The endoplasmic reticulum chaperone ERdj4 is required for survival, glucose metabolism and B cell development
A dissertation submitted to the University of Cincinnati
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
in the Immunobiology Graduate Program
of the College of Medicine
2012
by
Jill Marie Fritz
B.A., Miami University, Oxford, OH
Advisory Committee: Timothy Weaver, M.S., Ph.D., Chair George Deepe, M.D. Fred Finkelman, M.D. H. Leighton Grimes, Ph.D. Christopher Karp, M.D. Francis McCormack, M.D.
ABSTRACT
The ER-localized DnaJ homologue 4 (ERdj4) is a soluble ER chaperone induced by the unfolded protein response (UPR) to assist in the removal of unfolded/misfolded proteins from the ER lumen for proteasomal degradation. To elucidate the function of ERdj4 in vivo, ERdj4 gene trap (ERdj4GT/GT) mice were generated from embryonic stem cells harboring a gene trap cassette inserted into the ERdj4 locus. ERdj4GT/GT mice expressed hypomorphic levels of ERdj4 with a 10-100 fold reduction in all tissues and cell types examined. Approximately 30-50% of ERdj4GT/GT mice died perinatally in association with growth retardation and hypoglycemia. ERdj4GT/GT neonates exhibited signs of delayed pancreatic development, including abnormal distribution of pancreatic
α- and β-cells and reduced insulin and glucagon in the pancreas. Surviving adult
ERdj4GT/GT mice were glucose intolerant, resulting from hypoinsulinemia rather than insulin resistance. Pancreatic β-cells exhibited increased ER stress, including ER dilation and upregulation of the UPR. Proinsulin accumulated in the ER of β-cells from
ERdj4GT/GT mice consistent with our previous finding that proinsulin is a substrate for
ERdj4. The insulin processing enzymes, including Pcsk1, Pcsk2 and CPE, also associated with ERdj4, contributing to defects in proinsulin biosynthesis in ERdj4GT/GT mice. ERdj4 deficiency also resulted in pancreatic α-cell hyperplasia in association with increased ER stress. Since previous studies had demonstrated that the UPR is required for both early and late B lymphopoiesis, we hypothesized that ERdj4 deficiency would affect B cell development. Pro-B, pre-B, immature and mature B cell populations were significantly reduced in the bone marrow of ERdj4GT/GT mice in association with increased pro-B cell death. Further, mature B cell populations were reduced in the spleen and peritoneal cavity. ERdj4GT/GT donor cells transplanted into ERdj4+/+ recipients rescued all stages of B cell development, confirming a defect in the microenvironment. Osteogenic cells support B cell development and the UPR is required for osteoblast differentiation and function. Bone-lining cells, which differentiate from osteoblasts, were reduced in ERdj4GT/GT mice in association with increased ER stress. Further, primary bone cells from ERdj4GT/GT mice had reduced expression of osteogenic-specific markers. These data suggest that the loss of ERdj4 affects the maturation/survival of osteogenic cells, which in turn leads to a defect in B lymphopoiesis. Collectively, this work reveals an important role for ERdj4 in survival, glucose metabolism and B cell development.
iii iv ACKNOWLEDGMENTS
First and foremost, I wish to express sincere gratitude and appreciation to my scientific thesis advisor, Dr. Timothy Weaver. Without his dedication, guidance and encouragement, this work would not be possible.
I also wholeheartedly thank my committee members Dr. George Deepe, Dr. Fred
Finkelman, Dr. Lee Grimes, Dr. Chris Karp and Dr. Frank McCormack for their insight and support during my scientific training. I sincerely appreciate their efforts and direction in completing this work.
I acknowledge all the members of the Weaver lab, both past and present, for their friendship, motivation and expertise. I would especially like to thank Dr. Henry Akinbi for his careful supervision and support, especially during the first few years of my thesis project.
Pursuing my Ph.D. would not have been possible without my undergraduate mentor, Dr.
John Stevenson, who introduced me to the exciting world of scientific research.
This thesis is dedicated to my mother, Barbara A. Fritz and my father, Gerald W. Fritz, as well as my grandma, Barbara H. Bridge and my grandpa Edward D. Bridge. They instilled within me their passion, persistence and inquisitive mind. Their unconditional love and support make it possible to achieve any goal.
v TABLE OF CONTENTS
CONTENT PAGE
List of tables and figures xiii
List of abbreviations xvi
CHAPTER I Molecular Chaperones and the UPR in 1
Development and Metabolism: Background,
Significance and Hypothesis
1. Molecular Chaperones in the Endoplasmic Reticulum 2
1a. BiP: an ER-localized HSP70 chaperone 2
1b. DnaJ family of cochaperones 3
1bi. Structure and classification 5
1bii. Functionality 5
1biii. ER-localized DnaJ proteins 6
1biv. ERdj4 8
2. The Unfolded Protein Response 9
2a. Discovery 9
2b. Initial discovery of UPR signaling components in yeast 10
2c. The mammalian UPR 10
2ci. The ER stress sensors: ATF6, PERK and IRE1 11
2cii. ER stress-induced apoptosis 14
3. β-cell Stress, the UPR and Diabetes 14
3a. Inducers of ER stress in β-cells 15
3ai. Glucose 15
vi 3aii. Free fatty acids 16
3aiii. Proinflammatory cytokines 16
3aiv. Misfolded protein 18
3av. Islet amyloid peptide 18
3b. The UPR in β-cell dysfunction and diabetes 18
3bi. PERK/eIF2α 18
3bii. IRE1α/XBP1 19
3biii. ATF6α 21
3biv. ER chaperones 21
3c. The UPR and insulin resistance 22
4. B Cell Subsets and Effector Functions 22
4a. B1 cells 23
4b. B2 cells: Follicular and marginal zone B cells 24
4bi. Follicular B cells 24
4bii. Marginal zone B cells 24
5. B Cell Development 25
5a. B1 cell development 25
5b. B2 cell development 28
5bi. Early B2 cell differentiation in the bone marrow 28
5bii. Transcriptional regulation of early B2 cell development 30
5biii. Transitional B cells 31
5biv. Follicular verses marginal zone B cell fate 32
5c. The UPR in B cell development 34
vii 6. Growth Factors and Cellular Niches Required for B Cell Development 35
6a. B cell growth factors 36
6ai. CXCL12 36
6aii. IL-7 36
6aiii. FLT3L 38
6aiv. RANKL 38
6av. SCF 39
6b. Bone marrow niches that support B cell development 40
6bi. CAR niche: reticular cells expressing CXCL12 40
6bii. IL-7-expressing cells 40
6biii. Dendritic cells 41
6biv. Osteoblasts 41
7. The UPR in Osteoblast Differentiation 42
7a. PERK/eIF2α 42
7b. ATF4 44
7c. ATF6 45
7d. IRE1α/XBP1 45
8. Summary and Hypothesis 46
References 48
CHAPTER II Deficiency of ERdj4 is associated with perinatal 66
lethality, constitutive ER stress and glucose
intolerance
viii Abstract 68
Introduction 69
Results 72
Generation of ERdj4 gene trap mice 72
Elevated ER stress and increased susceptibility to cell death 72
in ERdj4GT/GT MEFs
Hypoglycemia and growth retardation in ERdj4GT/GT mice 73
Constitutive ER stress is associated with histological 74
abnormalities in pancreatic islets of ERdj4GT/GT mice
ERdj4 deficiency impairs insulin biosynthesis 75
Hypoinsulinemia causes glucose intolerance in ERdj4GT/GT 76
mice
Discussion 77
Materials and Methods 81
Acknowledgements 87
Figure Legends 88
Figures 93
References 105
CHAPTER III Deficiency of the ER stress chaperone ERdj4 is 110
associated with defects in osteogenesis and B cell
development
Abstract 112
ix Introduction 113
Results 117
Bone-lining cells are reduced in ERdj4GT/GT mice 117
ERdj4 deficiency affects early B lymphopoiesis 119
ERdj4 deficiency in the bone marrow microenvironment 119
impairs B cell development
Mature B cell populations are reduced in ERdj4GT/GT mice 120
Plasma cell differentiation and antibody secretion are normal 121
in ERdj4GT/GT mice
Discussion 122
Materials and Methods 126
Acknowledgements 130
Figure Legends 131
Figures 136
References 146
CHAPTER IV Discussion and Future Directions 151
Summary 152
1. Gene trapping ERdj4: a blessing in disguise? 154
2. What is the function of ERdj4 in perinatal survival? 155
3. ER stress and Type 2 Diabetes 157
3a. Do physiological stressors cause diabetes in ERdj4GT/GT mice? 158
3b. What causes β-cell failure in ERdj4GT/GT mice? 159
x 3c. Does rescue of ERdj4 expression in pancreatic β-cells result in 160
normal insulin secretion and glucose metabolism in ERdj4GT/GT mice?
3d. Is the chaperone activity of ERdj4 important in α-cells? 163
3di. Does loss of ERdj4 affect glucagon biosynthesis? 163
3dii. What causes α-cell hyperplasia in ERdj4GT/GT mice? 164
4. The role of ERdj4 in the bone marrow microenvironment 165
4a. Does the loss of ERdj4 in osteogenic cells impair B cell development 165
in ERdj4GT/GT mice?
4ai Do ERdj4GT/GT osteogenic cells support B cell maturation 165
in vitro?
4aii. Does administration of PTH rescue the defect in B cell 166
development in ERdj4GT/GT mice?
4aiii. Would rescue of ERdj4 expression in osteogenic cells result in 167
normal B cell development in ERdj4GT/GT mice?
4b. Which cell(s) of the osteogenic lineage affects B cell development? 168
4c. How does ERdj4 deficiency affect peripheral B cells? 169
4d. How does the loss of ERdj4 affect postosteoblasts and skeletal 170
development?
4di. Quantitation of osteogenic cells from ERdj4GT/GT mice 170
4dii. Why are postosteoblasts reduced in ERdj4GT/GT mice? 171
4diii. Does the loss of postosteoblasts affect skeletal development 171
in ERdj4GT/GT mice?
5. Molecular chaperones in the treatment of ER stress-related disease 172
xi 6. ERdj4 in cellular function and homeostasis 173
References 175
xii LIST OF TABLES AND FIGURES
CHAPER I PAGE
Figure 1 The BiP/HSP70 chaperone cycle 4
Figure 2 The mammalian ER-localized DnaJ proteins 7
Figure 3 The mammalian UPR 12
Figure 4 Mechanisms of ER stress-induced β-cell dysfunction 17
and apoptosis
Table 1 Diabetic phenotypes due to genetic alteration of ER 20
homeostasis
Figure 5 B1 and B2 development occur in distinct, overlapping waves 26
Figure 6 B1 cell development 27
Figure 7 B2 cell development in the bone marrow 29
Figure 8 Marginal and follicular B cell development 33
Table 2 B cell defects in mice that are deficient in microenvironmental 36
factors in the bone marrow
Figure 9 Microenvironmental niches for B cell development in the 37
bone marrow
Figure 10 The UPR in osteogenesis 43
CHAPER II
Figure 1 Generation of ERdj4GT/GT mice 93
Figure 2 Constitutive ER stress and decreased cell viability in 94
ERdj4GT/GT MEFs
xiii Figure 3 Hypoglycemia and growth retardation in ERdj4GT/GT mice 95
Figure 4 Elevated ER stress in pancreatic β-cells of ERdj4GT/GT mice 96
Figure 5 ERdj4 deficiency impairs insulin biosynthesis 97
Figure 6 Hypoinsulinemia causes glucose intolerance in ERdj4GT/GT mice 98
Suppl 1 Elevated ER stress in tissues of ERdj4GT/GT mice 99
Suppl 2 Ultrastructural evidence of ER stress in α-cells of ERdj4GT/GT mice 100
Suppl 3 ER stress in pancreatic β-cells of ERdj4GT/GT mice 101
Suppl 4 ER stress in pancreatic acinar cells of ERdj4GT/GT mice does 102
not impair function
Table 1. Genotype distribution of P21 progeny from heterozygous 103
intercrosses
Table 2. Genotype distribution of day 18.5 embryos from heterozygous 104
intercrosses
CHAPTER III
Figure 1 Bone-lining cells are reduced in ERdj4GT/GT mice 136
Figure 2 Primary osteogenic cells from ERdj4GT/GT mice have an 137
immature phenotype
Figure 3 B cell development is impaired in ERdj4GT/GT mice 138
Figure 4 ERdj4 deficiency in the bone marrow microenvironment 139
affects B cell development
Figure 5 Mature B cells are decreased in the spleen and peritoneal 140
cavity of ERdj4GT/GT mice
xiv Suppl 1 Skeletal development of ERdj4+/+ and ERdj4GT/GT embryos 141
Suppl 2 BMP2-treated ERdj4GT/GT MEFs have reduced osteoblast 142
differentiation potential
Suppl 3 B cells and erythrocytes are decreased in the bone marrow 143
of ERdj4GT/GT mice
Suppl 4 Bone marrow B cell proliferation and peripheral pro-B cells are 144
normal in ERdj4GT/GT mice
Suppl 5 Plasma cell differentiation and antibody production are normal 145
in ERdj4GT/GT mice
xv LIST OF ABBREVIATIONS
ALP Alkaline phosphatase
APRIL A proliferation-inducing ligand
ASK1 Apoptosis-signal-regulator 1
ATF Activating transcription factor
BAFF B cell activating factor
BAFFR B cell activating factor receptor
BCL10 B cell lymphoma 10
BCR B cell receptor
BiP Immunoglobulin binding protein
BMP2 Bone morphogenic protein 2
BTK Bruton’s tyrosine kinase bZIP basic leucine zipper
CHOP C/EBP-homologous protein
CLP Common lymphoid progenitor
CRE Cyclic AMP-responsive element
CTD Carboxy-terminal domain
CXCL12 CXC-chemokine ligand 12
CXCR4 CXC-chemokine receptor 4
DC Dendritic cell
EBF Early B cell factor eIF2α Eukaryotic initiation factor 2 alpha
ER Endoplasmic reticulum
xvi ERAD ER-associated degradation
ERSE ER stress response element
FACS Fluorescence activated cell sorting
FFA Free fatty acid
FLT3 Fms-related tyrosine kinase 3
GFP Green fluorescent protein
GLP Glucagon-like peptide
GRP Glucose-regulated protein
HPD His, Pro, Asp motif
HSC Hematopoietic stem cell
IAPP Islet amyloid polypeptide
IgH Immunoglobulin heavy chain
IgL Immunoglobulin light chain
IKKβ IκB kinase β
IRE1 Inositol-requiring enzyme 1
IRF Interferon regulatory factor
JNK c-Jun N-terminal kinase
MALT1 Mucosa-associated lymphoid tissue lymphoma translocation protein 1
MAML1 Mastermind-like 1
MIDY INS-gene-induced diabetes of youth
MIF1 Migration-inhibitory factor 1
MINT MSX2-interacting protein
MPP Multipotential progenitor
xvii MSC Mesenchymal stem cell
NBD Nucleotide binding domain
NEF Nucleotide exchange factor
Nemo NF-κb essential modulator
NF-κb Nuclear factor-kappa b
NIK NF-κb-inducing kinase
NKT Natural Killer T
NO Nitric oxide
OASIS Old astrocyte specifically induced substance
PAS Para-aortic splanchnopleura
PAX5 Paired box protein 5
PBA 4-phenyl butyric acid
PERK Protein kinase RNA (PKR)-like ER kinase
PI3K Phosphoinositide 3-kinase
PLCγ2 Phospholipase Cγ2
PTH Parathyroid hormone
RAG Recombination activating gene
RANKL Receptor activator of nuclear factor-κB ligand
RBP-Jκ Recombining binding protein suppressor of hairless
ROS Reactive oxygen species
SBD Substrate binding domain
SCF Stem cell factor
SHP Small heterodimer partner
xviii siRNA Small interfering RNA
SLC Surrogate light chain
SNP Single nucleotide pair
T1 Transitional 1
T2 Transitional 2
TI-2 T cell-independent type 2
TRAF2 TNF-receptor-associated factor 2
TRAP Tartrate-resistant acid phosphatase
TUDCA Tauroursodeoxycholic acid
XBP1 X-box-protein 1
UPR Unfolded protein response
UPRE Unfolded protein response element
VCAM1 Vascular adhesion molecule 1
xix
CHAPTER I
Molecular Chaperones and the UPR in Development and Metabolism:
Background, Significance and Hypothesis
1 1. Molecular Chaperones in the Endoplasmic Reticulum
Nearly one third of proteins destined for the cell surface, intracellular organelles, or secretion enter the endoplasmic reticulum (ER) to undergo protein folding and post- translational modifications1. Molecular chaperones are crucial components of the ER that direct protein transport across membranes, assist in folding/refolding of nascent proteins, regulate protein-protein interactions, and remove misfolded proteins from the
ER lumen for proteasomal degradation. The first identified ER chaperone, immunoglobulin binding protein (BiP), was discovered nearly three decades ago (refer to section 2a). Since then, BiP has been shown to cooperate with a diverse family of
DnaJ cochaperones, whose importance for ER homeostasis is only beginning to be appreciated.
Our laboratory identified the BiP cochaperone (ERdj4/DNAJB9) as one of the most highly upregulated genes in response to expression of two disease-associated mutations in surfactant protein C (SP-C) that are prone to misfolding and retention in the
ER lumen. Unlike other DnaJ proteins, ERdj4 preferentially associated with misfolded
SP-C, facilitating its removal from the ER via ER-associated degradation (ERAD)2. The significance of these findings prompted the study of ERdj4 in cellular homeostasis and function in vivo.
1a. BiP: an ER-localized HSP70 chaperone
BiP belongs to the HSP70 family of molecular chaperones, which are highly homologous in structure and function within and across species. HSP70 family members are a ubiquitous class of chaperones present in the cytosol, nucleus,
2 mitochondria and ER. BiP is involved in multiple ER processes, which include regulating the translocon pore3, folding nascent proteins4,5, targeting misfolded proteins for proteasomal degradation6,7, maintaining ER calcium stores8 and activating the unfolded protein response (UPR) under conditions of ER stress9,10. All of these functions, with the exception of maintaining ER calcium stores, require the ATPase activity of BiP, a conserved feature among HSP70 chaperones.
Like other HSP70 proteins, BiP contains an N-terminal nucleotide binding domain
(NBD) and C-terminal substrate binding domain (SBD), which actively communicate via an interdomain linker11. The SBD is in an open configuration when ATP is bound to the nucleotide binding domain (NBD), allowing for transient, low affinity interactions with hydrophobic residues of client proteins. ATP hydrolysis closes the SBD, resulting in tight association with bound substrate. When ADP is exchanged for ATP, the client is released and given the opportunity to fold. Several cycles of client-chaperone binding and release occur until the protein achieves a stable conformation. The ATPase activity of BiP requires cochaperones of the DnaJ family and nucleotide exchange factors
(NEFs) to stimulate client capture and dissociation, respectively (Figure 1). It has been postulated that the multifunctionality of BiP (and other HSP70 family members) is achieved through interaction with various DnaJ proteins12.
1b. DnaJ family of cochaperones
DnaJ proteins were discovered as cofactors for HSP70 in 1978 when the heat shock proteins GrpE and DnaJ were shown to modulate the ATPase activity of DnaK, an
HSP70 family member in Escherichia coli13-16. Since then, DnaJ proteins have emerged
3 Native protein
! DnaJ
Unfolded g SBD ! protein ! !
J domain ! ATP
a !
! BiP ! ADP ATP ATP f SBD ! !
b NBD ATP
ADP
! ! ATP ATP c ! ! Pi e ADP d
! NEF ! ! !
Figure 1. The BiP/HSP70 chaperone cycle. Unfolded protein interacts with the SBD of DnaJ proteins (a) or BiP (b). The J domain of DnaJ proteins associates with the NBD of BiP to stimulate ATPase activity (c), thereby resulting in ATP hydrolysis and closed conformation of the SBD of BiP (d). NEFs associate with BiP (e) to catalyze the exchange of ADP for ATP, which opens the SBD (f) and releases client protein. The client protein either achieves native conformation or reenters the chaperone cycle to continue folding (g). SBD, substrate binding domain; NBD, nucleotide binding domain;
NEF, nucleotide exchange factor.
4 as a structurally and functionally diverse family of cochaperones that play an essential role in many cellular processes.
1bi. Structure and classification
DnaJ proteins have been loosely categorized into three classes based on the presence or absence of structural domains. Class I members contain an N-terminal J domain, a glycine phenylalanine (Gly/Phe)-rich region, two zinc finger motifs and two carboxy- terminal domains (CTDI and CTDII). Class II members have a J domain and Gly/Phe- rich region, but do not possess zinc finger motifs, while class III members contain only a
J domain and do not fit the requirements for class I or class II17.
All DnaJ proteins encode the highly conserved J domain that contains a His, Pro,
Asp (HPD) motif necessary for the stimulation of the ATPase activity of BiP18,19. This interaction is also dependent on a positively charged helix located in the J domain that associates with a negatively charged loop on the NBD of BiP20. The functionality of the
Gly/Phe-rich region remains unclear, while the zinc finger motifs have been shown to be important for client binding and the ATPase activity of BiP21,22. The CTD domains have low sequence identity among DnaJ proteins, but are structurally similar, often by forming a hydrophobic pocket for association with client proteins23.
1bii. Functionality
The structural diversity of DnaJ proteins enables participation in a variety of cellular functions with or without binding to client proteins. Often their subcellular localization and J domain are sufficient; DnaJ proteins are positioned at ribosomes and translocons
5 of mitochondrial and ER membranes to recruit BiP and stimulate ATPase activity, which results in protein folding and/or polypeptide import. In addition to serving as a cochaperone to BiP, DnaJ proteins can interact with the client itself for many cellular processes, including transport across the ER membrane, folding of nascent proteins, remodeling of multimeric protein complexes and degrading unfolded/misfolded proteins12.
1biii. ER-localized DnaJ proteins
Yeast contain three ER-localized DnaJ proteins: Sec63p, Jem1p, and Scj1p. Sec63p is anchored in the ER lumen as a component of the translocon and functions to recruit BiP and assist in the translocation of nascent proteins18. Jem1p and Scj1p are luminal ER proteins upregulated under conditions of ER stress24. Yeast mutants deficient in both
Jem1p and Scj1p exhibited growth arrest, but deletion of either Jem1p or Scj1p individually did not affect cell growth25. Under conditions of ER stress, Scj1p interacts with BiP to facilitate protein folding26. Jem1p and Scj1p also cooperate with BiP to retrotranslocate misfolded proteins from the ER for degradation7,27.
