012b). The effect of GRP78 depletion on reducing HSC number
in the BM appears to be cell-intrinsic, as demonstrated by reciprocal BM transplantation.
In the apoptotic assay, only the 7-AAD
-
Annexin V
+
population was considered apoptotic
cells, as the 7-AAD
+
Annexin V
+
population could be resulted from the physical damage
during the process of isolating BM cells. Since leukemia cells are under pronounced ER
stress, it is speculated that loss of GRP78 would lead to more apoptosis in the PTEN-null
HSCs. However, flow cytometric analysis demonstrated comparable apoptosis in the
PTEN-null HSCs with or without Grp78 heterozygous knockout. This may be due to the
different levels of GRP78 in these HSCs. In the study of leukemia, heterozygous
knockout of Grp78 only leads to partial reduction in the GRP78 protein level, whereas in
the study of HSC homeostasis GRP78 protein was nearly completely depleted in the BM
cells. The remaining GRP78 in the leukemia study is sufficient to prevent additional cell
death. Consistently, mice with homozygous deletion of both Grp78 and Pten died during
the interval of pI.pC injection (Wey et al., 2012a), indicating the vital role of GRP78 in
the context of PTEN-null driven leukemia progression. In summary, studies in this
chapter demonstrated a differential role of GRP78 in regulating HSC homeostasis under
physiological and malignant conditions.
149
Chapter 6
Conclusions and Perspectives
ER homeostasis plays important roles in maintaining cellular function and
organization. Loss of ER homeostasis is involved extensively in a variety of diseases,
such as neurodegenerative disorders, inflammation, cancer, atherosclerosis, and diabetes
mellitus. However, the role of ER chaperones, particularly GRP94, in the adult
hematopoietic homeostasis and hematological disorders is still elusive. In this dissertation,
we set forth to elucidate the direct role of GRP94 in 1) the interaction of HSC with the
BM niche, 2) the regulation of HSC proliferation, and 3) the progression of multiple
myeloma (MM). In parallel with the studies on GRP94, this dissertation also includes a
portion of studies on the role of another major ER chaperone, GRP78, in HSC
homeostasis under physiological and malignant conditions. These studies expand our
understanding in the function of ER chaperones as well as regulatory mechanisms for
HSCs and hematopoietic cells.
In the first part of this dissertation, we investigated the requirement of GRP94 in
maintaining the interaction between HSCs and the adult BM niche. The model system
that we employed is the Grp94; Mx-1-Cre mouse model whereby GRP94 can be acutely
eliminated largely in the hematopoietic system in a temporally controlled manner in adult
mice. Our results showed that cGrp94
f/f
mice displayed increased number of
hematopoietic stem and progenitor cell pools, resulting from a loss of quiescence and an
150
increase in proliferation. This expansion of HSC pool upon GRP94 depletion can be
attributed to the impaired interaction of HSCs with the niche, evidenced by the enhanced
mobilization, defective homing and engraftment of cGrp94
f/f
HSCs. Reciprocal BM
transplantation between Grp94
f/f
and cGrp94
f/f
mice further demonstrated that GRP94 in
HSCs is required for their interaction with the BM niche, and depleting GRP94 in BM
microenvironment itself is not sufficient to affect HSC pool size. One explanation for the
loss of HSC-niche interaction upon GRP94 depletion is a complete loss of integrin α4
expression on the cell surface of cGrp94
f/f
HSCs. Collectively, in Chapter 2 we have
established GRP94 as a novel cell intrinsic factor required to maintain the interaction of
HSCs with their niche, and thus regulate their physiology.