To date, seven mammalian ER-localized DnaJ proteins have been identified and are ubiquitously expressed in vertebrate tissues28 (Figure 2). ERdj1 and ERdj2 are transmembrane proteins positioned at the translocon. ERdj1 recruits BiP to translating ribosomes, but also inhibits protein translation in the absence of BiP29-31. The function of ERdj2 at the translocon remains unclear; ERdj2 does not associate with translocation machinery in the presence of ribosomes32. ERdj3 and ERdj6 are soluble ER luminal proteins that bind unfolded/misfolded substrates and recruit BiP to assist in protein
6 Function UPR induction recruits BiP to ERdj1 No the translocon
ERdj2 unknown No
ERdj3 protein folding Yes
ERdj4 ERAD Yes
ERdj5 ERAD Yes
ERdj6 protein folding Yes
ERdj7 unknown No
J domain Tetratricopeptide repeat Gly/Phe region ER signal peptide Thioredoxin box Dimerization domain SANT domain Carboxy-terminal domain Sec63 domain Transmembrane domain
Figure 2. The mammalian ER-localized DnaJ proteins. Seven DnaJ proteins have been identified in the ER, playing an important role in transport, protein folding and degradation. The structural diversity among ERdj proteins likely drives diverse BiP- dependent and independent cellular processes. All members contain the highly conserved J domain; ERdj3 and ERdj4 are classified as class I and class II DnaJ proteins, respectively, while the remaining ERdjs are classified as class III members.
7 folding33,34. ERdj4 and ERdj5 are also soluble luminal proteins, but associate with
ERAD machinery to facilitate the removal of unfolded/misfolded proteins from the ER2,35-
37. ERdj7 is a transmembrane protein with unknown function, but reducing expression of ERdj7 did not induce ER stress, suggesting that it plays a minor role in protein folding38,39.
1biv. ERdj4
Hendershot et al. identified ERdj4 as a class II DnaJ family member through bioinformatic methods36. ERdj4 is a soluble ER luminal protein containing the signature
J domain, a Gly/Phe-rich region, and a C-terminal substrate binding domain36 (Figure
2). The HPD motif in the J domain of ERdj4 associates with BiP, thereby assisting BiP in achieving stable conformation with client proteins by stimulating ATP hydrolysis.
ERdj4 also interacts directly with unfolded (insulin2WT) and misfolded substrates
(insulin2C96Y, SP-CΔexon4), and reducing levels of ERdj4 slowed degradation of these substrates in vitro2,36. Further, ERdj4 associates with Derlin-1 and p97, components of
ERAD machinery2,35, to facilitate removal of clients for degradation by the cytosolic proteasome. The mechanism by which ERdj4 discriminates between unfolded and terminally misfolded substrates destined for ERAD remains unclear.
ERdj4 is ubiquitously expressed at low levels, but highly upregulated under conditions of ER stress2,36 by the UPR signal transducer X-box-protein 1 (XBP1)40,41.
The in vivo function of ERdj4 is entirely unknown, but given that ERdj4 mRNA is detected in secretory tissues28,36, it may play an important role in maintaining ER homeostasis in secretory cell types.
8 2. The Unfolded Protein Response
The UPR is an intracellular signaling pathway that is activated when protein load exceeds protein folding capacity in the ER lumen – a condition referred to as ER stress.
ER stress can result from pathological conditions that cause proteins to misfold, including genetic mutations, toxic compounds and oxidative stress. Physiologic stimuli such as nutrients, cytokines and hormones, can also cause ER stress by increasing the demand for protein synthesis. The UPR restores ER homeostasis by increasing ER size, attenuating protein translation and upregulating protein folding and degradation machinery.
2a. Discovery
The discovery of the UPR dates to 1974 when two 78 and 94 kDa proteins were induced in fibroblasts upon transformation with avian sarcoma virus42. These proteins were later designated as the glucose-regulated proteins GRP78 and GRP94, because their upregulation in tumor cells occurred in response to glucose deprivation in the media rather than an effect of cellular transformation43. Several other inducers of
GRP78 and GRP94 were identified including inhibitors of glycosylation, calcium ionophores and amino-acid analogues44. In 1983, Hass et al. independently discovered
GRP78 as an ER-localized protein that interacted with immunoglobulin heavy chain intermediates to facilitate assembly, thereby identifying the first ER chaperone termed
BiP, or immunoglobulin heavy chain binding protein45. The ‘unfolded protein response’ was finally coined in the early 1990’s by Gething and Sambrook46 after demonstrating that accumulation of malfolded influenza hemagglutinin protein in the ER efficiently induced GRP78/BiP and GRP9447. Conserved regulatory domains among GRPs
9 provided further evidence for a signaling pathway induced by ER stress47. However, despite identification of several UPR inducers, the molecular pathway(s) underlying the upregulation of ER chaperones was entirely unknown.
2b. Initial discovery of UPR signaling components in yeast
In Saccharomyces cerevisiae, BiP is encoded by the KAR2 gene, which contains an unfolded protein response element (UPRE) that induces BiP mRNA expression in response to accumulation of unfolded proteins48. Using the UPRE to select yeast mutants with defects in the UPR signaling pathway led to discovery of the first UPR signaling component, Ire1p49,50. Ire1p is a transmembrane receptor kinase, composed of an N-terminal domain located in the ER lumen that senses unfolded protein and a C- terminal protein kinase domain in the cytosol that activates transcription of KAR2 and other UPRE-containing genes. A basic leucine zipper (bZIP) transcription factor Hac1p was then identified as a downstream target of Ire1p by isolating genes that activated the
UPR when overexpressed in Ire1p-deleted yeast mutants51. Upon activation of the
UPR, cytosolic Hac1p mRNA is spliced51 by the endoribonuclease activity of Ire1p and re-ligated by Rlg1p, a tRNA ligase52. Spliced Hac1p mRNA is then translated and subsequently activates transcription of ER-resident genes including KAR2, PDI1a and
SCJ153, via direct binding to the UPRE51. Activation of the Ire1p/Rlg1p/Hac1p signaling pathway induces over 5% of the yeast genome, highlighting its importance in cellular function and homeostasis54.
2c. The mammalian UPR
10 2ci. The ER stress sensors: ATF6, PERK and IRE1
In contrast to yeast, which contains a single UPR signaling pathway
(Ire1p/Rlg1p/Hac1p), mammalian cells express three known UPR signal transducers:
ATF6, PERK and IRE1. These components were identified by Kaufman, Mori and Ron in the late 1990’s, laying the foundation for understanding how the mammalian UPR signaling pathway senses and responds to ER stress in order to maintain cellular homeostasis.
ATF6 is a type II transmembrane protein that contains an ER stress-sensing domain in the ER lumen. Following activation, ATF6 shuttles to the Golgi complex where it is cleaved by S1P and S2P proteases resulting in release of a cytosolic ATF6 fragment that contains a bZIP transcription factor55,56. The cytosolic ATF6 fragment then translocates to the nucleus to activate transcription of ER chaperones and components of ERAD by interacting with ER stress response elements (ERSE)57-59.
Mice ubiquitously express two isoforms of ATF6, ATF6α and ATFβ. Mice deficient in either isoform of ATF6 developed normally, while mice deficient in both isoforms were embryonic lethal, suggesting that ATF6α and ATFβ contain an overlapping function that is crucial for development58,60 (Figure 3).
The second ER stress sensor PERK is localized in the ER as a type I transmembrane protein with a luminal stress-sensing domain and a cytosolic protein kinase domain. In response to ER stress, oligomerization and trans- autophosphorylation of PERK results in phosphorylation of eukaryotic initiation factor 2
(eIF2α). eIF2α phosphorylation reduces protein load in the ER lumen by inhibiting the translation-initiation complex61. Further, phosphorylation of eIF2α upregulates the
11
ER lumen protein BiP
P IRE1 P P ! PERK P ATF6
P eIF2! ATF6 uXBP-1 cleavage ATF4 Golgi
Nutrient uptake, metabolism and apoptosis sXBP-1
Protein folding and degradation
Protein folding and degradation
Nucleus
Figure 3. The mammalian UPR. Unfolded/misfolded protein is detected by BiP in the
ER lumen; disassociation of BiP from the ER stress sensors IRE1α, ATF6 and PERK activates the UPR signaling cascade, ultimately resulting in an increase in ER size, attenuation of protein translation and upregulation of ER chaperones and ERAD machinery.
12 transcription factor ATF4, which induces genes involved in amino acid metabolism, redox status and apoptosis62. Approximately half of PERK-deficient mice died perinatally; surviving mice had postnatal growth restriction with defects in endocrine and exocrine pancreatic function and skeletal development63,64 (Figure 3).
Structurally similar to PERK, the third ER stress sensor IRE1 is also a type I transmembrane protein that contains a stress-sensing domain in the ER lumen and a protein kinase domain in the cytosol. IRE1 functions uniquely in the mammalian UPR by encoding a C-terminal endoribonuclease domain (similar to yeast Ire1p). Upon activation, IRE1 oligomerizes, trans-autophosphorylates and excises a 26 base pair intron in the mRNA of XBP1 (the mammalian homolog of Hac1p). Spliced XBP1 protein contains a C-terminal transactivation domain that interacts with UPREs of ER chaperones, ERAD machinery and genes involved in lipid biosynthesis65,66. The protein kinase activity of activated IRE1 also associates with TNF-receptor-associated factor 2
(TRAF2), which promotes apoptosis through JNK signaling67 (Figure 3).
There are two isoforms of IRE1: IRE1α and IRE1β. Although functionally identical, IRE1α is expressed ubiquitously, while IRE1β is predominantly expressed in the intestinal epithelium. IRE1α deficiency in mice resulted in embryonic lethality at
E10.5 due to defects in placental development68,69. XBP1-deficient mice also died during embryonic development at E12.5 due to liver hypoplasia70. When XBP1 was selectively expressed in the liver of XBP1-deficient mice to rescue embryonic lethality, postnatal lethality occurred within a few days of birth due to impaired production of pancreatic digestive enzymes71.
13 2cii. ER stress-induced apoptosis
During conditions of ER stress, the UPR restores cellular homeostasis by increasing the size of the ER (i.e. increased membrane synthesis), attenuating protein translation and upregulating protein folding/degradation machinery. Failure to resolve ER stress results in UPR-mediated activation of cell death signaling pathways. Phosphorylation of eIF2α by PERK induces transcription of the cell death mediator C/EBP-homologous protein
(CHOP)72, which downregulates the anti-apoptotic molecule BCL-273. IRE1α associates with TRAF2, ultimately resulting in activation of the proapoptotic molecule apoptosis- signal-regulator (ASK1) through c-Jun N-terminal kinase (JNK)67,74. IRE1α signaling through TRAF2 also activates caspase-12, which executes cell death under conditions of ER stress75.
3. β-cell Stress, the UPR and Diabetes
Pancreatic β-cells are highly secretory, insulin-producing cells, which frequently encounter physiological conditions that perturb ER homeostasis. Insulin biosynthesis occurs acutely at the translational level and maturation of proinsulin and insulin processing enzymes is a critical process that occurs within the ER76. Glucose can stimulate a 25-fold increase in insulin biosynthesis77,78, imposing a heavy load of unfolded/misfolded protein on the ER, leading to activation of stress pathways. The
UPR reduces ER burden of proinsulin biosynthesis by attenuating protein translation and increasing the expression of ER chaperones and ERAD machinery to assist in protein folding and degradation. However, excess nutrients (metabolic stress) and/or
14 insulin resistance induces chronic ER stress and hyperactivation of the UPR leading to
β-cell dysfunction and apoptosis, a hallmark of type 2 diabetes.
3a. Inducers of ER stress in β-cells
3ai. Glucose
Insulin resistance in type 2 diabetes is associated with chronic elevation of blood glucose levels, which compromises β-cell function and survival, a phenomenon known as glucotoxicity. Hyperglycemia results in an increase in reactive oxygen species
(ROS), which is associated with β-cell pathology79. High levels of glucose generate
ROS through several mechanisms, including glyceraldehyde autooxidation, glucosamine and hexosamine metabolism, sorbitol formation and oxidative phosphorylation80. Oxidative stress in β-cells is associated with a decrease in the insulin regulatory proteins, PDX-1 and MafA, resulting in reduced insulin gene expression, as well as impaired β-cell proliferation and survival81,82. Antioxidant treatment in rodent models of type 2 diabetes reduced circulating ROS and attenuated hyperglycemia79. Glucotoxicity is also associated with ER stress and UPR activation, which allows for increased proinsulin biosynthesis. However, upregulation of folding enzymes and chaperones by the UPR signaling pathway also contributes to ROS production and β-cell demise by increasing the oxidative protein folding machinery that drives disulfide bond formation83. Further, ROS production can inhibit protein folding by modifying substrates directly or inactivating chaperones. Since β-cells express low levels of antioxidant enzymes, proteins within the ER are particularly vulnerable to oxidation84 (Figure 4).
15 3aii. Free fatty acids
High fat diets and obesity are associated with increased levels of plasma free fatty acids
(FFAs)85. The saturated FFA palmitate causes ER stress and β-cell dysfunction by (1) reducing protein trafficking from the ER to the Golgi; (2) depleting ER calcium stores; (3) lowering fatty acid oxidation and triglyceride synthesis and (4) activating PKC-δ-JNK- mediated apoptosis86,87. Several studies have demonstrated that saturated FFAs activate the UPR, although the underlying mechanisms remain unclear. Palmitate activates the PERK/eIF2α signaling axis, resulting in upregulation of ATF4 and CHOP88, which mediates apoptosis under conditions of ER stress. There are discrepancies in the literature on whether palmitate activates the IRE1α/XBP1 and ATF6α signaling pathways89,90. However, Laybutt et al. reported activation of several XBP1-dependent genes in palmitate-treated insulinoma cells, including Edem1, Herpud1 and ERdj4, which assist in protein folding/degradation to reduce ER stress90-92 (Figure 4).
3aiii. Proinflammatory cytokines
Obesity is associated with chronic, low grade inflammation, due to proinflammatory cytokine production by adipocytes and leukocytes in adipose tissue93. Obesity causes an increase in circulating levels of FFAs, which can promote proinflammatory cytokine production by inducing the nuclear factor-κb (NF-κB) signaling pathway94.
Proinflammatory cytokines, including IL-1β, TNFα and IFNγ, stimulate the production of nitric oxide (NO) in pancreatic β-cells, which depletes intracellular calcium stores and causes ER stress95. Cytokine-induced NO production also activates the PERK/eIF2α signaling pathway, thereby inducing CHOP-mediated β-cell death96.
16
Figure 4. Mechanisms of ER stress-induced β-cell dysfunction and apoptosis.
Chronic glucose stimulation induces protein biosynthesis, which increases oxidative protein folding (generating ROS) and accumulation of unfolded/misfolded protein in the
ER. ROS production inhibits mobilization of calcium, ultimately resulting in mitochondrial-induced apoptosis. Hyperactivation of the UPR signaling cascade by either excess lipids or glucose results in degradation of proinsulin mRNA and ER stress- induced apoptosis (Modified from Back and Kaufman, Annual Review of Biochemistry,
2012).
17 3aiv. Misfolded protein
Mutations in the insulin gene that affect protein folding cause ER stress, β-cell failure and diabetes. The Akita and Munich diabetic mouse models have mutations in critical cysteine residues (C96Y and C95S) that are necessary for disulfide bond formation and proper folding of proinsulin97,98. Interestingly, 26 mutations in the human insulin gene are associated with autosomal dominant diabetes and are referred to as mutant INS- gene-induced diabetes of youth (MIDY). Similar to the mutations in mouse insulin,
MIDY mutations (one of which is C96Y) impair proinsulin folding in the ER often by disrupting disulfide bond formation. These insulin mutations in mice and humans resulted in proinsulin accumulation and unresolvable ER stress, leading to β-cell failure and apoptosis99 (Figure 4).
3av. Islet amyloid polypeptide
Deposition of islet amyloid polypeptide (IAPP) in pancreatic islets is strongly associated with the pathology of type 2 diabetes in humans. IAPP is a hormone that is normally co- secreted with insulin to regulate digestion. Human IAPP can form toxic amyloid deposits, which are associated with increased β-cell apoptosis through the activation of
CHOP and caspase-12100. Multiple studies have reported that human IAPP activated the UPR100,101; however, these findings are controversial as one study found that physiological levels of human IAPP did not induce ER stress102 (Figure 4).
3b. The UPR in β-cell dysfunction and diabetes
3bi. PERK/eIF2α
18 Genetic mutations in several components of the UPR are linked to diabetes in humans and mice103 (Table 1), suggesting that the UPR is a necessary component for β-cell development, homeostasis and survival. Humans with Wolcott-Rallison syndrome have an inactivating mutation in PERK that results in neonatal diabetes due to pancreatic hypoplasia and β-cell apoptosis104,105. A similar phenotype was observed in PERK- deficient mice; approximately 50% of PERK-deficient mice died neonatally, but surviving mice developed severe hyperglycemia within four weeks of birth due to impaired β-cell development63,64. PERK/eIF2α signaling was shown to be necessary for β-cell differentiation and proliferation during the fetal period in order to prevent postnatal diabetes. Further, conditional deletion of PERK after the neonatal stage did not affect
β-cell development or glucose homeostasis106. Mice harboring a homozygous mutation in the phosphorylation site of eIF2α (Ser51Ala; A/A mutants), the downstream effector of PERK signaling, die neonatally due to severe hypoglycemia associated with reduced glycogen storage, impaired gluconeogenesis and pancreatic β-cell deficiency107. The phenotype of PERK-deficient mice and eIF2α A/A mutants are different, suggesting that kinases other than PERK are important for eIF2α signal transduction.
3bii. IRE1α/XBP1
Several studies have indicated that the IRE1α/XBP1 signaling pathway is important for proinsulin biosynthesis and β-cell homeostasis. Under high glucose conditions for short periods of time (≤ 1 hour), IRE1α activation stimulated proinsulin biosynthesis. In contrast, prolonged elevation of glucose (≥ 24 hours) resulted in ER stress and IRE1α hyperactivation that suppressed proinsulin biosynthesis and upregulated CHOP, an
19 Table 1. Diabetes phenotypes due to genetic alteration of ER homeostasis Gene Mutation Genotype Species Effect Phenotype Client protein Ins Missense Het Human Misfolded proinsulin Neonatal diabetes Ins2 Akita/Munich Het Mouse Misfolded proinsulin Young adult onset diabetes Iapp hIAPP Transgene Mouse Aggregated hIAPP Young adult onset diabetes UPR signaling Perk Null Hom Mouse Deletion Young adult onset diabetes Perk Missense/deletion Hom Human Loss function Wolcott-Rallison syndrome Perk SNPs Polymorphism Human ? Type 1 diabetes eIF2! Ser51Ala Hom Mouse No phos. !-cell deficiency eIF2! Ser51Ala Het Mouse Reduced phos. High fat diet-induced diabetes Ire1! Null Hom Mouse Deletion Embryonic lethal, E10.5 Xbp1 Null Hom Mouse Deletion Embryonic lethal, E12.5 Xbp1 Null Het Mouse Deletion High fat diet-ins. resistance Xbp1 Null LivXBP1 transgene Mouse Xbp1 in liver only Neo. exocrine pancreas defects Xbp1 Conditional Xbp1 f/f;RIP-cre Mouse Xbp1 -/- !-cells !-cell deficiency and diabetes Atf6! Null Hom Mouse Deletion Normal Atf6" Null Hom Mouse Deletion Normal Atf6!/" Null Dual hom Mouse Deletion Embryonic lethal, E8 Atf6 SNPs Polymorphism Human ? Type 2 diabetes ER resident/chaperone Grp78 Null Het Mouse Deletion Attenuates HFD-induced obesity ERdj6/P58IPK Null Hom Mouse Deletion Adult diabetes Wfs1 Null Hom Mouse Deletion Adult diabetes Wfs1 Missense Hom Human Loss function Wolfram syndrome Het, Heterozygote; Hom, homozygote; Phos, phosphorylation; ?, unknown effect; ins, insulin; neo, neonatal Modifed from Scheuner and Kaufman, Endocrine Reviews, 2008 inducer of apoptosis108,109. Further, the endoribonuclease activity of IRE1α was capable of degrading insulin, insulin processing enzymes and ER chaperone mRNAs108,110.
Given that global deficiency of IRE1α or XBP1 was embryonic lethal in mice69,111, it has been difficult to assess the role of these signal transducers in vivo. Transgenic mice that selectively expressed XBP1 in the liver (XBP1-/-LivXBP1) to rescue embryonic lethality had normal islet structure and morphology at birth, but died shortly thereafter due to impaired production of pancreatic digestive enzymes71. Selective deletion of
XBP1 in β-cells of adult mice resulted in hyperglycemia associated with decreased production of proinsulin and β-cell deficiency. Interestingly, XBP1 deficiency in β-cells also resulted in hyperactivation of IRE1α, which negatively affected insulin production by stimulating degradation of insulin processing enzymes, ER chaperones and insulin itself110.
20 3biii. ATF6α
The role of ATF6α in β-cell homeostasis remains unclear. Two separate cohort studies had shown that single nucleotide pair (SNP) mutations in human ATF6α were linked to glucose intolerance and type 2 diabetes112,113. However, no β-cell dysfunction or diabetic phenotype has been described in ATF6α-deficient mice58,60. Interestingly, glucotoxicity upregulated ATF6α (as well as spliced XBP1, phosphorylated eIF2α and
CHOP), resulting in increased expression of a small heterodimer partner (SHP) that suppressed insulin gene expression114,115. Further, increased levels of activated ATF6α in β-cells caused apoptosis115. These data suggest that, under conditions of ER stress
(e.g. glucotoxicity), upregulation of the ATF6α signaling pathway contributes to β-cell dysfuncton and diabetes.
3biv. ER chaperones
ER chaperones also play an important role in maintaining β-cell homeostasis and function. Humans with a loss of function mutation in Wfs1, an ER-localized chaperone, developed Wolfram syndrome, a disease characterized by juvenile diabetes and neurological disorders. Wfs1 negatively regulates UPR signaling by facilitating the removal of ATF6α from the ER for proteasomal degradation116. ATF6α accumulated in the pancreas of Wfs1-deficient mice, which likely contributed to β-cell pathogenesis.
Wfs1-deficient mice were hyperglycemic, exhibiting increased ER stress and β-cell apoptosis117.
Mice deficient in another ER-resident protein, ERdj6/p58IPK, were also hyperglycemic due to increased β-cell apoptosis118. ERdj6/p58IPK has multiple functions
21 in the ER, including protein folding and negatively regulating eIF2α kinase. Since deficiency of ERdj6/p58IPK worsened the diabetic phenotype in Akita mice, which expressed mutant proinsulin119, it is likely that this chaperone plays an important role in proinsulin folding.
3c. The UPR and insulin resistance
Several UPR signal transducers have been associated with ER stress and insulin resistance in peripheral tissues. PERK, eIF2α and BiP were increased in liver and adipose tissue of either high-fat diet (HFD)-induced or genetic (ob/ob) mouse models of obesity. Increased ER stress was associated with impaired insulin signaling, which was mediated through the IRE1α-JNK signaling cascade. XBP1 heterozygous mice carrying a null mutation in one allele were both hyperglycemic and hyperinsulinemic after exposure to HFD, consistent with impaired insulin signaling in peripheral tissues120.
Interestingly, mice with a heterozygous null mutation in GRP78/BiP were resistant to
HFD-induced obesity due to increased energy expenditure and glucose metabolism121.
In summary, several physiological and pathological stimuli activate the UPR in order to alleviate the ER burden associated with insulin biosynthesis. However, hyperactivation of the UPR is linked to β-cell dysfunction and apoptosis. Disruption of the UPR causes diabetes in mice and humans due to defects in β-cell proliferation or survival, highlighting the importance of the UPR in β-cell homeostasis and function.
4. B cell Subsets and Effector Functions
22 B cells are a crucial component of the immune system, possessing both innate and adaptive properties. B cells present antigens to T cells and NK cells, as well as secrete cytokines and other soluble factors to modulate immune responses. During an adaptive immune response, B cells undergo clonal expansion to generate high affinity, antigen- specific antibodies of unique istoypes. B cells can be classified into B1 and B2 lineages, which are distinguished by anatomical location, cell surface phenotype and effector function.
4a. B1 cells
B1 cells make up approximately 5% of total B cells in mice and reside mainly in the pleural and peritoneal cavities, as well as the intestinal lamina propria. In the pleural and peritoneal cavities, B1 cells are classified as CD11b+IgMhiIgDlow and can be further divided into B1a (CD5+) and B1b (CD5-) subsets122,123. The B cell receptor (BCR) of B1 cells is more restricted due to reduced expression of terminal deoxynucleotidyl transferase (TdT), an enzyme that inserts nucleotides into D-J and V(DJ) regions of immunoglobulin heavy (IgH) and light (IgL) chains124. Upon activation, B1a cells migrate to the spleen and downregulate CD5 before differentiating into plasma cells125.
B1 cells are effectors of innate immunity, responding predominantly to TI-2 antigensa.
Haas et al. eloquently demonstrated the functional differences between B1a and B1b cells; after infection with Streptococcus pneumoniae, B1a cells rapidly secreted natural
IgM antibodies for early protection, while B1b cells generated antigen-specific antibodies necessary for pathogen clearance and long-term immunity126.
a T cell-independent type 2 (TI-2) antigens: polysaccharide antigens that engage the BCR to generate antigen-specific antibody responses
23 4b. B2 cells: Follicular and marginal zone B cells
B2 cells are subdivided into follicular and marginal zone B cells, which develop from transitional B cells and differ significantly in effector function. Both express the B cell lineage markers CD19 and B220, but otherwise are defined as IgMlowIgDhiCD21intCD23hi and IgMhiIgDlowCD21hiCD23low, respectively.
4bi. Follicular B cells
Follicular B cells continuously circulate through the follicles of the spleen and lymph nodes, responding to T cell-dependent and T cell-independent antigens. T cell- dependent antigens induce germinal center reactions in the follicles, where B cells rapidly proliferate while undergoing affinity maturation and class switch recombination of immunoglobulins. As a result, plasma cells and memory B cells exit the germinal center expressing high-affinity BCRs and immunoglobulins of different isotypes127.
4bii. Marginal zone B cells
Marginal zone B cells reside in the white pulp of the spleen adjacent to the marginal sinus, enabling quick responses to blood-borne pathogens in the absence of T cells.
They represent only 5-10% of total B cells in the spleen and differ from follicular B cells in their low threshold for activation, proliferation and differentiation to antigen. Similar to
B1 cells, marginal zone B cells respond to TI-2 antigens to differentiate into short-lived plasma cells that secrete IgM128. Marginal zone B cells can present protein antigens through MHC class II molecules to activate CD4+ T cells129 and lipid antigens through
CD1d (an MHC class I-like molecule) to natural killer T (NKT) cells130. Mice deficient in
24 marginal zone B cells had an increased susceptibility to bacterial infections, implicating their importance in innate and adaptive immunity131.
5. B Cell Development
B1 and B2 cell development occurs in sequential, overlapping waves beginning early in mouse embryogenesis (Figure 5). B1 cell progenitors can be generated from the yolk sac and para-aortic splanchnopleura (PAS) at day 9.5 of gestation before emergence of hematopoietic stem cells (HSCs); thus, the precursors that give rise to B1 cell progenitors in the yolk sac and PAS are unknown. Later in gestation, HSCs give rise to both B1 and B2 cells in the fetal liver132,133, but B1 cell development predominates.
Once HSCs begin to seed the bone marrow by day 15 of gestation, it becomes the primary location for B2 cell development throughout postnatal life122,134.
5a. B1 cell development
B1 cell development occurs predominantly during embryogenesis in the fetal liver. A B1 cell progenitor was recently identified as Lin-AA4.1+CD45-/lowCD19+, which was detected earliest in the yolk sac, peaked in number in the fetal liver and then declined postnatally135,136. B1 cell progenitors proliferated in response to thymic stromal lymphopoietin (TSLP) and differentiated only into B1a and B1b subsets136 (Figure 6). B cell progenitors in adult bone marrow that are CD19+B220- also gave rise to B1b cells, but not B2 cells136.
During the neonatal period, transitional B cells in the spleen primarily generate
B1 cells. Unlike transitional B2 cells (section 5biii), transitional B1 cells do not require B
25
Figure 5. B1 and B2 development occur in distinct, overlapping waves. The first developmental wave (1) initiates in the YS and the PSp before emergence of HSCs. It is unclear (dashed line) whether B1 progenitors in the YS and PSp develop into mature adult B1 cells. The second wave of development (2) begins in the fetal liver and fetal bone marrow where HSCs predominantly generate B1 progenitors and subsequently, mature B1 cells. Whether B1 and B2 cells develop from a single HSC or multiple stem cells remains unknown. Some B-ALLs, which are a B1 cell malignancy, may arise in utero during peak B1 cell development. The third developmental wave (3) occurs in adult bone marrow and results primarily in the production of B2 cells. YS, yolk sac; Psp, para-aortic splanchnopleura; HSCs, hematopoietic stem cells; CLP, common lymphoid progenitor; B-ALL, B cell acute lymphocytic leukemia134.
26 Fetal liver Neonatal Spleen Serous cavities
B1a IgM+ Transitional Natural IgM antibody strong BCR and production in response to T NF-!b signaling cell-independent antigens IgMhigh IgDlow AA4.1+ CD11b+ IgM+ CD5+ TSLP CD21int CD23-/+ B1b
Generate antigen-specific antibodies for long-term B1 immunity
progenitor high - IgM Lin low + IgD AA4.1 + -/low CD11b B220 CD5- CD19+
Figure 6. B1 cell development. B1 progenitors in the fetal liver respond to TSLP to differentiate into IgM+ B1 cells that are later detected in the neonatal spleen as transitional B1 cells. Transitional B1 cells are dependent on strong BCR engagement and classical NF-κb signaling in order to develop into B1a or B1b subsets, which home to the peritoneal and pleural cavities.
27 cell activating factor (BAFF) for survival137, but rather strong BCR engagement and the classical NF-κb signaling pathway136,138 (Figure 6).
5b. B2 cell development
5bi. Early B2 cell differentiation in the bone marrow
A seminal paper by Hardy et al. in the early 1990’s defined the stages of B2 cell development in adult mouse bone marrow based on the following: (1) cell surface phenotype, (2) differences in functional activity and (3) immunoglobulin gene rearrangement status139. Based on these criteria, Hardy described six different stages of B cell development in adult mouse bone marrow, referred to as fraction A through fraction F (Figure 7). Immediately prior to B lineage commitment, common lymphoid progenitors (CLPs) have the ability to differentiate into B cells, T cells and NK cells, but cannot differentiate into myeloid or erythroid lineages140. These cells lack lineage specific surface markers (B220, CD11b, Gr1 and Ter119), but express IL-7Rα and c-kit.
Pre-pro B cells (fraction A) are the earliest defined stage of B cell development based on the expression of the B lineage marker B220 (but not CD19) and the absence of immunoglobulin gene rearrangement. Since NK and T cells also express B220, the pre- pro B cells are distinguished from these cell types by the cell surface marker AA4.1, which is expressed on the earliest B cell progenitors through the immature stage of development. The pre-pro B cells then differentiate into the mitotically active pro-B cells
(fractions B/C), which undergo DJ or (V)DJ IgH rearrangements by upregulating expression of recombination activating genes (RAG). Progression from pro-B cells
(fraction B/C) to pre-B cells (fraction D) is dependent on a fully functional pre-B cell
28 Large Small Pre-Pro B Pro-B Pro-B Pre-B Pre-B Immature Mature
pre-BCR IgM IgM
A B C C’ D E F IgD
Figure 7. B2 cell development in the bone marrow. Fraction A through fraction F are defined based on cell surface phenotype, differences in functional activity and immunoglobulin gene rearrangement status. RAG, recombination activating gene; IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain.
29 receptor (pre-BCR). The pre-BCR complex consists of the rearranged IgH and a surrogate light chain (SLC), which is comprised of λ5 and V-preB, as well a non- covalent Igα and Igβ heterodimer. Signaling through the pre-BCR is a critical checkpoint during B cell development; pre-B cells expressing rearranged IgH that form poor BCRs are eliminated, while those that signal effectively temporarily downregulate
RAG and undergo rapid proliferation. Mice deficient in genes that are crucial for IgH gene rearrangement (RAG1, RAG2, scid) or components of the pre-BCR signaling complex (λ5, Igβ) have a block in B cell development at the pro-B cell stage (Figure 7).
After proliferation, resting pre-B cells (fraction D) increase RAG expression to undergo
IgL gene rearrangement and differentiate into immature B cells (fraction E), which express IgM on the cell surface. The transition from immature (fraction E) to mature
(fraction F) B cells is another critical checkpoint where the B cell undergoes selection with self-antigen. Crosslinking the BCR with antigen results in elimination, anergy, or receptor editing of autoreactive clones. Receptor editing involves an additional round(s) of IgL gene rearrangement to eliminate self-reactivity. The degree to which the BCR binds antigen ultimately determines B cell fate; high affinity binding to self-antigen results in elimination, whereas low affinity or no interaction with self-antigen typically results in survival, anergy, or receptor editing (Figure 7).
5bii. Transcriptional regulation of early B2 cell development
Several transcription factors are important for B2 cell development by influencing lineage commitment, immunoglobulin gene rearrangement and induction of signaling receptors141. The transcription factors PU.1 and Ikaros are essential for the
30 differentiation of CLPs. Low levels of PU.1 drive B lineage commitment and regulate the expression of fms-related tyrosine kinase 3 (FLT3), IL-7Rα and c-kit, key signaling receptors necessary for B cell development. Ikaros directly regulates TdT and λ5, genes essential for immunoglobulin gene rearrangement and pre-BCR signaling, respectively. The transcription factors E2A, early B cell factor (EBF), interferon regulatory factor 4 (IRF4), IRF8 and paired box protein 5 (PAX5) are necessary for immunoglobulin gene rearrangement and pre-BCR signaling. E2A induces recombination at the IgH locus through chromatin remodeling, while EBF influences the expression of Igα, Igβ and VpreB genes, components of the pre-BCR complex. IRF4 and IRF8 bind directly to the IgL locus to induce DNA gene rearrangement142. PAX5 is crucial for V-DJ IgH gene rearrangement and also regulates B cell-specific genes including CD19 and Igα. Expression of PAX5 is also necessary for B lineage commitment; deletion of PAX5 resulted in the ability of pro-B cells to differentiate into osteoclasts, macrophages, dendritic cells (DCs) and NK cells143.
5biii. Transitional B cells
Immature B cells leave the bone marrow and migrate to the spleen where they go through transitional stages (T1 and T2) before differentiation into mature follicular or marginal zone B cells. T1 and T2 cells are defined by cell surface phenotype, a short half-life and an increased susceptibility to apoptosis. T1 cells (AA4.1+IgM+IgD-
CD21lowCD23-) enter the spleen through the central arterioles and marginal sinus, where they migrate to B cell follicles in response to CXCL13144. In the follicles, T2 cells
31 (AA4.1+IgMhiIgDhiCD21midCD23+) gain the ability to recirculate and upregulate IgD and
CD23, but still express the immature cell surface marker AA4.1145.
T1 cell differentiation into T2 cells is dependent on BCR engagement, BAFF and non-canonical NF-κb signaling (Figure 8). BAFF production by stromal and myeloid cells promotes survival by inducing expression of anti-apoptotic proteins, including BCL-
2, BCL-xL and MCL-1146. BAFF deficiency blocks T1 to T2 differentiation, resulting in dramatic deficiencies in mature B cells147. Signaling through the BAFF receptor
(BAFFR) predominantly activates the noncanonical NF-κb signaling pathway, leading to induction of p52 through NF-κb-inducing kinase (NIK) and IκB kinase α (IKKα). NIK- deficient bone marrow chimeras lacked marginal zone B cells, while IKKα-deficient bone marrow chimeras had reduced follicular B cells148,149, suggesting that the noncanonical
NF-κb signaling pathway plays a role in follicular and marginal B cell development.
5biv. Follicular versus marginal zone B cell fate
Follicular versus marginal zone B cell development from the T2 cell stage is dependent on BCR engagement and signaling through Notch2, NF-κb and BAFFR (Figure 8).
Defects in the BCR signaling components Bruton’s tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2) were associated with reduced numbers of follicular B cells, but normal numbers of marginal zone B cells150,151. In contrast, genetic alterations that increased BCR signaling, such as CD22b-deficient mice, drastically reduced marginal zone B cells152. Thus, weak BCR signaling favors marginal zone B cell development, while stronger BCR signaling favors follicular B cell development153.
b CD22 inhibits BCR signaling
32 Adult Spleen
- Antigen presentation MZ - Respond to TI-2 antigens of blood-borne pathogens to Tonic BCR secrete IgM BAFF and NF-!b signaling weak BCR, BAFF IgMhigh !b and Notch signaling low NF- IgD T1 T2 CD21high strong BCR CD23low BAFF and NF- AA4.1+ AA4.1+ !b IgM+ IgMhigh signaling - Germinal center reactions in IgD- IgDhigh FO response to T cell-dependent CD21low CD21int antigens CD23- CD23+ - Respond to T cell- low IgM independent antigens IgDhigh CD21int CD23high
Figure 8. Marginal and follicular B cell development. Immature B cells from the bone marrow migrate to the spleen to undergo further differentiation into mature B cell subsets. Transitional B cell differentiation requires tonic BCR engagement, BAFF survival signals and the alternative NF-κb pathway. Marginal zone or follicular B cell development depends on the strength of BCR engagement and signaling through
BAFF, NF-κb and/or Notch. This cell fate decision results in two B cell subsets that differ in anatomical location, cell marker expression and function. T1 or T2, transitional
B cell; BAFF, B cell activating factor; MZ, marginal zone B cell; FO, follicular B cell.
33 Once BCR signal strength determines B cell fate, NF-κb, Notch and BAFFR signaling drive the development of follicular or marginal zone B cells. In follicular B cells, strong BCR signaling through BTK and phosphoinositide 3 kinase (PI3K) contributes to activation of the canonical NF-κb signaling components p50 and RelA.
Null mutations in the canonical NF-κb signaling pathway, including the NF-κb essential modulator (Nemo) and IKKβ, led to a dramatic loss in all peripheral B cell subsets, implicating the canonical NF-κb signaling pathway in mature B cell development154.
Marginal B cell development also requires BAFFR signaling and the BCL10-MALT1c-
CARMA1 complex to activate the canonical NF-κb signaling pathway. Mice deficient in
BCL10, MALT1, or CARMA1 had impaired development of B1 and marginal zone B cells155-157.
Notch signaling is also an important signal transduction pathway in the development of marginal zone B cells. Mice deficient in components of the Notch signaling pathway, including Notch2, recombining binding protein suppressor of hairless
(RBP-Jκ) and mastermind-like 1 (MAML1), had defects in marginal zone B cell development131,158-160, while inactivation of the Notch suppressor MSX2-interacting protein (MINT) resulted in increased marginal zone B cells161.
5c. The UPR in B cell development
Several components of the UPR influence both early and late stages of B cell development. A seminal paper by Reimold and Glimcher described a physiological role for the IRE1α/XBP1 signaling pathway in plasma cell differentiation and antibody secretion. XBP1-deficient B cells became activated and proliferated normally in c Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1)
34 response to antigen, but failed to differentiate into plasma cells. Plasma cell deficiency resulted in lower levels of basal immunoglobulins, as well as an inability to generate immunoglobulins against T cell-dependent or T cell-independent antigens70. IL-4, ATF6 and BLIMP1 induced the expression of spliced XBP1 in primary B cells, driving plasma cell differentiation162-164. Activation of IRE1α/XBP1 in primary B cells promoted ER expansion by upregulating chaperones and enzymes involved in transport, folding, glycosylation and trafficking165. Spliced XBP1 also induced expression of IL-6, a plasma cell growth factor162.
Interestingly, IRE1α signaling has also been shown to be important for early B cell development; pre-B and mature B cells were reduced in the bone marrow of IRE1α-
/-RAG2-/- chimeric mice. The developmental block at the pro-B cell stage was associated with defective IgH gene rearrangement due to reduced expression of TdT,
RAG1 and RAG269. Deficiency of TRAF2, a downstream effector molecule of IRE1α, also caused a block at the pro-B cell stage of development, but a causal relationship with impaired immunoglobulin gene rearrangement was not ascertained166. Spliced
XBP1 was also detected at the pro- and pre-B cell stage of development, but the significance of this finding remains unknown167.
6. Growth Factors and Cellular Niches Required for B Cell Development
Microenvironmental niches in the bone marrow support B cell lineage commitment and differentiation from HSCs. This is a highly dynamic process that depends upon stromal cell contact, soluble growth factors and cytokines throughout each developmental stage168 (Table 2 and Figure 9).
35 6a. B cell growth factors
6ai. CXCL12
Eqawa et al. identified CXC-chemokine ligand 12 (CXCL12) as the first soluble factor required after commitment to the B lineage. CXCL12 is highly expressed by stromal cells in the bone marrow. The primary receptor for CXCL12 is CXC-chemokine receptor
4 (CXCR4)169,170, whose expression is Table 2. B cell defects in mice that are deficient in 167 primarily limited to brain and lymphoid microenvironmental factors in the bone marrow . tissues171,172. Both CXCL12 and
CXCR4-deficient mice died perinatally and had identical defects in neural and vascular tissue173,174. Deficiency of either CXCL12 or CXCR4 resulted in severely reduced numbers of pre-pro-
B cells in the bone marrow of fetal and adult mice173-175 (Table 2). Ma et al. further demonstrated that B cell precursors were generated in the bone marrow of CXCR4- deficient mice, but egressed to the periphery before maturation was complete176. Thus,
CXCL12 secretion by stromal cells attracts and retains B cell precursors for development in the bone marrow. Further, plasma cell localization in the bone marrow is also dependent on CXCL12177,178.
6aii. IL-7
IL-7 was identified in 1988 by Namen et al. as the first environmental factor to induce rapid proliferation of B cell precursors179. The IL-7 receptor is comprised of the IL-7Rα
36 Bone marrow *!+%
Dendritic cells Mature Immature B B cells
Blood Megakaryocyte vessel IL-7-secreting cell
Pre-B
Eosinophil !"#&% !"#$% Plasma cell
'()!"%
,-,"./% Pro-B CAR cell
Pre-pro-B
HSC CLP
Osteoblast Bone
Figure 9. Microenvironmental niches for B cell development in the bone marrow.
Stromal and immune cell types support B cell development by expressing cell-surface molecules and secreting growth factors and cytokines that promote retention, survival and differentiation in the bone marrow. CAR, CXCL12-expressing reticular cells; HSCs, hematopoietic stem cells; CLP, common lymphoid progenitor; MIF, migration inhibitory factor; APRIL, a proliferation-inducing ligand.
37 chain and the common γ chain, which also signals for IL-2, IL-4, IL-9 and IL-15. IL-7 signaling in B cell precursors induces IgH gene rearrangement180, the pro-survival factor
MCL-1181 and EBF to maintain B cell differentiation182. Deficiency of either IL-7 or IL-
7Rα resulted in severe defects in both T and B cell development; B cell precursors were drastically reduced starting at the pro-B cell stage (Table 2)183,184. Although the number of CLPs and pre-pro-B cells were normal in IL-7 and IL-7Rα-deficient mice, their ability to differentiate in vitro was severely impaired185, implicating that IL-7 signaling is necessary starting at the CLP stage of development.
6aiii. FLT3L
The fms-like-tyrosine-3 ligand (FLT3L) is a cell surface protein that often undergoes proteolytic cleavage to generate a soluble form, both of which are active in vivo186,187.
FLT3L-deficient mice had drastically reduced CLPs in the bone marrow188, suggesting that FLT3L promotes survival of CLPs (Table 2). The receptor for FLT3L is highly expressed during early lymphoid development through the pre-B cell stage187,189,190.
FLT3 receptor-deficient mice had a milder defect in B lymphopoiesis; pre-pro-B, pro-B and pre-B cells were significantly reduced, while immature and mature B cell populations were normal191. Interestingly, mice double deficient in IL-7Rα and FLT3L had a more severe defect in the early and late stages of B cell development than single deficient mice, suggesting that IL-7 and FLT3L synergistically regulate B lymphopoiesis192.
6aiv. RANKL
38 The receptor activator of nuclear factor-κB ligand (RANKL) is predominantly expressed by osteocytes193 and drives osteoclast differentiation by interacting with the RANKL receptor (RANK) on osteoclast precursors. Mice deficient in either RANK or RANKL had osteopetrosis due to defective osteoclastogenesis, but also lacked all lymph nodes and had severe defects in early T and B cell development194,195 (Table 2). B cell development is dependent on RANKL expression by cells of the hematopoietic lineage195, despite much lower RANKL expression than that of bone cells193.