The second part of this dissertation is focusing on the role of GRP94 in one
specific aspect of HSC homeostasis: the regulation of proliferation. Since in Chapter 2
BM transplantation experiments demonstrate that the expansion of GRP94-null HSC pool
size is cell autonomous, we further investigated into the underlying mechanisms for these
phenomena using the same mouse models. Interestingly, we discovered that GRP94
depletion in the LSK cells led to increase in AKT activation and higher production of
PI(3,4,5)P3, and the elevated AKT activation is required for the hyperproliferation of
GRP94-null HSCs. Microarray analysis on cGrp94
f/f
and Grp94
f/f
LSK cells revealed a 97%
reduction in the expression of the hematopoietic cell cycle regulator Ms4a3 in the
GRP94-null LSK cells, and further study revealed this downregulation is restricted to BM
LSK cells. When examining cell surface proteins that are known to regulate stem cell
151
proliferation, a reduced expression of cell surface Cx32 plaques was observed in
GRP94-null HSCs. However, suppression of Cx32 hemichannel activity in WT HSCs did
not recapitulate the HSC expansion caused by GRP94 depletion in vitro. Collectively,
studies in Chapter 3 uncover novel and unique roles of GRP94 in regulating HSC
proliferation.
Studies in Chapter 2 and Chapter 3 established the unique function of GRP94 as
an intrinsic regulator of HSC homeostasis, specifically, their proliferation, differentiation
and interaction with the BM niche. So far most intrinsic regulators of HSC homeostasis
can be categorized as transcription factors, cell cycle regulators, chromatin-associated
proteins as well as genetic and epigenetic regulators during development. In addition to
its roles in innate and adaptive immunity demonstrated by extensive studies, GRP94 is
the first ER chaperone that is identified to regulate HSC pool size and functions. Studies
in this dissertation investigate in detail on how loss of GRP94 affects the regulation of
HSCs, and expand our understanding about the functions of GRP94.
The effect of GRP94 depletion on HSC homeostasis and hematopoiesis in our
model resembles the phenotypes observed in mouse models in which the gain-of-function
and loss-of-function of other HSC regulators were studied. However, the phenotypes in
cGrp94
f/f
mice do not fully recapitulate the phenotypes in those models, suggesting the
comprehensive role of GRP94 in regulating HSC homeostasis. For example, similar to
GRP94 depletion, loss of integrin α4 leads to impaired HSC homing and engraftment
(Jiang et al., 2009). In Chapter 2 we identified the loss of integrin α4 on the surface of
152
GRP94-null HSCs. However, acute or chronic loss of integrin α4 does not lead to
increased HSCs or increased HSC cycling (Priestley et al., 2006; Jiang et al., 2009).
Therefore, loss of integrin α4 could be one mechanism of how GRP94-null HSCs lose
their physical and functional interaction with the BM niche, but other players in this
process remain to be determined. Another example is the PI3K-AKT pathway. Loss of
PTEN or constitutive activation of AKT in the hematopoietic system results in
myeloproliferative diseases as well as short-term increased HSCs through
hyperproliferation (Zhang et al., 2006; Yilmaz et al., 2006; Kharas et al., 2010). Studies
in Chapter 3 demonstrated elevated AKT activation in GRP94-null HSCs, suggesting the
possibility of AKT in mediating the effect of GRP94 depletion on HSC homeostasis.
However, notable differences exist in the Grp94 knockout and Pten knockout/AKT
constitutive activation models. For instance, in the BM chimera mice where GRP94 was
depleted only in the hematopoietic cells, the myeloproliferative diseases did not further
progress to leukemia up to 9 months after pI.pC injection (consulted with hematologist).
In addition, the hyperproliferation of GRP94-null HSC appears to be transient, detectable
at 11 days post the first pI.pC injection but not at 21 days post the first pI.pC injection. In
contrast, Pten knockout mice exhibit persistent activation of AKT and eventually HSC
pool depletion. These data imply a different nature of GRP94 knockout from simply loss
of a surface molecule or activation of growth signaling pathways. There may be
regulatory mechanisms in cGrp94
f/f
mice to prevent uncontrolled HSC proliferation, and
153
the elevated activation of AKT in GRP94-null HSCs is required for the initial
hyperproliferation but not sufficient to overcome these regulatory mechanisms.
How might GRP94 regulate HSC homeostasis? Although we have discovered
several potential mechanisms that may mediate the regulation of GRP94 on HSCs,
questions remain on whether these are really the primary cause of the increased HSCs in
the GRP94-null BM, and whether there are other factors contributing to this phenomenon.
For example, we have discovered that the elevated AKT activation is required for the
hyperproliferation of GRP94-null HSCs. However, the activation of PI3K-AKT pathway,
or specifically the increased PI(3,4,5)P3 production and AKT activation could be a direct
effect of GRP94 deficiency, or a secondary effect resulted from the change of other
molecules upon GRP94 depletion. Further more, if the elevated AKT activation is a
secondary effect, the primary causes of this phenomenon could be BM niche-dependent
or/and independent. Although we have determined by reciprocal BM transplant that the
regulation of GRP94 is HSC autonomous, this regulation could still be niche-dependent,
as loss of GRP94 could affect the expression of surface proteins that are important for
HSC-niche interaction. Co-culturing GRP94-null LSK cells with BM stromal cells
resulting in increased cell expansion at similar magnitudes, implying the dispensability of
BM microenvironment in this regulation. Nonetheless, GRP94-null HSCs may have
already entered cell cycle and passed the cell cycle checkpoints in vivo before they were
isolated and co-cultured with the BM stromal cells. A more definitive experiment to
address this question is to isolated HSCs from Grp94
f/f
and cGrp94
f/f
mice before pI.pC
154
injection, activate the Cre by the administration of pI.pC or interferon α, and then
compare their proliferation. However, the results of this short-term culture experiment
could be confounded by the time required for Cre activation and GRP94 depletion, as
well as the time for the effect of GRP94 depletion on HSC proliferation to take place. A
long-term cobblestone assay to culture HSCs could be an alternative solution.
We identified that GRP94 depletion results in loss of cell surface expression of
integrin α4, which is required for HSC homing and retention in the BM. Therefore, the
regulation of GRP94 on HSC homeostasis could be partially attributed to its function as
an ER chaperone protein. However, GRP94 could also function beyond folding and
processing secretory and surface proteins. Studies in this dissertation cannot rule out this
possibility, as whether the requirement of GRP94 in HSC homeostasis is BM
niche-dependent or independent remains to be determined. While the mechanisms
whereby GRP94 deficiency promotes PI(3,4,5)P3 production and AKT activation
remains unclear, it is noted that as in the case of several other ER chaperones, GRP94 can
also be localized on the cell surface of specific cell types (Luo and Lee, 2013). It is
speculated that cell surface GRP94 could act as a suppressor for PI3K signaling and its
ablation will relieve the inhibition, resulting in increase of PI(3,4,5)P3 production and
AKT activation.
In parallel with studies on GRP94, Chapter 5 of this dissertation also includes
studies on the role of GRP78 in regulating HSC proliferation and apoptosis under both
physiological condition and PTEN-null driven leukemia progression. Interestingly, the
155
regulation of GRP94 and GRP78 on HSC homeostasis appears to be chaperone specific,
as deficiency of GRP78 does not result in expanded HSC pool size or increased
proliferation, but rather, leads to elevated apoptosis. These studies enrich our
understanding about the functions of GRP94 in comparison to other ER chaperones such
as GRP78. The distinct roles of GRP94 and GRP78 in regulating HSCs under different
circumstances reflect their unique characteristics. The specificity of client proteins that
mediate HSC-niche interaction could be one easy explanation for the phenomenon we
observed in GRP94-deficient mouse hematopoietic system. For example, a recent study
demonstrated the requirement of GRP94 in intestinal homeostasis via its requirement for
chaperoning the co-receptor LRP6 in canonical Wnt signaling pathway (Liu et al., 2013).