Reconstitution of RAG1-deficient mice with RANKL-deficient bone marrow resulted in reduced pre-B and immature B cells, while wild-type bone marrow transplanted into
RANKL-deficient recipients rescued B cell development195. The cellular source of
RANKL and downstream effects of RANKL signaling in B cell development remains entirely unclear.
6av. SCF
Stem cell factor (SCF) is a ligand for the tyrosine kinase receptor c-kit, which is highly expressed by hematopoietic progenitor cells and fully differentiated mast cells. The
SCF-c-kit signaling axis is crucial for the development of many hematopoietic cell lineages including multipotential progenitors (MPPs), T cells, mast cells and erythrocytes. Although B cell development in c-kit-deficient mice was normal during the fetal and neonatal period196, several studies indicate a role for SCF-c-kit signaling in adult B lymphopoiesis. Mice with spontaneous null mutations in c-kit and SCF died from anemia within 10 days of birth197, but overexpression of erythropoietin in c-kit- deficient mice (Wepo mice) rescued lethality198. Adult Wepo mice had normal numbers
39 of pre-pro B cells, but reduced CLPs, pro-B and pre-B cells in the bone marrow198
(Table 2). Further, discovery of a viable and healthy c-kit-deficient mouse that arose from heterozygous crossing of c-kit mutants had a similar phenotype as Wepo mice198.
6b. Bone marrow niches that support B cell development
6bi. CAR niche: reticular cells expressing CXCL12
The CXCL12-CXCR4 signaling axis is crucial for both early and late B cell development
(section 6ai). Mice containing a green fluorescent protein (GFP) gene knocked into the
CXCL12 locus (CXCL12-GFP mice) were used to identify the distribution of cells that expressed CXCL12 in the bone marrow during B cell development. CXCL12-GFP expression was detected on vascular adhesion molecule 1 (VCAM-1)-positive reticular cells, termed CAR cells, which make up approximately 0.27% of all nucleated cells in the bone marrow. CAR cells are uniformly distributed throughout the bone marrow and are morphologically similar to dendritic cells, forming a matrix with their wide net of cytoplasmic processes178,199. CXCL12-expressing cells are found in tight association with pre-pro B cells and plasma cells. Conditional depletion of CAR cells impaired
CXCL12 and SCF production, which was associated with lower numbers of HSCs,
CLPs and pro-B cells200. Secretion of CXCL12 by CAR cells recruits eosinophils and megakaryocytes, which also support the plasma cell niche by secreting a proliferation- inducing ligand (APRIL) and/or IL-6201,202 (Figure 9).
6bii. IL-7-expressing cells
40 IL-7 signaling plays an important role in early B cell development by influencing CLP and pro-B cell differentiation (section 6aii). Similar to CAR cells, IL-7-expressing cells are scattered throughout the bone marrow and are VCAM-1-positive, but they have a fibroblast-like morphology, lacking the long dendrites of CAR cells. While CAR cells interact specifically with pre-pro B cells and plasma cells, IL-7-expressing cells associate tightly with pro-B cells in the bone marrow178 (Figure 9).
6biii. Dendritic cells
Resident dendritic cells are clustered around blood vessels in the blood marrow, secreting migration-inhibitory factor 1 (MIF1), which supports the survival of recirculating mature B cells203 (Figure 9). Ablation of bone marrow dendritic cells resulted in reduced levels of mature B cells, which was rescued by expression BCL-2, an anti- apoptotic protein.
6biv. Osteoblasts
Bone-forming cells of the osteoblast lineage express VCAM-1, CXCL12 and IL-7, which are sufficient to support all stages of B cell development from HSCs in vitro204 (Figure
9). Induction of CXCL12 and IL-7 in primary osteoblasts was dependent on parathyroid hormone (PTH)204,205 and ablation of the PTH signaling pathway in osteoprogenitors reduced pro-B and pre-B cells in the bone marrow206. Conditional depletion of osteoblasts decreased bone marrow cellularity, which was associated with reduced numbers of HSCs, B cells and erythrocytes207.
41 Osteoblasts can differentiate into osteocytes, which regulate bone resorption by osteoclasts through RANKL193. Inhibiting bone resorption in mice with zoledronic acid treatment impaired all stages of B cell development in the bone marrow, while HSCs and CLPs were normal208. However, treatment with zoledronic acid was also associated with reduced numbers of osteoblasts, which likely contributed to the B cell phenotype208.
7. The UPR in Osteoblast Differentiation
Osteoblasts develop from mesenchymal stem cells (MSCs) and commitment to the osteoblast lineage requires the transcription factors Runx2 and Osterix and the canonical Wnt signaling pathway. The primary function of osteoblasts is the synthesis, deposition and mineralization of bone, although they also play an important role in hematopoiesis (section 6biv). Mature osteoblasts can become either quiescent bone- lining cells, undergo apoptosis, or differentiate into osteocytes, which are surrounded by extracellular matrix and regulate bone remodeling. Several components of the UPR are essential for osteoblast differentiation and function (Figure 10).
7a. PERK/eIF2α
A role for the UPR (section 2ci) in skeletal development was revealed when a loss of function mutation in human PERK resulted in growth retardation, epiphyseal dysplasia and osteoporosis104,105. A PERK-deficient mouse model duplicated the skeletal defects observed in humans, providing a resource to study the underlying mechanisms of PERK in skeletal development. Neonatal PERK-deficient mice had reduced mineralization in
42 Runx2
MSC Osteoblast Osteocyte ATF6 Bone-lining cell
Osterix
IRE1!/XBP1 ATF4 PERK/eIF2!
Figure 10. The UPR in osteogenesis. The UPR drives osteoblast differentiation by regulating Osterix, a transcription factor necessary for mature osteoblast development.
ATF6 induction by Runx2 influences osteoblast function by controlling the expression of osteocalcin, an extracellular matrix protein. Genetic alterations affecting the UPR cause growth retardation and skeletal dysplasias in mice and humans.
43 flat and long bones, implicating impaired ossification. Osteoblasts were decreased in bone tissue of PERK-deficient mice, which was associated with impaired proliferation and differentiation of primary osteoblasts in vitro209. PERK-deficient osteoblasts also had signs of increased ER stress including ER distention and retention of type I collagen, which surprisingly, did not activate the UPR or increase cell death64,209.
7b. ATF4
ATF4 is a downstream mediator of PERK/eIF2α signaling and a crucial component of osteoblast biology. ATF4 influences bone formation by regulating osteoblast-specific gene expression, amino acid transport and synthesis of type I collagen210. Osteoblast differentiation is also dependent on ATF4, which regulates the expression of Osterix
(Figure 10), an essential transcription factor of mature osteoblast development211.
ATF4-deficient osteoblasts had defects in proliferation and survival, implicating that
ATF4 is necessary for osteoblast homeostasis211,212.
Although PERK/eIF2α can induce ATF4, there is conflicting data on whether
ATF4 functions downstream of PERK in osteoblasts. Downstream target genes of
ATF4 that regulate amino acid metabolism (and ultimately, osteoblast function) were decreased in ATF4-deficient osteoblasts, but were unchanged in PERK-deficient osteoblasts209. Further, PERK deficiency resulted in accumulation of type I collagen209, while ATF4-deficient osteoblasts had impaired production of type I collagen210.
However, other studies reported that ATF4 activation was impaired in PERK-deficient osteoblasts and ATF4 rescued the defects in mineralization and osteoblast differentiation observed in PERK-deficient osteoblasts213. Further studies are needed to
44 identify the underlying mechanisms involving PERK and ATF4 in osteoblast differentiation and function.
7c. ATF6
A recent study identified a role for the UPR signal transducer ATF6 in osteoblast differentiation. The transcription factor Runx2, which induces the differentiation of
MSCs into osteoblasts, increases expression of ATF6 by binding to the ATF6 promoter region. Activation of ATF6 stimulates osteoblast differentiation through upregulation of osteocalcin and increases extracellular matrix production214. However, defects in skeletal development in ATF6-deficient mice have not been described.
The old astrocyte specifically-induced substance (OASIS) is structurally homologous to ATF6 and modulates UPR signaling by activating the transcription of genes containing ERSE and cyclic AMP-responsive elements (CRE)215. OASIS regulates type I collagen production by binding to an ERSE in the type I collagen promoter. OASIS deficiency impaired osteoblast differentiation and function in vitro and
OASIS-deficient mice were osteopenic in association with decreased type I collagen in bone tissue216.
7d. IRE1α/XBP1
The IRE1α/XBP1 signaling pathway is upregulated during bone morphogenetic protein
2 (BMP2)-induced osteoblast differentiationd. XBP1 induces the expression of Osterix, a transcription factor necessary for osteoblast differentiation217. IRE1α or XBP1
d BMP2 induces osteoblast differentiation by upregulating osteoblast-specific genes including Runx2 and Osterix
45 deficiency impaired osteoblast differentiation in vitro. However, given that both IRE1α and XBP1-deficient mice were embryonic lethal69,70, it remains unclear whether this arm of the UPR plays a role in postnatal skeletal development in vivo.
8. Summary and Hypothesis
Highly secretory cells, including pancreatic β-cells, plasma cells and osteoblasts, require the UPR to maintain ER homeostasis in the face of increased protein synthesis.
The UPR signal transducers PERK/eIF2α, ATF6 and IRE1α/XBP1 reduce protein load by attenuating protein translation and upregulating protein folding and degradation machinery. Genetic mutations affecting the UPR cause defects in glucose metabolism, skeletal development and antibody-mediated immune responses in mice and humans, highlighting the importance of this signaling pathway in highly secretory cells.
ERdj4 was identified a decade ago by Shen et al. as a BiP/GRP78 cochaperone expressed most highly in secretory tissues and significantly upregulated in response to
ER stress36. Since then, our laboratory and others have demonstrated that ERdj4 is induced by the UPR to assist in the removal of unfolded and misfolded substrates from the ER lumen. This conclusion is supported by: (1) upregulation of ERdj4 by
IRE1α/XBP1 signaling in response to terminally misfolded protein, (2) association of
ERdj4 with unfolded (insulin2WT) and misfolded (insulin2C96Y, SP-CΔexon4) substrates, (3) interaction of ERdj4 with ERAD machinery and (4) accumulation of unfolded/misfolded protein in the absence of ERdj42,35.
Although previous studies identified the function of ERdj4 in vitro, the biological relevance of ERdj4 was entirely unknown. The essential role of the UPR in highly
46 secretory cells, as well as prominent expression of ERdj4 in secretory tissues, led to the generation of the central hypothesis that underlies the studies in this thesis: the chaperone activity of ERdj4 is required for homeostasis and function of secretory cells in vivo.
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65
CHAPTER II
Deficiency of ERdj4 is associated with perinatal lethality,
constitutive ER stress and glucose intolerance
66 Deficiency of ERdj4 is associated with perinatal lethality, constitutive ER stress and glucose intolerance
Jill M. Fritz*, Mei Dong*, Emily P. Martin, Cheng-Lun Na and Timothy E. Weaver
Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, and the
University of Cincinnati College of Medicine, Cincinnati, OH 45229
Correspondence: Timothy E. Weaver, 3333 Burnet Ave., Cincinnati, OH 45229; Phone:
513-636-7223; Email: [email protected]
Supported by NIH awards HL103923 and HL086492
*These authors contributed equally to this study
Manuscript in preparation
67 Abstract
ERdj4 maintains homeostasis during conditions of ER stress by facilitating
removal of unfolded/misfolded substrates from the ER lumen for degradation by the
proteasome. To elucidate the function of ERdj4 in vivo, the ERdj4 allele was disrupted
by a gene trap (ERdj4+/GT) leading to hypomorphic expression of the encoded protein in
mice homozygous for the trapped allele. ERdj4GT/GT mice exhibited significant perinatal lethality associated with growth retardation and hypoglycemia. Immature islets and reduced glucagon and insulin in pancreas of newborn ERdj4GT/GT mice indicated a delay in endocrine pancreatic development. Surviving adult mice exhibited constitutive ER stress in both non-secretory and secretory cell types and an increased susceptibility to cell death following induction of ER stress. ERdj4 deficiency resulted in altered morphology of pancreatic islets including cytoplasmic vacuolation and α-cell hyperplasia. Pancreatic β-cells from ERdj4GT/GT mice exhibited evidence of ER stress
including ER dilation and increased expression of spliced XBP1. Accumulation of
proinsulin in the ER and an increased number of immature granules in β-cells from
ERdj4GT/GT mice indicated a defect in the insulin secretory pathway. In addition to
insulin, ERdj4 associated with the insulin processing enzymes Pcsk1, Pcsk2 and CPE.
ERdj4GT/GT mice were glucose intolerant, resulting from hypoinsulinemia. Exogenous insulin effectively lowered blood glucose levels consistent with a disruption in the insulin secretory pathway. Together, these results indicate that the chaperone activity of ERdj4 is required for normal growth, development, and metabolism.
68 Introduction
Nascent proteins destined for the cell surface, intracellular organelles or
secretion enter the endoplasmic reticulum (ER) where they undergo chaperone-
mediated protein folding to achieve a stable conformation. Highly secretory cells,
including pancreatic acinar and β-cells, plasma B cells, and serous and mucous cells of
the salivary gland, invoke the unfolded protein response (UPR) to increase ER folding
capacity and maintain homeostasis in the face of increased protein load and
consequent ER stress1-4. The UPR reduces ER burden by attenuating protein translation and upregulating the machinery involved in protein folding and ER-
associated degradation (ERAD). The ER transmembrane sensors, protein kinase RNA
(PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol
requiring kinase 1 (IRE1) detect accumulation of unfolded or misfolded protein in the ER
lumen and transduce signals across the ER membrane to alleviate ER stress. PERK
kinase activation results in phosphorylation of eukaryotic translation initiation factor-2
(eIF2α), which inhibits translation initiation, thereby reducing the load of newly
synthesized proteins within the ER. Activation of ATF6 results in relocation to the Golgi,
proteolytic cleavage and release of a cytosolic ATF6 fragment that translocates to the
nucleus to activate transcription of UPR target genes including ER chaperones. The
activated endoribonuclease domain of IRE1 removes an unspliced intron from X-box
binding protein-1 (XBP1) mRNA leading to translation of a transcription factor that
upregulates chaperones involved in protein folding as well as components of the ERAD
machinery. Failure of the UPR to resolve ER stress can trigger apoptosis5,6.
69 Molecular chaperones are critical components of the ER quality control
machinery that distinguish between unfolded, correctly folded and terminally misfolded proteins within the ER. BiP, an ER-localized member of the Hsp70 family, is a multifunctional chaperone that assists in the folding of nascent proteins7,8, facilitates
targeting of misfolded proteins for proteasomal degradation9,10 and plays a key role in activating the UPR in response to ER stress11,12. BiP contains a C-terminal substrate- binding domain (SBD) that binds unfolded substrates with low affinity via interaction with exposed hydrophobic regions. Hydrolysis of ATP bound to the N-terminal nucleotide- binding domain (NBD) of BiP results in high affinity binding of the SBD with substrate.
Exchange of ADP for ATP releases the unfolded substrate from BiP allowing the substrate to continue folding13. A critical arginine residue located on the surface of the
NBD in the ATP-bound form is necessary for communication with the SBD, as well as
interaction with cochaperones of the DnaJ family14,15.
ER-localized DnaJ (ERdj) homologues induce high affinity substrate binding by
interacting with the NBD of BiP and stimulating ATP hydrolysis. While some ERdjs
interact with BiP independently of substrate, other ERdjs bind substrates directly and
recruit BiP16-19. ERdjs facilitate protein folding and/or degradation of newly translocated
(ERdj1-3), unfolded (ERdj3 and ERdj6) or misfolded (ERdj4 and ERdj5) substrates,
often by interacting with translocation or ERAD machinery20. ERdj4 is a soluble type II
DnaJ protein that binds BiP through a His-Pro-Asp (HPD) motif in the J domain and likely binds directly to misfolded substrates through a C-terminal glycine/phenylalanine- rich region21-23. ERdj4 is ubiquitously expressed at very low levels24,25, but is highly upregulated in response to ER stress21,23 by the UPR signal transducer XBP126,27.
70 ERdj4 binds to unfolded (insulin2WT) or misfolded (insulin2C96Y, SP-CΔexon4 and SP-
CL188Q) substrates to facilitate their removal from the ER for degradation by the proteasome21,22. In the current study, we report that deficiency of ERdj4 is associated with decreased survival, growth retardation and delayed endocrine pancreatic development in newborn mice, and hypoinsulinemia and glucose intolerance in adult
mice. Consistent with these findings, constitutive ER stress was observed in a variety
of tissues and cell types in mutant mice. Taken together, these results reveal an
important and unexpected role for ERdj4 in adaptation to ER stress associated with
growth, development and metabolism.
71 Results
Generation of ERdj4 gene trap mice⎯Since previous studies demonstrated significant expression of ERdj4 in secretory tissues23, we hypothesized that the chaperone activity
of ERdj4 would be important for the homeostasis and function of secretory cells in vivo.
Chimeric mice were generated from embryonic stem (ES) cells harboring a gene trap
(GT) cassette inserted into the ERdj4 locus (Fig. 1A). Offspring carrying the GT allele
were backcrossed eight generations to the 129P2 or C57BL/6 strains. Tail genomic
DNA (gDNA) isolated from the progeny of ERdj4+/GT intercrosses was used to confirm the presence of WT and/or GT alleles (Fig. 1B). Only one or two copies of the GT cassette were present in ERdj4+/GT or ERdj4GT/GT mice, respectively, confirming that
ERdj4 was the only locus that was disrupted (Fig. 1C). Insertion of the GT cassette into intron 1 of the ERdj4 locus (confirmed by sequencing) resulted in decreased expression of ERdj4 mRNA (Fig. 1D) and a corresponding increase in beta-galactosidase mRNA from the GT cassette (Fig. 1E) in MEFs isolated from E13.5 ERdj4GT/GT mice. Variable expression of ERdj4 mRNA in tissues from adult mice indicated that the insertion of the
GT cassette resulted in a hypomorphic allele (Fig. 1F).
Elevated ER stress and increased susceptibility to cell death in ERdj4GT/GT MEFs⎯To
determine if ERdj4 deficiency enhances ER stress, splicing of XBP1 mRNA was
assessed in control and tunicamycin-treated MEFs isolated from ERdj4+/+ and
ERdj4GT/GT mice. The spliced form of XBP1 mRNA was elevated at baseline in
ERdj4GT/GT MEFs compared to ERdj4+/+ controls (Fig. 2A, 0 hrs). Following treatment
with tunicamycin, which induced ER stress by inhibiting N-linked glycosylation, splicing
of XBP1 mRNA was enhanced in both ERdj4GT/GT and ERdj4+/+ MEFs (Fig. 2A).
72 Consistent with constitutive ER stress in ERdj4GT/GT MEFs, BiP and IRE-1α proteins
were elevated at baseline (Fig. 2B, 0 hrs). Treatment with tunicamycin or the proteasome inhibitor MG-132 increased the expression of BiP and IRE-1α in both
ERdj4GT/GT and control MEFs, but the effect was clearly more pronounced in ERdj4GT/GT
MEFs. In contrast, the concentration of another ER chaperone, calnexin, was similarly
regulated in control and ERdj4GT/GT MEFs under both unstressed and stressed
conditions (Fig. 2B). Ultrastructural analyses of ERdj4GT/GT MEFs revealed ER
distention and large Golgi inclusions providing further evidence of constitutive ER stress
(Fig. 2C). In the absence of exogenous stressors, survival of ERdj4GT/GT MEFs and control cells was similar (Fig. 2D). However, when ERdj4GT/GT MEFs were treated with
MG-132, tunicamycin or thapsigargin (an inhibitor of calcium transport) for 48 hours, cell viability was significantly decreased in a dose-dependent manner compared to ERdj4+/+
controls (Fig. 2D). In keeping with this finding, overexpression of ERdj4 was previously
reported to protect against cell death induced by ER stress28.
Hypoglycemia and growth retardation in ERdj4GT/GT mice⎯Perinatal lethality was
observed in ERdj4GT/GT progeny derived from ERdj4+/GT intercrosses of 129P2xC57BL/6,
129P2 and C57BL/6 strains (Table 1). Genotype distribution at E18.5 approximated
Mendelian ratios indicating that lethality resulting from homozygosity of the GT allele
occurred perinatally (Table 2). ERdj4GT/GT embryos were smaller (Fig. 3A) and E18.5 body weights were significantly decreased compared to control animals (Fig. 3B).
Analysis of blood glucose levels in newborn mice indicated that ERdj4GT/GT mice were
severely hypoglycemic (Fig. 3C), something that likely contributed to death. Pancreatic
islets in newborn ERdj4GT/GT mice formed immature cord-like structures in contrast to
73 spherical islets observed in ERdj4+/+ mice (Fig. 3D)29. Glucagon and insulin were also significantly decreased in pancreatic tissues of ERdj4GT/GT neonates (Fig. 3E-F),
providing further evidence of delayed endocrine pancreatic development. Surviving
male and female mice often displayed signs of a vestibular disorder, which included a
head tilt accompanied by abnormal circling behavior and hyperactivity. Surviving
female ERdj4GT/GT mice were infertile. All subsequent experiments were conducted using littermates derived from heterozygous crosses in the mixed 129P2xC57BL/6 genetic background, unless otherwise indicated.
Constitutive ER stress is associated with histological abnormalities in pancreatic islets of ERdj4GT/GT mice⎯In order to determine if ERdj4 deficiency enhanced ER stress in vivo, ERdj4+/GT mice were crossed to ER stress-activated indicator (ERAI) transgenic mice30. GFP protein (resulting from XBP1 splicing and expression of XBP1/GFP fusion
protein) was detected in multiple organs including the lungs and kidneys of
ERdj4GT/GTERAI+ mice (Fig. S1A). Ultrastructural analyses revealed ER distention and
Golgi inclusions in renal tubular epithelial cells (Fig. S1B) and serous cells of the
salivary gland (Fig. S1C). GFP and IRE-1α proteins were also increased in pancreatic
lysates from ERdj4GT/GTERAI+ mice compared to ERdj4+/+ERAI+ controls (Fig. 4A). GFP
immunofluorescence localized to β-cells in the islets of pancreatic sections from both
ERdj4+/+ERAI+ and ERdj4GT/GTERAI+ mice (Fig. 4B), with more GFP-positive cells and
increased fluorescence intensity in mutant mice. GFP fluorescence was not detected in pancreatic α-cells of ERdj4+/+ERAI+ or ERdj4GT/GTERAI+ mice (Fig. S2A) although ultrastructural evidence of ER stress was detected (Fig. S2B). Histological analyses of pancreatic sections from ERdj4GT/GT mice identified vacuolated cells in the islets (Fig.
74 4C). Ultrastructural analyses of islets revealed ER distention, enlarged mitochondria
and Golgi inclusions in both α- and β-cells of ERdj4GT/GT mice (Fig. S2B, S3A). An increased number of glucagon-positive α-cells were detected in the mantle and core of
islets from ERdj4GT/GT mice compared to controls (Fig. 4D and S2A). Glucagon protein was significantly increased in pancreatic extracts of adult ERdj4GT/GT mice (Fig. 4E)
consistent with α-cell hyperplasia and increased glucagon granules (Fig. 4D and S2B).
Further, plasma glucagon was significantly increased in ERdj4GT/GT mice compared to
controls (Fig. 4F). Hypomorphic expression of ERdj4 mRNA was confirmed in islets
isolated from ERdj4GT/GT mice (Fig. 4G), linking the loss of ERdj4 in pancreatic islets to
elevated ER stress.
While histology of ERdj4GT/GT acinar cells appeared normal at 8 weeks of age
(Fig. S4A), cytoplasmic vacuolation was observed in pancreatic acinar cells from 8-
month-old ERdj4GT/GT mice (S4B). Ultrastructural analyses of pancreatic acinar cells revealed dilated ER and multiple immature zymogen granules in ERdj4GT/GT mice (Fig.
S4C), although this did not appear to interfere with acinar cell function, as reflected by similar serum lipase and amylase levels in ERdj4GT/GT and control mice (Fig. S4D).
ERdj4 deficiency impairs insulin biosynthesis⎯Since ERdj4 was previously reported to
associate with insulin21, we assessed whether ERdj4 deficiency caused β-cell stress by
increasing proinsulin retention in the ER. Although total insulin protein in pancreatic
extracts of ERdj4GT/GT mice was normal (Fig. S3B), proinsulin and BiP colocalization was enhanced in pancreatic β-cells, indicating accumulation of proinsulin in the ER (Fig.
5A). Metabolic labeling of islets isolated from ERdj4GT/GT mice showed a modest increase in proinsulin protein compared to controls (Fig. 5B); however, proinsulin was
75 likely underrepresented due to significant α-cell hyperplasia in ERdj4GT/GT islets. To
determine whether ERdj4 associates with components of the insulin/glucagon secretory
pathways, HEK293 cells were cotransfected with ERdj4 and Pcsk1, Pcsk2, glucagon or
CPE. In contrast to insulin, ERdj4 did not associate with glucagon (Fig. 5C), but
consistently formed complexes with the insulin processing enzymes Pcsk1, Pcsk2 and
CPE (Fig. 5C), which generate mature insulin in secretory granules. Ultrastructural analyses of β-cells in ERdj4GT/GT mice revealed an increase in translucent (immature)
insulin granules (Fig. S3A), suggesting a disruption in proinsulin processing by Pcsk1,
Pcsk2 and/or CPE.
Hypoinsulinemia causes glucose intolerance in ERdj4GT/GT mice⎯Since increased ER stress and defective proinsulin processing were observed in β-cells of ERdj4GT/GT mice, endocrine pancreatic function was evaluated by measuring blood glucose and insulin after fasting. Although basal blood glucose levels were normal (Fig. 6A), glucose remained elevated in ERdj4GT/GT mice following glucose administration (Fig. 6B).
Importantly, insulin effectively lowered blood glucose levels in ERdj4GT/GT mice,
suggesting that ERdj4 deficiency leads to a decrease in mature insulin rather than a
defect in the insulin signaling pathway (Fig. 6C). Plasma insulin was significantly
decreased at baseline in ERdj4GT/GT mice (Fig. 6D, 0 min), but did not affect basal blood glucose levels (Fig. 6A). Following glucose administration, plasma insulin increased slightly in ERdj4GT/GT mice, but remained significantly lower than controls (Fig. 6D, 15
and 30 min). Plasma proinsulin:insulin ratios were also significantly increased in
ERdj4GT/GT mice (Fig. 6E), providing further evidence of a disruption in proinsulin processing coincident with ER stress.
76 Discussion
ERdj4 is a soluble ER chaperone protein expressed at low levels in a wide range of cells22-24. In response to ER stress, expression of ERdj4 is rapidly upregulated to assist in the disposal of an as yet poorly defined subset of misfolded proteins via ERAD21,23.
In this study, we showed that hypomorphic expression of ERdj4 in ERdj4GT/GT mice
resulted in perinatal lethality associated with growth retardation and hypoglycemia.
Immature islets and decreased glucagon and insulin in the pancreas of newborn
ERdj4GT/GT mice indicated a delay in endocrine pancreatic development. Constitutive
ER stress was observed in multiple cell types and tissues of surviving ERdj4GT/GT mice
in association with accumulation of immature granules in secretory cells and increased
susceptibility to cell death. These results strongly suggest that ERdj4 plays an
important role in ER homeostasis beyond facilitating disposal of terminally misfolded
proteins. Consistent with this hypothesis, we previously reported that ERdj4 interacts
with wild-type insulin21; in keeping with this observation, insulin accumulated in the ER of β-cells from ERdj4GT/GT mice. A disruption in the insulin secretory pathway elevated
ER stress leading to hypoinsulinemia and glucose intolerance in ERdj4GT/GT mice.
Collectively, these results demonstrate an important and unanticipated role for ERdj4 in
adaptation to ER stress associated with growth, development and metabolism.
Hypomorphic expression of ERdj4 resulted in normal Mendelian distribution of
genotypes at E18.5; in contrast, survival of newborn ERdj4GT/GT pups was significantly
decreased in a strain-dependent manner. Neonatal lethality can arise from defects in
respiration, suckling or metabolism31. ERdj4GT/GT pups did not display overt signs of
respiratory distress and contained milk in their stomachs, consistent with normal
77 respiration and suckling. However, newborn ERdj4GT/GT pups were significantly smaller
and severely hypoglycemic, suggesting that a defect in glucose homeostasis
contributed to lethal nutrient deprivation in the perinatal period. Immature islet
morphology and reduced levels of insulin and glucagon indicated a delay in endocrine
pancreatic development in newborn ERdj4GT/GT mice. Overall, the loss of perinatal
ERdj4 expression was strongly associated with neonatal demise, although the molecular pathway(s) underlying the perinatal survival function of this chaperone remains unclear.
Adult ERdj4GT/GT mice exhibited glucose intolerance resulting from
hypoinsulinemia. Proinsulin accumulated in the ER consistent with ERdj4 associating with insulin and the insulin processing enzymes Pcsk1, Pcsk2 and CPE. Notably, immature insulin granules and plasma proinsulin:insulin ratio were increased suggesting that, in addition to proinsulin, ERdj4 may also facilitate maturation of insulin processing enzymes. A similar phenotype (hypoinsulinemia and increased proinsulin:insulin ratio) was recently described for mice in which XBP1 was selectively deleted in β-cells32.
Dysregulation of proinsulin processing was attributed to degradation of mRNAs
encoding insulin processing enzymes as well as loss of chaperones, including ERdj4.
Thus, retention of proinsulin and possibly its processing enzymes may contribute to β-
cell stress and, ultimately hypoinsulinemia.
In contrast to newborn mice, glucagon was significantly elevated in pancreatic
tissues and plasma of ERdj4GT/GT mice in association with α-cell hyperplasia and ultrastructural evidence of increased glucagon granules. It remains unclear whether
ERdj4 deficiency disrupts proglucagon processing in α-cells. In contrast to insulin,
78 ERdj4 did not associate with glucagon; however, it did interact with Pcsk2 and CPE,
which are responsible for proglucagon processing in α-cells33. To date, we have not
identified any metabolic derangements arising from altered α-cell structure/function in
adult ERdj4GT/GT mice. However, a recent study revealed a possible explanation for α-
cell hyperplasia in ERdj4GT/GT mice by demonstrating that increased metabolic stress
caused β-cells to dedifferentiate into progenitor cells that subsequently adopted the α-
cell fate34.
ERdj4 can directly bind client proteins21, and recruit BiP to promote substrate solubilization and/or folding23. Several lines of evidence previously suggested that
ERdj4 is an ERAD-specific chaperone. First, expression of ERdj4 is normally low and rapidly upregulated in response to ER stress23,28, including stress resulting from
expression of a terminally misfolded protein21. Second, ERdj4 exhibited prolonged association with misfolded SP-C but not with the corresponding wild-type protein21.
Third, misfolded SP-C co-precipitated in a complex containing ERdj4 and the cytosolic
ATPase p97/VCP21. Fourth, ERdj4 was shown to transiently associate with derlin-122.
Taken together, these results suggest that ERdj4 may function in cooperation with the
retrotranslocation machinery late in the ERAD pathway. The results of the current
study, demonstrating widespread ER stress in cells/tissues of ERdj4GT/GT mice, were therefore unexpected. One possible explanation is that ERdj4 associates with proteins that are abnormally prone to misfolding and/or fold more slowly than other substrates, leading to prolonged ERdj4/client interaction and elimination via ERAD under conditions of stress. Alternatively, it is possible that ERdj4 is required for productive folding of
79 some wild-type substrates as well as elimination of unfolded/misfolded substrates from
the ER lumen.
Diabetes pathogenesis has been linked to failure of the UPR to properly manage
ER stress, resulting in β-cell failure or insulin resistance in peripheral tissues. The UPR
is mediated by ER stress sensors that maintain ER homeostasis by attenuating protein
translation (PERK/eIF2α) and upregulating expression of chaperones to assist in protein
folding, maturation and disposal of misfolded substrates via ERAD (IRE-1α/XBP1 and
ATF6)5,6. Mutations in PERK and ATF6α are associated with disrupted glucose homeostasis and diabetes in humans35-39. Furthermore, deficiency of XBP1, PERK or
ERdj6/p58IPK (an XBP1 target gene and ERdj/HSP40 family member) resulted in spontaneous diabetes in mice due to a failure in development and/or function of β- cells3,32,40-42. In this study, we showed that decreased expression of another XBP1
target gene and HSP40 family member, ERdj4, caused glucose intolerance associated
with increased ER stress in pancreatic β-cells. Administration of insulin effectively
reduced blood glucose levels in ERdj4GT/GT mice, indicating that ERdj4 deficiency did
not impair insulin signaling or glucose uptake in peripheral tissues and confirming a
pathologic role for β-cell dysfunction in these mice. Further, increased expression of
sXBP1 was localized to β-cells of ERdj4GT/GT mice, similar to findings in pancreatic islets of type 2 diabetic patients and diabetic mouse models43-45. Overall, ERdj4 plays an important role in β-cell homeostasis by promoting maturation of insulin and possibly insulin processing enzymes. Given the evidence of constitutive ER stress in other cells and organs of ERdj4GT/GT mice, it is likely that the chaperone activity of ERdj4 also
extends to a broad range of unfolded protein substrates.
80 Materials and Methods
Mice. ES cells harboring a gene trap (GT) cassette inserted into the ERdj4 locus (Bay
Genomics, ES cell line KST256) were purchased from the Mutant Mouse Regional
Resource Center. GT ES cells were injected into blastocysts to generate chimeras at
the University of Cincinnati Gene Targeting Facility. Animals were backcrossed eight
generations onto the 129P2 or C57BL/6 genetic backgrounds. With the exception of
perinatal lethality studies, all experiments in this study used 129P2×C57BL/6
littermates. The presence of the GT cassette was confirmed by PCR using primers
designed for WT and GT alleles (WT: 5’-AAG CCC AGT AAT AAC CCA ACT TAT C-3’;
GT: 5’-TGC TGG AGT CTA GCT ACT TAT CCA C-3’; WT/GT: 5’-CTG TCT TTT CTG
TGT TTG GCT AGA A-3’). ERAI mice were provided by RIKEN BRC and the presence
of the ERAI transgene was confirmed by PCR using primers designed by the supplier
(5’-GAA CCA GGA GTT AAG ACA GC-3’; 3’-GAA CAG CTC CTC GCC CTT GC-5’).
All mice were housed in a pathogen-free barrier facility with free access to food and
water. Animal procedures were performed under protocols approved by Cincinnati
Children’s Hospital Medical Center Institutional Animal Care and Use Committee.
RNA Isolation and RT-PCR. Total RNA was isolated from cells and tissues using the
RNeasy Plus Mini Kit (Qiagen) and cDNA was synthesized using iScript cDNA
Synthesis Kit (Bio-Rad). XBP1 was detected in MEFs by standard RT-PCR using
XBP1-specific primers (5’-GTG GTT GAG AAC CAG GAG TTA AGA-3’; 3’-AGA ATC
TGA AGA GGC AAC AGT GTC-3’). PCR products were separated on 4% agarose gels and visualized by ethidium bromide staining. Quantitative RT-PCR was performed with
25 ng of cDNA per reaction on the ABI 7300 system with TaqMan Assays (Applied
81 Biosystems) for mouse ERdj4 (Mm01622956_s1), a custom-designed beta-
galactosidase/neomycin resistance region of the GT cassette and mouse β-actin
(endogenous control, Mm00607939_s1). Relative quantitation was assessed using the
SDS Software (Applied Biosystems).
Gene Copy Number Assay. Tail genomic DNA (gDNA, 20 ng) was prepared in
quadruplicate as recommended by Applied Biosystems. The reaction mix included a
custom-designed TaqMan Copy Number Assay targeted for the beta-galactosidase
region of the GT cassette and a TaqMan Copy Number Reference Assay specific for
the mouse transferrin receptor (Applied Biosystems). Absolute quantitation was performed with the ABI 7300 system followed by gene copy number analysis with SDS and CopyCaller software (Applied Biosystems).
Immunoblot Analysis. Tissue lysate preparation and immunoblot analyses were performed as previously described46. Briefly, tissue was harvested and homogenized in
RIPA buffer (Teknova) containing protease inhibitor cocktail (Sigma). Homogenates were centrifuged at 5,000 g for 10 minutes to clear tissue debris and protein
concentration in the supernatants was assessed using the Micro BCA Kit (Pierce).
Lysates were analyzed by SDS-polyacrylamide gels under reducing conditions,
transferred to nitrocellulose membranes and immunoblotted with antibodies specific for
IRE-1α (Cell Signaling), GFP (Invitrogen), BiP (Cell Signaling), calnexin (Cell Signaling),
and β-actin (Seven Hills Bioreagents).
Cell Culture, Transfection and Coimmunoprecipitation. MEFs were isolated from
ERdj4+/+ and ERdj4GT/GT embryos on day 13.5 as described by Conner47. Cells were
cultured in DMEM with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml
82 streptomycin and 200 mM L-glutamine and passaged at least three times before use in
experiments. Cell viability was assessed (CellTiter 96 AQueous One Solution Cell
Proliferation Assay, Promega) following treatment with tunicamycin (CalBiochem), thapsigargin (CalBiochem) or MG-132 (CalBiochem) for 48 hours.
Human ERdj4-HA was cloned and inserted into a pIRES-EGFP vector
(Clontech), as previously described21. Human Pcsk1 (NM_000439), Pcsk2
(NM_002594), GCG (NM_002054) and CPE (NM_001873) containing C-terminal Myc-
DDK (FLAG) tags in a pCMV6-Entry vector were purchased from OriGene. HEK293 cells per 10cm plate were transfected with 1ug of each construct using Lipofectamine
2000 (Invitrogen) per manufacturer recommendations. Cells were harvested 48 hours after transfection in 50mM Tris, 150 mM NaCl and 1% Triton X-100 with 1X mammalian protease inhibitors (Sigma). Cell lysates were briefly sonicated, centrifuged at 20,000 g for 10 minutes and quantitated using the Direct Detect system (Millipore). Cell lysates were immunoprecipitated with α-FLAG M2 Affinity Gel (Sigma) and eluted proteins were analyzed by SDS-PAGE under reducing electrophoretic conditions followed by Western blotting with antibodies directed against HA (Santa Cruz).
Histology and Electron Microscopy. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin and 5µm sections were cut for H&E staining and immunofluorescence as previously described46. For immunofluorescence, slides underwent antigen retrieval by boiling in 10mM citrate buffer (pH 6.0) for 20 minutes followed by blocking in normal donkey serum (Jackson ImmunoResearch) for two hours. Sections were stained with guinea pig anti-insulin (ab7842, Abcam), rabbit anti- glucagon (ab932, Millapore) and anti-chicken GFP (ab13970, Abcam) followed by
83 application of secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594
(Invitrogen). Image stacks were acquired by 0.25 µm step distance using a Nikon C1 Si
Confocal Imaging System and processed for deconvolution to remove image blurring
using Autoquant X 2.2 (Media Cybernetics).
For transmission electron microscopy, cells and tissues were processed as previously described48. Briefly, samples were fixed in 2% paraformaldehyde (Electron
Microscopy Sciences ‘EMS’), 2% glutaraldehyde (EMS) and 0.1% calcium chloride in
0.1 M sodium cacodylate buffer (EMS), pH 7.3 for 30 min, followed by transfer to fresh fixative at 4°C overnight. Fixed cells and tissues were cut into 1-2 mm blocks, incubated with 1% aqueous osmium tetroxide (EMS) and 1.5% potassium ferrocyanide
(Sigma) in 0.1M sodium cacodylate buffer, pH 7.3 for 2 hrs, stained en bloc with 4% aqueous ureanyl acetate (EMS) at 4 °C overnight, dehydrated with a graded series of alcohol and embedded in expoxy resin EMbed 812 (EMS) or Quetol 651 (EMS).
Ultrathin sections 90 nm were cut using a Reichert Ultracut E ultramicrotome (Leica).
Electron micrographs were collected using a Hitachi H-7650 TEM (Hitachi High
Technologies America) equipped with a 2,000 by 2,000 pixel AMT TEM CCD camera
(Advanced Microscopy Techniques).
Islet Isolation and Metabolic Labeling. Pancreatic islets were isolated from 6-8-week old mice as previously described49. Briefly, the pancreas was perfused with liberase
(Roche, 0.5 mg/mL) and removed for digestion in a 37°C water-bath for 20 minutes.
After washing and filtering the digest, islets were separated from exocrine cells by
density gradient centrifugation using Histopaque 1077 (Sigma). Islets were collected
from the histopaque/media interface and washed several times before culturing
84 overnight in CRML 1066 (Cellgro) containing 10% FBS, 100 U/ml penicillin and 100
µg/ml streptomycin. For metabolic labeling experiments, 100 islets were hand-picked
from each genotype and repeatedly washed in Kreb’s Ringer-bicarbonate buffer (KRB)
containing 1% BSA. To stimulate insulin biosynthesis, islets were incubated in KRB
containing 2.8 mM glucose for 1 hour at 37°C and then resuspended in KRB containing
16.7 mM glucose and incubated for another hour50,51. During the last 20 minutes of incubation, islets were labeled with 0.5 mCi/ml [35S]methionine/cysteine (MP
Biomedicals) for 60 minutes. Cells were lysed and immunoprecipitated with guinea pig
anti-insulin (abcam, 7842) and protein G plus agarose beads (Santa Cruz) at 4°C
overnight. Immunoprecipitates were analyzed by SDS-PAGE under reducing conditions
and visualized by autoradiography.
Serum Lipase and Amylase Activity. Blood was collected from mice through the
retroorbital sinus into serum separator tubes (BD Biosciences). Serum was submitted
to the Clinical Laboratory at Cincinnati Children’s Hospital for enzyme analyses.
Insulin and Glucagon Assays. Insulin and glucagon were extracted from the
pancreas by repeated homogenization in acid-ethanol (0.18 M HCl in 70% ethanol)52.
Mouse insulin (Crystal Chem) and glucagon (R&D) were assessed by ELISA and
results were normalized to total protein in the pancreatic extracts. Plasma proinsulin
(Alpco), insulin and glucagon were determined by ELISAs in 6 hour-fasted mice at
baseline or after intraperitoneal injection with 10% glucose.
Glucose and Insulin Tolerance Tests. Mice were fasted for 16 hours with free access
to water. Blood glucose levels were determined by collecting blood from the tail vein at
baseline using an ACCU-CHEK Aviva glucose monitor (Roche) before intraperitoneal
85 injection of 10% glucose (1 unit/g of body weight). Blood glucose levels were monitored
overtime following injection. Insulin tolerance tests were performed on 6 hour-fasted
mice by intraperitoneal injection of human insulin (0.75 U/kg, Eli Lilly) followed by blood glucose measurements as described above.
Statistical analyses. All data represent mean ± SEM with a significance difference reported as P ≤ 0.05. Two-way comparisons were analyzed by two-tailed, unpaired
Student’s t test using GraphPad Prism Software.
86 Acknowledgements
We thank Eileen Elfers for assistance with islet isolation, Mary Falconieri for metabolic labeling experiments and Karen Apsley for transfection and coimmunoprecipitation experiments. This study was supported by National Institute of Health Grants
HL103923 (T.E.W.) and HL086492 (T.E.W.).
87 Figure Legends
Figure 1. Generation of ERdj4GT/GT mice. (A) Diagram of the GT allele. The GT
cassette was inserted immediately after adenosine at position 1151 within intron 1 of the
ERdj4 locus (NC_000078.5). SA, splicing acceptor; TM, transmembrane region, β-GEO
(β-galactosidase-neomycin resistance fusion gene); IRES, internal ribosome entry site;
PLAP, placental alkaline phosphatase; pA, polyadenylation signal. (B) PCR genotyping of the WT and GT alleles in tail DNA isolated from the progeny of heterozygous intercrosses. (C) Gene trap copy number was determined in tail gDNA by the TaqMan
Gene Copy Number assay. N = 4 mice/genotype, ***P ≤ 0.001. (D-E) qRT-PCR of
ERdj4 and β-galactosidase mRNAs in ERdj4+/+ and ERdj4GT/GT MEFs. RQ, Relative quantitation. N = 3 samples/group, ***P ≤ 0.001. (F) qRT-PCR of ERdj4 mRNA in tissues isolated from 6-week-old ERdj4+/+ and ERdj4GT/GT littermates; samples were normalized to β-actin. N = 4 mice/genotype, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Figure 2. Constitutive ER stress and decreased cell viability in ERdj4GT/GT MEFs.