It is possible that GRP94 and GRP78 process distinct client proteins and thereby exert
different effects in the same experimental system. However, the differential regulation of
GRP94 and GRP78 on HSCs could also reflect the distinct function of individual ER
chaperones besides their common capacity of assisting protein folding, and this could be
attributed to their unique localization and other possible functions such as mediating
signal transduction. For example, GRP94 and GRP78 deficiency exert distinct influences
in the activation of PI3K-AKT pathway. While GRP78 deficiency suppresses the
activation of AKT in both PTEN-null leukemia model and in vitro cell culture upon
serum stimulation (Fu et al., 2008; Wey et al., 2012a), GRP94 deficiency in the BM leads
to increased PI(3,4,5)P3 production and elevated AKT phosphorylation in HSCs but did
not alter AKT activation in cell lines upon serum stimulation in vitro (data not shown). It
156
is speculated that a small portion of GRP78 located on the cell surface interacts with
signaling receptors, possibly as part of the receptor complex or stabilizing the receptor
complex via its chaperoning capability. Similar scenario could apply to GRP94, as
GRP94 is also detected on the surface of specific cell types via its function in antigen
presentation. However, it is also possible that the interaction of GRP94 with the
PI3K-AKT signaling pathway is dependent on the context. The increased activation of
AKT in GRP94-null HSCs could be intrinsically restricted to primitive hematopoietic
cells, but it could also be simply a consequence of HSCs escaping from the quiescent
signals from the BM endosteal niche due to their detachment from the BM niche. The
specific underlying mechanisms for the regulation of HSC and hematopoiesis by
individual ER chaperone are still elusive, and await future investigation.
Based on the discovery in Chapter 2, we hypothesized that loss of GRP94 will
suppress the progression of MM through interrupting their interaction with the BM
microenvironment. The studies were reported in Chapter 4 of this dissertation. The core
ideas of this study is to knockdown Grp94 in the well-characterized 5TGM1 syngeneic
mouse MM model, and see if GRP94 deficiency impairs the homing of MM cells to the
BM and thereby suppresses cancer progression in the recipient C57BL6/KaLwRijHsd
mice. We established the protocol of producing lentivirus-mediated shRNA targeting
mouse and human GRP94, and discovered that knockdown GRP94 in the 5TGM1 cells
leads to increased apoptosis. Moreover, GRP94 expression level was gradually restored
despite the presence of puromycin selection, possibly due to the survival selection
157
pressure against GRP94-depleted 5TGM1/luc cells in culture. We have also established
the C57BL6/KaLwRijHsd syngeneic MM model to test the effect of GRP78 and GRP94
inhibitors in disease progression. Quantitative measurement can be further applied to
evaluate MM disease progression, including flow cytometry measuring the percentage of
MM cells in the BM, ELISA detecting serum IgG2bκ level, and Micro-CT determining
bone destruction.
Based on the studies demonstrating the requirement of GRP94 and GRP78 in
regulating HSC homeostasis and hematopoiesis, it is tempting to speculate the
development of potential therapeutic strategies targeting GRP94 or GRP78 for
hematological diseases. While extensive studies have been focusing on GRP78 inhibitors
and their application in cancer treatment, manipulating GRP94 for clinical application is
still a relatively new territory, except for clinical trials utilizing genetically engineered
GRP94 as in anti-cancer vaccination. Studies in this dissertation on GRP94 shed lights on
additional potentials of GRP94 as a therapeutic target. For example, one primary source
of HSCs for clinical transplantation is circulating HSCs isolated from peripheral blood.
Since GRP94 depletion in HSC promotes their mobilization, it is promising to develop
agents that can alter GRP94 expression or activity to increase HSC mobilization as
source for blood stem cell therapy. Another potential application for targeting GRP94 is
non-myeloablative conditioning, in which less-toxic reagents are used to deplete recipient
hematopoietic system prior HSC transplantation. Inhibiting key molecules for HSC
retention in the niche has recently been employed as an effective strategy for
158
non-myeloablative conditioning before transplantation (Czechowicz et al., 2007). Since
GRP94 depletion impairs the ability of HSC to retain in the niche, it is tempting to study
the application of GRP94 inhibitors in non-myeloablative conditioning for clinical HSC
transplantation. While traditional GRP94 inhibitor 17-AAG has been shown to target
both GRP94 and HSP90, a novel GRP94 inhibitor has been reported to specifically block
the function of GRP94 (Duerfeldt et al., 2012). It is will be exciting to investigate the
effect of this inhibitor in the treatment of hematological diseases.
GRP94 can also be a promising therapeutic target for the treatment of MM. On
one hand, the survival and proliferation of MM cells in vivo requires interaction with the
BM microenvironment via similar mechanisms employed by HSCs; on the other hand,
MM cells undergo chronic ER stress because of the hypoxic BM environment as well as
the huge demand for protein secretion in tumor cells. Although studies in this dissertation
was not able to establish the requirement of GRP94 in MM progression due to the failed
attempt in knocking down GRP94 in vitro, the potential of targeting GRP94 in MM
progression is still alive. The establishment of 5TGM1 syngeneic mouse model enables
us to further test the requirement of GRP94 for MM progression in vivo. Inducible
lentivirus-mediated Grp94 deletion by tamoxifen can be utilized to circumvent the
problem of knocking down GRP94 in vitro. Chemical inhibitors of GRP94 can also be
tested for their effect of targeting MM tumor cells in vivo. Since MM cells are under
chronic ER stress, targeting GRP78 can also be a promising strategy to induce tumor
apoptosis and suppress disease progression. The 5TGM1 syngeneic mouse model
159
established in this dissertation also enables us to test the requirement of GRP78 for MM
progression in vivo, and may serve as an animal model to test the toxicity and efficacy of
therapeutic regimen targeting GRP78. For example, we are currently collaborating with
Dr. Parkash Gill at USC who discovered the production of an anti-GRP78 antibody that
captures cell surface GRP78. Since MM cells are under chronic ER stress, they are very
likely to express high level of GRP78, which promotes cell surface GRP78 expression
(Zhang et al., 2010). This anti-GRP78 antibody, once capturing cell surface GRP78, will
attract macrophages and other immune cells via its Fc domain and thereby activate the
anti-tumor immune response (data from Dr. Gill’s lab). The antibody has been shown to
be effective in the treatment of PTEN-null driven leukemia (Wey et al., 2012a), and it is
tempting to investigate its potential in MM treatment.
While therapeutic regimens targeting GRP94 in hematological diseases exhibit
pronounced potential, possible side effects remain to be evaluated. For instance, targeting
GRP94 in the treatment of MM might cause myeloproliferative diseases and/or increased
mobilization of HSCs, as shown in studies on cGrp94
f/f
mice in Chapter 2. In addition,
GRP94 deficiency in the hematopoietic system causes impaired platelet maturation
(Staron et al., 2011), which could be another side effect in treatments targeting GRP94.
Since loss of GRP94 blocks HSC homing and engraftment, targeting GRP94 to mobilize
HSCs as a source of HSC transplant requires transient GRP94 inhibitors whose inhibitory
effect can reversed before transplantation. Additionally, a recent study discovered that
160
GRP94 deficiency disrupts intestinal homeostasis (Liu et al., 2013), which could be
another source of side effects in the treatment targeting GRP94. One thing to remember is
that, all of these potential side effects of GRP94 inhibitors are speculated from studies
with complete loss of GRP94, however, the requirement of GRP94 or the sensitivity to
GRP94 inhibitors in different organs and cellular compartments may be different. Studies
in this dissertation open a door to the possibilities of utilizing GRP94 as a therapeutic
target in the treatment of hematological diseases, and effective ways of targeting GRP94
remain to be determined in the future.
In summary, studies in this dissertation revealed the pleiotropic roles of ER
chaperones, specifically GRP94 and GRP78, in regulating HSC homeostasis,
hematopoiesis and hematological malignancies. These studies have not only expand our
understanding on the function of ER chaperones as well as the diverse mechanisms of
how stem cells is regulated, but also shed light on future therapeutic regimens that target
ER chaperones for the treatment of hematological diseases.