(A) RT-PCR of XBP1 mRNA in ERdj4+/+ and ERdj4GT/GT MEFs treated with tunicamycin
(10 ug/ml) for the indicated periods of time. The PCR products represent hybrid (h),
unspliced (u) and spliced (s) XBP1 species. (B) Western blot analyses of BiP, IRE1α,
calnexin and β-actin (loading control) in ERdj4+/+ and ERdj4GT/GT MEFs treated with indicated doses of tunicamycin or MG-132 (MG, 5 nM, 4 hours). (C) Electron micrographs of ERdj4+/+ and ERdj4GT/GT MEFs. Note dilated ER (arrows) and large inclusions (*) near the Golgi complex in ERdj4GT/GT MEFs. ER: endoplasmic reticulum;
G: Golgi complex; MT: mitochondria; NUC: nucleus. (D) Cell viability of ERdj4+/+ and
ERdj4GT/GT MEFs treated with indicated doses of MG-132, tunicamycin or thapsigargin
88 for 48 hours. Relative values were determined by setting the absorbance of untreated
ERdj4+/+ MEFs to 100%. N = 3 samples/group, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Figure 3. Hypoglycemia and growth retardation in ERdj4GT/GT mice. Studies assessing perinatal lethality were performed using C57BL/6 littermates. (A)
Comparison of E18.5 embryos from ERdj4+/GT and ERdj4GT/GT mice. (B) Body weights of E18.5 embryos. N = 6-14 mice/genotype, **P ≤ 0.01. (C) Blood glucose levels of
naturally born or caesarean delivered, nonsuckling neonates. N = 10-11
mice/genotype, **P ≤ 0.01, ***P ≤ 0.001. (D) Immunofluorescence of insulin (red) and
glucagon (green) in pancreatic tissue sections from newborn mice. Scale bars are 100
µm. N = 2 mice/genotype. (E-F) Quantitation of insulin and glucagon in pancreatic
extracts from newborn mice. N = 6 mice/genotype, *P ≤ 0.05, **P ≤ 0.01.
Figure 4. Elevated ER stress in pancreatic β-cells of ERdj4GT/GT mice. (A) Western blot analyses of GFP (reporter for XBP1 splicing), IRE-1α and β-actin (loading control) proteins in pancreatic lysates from 6-week-old ERdj4+/+ERAI+ and ERdj4GT/GTERAI+
mice. N = 2-5 mice/genotype. (B) Confocal microscopy of insulin (red) and GFP
(green) protein in pancreatic tissue sections from 8-week-old ERdj4+/+ERAI+ and
ERdj4GT/GTERAI+ mice. Scale bars are 10 µm. N = 4-5 mice/genotype. (C)
Hematoxylin and eosin staining of pancreatic tissue sections from 16-20-week-old
ERdj4+/+ and ERdj4GT/GT mice. Note vacuolated cells in the islets of ERdj4GT/GT mice
(arrows and inset). Scale bars are 10 µm. N = 5 mice/genotype. (D)
Immunofluorescence of insulin (red) and glucagon (green) in pancreatic tissue sections
from 16-20-week-old ERdj4+/+ and ERdj4GT/GT mice. Scale bars are 10 µm. N = 5 mice/genotype. (E) Quantitation of glucagon in pancreatic extracts of 16-20-week-old
89 ERdj4+/+ and ERdj4GT/GT mice. N = 5 mice/genotype, **P ≤ 0.01. (F) Plasma glucagon levels in fasted 8-16-week-old ERdj4+/+ and ERdj4GT/GT mice. N = 11-12 mice/genotype,
*P ≤ 0.05. (G) qRT-PCR of ERdj4 mRNA in islets isolated from 6-week-old, C57BL/6
ERdj4+/+ and ERdj4GT/GT littermates; samples were normalized to β-actin. N = 2-3
samples/genotype.
Figure 5. ERdj4 deficiency impairs insulin biosynthesis. (A) Immunofluorescence of insulin (red) and BiP (green) in pancreatic tissue sections from 16-20-week-old
ERdj4+/+ and ERdj4GT/GT mice. Scale bars are 5 µm. N = 3 mice/genotype. (B)
Metabolic labeling of islets stimulated with 16.7 mM glucose from 6-week-old, C57BL/6 littermates. Equal numbers of trichloroacedic-precipitable cpms were immunoprecipitated with insulin antibody. N = 3 samples/genotype. (C) HEK293 cells were transfected with empty vector or cotransfected with plasmid expressing ERdj4-HA and plasmid encoding Pcsk1, Pcsk2, GCG or CPE with FLAG. Cell lysates were immunoprecipitated with FLAG antibody followed by Western blotting with HA antibody to detect ERdj4.
Figure 6. Hypoinsulinemia causes glucose intolerance in ERdj4GT/GT mice. (A)
Fasting blood glucose levels in 16-20-week-old mice. N = 12-14 mice/genotype. (B)
Blood glucose levels over time after administration of glucose to fasted 12-16-week-old mice. N = 14 mice/genotype, *P ≤ 0.05, **P ≤ 0.01. (C) Blood glucose levels over time after administration of human insulin to fasted 16-20-week-old mice. N = 5 mice/genotype. (D) Plasma insulin levels before and after glucose administration to fasted 12-week-old mice. N = 8 mice/genotype, **P ≤ 0.01, ***P ≤ 0.001. (E) Plasma
proinsulin/insulin ratio in 12-week-old, fasted mice. N = 8 mice/genotype, *P ≤ 0.05.
90 Supplemental Figure 1. Elevated ER stress in tissues of ERdj4GT/GT mice. (A)
Western blot analyses of GFP (reporter for XBP1 splicing), IRE-1α and β-actin (loading control) proteins in kidney and lung lysates from 8-week-old ERdj4+/+ERAI+ and
ERdj4GT/GTERAI+ mice. N = 2-5 mice/genotype. (B) Transmission electron micrographs
of renal tubular epithelial cells in the kidneys of ERdj4+/+ and ERdj4GT/GT mice. Dilated
ER (arrows) was detected in ERdj4GT/GT cells. Scale bars are 1 µm. N = 2 mice/genotype. (C) Transmission electron micrographs of serous cells in the salivary glands of ERdj4+/+ and ERdj4GT/GT mice. Dilated ER (arrows) and Golgi inclusions (*)
were detected in ERdj4GT/GT mice. Scale bars are 1 µm. N = 2 mice/genotype. ER: endoplasmic reticulum; G: Golgi complex; LYS: lysosome; MT: mitochondria; NUC: nucleus.
Supplemental Figure 2. Ultrastructural evidence of ER stress in α-cells of
ERdj4GT/GT mice. (A) Immunofluorescence of glucagon (red) and GFP (green) proteins in pancreatic islets of 16-20-week-old ERdj4+/+ and ERdj4GT/GT mice. Autofluorescence
(yellow in merged panel) was detected in red blood cells. Scale bars are 10 µm. N = 5
mice/genotype. (B) Transmission electron microscopy of α-cells in pancreatic islets of
6-week-old ERdj4+/+ and ERdj4GT/GT mice. Note the pronounced ER dilation and Golgi inclusions in α-cells of ERdj4GT/GT mice. Scale bars are 2 µm. N = 2 mice/genotype.
ER: endoplasmic reticulum; G: Golgi complex; MT: mitochondria; NUC: nucleus; RBC: red blood cell.
Supplemental Figure 3. ER stress in pancreatic β-cells of ERdj4GT/GT mice. (A)
Transmission electron microscopy of β-cells in pancreatic islets of 6-week-old ERdj4+/+
and ERdj4GT/GT mice. Note the pronounced ER dilation, Golgi inclusions and electron
91 translucent secretory granules (*) in β-cells of ERdj4GT/GT mice. Scale bars are 2 µm. N
= 2 mice/genotype. ER: endoplasmic reticulum; G: Golgi complex; MT: mitochondria;
NUC: nucleus; RBC: red blood cells. (B) Quantitation of insulin in pancreatic extracts of
16-20-week-old ERdj4+/+ and ERdj4GT/GT mice. N = 5 mice/genotype.
Supplemental Figure 4. ER stress in pancreatic acinar cells of ERdj4GT/GT mice does not impair function. (A-B) Hematoxylin and eosin staining of pancreatic tissue sections from 8-week-old (A) or 8-month-old (B) ERdj4+/+ and ERdj4GT/GT mice. Note
cytoplasmic vacuolation in the acinar cells of ERdj4GT/GT mice (arrows). Scale bars are
20 µm. N = 3-5 mice/genotype. (C) Transmission electron microscopy of pancreatic acinar cells from 8-week-old ERdj4+/+ and ERdj4GT/GT mice. Note ER dilation (arrows),
Golgi inclusions (*) and immature zymogen granules (IZ). Scale bars are 1 µm. N = 2 mice/genotype. ER: endoplasmic reticulum; Z: zymogen granules; IZ: immature zymogen granules. (D) Amylase and lipase enzyme activity in the serum of 8-week-old
ERdj4+/+ and ERdj4GT/GT mice. N = 4-6 mice/genotype.
92 A A1151 G1152
SA SA TM !-GEO IRES PLAP pA
1 2 3 non-coding coding C
B
+/GT +/GT +/+ +/GT GT/GT
690bp trap 258bp wt
D E
F
Figure 1 93 A XBP-1 mRNA ERdj4+/+ ERdj4GT/GT 0 0.25 0.5 1 2 4 8 0 0.25 0.5 1 2 4 8 hrs h u s
B ERdj4+/+ ERdj4GT/GT TM 0 0.25 0.5 1 4 8 16 MG 0 0.25 0.5 1 4 8 16 MG
BiP
IRE1"
Calnexin
!-actin
C D MEFs ERdj4+/+ ) 125 d *** GT/GT e ERdj4 MT ER t a *** e 100 +/+ r NUC G G t n
u *** 75
% *** ( ** ERdj4
y * t i l
i 50 b a i v
l 25 l e ER C 0 M M M M g/mL g/mL
GT/GT GT/GT TG 1 µ MT Untreated TG 0.5 µ NUC TM 2 µ TM 8 µ * MG-132 1 µ G MG-132 0.5 G µ ERdj4 *
Figure 2 94 A B C ** ERdj4+/GT 1.6 ** ** 100 *** ) g (
t ) 80 l h 1.4 d / g i g e m
w 60 (
y 1.2 e d s o o b
c 40
u l 5 . G 8 1.0
1 20 E 0.8 0
GT/GT +/+ +/+ ERdj4 +/GT +/GT GT/GT GT/GT
ERdj4 ERdj4 ERdj4 ERdj4 ERdj4 ERdj4
D ERdj4+/GT ERdj4GT/GT insulin / glucagon
E F 20 150 ) g
) ** *** µ / g g µ / p g 15 (
t n
( 100
n t e n t e n t o
n 10 c
o c n
o
n 50 i g l a u 5 c s u n l i g 0 0 ERdj4+/GT ERdj4GT/GT ERdj4+/GT ERdj4GT/GT
Figure 3 95 A E 80 Pancreas ) ** g µ /
ERdj4 + + GT GT GT GT GT g n
( 60
t n e GFP t n
o 40 c
n
IRE-1" o g
a 20 c u l
!-actin G 0 ERdj4+/+ ERdj4GT/GT ERdj4+/+ ERdj4GT/GT B F *
) 500 L m /
g 400 p (
n
o 300 g GFP insulin / GFP a c u l 200 g
a
C m s 100 a l p 0 ERdj4+/+ ERdj4GT/GT H&E
G 101
D 0
) 10 Q R (
4 -1 j 10 d R E 10-2 insulin / glucagon 10-3 ERdj4+/+ ERdj4GT/GT
Figure 4 96 A B insulin/BiP ** 1.8
1.5
) +/+ Q
R 1.2 (
n i l ERdj4
u 0.9 s n i o
r 0.6 p 0.3
0.0 ERdj4+/+ ERdj4GT/GT
Islets GT/GT ERdj4
C
FL FL FL FL FL FL FL FL FL + Pcsk1 + GCG + CPE + Pcsk2
+ Pcsk1 + Pcsk2 + GCG + CPE
HA HA HA HA HA HA HA HA HA HA FL FL FL FL ERdj4 Vector Vector ERdj4 ERdj4 ERdj4 ERdj4 ERdj4 ERdj4 Vector Vector GCG ERdj4 Pcsk1 ERdj4 Pcsk2 ERdj4 CPE
!-HA ERdj4 --22 Input IP !-FLAG
Figure 5 97 A B 250 200 ** * ** **
) 200 ** l ) l d / d 150 / g g
m 150 m (
(
e e
100 s s o o
c 100 c +/+ u l u ERdj4 l G G 50 50 ERdj4GT/GT
0 0 ERdj4+/+ ERdj4GT/GT 0 15 30 60 90 120 Time post-glucose injection (min) C D 1.2 175 ERdj4+/+ ) L
) 150 GT/GT l ERdj4 m d
/ 0.8 /
125 g g n ( m
(
100 n
i ** e l s
u ***
o 75 ** s 0.4 c n I u l 50 ERdj4+/+ G 25 ERdj4GT/GT 0 0.0 0 15 30 60 90 120 0 15 30 Time post-insulin injection (min) Time post-glucose injection (min) E 0.8 * n i
l 0.6 u s n i / n i l 0.4 u s n i o r
p 0.2
0.0 ERdj4+/+ ERdj4GT/GT
Figure 6 98 A
Kidney Lung
ERdj4 + + GT GT GT GT GT + + GT GT GT GT GT
GFP
IRE1"
!-actin
B ERdj4+/+ ERdj4GT/GT Kidney
C
G ! ! Salivary Gland
Supplemental Figure S1 99 A Glucagon GFP (sXBP-1) Merge +/+
GT/GT
B ERdj4+/+ ERdj4GT/GT
NUC NUC MT G MT ! -cells ER MT RBC MT
Supplemental Figure S2 100 B Supplemental FigureS3 A
"-cells insulin content (ng/µg) 0.0 0.1 0.2 0.3 0.4 0.5 MT NUC ERdj4 ERdj4 +/+ ERdj4 NUC +/+
GT/GT 101 NUC * * ER G ERdj4 ER
ER MT GT/GT *
* MT A ERdj4+/+ ERdj4GT/GT 8 weeks
B 8 months
C Z Z ER Z ER ER IZ IZ * Z Z
D
Supplemental Figure S4 102 Table 1. Genotype distribution of P21 progeny from heterozygous intercrosses.
103 Table 2. Genotype distribution of day 18.5 embryos from heterozygous intercrosses.
104 References
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109
CHAPTER III
Deficiency of the ER stress chaperone ERdj4 is associated
with defects in osteogenesis and B cell development
110 Deficiency of the ER stress chaperone ERdj4 is associated with defects in
osteogenesis and B cell development
Jill M. Fritz, Mei Dong, Cheng-Lun Na and Timothy E. Weaver
Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center and the
University of Cincinnati College of Medicine, Cincinnati, OH 45229
Correspondence: Timothy E. Weaver, 3333 Burnet Ave., Cincinnati, OH 45229; Phone:
513-636-7223; Email: [email protected]
Supported by NIH awards HL103923 and HL086492
Manuscript in preparation
111 Abstract
Highly secretory cells, including bone-forming osteoblasts and antibody-secreting plasma cells, require the unfolded protein response (UPR) to maintain ER homeostasis during increased protein synthesis. Expression of the UPR chaperone ERdj4 is
upregulated in response to ER stress arising from accumulation of unfolded/misfolded
proteins in the ER lumen. In the current study, we investigated the role of ERdj4 in
osteoblast differentiation and function in hypomorphic ERdj4 gene trap (ERdj4GT/GT) mice. Bone-lining cells, which differentiate from osteoblasts, were reduced in association with increased ER stress in ERdj4GT/GT mice. Primary bone cells isolated from ERdj4GT/GT mice had reduced expression of osteogenic-specific markers. Reduced
osteogenesis, in turn, was associated with decreased numbers of bone marrow B cells
related to increased cell death. Further, numbers of mature B cells in the spleen and
peritoneal cavity were also compromised by ERdj4 deficiency. ERdj4GT/GT bone marrow
transplanted into ERdj4+/+ hosts rescued all B cell compartments, confirming a B cell-
extrinsic defect. Collectively, these data suggest that the loss of ERdj4 impairs the
maturation/survival of osteogenic cells in association with increased ER stress. Further,
deficiency of osteogenic cells in ERdj4GT/GT mice is linked to a defect in B cell development in the bone marrow.
112 Introduction
Highly secretory cells, including bone-forming osteoblasts and antibody-secreting plasma cells, utilize the UPR to alleviate the ER burden imposed by increased protein synthesis. The UPR maintains ER homeostasis by attenuating protein translation, increasing protein folding capacity and removing unfolded/misfolded proteins from the
ER lumen. The UPR signal transducers protein kinase RNA (PKR)-like ER kinase
(PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 alpha
(IRE1α) are localized in the ER membrane where they trigger the UPR in response to accumulation of unfolded or misfolded protein in the ER lumen. Activation of UPR sensors occurs when the ER chaperone BiP/GRP78 is recruited away from the luminal domain of PERK, ATF6 and IRE1α to unfolded/misfolded substrates. PERK phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α) leading to inhibition of protein translation, thereby preventing further protein accumulation in the
ER1. ATF6 results in translocation to the Golgi, where site-specific serine proteases
generate a cytosolic fragment that, in turn, translocates to the nucleus and increases
the expression of ER chaperones2,3. The endocribonuclease activity of IRE1α splices an intron from the mRNA of X-box-protein 1 (XBP1) resulting in translation of a transcription factor that increases the expression of ER chaperones and ER-associated degradation (ERAD) machinery4,5. Failure to restore ER homeostasis results in
apoptosis through PERK/eIF2α-mediated induction of activating transcription factor 4
(ATF4) and C/EBP homologous protein (CHOP)6.
The HSP70 family member BiP/GRP78 is a soluble ER chaperone with multiple functions, including regulation of the translocon pore7, maintenance of ER calcium
113 stores8, protein folding and degradation9-12, and induction of the UPR13,14. The
multifunctionality of BiP is modulated by ER-localized DnaJ (ERdj) proteins, a diverse
class of cochaperones from the HSP40 family. ERdjs stimulate the ATPase activity of
BiP, leading to conformational changes that stabilize client interaction. Although some
ERdjs interact with BiP independently of substrate, others bind substrates directly to
recruit BiP15-18. ERdjs promote protein folding and/or degradation of newly translocated
(ERdj1, ERdj2, ERdj3), unfolded (ERdj3, ERdj4, ERdj6) or misfolded substrates (ERdj4,
ERdj5), often through association with translocation or ERAD machinery19,20.
ERdj4 is a soluble ER luminal protein, containing a J domain that associates with
BiP to stimulate ATP hydrolysis, a glycine-phenylalanine rich region of unknown
function and a substrate binding domain that interacts with misfolded (SP-CΔexon4,
insulin2C96Y) and unfolded (insulin2WT, Pcsk1, Pcsk2, CPE) substrates. ERdj4 is an ER stress-related chaperone21 that facilitates the removal of unfolded/misfolded substrates
from the ER lumen by interacting with ERAD machinery22,23. Although ERdj4
expression is upregulated in response to ER stress arising from expression of terminally
misfolded substrates, recent studies in our laboratory revealed that ERdj4 also plays an
important role in adaptation to ER stress associated with normal growth, development
and metabolism. Hypomorphic expression of ERdj4 in mice resulted in perinatal
lethality associated with growth restriction and hypoglycemia, while surviving adult mice
were glucose intolerant and hypoinsulinemic, with defects in the pancreatic β-cell secretory pathway (Chapter 2).
Osteoblasts are highly secretory cells that are responsible for the synthesis and secretion of extracellular bone matrix. Several components of the UPR regulate the
114 expression of osteoblast-specific genes that influence cell differentiation and function.
ATF4 and XBP1 increase the expression of Osterix, a transcription factor required for
osteoblast maturation. ATF6 and old astrocyte specifically-induced substance (OASIS), an ATF6 structural homologue, regulate expression of osteocalcin and type I collagen, respectively, leading to an increase in extracellular matrix production24,25. Further, the
UPR in osteoblasts is essential for skeletal development in humans and mice. An
inactivating mutation in human PERK caused growth retardation, epiphyseal dysplasia
and osteoporosis26,27. A similar phenotype was observed in PERK-deficient mice and
was associated with decreased numbers of osteoblasts in bone tissue and impaired
osteoblast proliferation and differentiation in vitro28,29. Mice deficient in ATF4, a
transcription factor activated by the PERK signaling cascade, exhibited a delay in
skeletal development during embryogenesis30 and low postnatal bone density due to
impaired osteoblast differentiation and function31.
In addition to bone formation, osteoblasts are an important component of the bone marrow microenvironment, influencing hematopoiesis through cell-surface expression of signaling and adhesion molecules, and secretion of growth factors and cytokines32. Conditional depletion of osteoblasts in vivo reduced hematopoietic stem cells (HSCs), B cells and erythrocytes in the bone marrow, and increased extramedullary hematopoiesis33. Calvarial osteoblasts were shown to support B cell
commitment and maturation from HSCs in vitro through the expression of IL-7, CXC-
chemokine ligand 12 (CXCL12) and vascular adhesion molecule-1 (VCAM-1)34.
Parathyroid hormone (PTH), which regulates osteoblast differentiation, function and
115 survival35, induced IL-7 and CXCL12 production by osteoblasts in vitro34,36,37 and impaired PTH signaling on osteogenic cells reduced B cell development in vivo38.
Since the UPR is required for osteogenesis, we hypothesized that ERdj4
deficiency would impair osteoblast differentiation and/or function. Bone-lining cells of
the osteogenic lineage were reduced in association with increased ER stress in ERdj4
gene trap (ERdj4GT/GT) mice. Primary osteogenic cells isolated from the long bones of
ERdj4GT/GT mice exhibited decreased osteogenic-specific markers consistent with impaired osteogenesis. Deficiency of osteogenic cells in ERdj4GT/GT mice was also associated with reduced B cell development in the bone marrow. Further, ERdj4 deficiency affected mature B cell populations in the spleen and peritoneal cavity.
Transfer of ERdj4GT/GT bone marrow into ERdj4+/+ hosts rescued the defect in all B cell
compartments. Collectively, these results suggest that the loss of ERdj4 in osteogenic
cells impairs maturation and/or survival, which is associated with a defect in B cell
development.