161
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Abstract (if available)
Abstract
The endoplasmic reticulum (ER) is a cellular organelle essential for protein folding and transportation, as well as lipid synthesis and Ca2⁺ storage. ER chaperones play critical roles in assisting protein folding and processing. Two most abundant ER chaperones are GRP94 and GRP78. ❧ Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. The adult BM niche maintains HSC quiescence via soluble cues and direct contact. Traditionally regarded as an ER chaperone that assists the folding and processing of secretory and membrane bound proteins, GRP94 is speculated to play an important part in regulating HSC homeostasis. To directly elucidate the requirement of GRP94 in HSC homeostasis, we employed an inducible conditional knockout strategy to eliminate GRP94 in the hematopoietic system. The mutant mice displayed a 2-fold increase in HSC pool size resulted from a loss of quiescence and an increase in proliferation. The expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the BM niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT controls, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Furthermore, GRP94-null HSCs exhibited a near complete loss of integrin α4 expression on the cell surface and showed impaired binding with fibronectin, which are key molecules known to mediate HSC-niche interactions, providing a possible explanation for the loss of interaction of GRP94-null HSCs with the adult BM niche. ❧ Further investigation into the underlying mechanisms for this intrinsic hyperproliferation in GRP94-defiecient BM HSCs discovered an 1.8-fold increase in AKT activation, corresponding with higher production of PI(3,4,5)P3, indicating elevated PI3K activation. Treatment of GRP94-null HSCs with AKT inhibitors demonstrated that this elevated AKT activation is required for the increased proliferation in GRP94-null HSCs. Microarray analysis on freshly isolated Lin⁻ c-Kit⁺ Sca-1⁺ (LSK) cells demonstrated a 97% reduction in the expression of the hematopoietic cell cycle regulator Ms4a3 in the GRP94-null LSK cells. Furthermore, we observed a reduced expression of cell surface connexin 32 plaques in GRP94-null LSK cells. However, suppression of connexin 32 hemichannel activity in WT LSK cells through mimetic peptides did not lead to increased LSK proliferation in vitro. In addition, two other important cell surface proteins that mediate HSC-niche interactions, specifically Tie2 and CXCR4, were not impaired by Grp94 deletion. ❧ The requirement of GRP94 for maintaining HSC-niche interaction provides a therapeutic strategy of targeting GRP94 in the treatment of multiple myeloma (MM), the progress of which relies closely on the interaction of tumor cells with the BM microenvironment. We utilized a syngeneic MM mouse model to test whether GRP94 knockdown in tumor cells suppresses disease progression. Knockdown of GRP94 in mouse MM cells with lentivirus-mediated shRNA led to increased apoptosis in vitro, and GRP94 protein expression was gradually restored despite the presence of puromycin selection. Despite the unsuccessful knockdown of GRP94 in vitro, the syngeneic MM mouse model was established via tail vein tumor cell inoculation. MM mice displayed clusters of cells with lymphocyte-like morphology in the BM, as well as lesions of bone resorption. Further analysis at late stage MM mice demonstrated multiple tumor formation in the BM, accompanied by significant weight loss and hunched posture. ❧ In parallel with the study on GRP94 in HSC homeostasis, this dissertation also includes studies on the role of GRP78 in regulating HSC proliferation and apoptosis under both physiological condition and PTEN-null driven leukemia progression. Unlike GRP94 whose depletion causes HSC hyperproliferation, GRP78 depletion did not affect the cell cycle distribution of HSCs. However, in the context of PTEN-null driven leukemia, partial reduction of GRP78 suppressed the hyperproliferation induced by PTEN inactivation. Examining the requirement of GRP78 for HSC survival and apoptosis revealed an increased HSC apoptosis upon complete loss of GRP78, whereas heterozygous deletion of Grp78 did not lead to increased HSC apoptosis even in the context of cancer. ❧ In summary, studies in this dissertation revealed the comprehensive roles of ER chaperones, namely GRP94 and GRP78, in regulating HSC homeostasis and hematological malignancies.
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Luo, Biquan
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Core Title
The role of endoplasmic reticulum chaperones in regulating hematopoietic stem cells and hematological malignancies
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Keck School of Medicine
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Doctor of Philosophy
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Genetic, Molecular and Cellular Biology
Publication Date
08/03/2014
Defense Date
08/03/2013
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bone marrow niche,endoplasmic reticulum,GRP78,GRP94,hematopoietic stem cells,multiple myeloma,OAI-PMH Harvest,proliferation
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Tags
bone marrow niche
endoplasmic reticulum
GRP78
GRP94
hematopoietic stem cells
multiple myeloma
proliferation