116 Results
Bone-lining cells are reduced in ERdj4GT/GT mice⎯Since several components of the
UPR are essential for skeletal development and osteoblast differentiation28,29,39,40, cells
of the osteogenic lineage were assessed in long bones of adult mice. Bone-lining cells, which are quiescent osteoblasts, were markedly reduced in ERdj4GT/GT mice compared to controls (Fig. 1A). Since ERdj4 deficiency caused constitutive stress in a variety of non-secretory and secretory cell types (Chapter 1), ER stress was assessed in osteogenic cells in vivo by crossing ERdj4+/GT mice to ER stress-activated indicator
(ERAI) transgenic mice41. GFP fluorescence (resulting from XBP1 splicing and
expression of XBP1/GFP fusion protein) was increased in osteocalcin-positive bone-
lining cells of ERdj4GT/GTERAI+ mice (Fig. 1B). These data indicate that bone-lining cells
are reduced in association with increased ER stress in ERdj4GT/GT mice.
Genetic alterations in the UPR caused defects in growth and skeletal development due to impaired osteoblast differentiation and function28,31. Although previous studies in our laboratory demonstrated growth restriction in ERdj4GT/GT
embryos, postnatal compensatory growth resulted in normal weights by 8 weeks of age
(data not shown). Further, bone and cartilage staining of E18.5 embryos revealed no
gross defects in skeletal development in ERdj4GT/GT mice (Supplemental Fig. S1).
Collectively, these data suggest that ERdj4 deficiency does not affect postnatal growth
or osteoblast function.
Primary cells isolated from the long bones of ERdj4+/+ and ERdj4GT/GT mice
varied morphologically in size and staining intensity (Fig. 2A), but were osteocalcin-
positive (Fig. 2B), suggesting that these cells were of the osteogenic lineage.
117 Osteocalcin protein was modestly reduced in osteogenic cells of ERdj4GT/GT mice (Fig.
2B) in association with decreased osteocalcin mRNA (Fig. 2C). Other osteoblast-
specific markers were also significantly reduced in osteogenic cells of ERdj4GT/GT mice, including Osterix and type I collagen (Fig. 2C). RANKL mRNA, an osteocyte marker42,
was also reduced in ERdj4GT/GT mice (Fig. 2C). Treatment with ascorbic acid and beta-
glycerol phosphate, which induces osteoblast differentiation, significantly upregulated
osteocalcin mRNA in ERdj4GT/GT cells (Fig. 2D). Similar to findings in vivo, osteogenic
cells from ERdj4GT/GT mice exhibited increased levels of ER stress (Fig. 2E). Overall, these data suggest that osteogenic cells isolated from the long bones of ERdj4GT/GT
mice were predominantly immature bone cells.
Recent studies demonstrated that UPR deficiency impaired bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation39,40. BMP2 induces osteogenesis by
activating the Smad-Runx2 signaling pathway that regulates osteoblast-specific genes,
including alkaline phosphatase (ALP), type I collagen and osteocalcin43,44. ERdj4 mRNA was upregulated in a time-dependent manner following BMP2 treatment of
ERdj4+/+ mouse embryonic fibroblasts (MEFs) (Supplemental Fig. S2A). Similarly, BiP
and spliced XBP1 mRNAs were also induced in ERdj4+/+ MEFs after 96 hours of BMP2
treatment (Supplemental Fig. S2B-C), indicating that BMP2 signaling of osteoblast
differentiation upregulates the UPR in ERdj4+/+ MEFs. BMP2 treatment induced expression of BiP and spliced XBP1 mRNAs to a greater extent in ERdj4GT/GT MEFs compared to controls (Supplemental Fig. S2B-C). ALP activity and osteocalcin, Osterix and type I collagen mRNAs, were all increased in BMP2-treated ERdj4GT/GT MEFs
(Supplemental Fig. S2D-E); however, the magnitude of the increase was significantly
118 blunted in ERdj4GT/GT MEFs compared to ERdj4+/+ MEFs. Overall, these results are consistent with a defect in BMP2-induced osteoblast differentiation associated with increased levels of ER stress in ERdj4GT/GT MEFs.
ERdj4 deficiency affects early B lymphopoiesis⎯Several studies have reported that osteoblasts support hematopoiesis33,34,36,38. Since osteogenic cells were reduced in
ERdj4GT/GT mice, hematopoiesis was assessed in the bone marrow of adult mice by flow
cytometry. Bone marrow cellularity was reduced in ERdj4GT/GT mice (Fig. 3A), which correlated with a significant decrease in erythrocytes (Ter119+) and B cells (B220+), but
not macrophages (F4/80+) or granulocytes (Gr-1+) (Supplemental Fig. S3A-C). Based
on the Hardy classification method of B lymphopoiesis45, the frequency and absolute number of pro-B, pre-B, immature and mature B cells was decreased in the bone marrow of ERdj4GT/GT mice compared to controls (Fig. 3B-C). When pro-B cells were further classified into fractions A through C based on HSA and BP-1 expression, there was a significant decrease in the number of cells in fractions B and C, but not fraction A
(Fig. 3D). The frequency of pro-B cells in the blood and proliferation of developing B cells in the bone marrow were normal (Supplemental Fig. S4A-B), but the percentage of
7-AAD+ pro-B cells was significantly increased compared to controls (Fig. 3E).
Collectively, these results indicate a defect in B cell development starting at the pro-B cell stage, which is associated with an increase in pro-B cell death.
ERdj4 deficiency in the bone marrow microenvironment impairs B cell development⎯To
confirm that the defect in B lymphopoiesis was not cell autonomous, ERdj4GT/GT bone
marrow was transplanted into irradiated ERdj4+/+ hosts and B cell development was analyzed 8 weeks post-transplantation. ERdj4GT/GT bone marrow transplanted into
119 ERdj4+/+ hosts rescued the defect in B cell development starting at the pro-B cell stage
when compared to B cell development in ERdj4GT/GT recipients (Fig. 4A-B).
Furthermore, pre-B colony forming units generated from total bone marrow in media containing IL-7 were normal in ERdj4GT/GT mice, suggesting that ERdj4GT/GT B cells
develop normally in the proper microenvironment (Fig. 4C).
Mature B cell populations are reduced in ERdj4GT/GT mice⎯The defect in early B cell
development in the bone marrow prompted analysis of mature B cell subsets in the
periphery. Immature B cells migrate to the spleen where they undergo further
differentiation into follicular and marginal zone B cell subsets46. Total splenocytes were
reduced in ERdj4GT/GT mice (Fig. 5A), which was associated with a decrease in mature
(B220+CD93-) B cells (Fig. 5B-C). Interestingly, immature (B220+CD93+) B cells were
normal in ERdj4GT/GT mice (Fig. 5B-C), despite lower numbers of immature
(B220+IgMlow) B cells in the bone marrow (Fig. 3B-C). The mature B cell subset affected by ERdj4 deficiency was the follicular B cells (B220+CD21lowCD23+), while
marginal zone B cells (B220+CD21+CD23low) were normal (Fig. 5B-C). Transfer of
ERdj4GT/GT bone marrow into irradiated ERdj4+/+ hosts rescued follicular B cell
development, supporting a defect in the microenvironment (Fig. 5D).
B1 cell development occurs predominantly in the fetal liver and neonatal spleen
to generate self-renewing, mature B1 cells that are localized in the pleural and
peritoneal cavities throughout life47. Total B1 cells (CD19+CD11b+) were decreased in the peritoneal cavity of ERdj4GT/GT mice, resulting from lower numbers of B1b (CD5-) cells, while B1a cells were normal (Fig. 5E).
120 The B cell activating factor (BAFF), which influences mature B cell differentiation
and survival in the periphery, was modestly but significantly increased in the serum of
ERdj4GT/GT mice (Fig. 5F). Collectively, these data suggest that mature B cell survival or proliferation is defective in the periphery of ERdj4GT/GT mice even in the face of increased BAFF.
Plasma cell differentiation and antibody secretion are normal in ERdj4GT/GT mice⎯The
UPR signal transducer XBP1 regulates immunoglobulin synthesis and secretion by plasma cells48,49. Given that ERdj4 expression was reduced in LPS-treated XBP1-/- B
cells50, plasma cell differentiation and function was examined in ERdj4GT/GT mice. The
frequency of splenic plasma cells (CD19+CD138+) was normal at baseline in ERdj4GT/GT
mice and treatment with gαmIg-D antibody induced plasma cell differentiation in vivo
(Supplemental Fig. S5A). Basal immunoglobulins, including IgM, IgG, IgA and IgE,
were normal in the serum of ERdj4GT/GT mice (Supplemental Fig. S5B) and ERdj4GT/GT
mice efficiently produced TNP-specific IgM and IgG antibodies to T cell-dependent and
T cell-independent antigens (Supplemental Fig. S5C-D). Therefore, ERdj4 deficiency
does not affect plasma cell differentiation or antibody secretion even though ERdj4 is an
XBP1 target in vivo.
121 Discussion
Osteoblasts are highly secretory cells that require the UPR to induce transcription of genes essential for maturation and function. In the current study, we
demonstrate that ERdj4, a chaperone regulated by the UPR, influences cells of the
osteogenic lineage in vitro and in vivo. ERdj4 expression was upregulated in response
to BMP2, consistent with previous studies demonstrating induction of the UPR during
BMP2-induced osteoblast differentiation39,40. BMP2 supports osteogenesis by activating Runx2 and Osterix, thereby inducing the transcription of several osteoblast- specific genes, including ALP, osteocalcin and type I collagen43,44. The UPR can upregulate transcription of Osterix (XBP1 and ATF4), or function downstream of Runx2
(ATF6) to induce extracellular matrix production24,40,51. BMP2-induced osteoblast
differentiation was impaired in ERdj4GT/GT MEFs in association with increased ER stress.
Previous studies demonstrated that inducers of ER stress decreased cell viability of
MEFs in the absence of ERdj4 (Chapter 1). The loss of ERdj4 chaperone activity likely
resulted in accumulation of unfolded/misfolded protein within the ER lumen, although
these substrates have yet to be identified. Overall, these findings highlight the importance of ERdj4 in maintaining ER homeostasis during BMP2-induced osteoblast differentiation in vitro.
Primary osteogenic cells isolated from the long bones of adult mice were
morphologically heterogeneous, but osteocalcin-positive, suggesting that these cells
were of the osteogenic lineage. Expression of osteoblast-specific markers, including
Osterix, osteocalcin and type I collagen, were decreased in ERdj4GT/GT osteogenic cells,
but upregulated following treatment with differentiation media. These findings suggest
122 that the frequency of immature bone cells was increased in primary osteogenic cells
from ERdj4GT/GT mice. Although these cells can be found within cortical bone52,53,
osteocytes are the predominant postosteoblastic cell type encased within extracellular
bone matrix. Thus, it is possible that osteocytes are decreased in ERdj4GT/GT mice.
Several lines of evidence support this hypothesis, including (1) reduced expression of
RANKL, a marker most highly expressed by osteocytes of the osteogenic lineage42 and
(2) reduced bone-lining cells, another postosteoblast cell type. The majority of osteoblasts undergo apoptosis after bone formation, but some differentiate into osteocytes or bone-lining cells54. Given that ER stress was increased in osteogenic cells of ERdj4GT/GT mice, it is possible that reduced bone-lining cells were the result of
increased apoptosis.
The role of the IRE1α/XBP1 signaling pathway in postnatal growth and skeletal
development has yet to be explored in vivo, impeded by embryonic lethality in both
IRE1α and XBP1-deficient mice55,56. ERdj4, a chaperone induced by the IRE1α/XBP1 signaling pathway, was previously demonstrated to be important for growth during embryogenesis (Chapter 1). Although ERdj4GT/GT embryos were smaller, compensatory
growth during the postnatal period corrected the phenotype. ERdj4GT/GT mice reached normal weight by 8 weeks of age and did not display any gross skeletal defects. In contrast, deficiency of other UPR components, including PERK and ATF4, resulted in multiple skeletal dysplasias due to impaired osteoblast differentiation and function28,31.
Collectively, these data suggest that the loss of ERdj4 does not affect bone formation by
osteoblasts, but instead influences postosteoblasts. Given that postosteoblasts support
123 hematopoiesis52, deficiency of bone-lining cells in ERdj4GT/GT mice may be responsible
for the defect in B cell development.
Osteoblasts are reported to influence B cell development; however, this
conclusion is based on studies that used either heterogeneous osteogenic cultures in
vitro or ablation of multiple cell types within the osteogenic lineage in vivo33,34. In our study, deficiency of postosteoblasts in ERdj4GT/GT mice was associated with reduced B cells and erythrocytes, the same cell lineages affected by conditional depletion of osteoblasts and postosteoblasts in transgenic mice33. As osteoblasts are transient and
short-lived, it is unlikely that they provide sustained support of the hematopoietic
niche52; in contrast, postosteoblasts cover approximately 90% of all bone surfaces and
survive for months to years in humans and mice54. These observations suggest that postosteoblasts influence B cell development, although this hypothesis remains to be directly tested.
Other cellular niches have been described to support B cell development in the bone marrow. Elegant studies by Tokoyoda et al. using CXCL12-GFP reporter mice demonstrated that the earliest and latest stages of B cell development, the pre-pro B cells and plasma cells, respectively, associated with reticular cells that expressed high levels of CXCL12, termed CAR cells. The pro-B cells moved away from CAR cells and interacted with fibroblast-like cells that expressed IL-7, while the pre-B and immature B cells did not associate with either of these stromal cell types57. CAR cells were located
away from the endosteal surface and did not express osteopontin57, indicating that
these were not osteogenic cells. However, the IL-7-expressing cells may be from the
osteogenic lineage38, given that IL-7 expression was induced in osteogenic cells by
124 parathyroid hormone (PTH)34,37 and disruption of the PTH signaling pathway in
osteogenic cells impaired B cell development at the pro-B cell stage38.
Follicular B cells in the spleen were also compromised by ERdj4 deficiency due
to a defect in the microenvironment. Follicular B cells continuously recirculate
throughout the spleen, liver, lymph nodes and bone marrow58,59. In the bone marrow,
mature recirculating B cells occupy the sinusoidal niche and are supported by survival
factors secreted by resident dendritic cells60. Notably, mature recirculating B cells were reduced in the bone marrow of ERdj4GT/GT mice. Sinusoidal formation in the bone marrow is driven by osteocytes through osteoclastogenesis52. Thus, it is possible that
osteocyte deficiency in ERdj4GT/GT mice affected recirculating B cells in the sinusoidal
niche. It remains to be defined whether peritoneal B1b cells were reduced in ERdj4GT/GT
mice due to a defect in the microenvironment. Although B1b cell development occurs
predominantly during embryogenesis47, they can be generated in the adult bone marrow61. Thus, a defect in the bone marrow microenvironment of ERdj4GT/GT mice may contribute to the loss of B1b cells in the peritoneal cavity. Collectively, these data suggest that the loss of ERdj4 affects the maturation/survival of postoosteoblasts in association with increased ER stress. Deficiency of postosteoblasts is linked to a defect in B cell development in the bone marrow.
125 Materials and Methods
Mice. Hypomorphic ERdj4GT/GT mice were generated from an ES cell line harboring a gene trap cassette (GT) in intron 1 (Bay Genomics), as previously described (Chapter
1). Unless otherwise indicated, all mice used in these experiments were 6 to 8-week- old littermates on a mixed 129P2-C57BL/6 genetic background. ERAI mice were obtained from RIKEN BRC. All mice were housed in a pathogen-free barrier facility and studies were conducted with approval from Cincinnati Children’s Hospital Medical
Center Institutional Animal Care and Use Committee.
Histology and Immunohistochemistry. Bone tissue was fixed in 4% paraformaldehyde, decalcified, embedded in paraffin and 5 µm sections were cut for
H&E staining and immunohistochemistry, as previously described62. For immunohistochemistry, endogenous peroxidase activity was quenched in bone tissue/cells by treatment with 1.5% hydrogen peroxide in methanol for 15 minutes.
Slides then underwent antigen retrieval by boiling in 10 mM citrate buffer (pH 6.0) for 20 minutes before blocking in normal goat serum (Jackson ImmunoResearch) for two hours. Bone tissue/cells were incubated with rabbit anti-mouse osteocalcin (1:100,
Enzo Life Sciences), followed by application of biotinylated goat anti-rabbit IgG (1:200,
Vector Laboratories). Bone tissue/cells were visualized with the Vectastain Elite ABC kit (Vector Laboratories) using nickel-diaminobenzidine as a substrate followed by counterstaining with 0.1% nuclear fast red. Images were obtained using a Axio
Imager.A2 microscope (Zeiss) and AxioCam MRc5 digital camera (Zeiss).
For immunofluorescence, slides underwent antigen retrieval followed by blocking in normal donkey serum (Jackson ImmunoResearch) for two hours. Sections were
126 stained with rabbit anti-osteocalcin and anti-mouse GFP (Invitrogen) followed by
application of secondary antibodies conjugated to Alexa Fluor 555 or Alexa Fluor 647
(1:100, Invitrogen). To remove autofluorescence, slides were incubated in 0.1% Sudan
Black in 70% ethanol for 20 minutes. Image stacks were acquired by 0.25 µm step
distance using a Nikon C1 Si Confocal Imaging System and processed for deconvolution to remove image blurring using Autoquant X 2.2 (Media Cybernetics).
Tissue harvest and cell isolation. Mouse femurs were harvested, trimmed and
flushed with media using a 3 cc syringe and a 21 gauge (g) needle to release the bone
marrow. Single cell suspensions were obtained by repeatedly passing the cells through
a syringe. Mouse spleens were harvested and placed on a nylon mesh strainer (100
µm, BD Biosciences) in a 50 mL conical tube. Spleens were gently massaged with the
blunt end of a plunger from a 3 cc syringe and the strainer was then rinsed with media
to release cells. Peritoneal cavity cells were harvested from mice by injecting 3 mL of
cold PBS with a 27 g needle into the peritoneal cavity, gently massaging the peritoneum
and retrieving the fluid with an 18 g needle63.
Flow cytometry. Bone marrow, spleen and peritoneal cavity cells (1x106 cells/100 µl) were incubated in FACS buffer (1X PBS, 1% FBS, 0.05% NaN3) containing CD16/32
Fc-blocking antibody (Biolegend, 93) for 30 minutes at 4°C and then stained with
fluorescently-labeled antibodies for 30 minutes at 4°C. Data was acquired using the
LSR II flow cytometer (BD Biosciences) and analyzed on FlowJo software (TreeStar,
Inc.). The following mouse-specific monoclonal antibodies were used for flow cytometry
with the source and clone indicated in parentheses: B220-Pacific Blue (Biolegend,
RA3-6B2), IgM-PE (Biolegend, RMM-1), CD43-PE/Cy5 (Biolegend, 1B11), CD24-FITC
127 (Biolegend, M1/69), BP-1-Alexa Fluor 647 (Biolegend, 6C3), CD93-APC (Biolegend,
AA4.1), CD21-Pacific Blue (Ebioscience, eBio4E3), CD23-PE (Ebioscience, B3B4),
CD138-APC (BD Pharmingen, 281-2), CD5-PerCP-Cy5.5 (Ebioscience, 53-7.3), CD19-
PE (Ebioscience, eBio1D3), CD11b-eFluor450 (Ebioscience, M1/70), TER119-FITC
(Biolegend, TER119), Gr-1-PE (Biolegend, RB6-8C5) and F4/80-PerCP (Biolegend,
BM8).
Bone marrow transplantation. ERdj4+/+ and ERdj4GT/GT mice were exposed to two
doses of irradiation (7 and 4.75 Gy) and intravenously transplanted with ERdj4GT/GT
bone marrow (5×106 cells/200 µl). B cell populations in the bone marrow and spleen
were analyzed 8 and/or 16 weeks after transplantation by flow cytometry. Bone marrow
aspiration was performed 8 weeks after transplantation by the Cincinnati Children’s
Comprehensive Mouse and Cancer Core.
Quantitative Real-Time PCR. Total RNA was extracted from cells using the RNeasy
Plus Mini Kit (Qiagen) and cDNA was synthesized using iScript cDNA Synthesis Kit
(Bio-Rad). Quantitative RT-PCR was performed with 25 ng of cDNA per reaction on the
ABI 7300 system with TaqMan Assays (Applied Biosystems) for mouse ERdj4
(Mm01622956_s1), custom-designed spliced XBP1 (5’-GGC CGG GTC TGC TGA GT-
3’; 3’-CTG AAG AGG CAA CAG TGT CAG AGT; probe, CGC AGC AGG TGC AGG
CCC A), BiP (Hspa5, Mm00517690_g1), osteocalcin (Bglap1, Mm03413826_mH),
Osterix (Sp7, Mm04209856_m1), type 1 collagen (Col1a1, Mm00801666_g1), RANKL
(Tnfsf11, Mm00441906_m1) and mouse β-actin (endogenous control,
Mm00607939_s1). Relative quantitation was determined using the SDS software
(Applied Biosystems).
128 Cell culture. MEFs were isolated from ERdj4+/+ and ERdj4GT/GT embryos and cultured in DMEM, as previously described (Chapter 1). For osteoblast differentiation studies,
MEFs were plated in 24- or 96-well plates and treated with recombinant human BMP2
(Roche, 200 ng/mL) for up to 96 hours. ALP activity was determined by incubating cell lysates with p-nitrophenyl phosphate (Pierce) at room temperature for 30 minutes. The reaction was stopped with addition of 2N NaOH and absorbance was measured at 405 nm on a Synergy 2 microplate reader (BioTek Instraments, Inc.). For pre-B colony forming assays, total bone marrow (1×105 cells) was cultured in MethoCult media (Stem
Cell Technologies, M3630) for 7 days at 37°C before colonies were counted under a light microscope (Zeiss) using low magnification (4X).
Statistical analysis. All experiments were performed at least twice, unless otherwise stated. Data are presented as mean ± SEM and were analyzed by a two-tailed
Student’s t test using GraphPad Prism software, where P ≤ 0.05 was considered statistically significant.
129 Acknowledgements
We thank Jessica Allen for preliminary B cell experiments, Fred Finkelman for providing the IgD antibody and Sheila Bell for skeletal staining. This study was supported by
National Institute of Health Grants HL103923 (T.E.W.) and HL086492 (T.E.W.).
130 Figure Legends
Figure 1. Bone-lining cells are reduced in ERdj4GT/GT mice. (A) H&E staining of
femur tissue from 6-8-week-old mice. Bone-lining cells are present on the cortical bone
surface (arrows). Inset is 100X and scale bars are 20 µm. (B) Confocal microscopy of
osteocalcin (red) and GFP (green) protein in femur tissue sections from 6-8-week-old
ERAI+ mice. Scale bars are 10 µm.
Figure 2. Primary osteogenic cells from ERdj4GT/GT mice have an immature
phenotype. (A) Diff-Quik staining of osteogenic cells cultured in growth media for 16-
18 days. Scale bars are 20 µm. (B) Osteocalcin immunohistochemistry of osteogenic
cells cultured in growth media for 16-18 days. Scale bars are 20 µm. (C) Quantitation of
osteocalcin, Osterix, type 1 collagen (Col1a1) and RANKL mRNAs in osteogenic cells
cultured in growth media for 16-18 days. Mouse β-actin was used as an endogenous
control. N = 3 samples/group, representative of two independent experiments, *P ≤
0.05, **P ≤ 0.01, ***P ≤ 0.001. (D) Quantitation of Osterix and osteocalcin mRNAs in
ERdj4GT/GT osteogenic cells cultured in growth media for 16-18 days (d0) or treated with differentiation media for an additional 21 days (d21). Mouse β-actin was used as an endogenous control. RQ, relative quantitation. N = 3 samples/group, **P ≤ 0.01. (E)
Quantitation of BiP and sXBP1 mRNAs in osteogenic cells cultured in growth media for
16-18 days. Mouse β-actin was used as an endogenous control. N = 8 samples/group, representative of two independent experiments, *P ≤ 0.05, ***P ≤ 0.001.
Figure 3. B cell development is impaired in ERdj4GT/GT mice. (A) Total bone marrow
cells isolated from the femurs of 6-week-old mice. N = 20 mice/genotype, *P ≤ 0.05.
(B) Bone marrow cells isolated from the femurs of 6-week-old mice were stained
131 antibodies against IgM, B220 and CD43. Frequency of bone marrow pro-B (IgM-
B220+CD43+), pre-B (IgM-B220+CD43-), immature (IMB, B220lowIgM+) and mature (MB,
B220highIgM+) B cells were analyzed by flow cytometry. Contour plots are representative of each genotype. (C) Absolute numbers of each bone marrow B cell subset. N = 20 mice/genotype pooled from four independent experiments, *P ≤ 0.05, **P ≤ 0.01, ***P ≤
0.001. (D) Pro-B cells were further differentiated based on HSA and BP-1 expression
(fractions A through C). N = 20 mice/genotype pooled from four independent experiments, *P ≤ 0.05, ***P ≤ 0.001. (E) B cells were isolated from the femurs of 6- week-old mice, stained with 7-AAD and analyzed by flow cytometry. N = 6 mice/genotype, *P ≤ 0.05, **P ≤ 0.01.
Figure 4. ERdj4 deficiency in the bone marrow microenvironment affects B cell development. Recipient mice received two doses of lethal irradiation followed by intravenous injection of ERdj4GT/GT bone marrow cells. (A) Bone marrow pro-B (IgM-
B220+CD43+), pre-B (IgM-B220+CD43-), immature (IMB, B220lowIgM+) and mature (MB,
B220highIgM+) B cells were quantified by flow cytometry 8 weeks after transplantation. N
= 5-6 mice/genotype, *P ≤ 0.05, **P ≤ 0.005. (B) Frequencies of fraction A (BP-1-HSA-),
fraction B (BP-1+HSA-) and fraction C (BP-1+HSA+) were determined by gating on pro-B
cells. N = 5-6 mice/genotype, *P ≤ 0.05. (C) Pre-B colony forming units were assayed
by culturing total bone marrow in MethoCult media containing IL-7. N = 5-7
mice/genotype.
Figure 5. Mature B cells are decreased in the spleen and peritoneal cavity of
ERdj4GT/GT mice. (A) Total splenic leukocytes were isolated from 6-week-old mice. N =
9-12 mice/genotype pooled from two independent experiments, *P ≤ 0.05. (B)
132 Frequency of splenic immature (IMB, B220+CD93+), mature (MB, B220+CD93-),
follicular (FOB, B220+CD21lowCD23+) and marginal zone (MZB, B220+CD21+CD23low) B cells were analyzed by flow cytometry. Contour plots are representative of each genotype. (C) Absolute numbers of splenic B cell subsets. N = 9-12 mice/genotype, **P
≤ 0.01. (D) Recipient mice received two doses of lethal irradiation followed by intravenous injection of ERdj4GT/GT bone marrow cells. Splenic B cell populations were
quantified by flow cytometry 16 weeks after transplantation. N = 5-6 mice/genotype,
***P ≤ 0.001. (E) Peritoneal B1 (CD19+CD11b+) B cells were quantified in 6-week-old mice by flow cytometry. B1a and B1b subsets were differentiated based on CD5 expression. N = 16 mice/genotype pooled from three independent experiments, *P ≤
0.05. (F) Serum BAFF levels were determined in 6-week-old mice by ELISA. N = 6-7
mice/genotype, *P ≤ 0.05.
S1. Skeletal development of ERdj4+/+ and ERdj4GT/GT embryos. Alizarin red (bone)
and Alcian blue (cartilage) staining of mouse skeletons at E18.5. Scale bars are 0.25
cm. N = 4 mice/genotype.
S2. BMP2-treated ERdj4GT/GT MEFs have reduced osteoblast differentiation
potential. (A) Quantitation of ERdj4 mRNA in ERdj4+/+ MEFs treated with rhBMP2.
Mouse β-actin was used as an endogenous control. RQ, relative quantitation. N = 3
samples/group, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (B-C) BiP and sXBP1 mRNA
expression was assessed in ERdj4+/+ and ERdj4GT/GT MEFs treated with rhBMP2 for 96
hours. Mouse β-actin was used as an endogenous control. N = 3 samples/group, *P ≤
0.05, ***P ≤ 0.001. (D) ALP activity was determined in ERdj4+/+ and ERdj4GT/GT MEFs treated with rhBMP2 for 96 hours. N = 3 samples/genotype, **P ≤ 0.01. (E)
133 Quantitation of osteocalcin, Osterix and type 1 collagen (Col1a1) mRNAs by qRT-PCR
in ERdj4+/+ and ERdj4GT/GT MEFs treated with rhBMP2 for 96 hours. Mouse β-actin was used as an endogenous control. ND, not detected. Mean +/- SEM of triplicate cultures are depicted, *P ≤ 0.05, **P ≤ 0.01.
S3. B cells and erythrocytes are decreased in the bone marrow of ERdj4GT/GT
mice. (A-B) Bone marrow cells isolated from the femurs of 6-week-old mice were
stained with antibodies against Gr-1, F4/80, Ter119 and B220 and analyzed by flow
cytometry. N = 6-21 mice/genotype pooled from at least two independent experiments,
*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (C) Absolute number of bone marrow B cells from
6-week-old mice. N = 20 mice/genotype pooled from four independent experiments, **P
≤ 0.01, ***P ≤ 0.001.
S4. Bone marrow B cell proliferation and peripheral pro-B cells are normal in
ERdj4GT/GT mice. (A) Mice were intraperitoneally injected with BrdU (1 mg) 24 and 12
hours before sacrifice. Bone marrow B cell incorporation of BrdU was assessed by flow
cytometry. N = 9-10 mice/genotype. (B) Pro-B cells (IgM-B220+CD43+) were assessed
in the peripheral blood of 6-week-old mice by flow cytometry. N = 6 mice/genotype.
S5. Plasma cell differentiation and antibody production are normal in ERdj4GT/GT
mice. (A) Mice were intraperitoneally injected with 200 µl of goat anti-IgD antiserum and
the frequency of splenic plasma cells was determined 8 days post-immunization by flow
cytometry. N = 5-8 mice/genotype, **P ≤ 0.01. (B) Baseline immunoglobulin levels
were determined by ELISA in 4-6-month-old mice. N = 5-9 mice/genotype. (C-D) Mice
were intraperitoneally injected with TNP-CGG (C, 100 µg) or TNP-Ficoll (D, 50 µg) and
134 TNP-specific IgM and IgG antibody production was determined by ELISA. N = 5-7 mice/genotype.
135 A ERdj4+/+ ERdj4GT/GT
B ERAI Osteocalcin Merge +/+ ERdj4 GT/GT GT/GT ERdj4
Figure 1 136 A ERdj4+/+ ERdj4GT/GT
Quik Diff-
B Osteocalcin
C 1.0 ** ***** +/+ ) * * * ERdj4 Q * GT/GT
R 0.8 ERdj4 (
) 0.8 n Q o i R
( 0.6 s
s A 0.6 e N r p R 0.4 x m e
0.4
e 0.2 v i t a
l 0.2
e 0.0
R Osteocalcin Osterix Col1a1 RANKL 0.0 Osteocalcin Osterix Col1a
D 8 E *** d0 ** 2.5 ERdj4+/+ GT/GT
) d21 6 ERdj4 Q 2.0 ) R ( Q
R A *
4 ( 1.5
N A R N
m 1.0 2 ns R m 0 0.5 Osterix Osteocalcin 0.0 ERdj4GT/GT BiP sXBP-1
Figure 2 137 A B Gated on: IgM- Live cells Pre-B Pro-B 40 * 2.6% 21.6% 4.0% MB ) 6 0 1
30 +/+
x ERdj4 (
s
l 8.0% IMB l e
c 20
M B
l a B220 t 10 Pre-B Pro-B o MB T 2.0% 11.4% 1.6%
0 +/+ GT/GT ERdj4 ERdj4 ERdj4GT/GT 5.8% IMB
CD43 IgM C *** ** 30 64 8 * 12 ** 56
) 48
5 6
0 20 8
1 40
x ( 32 4 s l l e 24 10 4 C 16 2 1.0 ** +/+ 8 ) * ERdj4 Q * GT/GT
0 0 R 0 ERdj4
( 0 0.8
Pro-B Pren -B IMB MB o i s
s 0.6
D e r
3.0 p 15 x * 50 *** e
0.4 ) ) ) 4 5 5 2.5 e 0 0 0 v 40 i 1 1 1 t
x x x a ( ( l
( 2.0
10 0.2
- + e + 30 A A A 1.5 R S S S H H H 0.0 - - 20 + 1 1.0 1 5 1 -
- Osteocalcin Osterix Col1a - P P P +/+ B 1.0B 10 ** ) 0.5 B * ERdj4 Q * GT/GT
0.0 R ERdj4 ( 0 0 0.8
Fraction A n Fraction B Fraction C o i s s 0.6 ns E e r
20 ** p * 16 * x e
0.4 e ) v ) 15 i 12 t % % ( a
( l 0.2 + + e D D
10 R 8 A A A - A
0.0 - 7 5 Osteocalcin Osterix7 4 Col1a 1.0 ** +/+ ) * ERdj4 Q * GT/GT
0 R 0 ERdj4 ( Total B220B220++ B220B2200.8lolow B220B220highhi Pro-B n o i s
s 0.6 e r
Figure 3 p x e
0.4
e 138 v i t a
l 0.2 e R 0.0 Osteocalcin Osterix Col1a A B )
GT GT - 15 ! 10 GT ! GT * M *
GT ! WT g I 8 GT ! WT % t (
n 10 ** + e
r 6 3 a 0.07 4 P
D 4 * % 5 C +
0 2 * 2 2
0 B 0 Pro-B Pre-B IMB MB A B C
C
150 s l l e c
5 0
1 100 ! 1 / s U F
C 50
B - e r P 0 ERdj4+/+ ERdj4GT/GT
Figure 4 139 A B ERdj4+/+ ERdj4GT/GT MB MB
) 32.7% 26.8%
7 15 * 0 1
x ( B220
s IMB IMB
e 11.7% 11.5% t 10 y c o k CD93 u
e MZB MZB l 5 4.8% 6.3% c i n e l p CD21 S 0 FOB FOB +/+ GT/GT ERdj4 ERdj4 59.0% 43.5%
CD23 C D *** 50 +/+ GT GT ) ERdj4 60 ! 6 ** ** 0 GT/GT GT ! WT
1 ERdj4
40 x
( 45
s l
l *** 30 e v e i c
L
30 B
20 % c i n e l 10 15 p S 0 0 IMB MB FOB MZB IMB MB FOB MZB
1.0 E ** +/+ F ) * ERdj4 Q * GT/GT
R ERdj4 * ( 0.8 8 n o i * s 35 s 0.6 e ) r * 4 p 30 ) 6 0 x l 1 e
0.4 m x / e
( 25
g v i t s n l ( a l l 0.2 20 4 e e F c
R F 1 0.0 15 A B B Osteocalcin Osterix Col1a
C 10 2 r e
P 5 0 0 B1 B1-a B1-b ERdj4+/+ ERdj4GT/GT
Figure 5 140 A ERdj4+/+ ERdj4GT/GT
Supplemental Figure S1 141 +/+ A MEFs B 2.5 *** ERd+j/4+ 2.5 *** * ERdj4 GT/GT 2.8 * * ERdGjT4/GT ) ERdj4
) 2.0 ) 2.4 Q Q ** 2.0 Q R ( R
( R ***
2.0 ( A *** 1.5 *** A A N 1.5 N 1.6 N R R R
m 1.0 m
1.2
m 1.0
P 4 i j P d i 0.8 B
R 0.5 B
E 0.4 0.5 0.0 0.0 0.0 Untreated BMP2 0 24 48 72 96 Untreated BMP2 Time after BMP2 treatment (hours) MEFs MEFs
C D +/+ 20 4 *** ERdj4 ERdj4+/+ **
GT/GT ) ) *** ERdj4 GT/GT g
Q ERdj4 m R / ( 3 15
U A
*** (
N y
*** t R i
2 v
m 10 i
t 1 c - a P
B 1 P 5 X L s A 0 ** Untreated BMP2 0 Untreated BMP2 MEFs MEFs
E
** * ) 250 +/+ ** 1000 ERdj4+/+ ERdj4 4 +/+ Q
) ERdj4 GT/GT GT/GT ) R
Q ERdj4 GT/GT ( ERdj4 Q 200 ERdj4 800 R R ( A (
3 N A A R N 600 150 N m R R 2 n m m i
c x l 100 400 1 i r a a c e 1 t l
o 1 s o
e 200 50 t O C s *
O ND 0 0 0 Untreated BMP2 Untreated BMP2 Untreated BMP2 MEFs MEFs MEFs
Supplemental Figure S2 142 A 1.0B ** +/+ ) * ERdj4
Q *** 1.0 ** +/+ * GT/GT ) 80 ERdj4 R ERdj4
* (
Q * GT/GT 0.8 30 n
R ERdj4 o ( i
) ** 0.8 s n ) M s 0.6 o 25 e i 60 B r
% s p (
s * e
0.6 x e v s e i r 20
0.4 l l p L e x v e i t
e 40 c %
0.4
a
( 15
l 0.2 e e e v + i v t R 0 i a l 0.2 2 10 L 0.0 e 20 2 Osteocalcin Osterix Col1a ** R B 0.0 5 Osteocalcin Osterix Col1a 0 0 Gr-1+ F4/80+ Ter119+ Total B220+ B220low B220high
1.0 C ** +/+ ) * ERdj4 Q * *** GT/GT
R 100 ERdj4 ( 0.8 n o ** i s
) 80 s
0.6 5 e r 0 p 1 x x e
( 60
0.4 e s l v l i t e a l 0.2 c 40 e M R 0.0 B ** Osteocalcin20 Osterix Col1a 0 Total B220+ B220low B220high
Supplemental Figure S3 143 1.0 ** +/+ A ) * ERdj4 Q * GT/GT
R ERdj4 ( 0.8 72 60 n o i
s 60 s 0.6 e r ) 45 p x
) 48 % e (
0.4
% ( e
+ v +
i 36 U 30 t U d a l r
d 0.2 r e B
B 24 R 15 0.0 Osteocalcin12 Osterix Col1a 0 0 Total B220+ B220low B220high Pro-B Pre-B
B 0.5 ) e v i L 0.4 % (
+
3 0.3 4 D C
+ 0.2 0 2 2 B - 0.1 M g I 0.0 ERdj4+/+ ERdj4GT/GT
Supplemental Figure S4 144 1.0 ** +/+ +/+ ) A * ERdj4 B 2.5 ERdj4
Q * GT/GT GT/GT
R ERdj4
( ERdj4 0.8
n 2.0 o )
i ** l s m s
0.6 ** 4 / 2.5 e 1.5 r
2.5 g ) p n e ) ( x v l
i e
0.4 2.0 E L
m 1.0
e 2.0 / 3 g v I g i % t ( a L m l 0.2 + ( 0.5 1.5 e 1.5 m 8 / g R 3 I 2 g
1
0.0 n D 1.0 m 0.0 1.0
Osteocalcin Osterix Col1a u C r IgE + e
9 1 S 1 0.5 0.5 D C 0.0 0 0.0 Untreated g!mIg-D IgM IgG IgA IgE
ERdj4+/+ GT/GT C ERdj4 2.5 ERdj4+/+ ERdj4GT/GT 2.0 ) l 2.5 ) ) 2.0 m m /
m 1.5 g n n
n
( 0
0 2.0 5 5 1.5 E 1.0 4 4 g I ( (
1.5 M G 0.5 g g I I
1.0 G 1.0 G 0.0 G G
C IgE - C
- 0.5 P 0.5 P N N T 0.0 T 0.0 -3 7 14 21 -3 7 14 21 Time (days) Time (days) D 2.5 ERdj4+/+
) 3.0 ERdj4GT/GT ) 2.5 m 2.0 n ) m l
n 0
m / 5 0 1.5 2.0 g 4 5 n ( (
4
2.0 (
E G 1.0 g g M 1.5 I I
g l I l
l 0.5 o l c
o 1.0 i 1.0 c i F
- 0.0 F - 0.5 P IgE P N N T
T 0.0 0.0 -3 7 14 21 -3 7 14 21 Time (days) Time (days)
Supplemental Figure S5 145 References
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150
CHAPTER IV
Discussion and Future Directions
151 Summary
ERdj4 facilitates the removal of unfolded/misfolded substrates from the ER lumen
for proteasomal degradation. Previous studies identified ERdj4 as a chaperone that
was ubiquitously expressed at low levels under normal conditions, but rapidly
upregulated in response to stress. However, herein we identify an important role for
ERdj4 in postnatal survival, glucose metabolism and B cell development in vivo.
Approximately half of ERdj4 gene trap (ERdj4GT/GT) mice died perinatally in association with growth retardation and hypoglycemia. Altered distribution of pancreatic
α- and β-cells, as well as decreased insulin and glucagon in the pancreas, indicated a
delay in pancreatic development in ERdj4GT/GT neonates. Other common causes of
perinatal lethality, including defects in respiration and suckling, were excluded in
ERdj4GT/GT neonates. Collectively, these results suggest that ERdj4 is required for adaptation to metabolic stress in the neonatal period.
Mice that survived into adulthood were glucose intolerant due to hypoinsulinemia, rather than insulin resistance. Pancreatic β-cells from ERdj4GT/GT mice exhibited
increased levels of ER stress, as indicated by cytoplasmic vacuolation, ER dilation and upregulation of the UPR. A previous study in our laboratory identified insulin as a
substrate for ERdj41; the current work identified other ERdj4 substrates including the
insulin-processing enzymes, Pcsk1, Pcsk2 and carboxypeptidase E (CPE).
Furthermore, the loss of ERdj4 resulted in accumulation of proinsulin in the ER, which
likely contributed to β-cell stress and dysfunction. Overall, these results indicate that
the chaperone activity of ERdj4 is essential for β-cell homeostasis, insulin biogenesis
and glucose metabolism.
152 Since the UPR influences several stages of B cell development2,3, we hypothesized that B cell maturation would also be impaired in ERdj4GT/GT mice. The
loss of ERdj4 resulted in fewer developing B cells in the bone marrow in association
with increased cell death. Further, several mature B cell populations, including follicular
and B1b B cells, were reduced in the spleen and peritoneal cavity, respectively.
Surprisingly, ERdj4 deficiency did not affect antibody secretion by plasma cells, even
though expression of ERdj4 was increased during plasma cell differentiation4.
Bone marrow chimeras revealed that a defect in the microenvironment of
ERdj4GT/GT mice affected B cell development in the bone marrow and periphery. Given
the role of the UPR in osteoblast differentiation and function, we hypothesized that
osteogenic cells were the defective stromal cell type in ERdj4GT/GT mice. Osteogenic
cells support B lymphocyte commitment and development through the expression of
adhesion molecules and cytokines5,6. Osteogenic cells isolated from ERdj4GT/GT mice
exhibited reduced expression of osteoblast-specific markers and increased levels of ER
stress. Importantly, bone-lining cells, which differentiate from osteoblasts, were
reduced in femur tissue sections of ERdj4GT/GT mice. These data support the hypothesis that reduced osteogenic cells in ERdj4GT/GT mice are responsible for the defect in B cell development.
Although previous studies demonstrated that ERdj4 facilitates the removal of unfolded/misfolded proteins from the ER lumen in vitro1,7,8, the biological relevance of
ERdj4 was unknown. This study highlights the significance of ERdj4 in vivo, revealing
an important role in survival, glucose metabolism and B cell development. The
questions raised by these findings will be addressed in the remainder of this discussion.
153 1. Gene trapping ERdj4: a blessing in disguise?
To determine the role of ERdj4 in vivo, ERdj4GT/GT mice were generated from embryonic stem cells harboring a gene trap vector randomly inserted into the ERdj4 locus. Gene trapping is a cost-effective and efficient experimental strategy for gene ablation. The International Gene Trap Consortium (IGTC) provides embryonic stem cells with identified trapped genes, eliminating the need to generate a targeting construct for homologous recombination in embryonic stem cells9,10. When the gene trap is inserted into an intron, the promoter of the “trapped” gene drives transcription of a fusion transcript, consisting of the upstream exon(s) spliced with the gene trap.
Sequencing analyses determined that the gene trap cassette inserted into intron 1 of the ERdj4 locus, which resulted in a hypomorphic allele. We hypothesized that splicing around the gene trap cassette led to transcription of some full-length, wildtype ERdj4; however, the presence of multiple transcripts in ERdj4GT/GT mice remains to be confirmed. A gene copy number assay determined that only one copy of the gene trap was present per haploid genome (Chapter 2, Figure 1C). This data was critical for the credibility of the ERdj4GT/GT phenotype, confirming that the gene trap disrupted only the
ERdj4 locus and not any other unknown loci.
In mice homozygous for the gene trap allele, expression of ERdj4 mRNA differed
among tissues and cell types examined by quantitative RT-PCR, but was 10-100 fold
lower compared to ERdj4+/+ controls (Chapter 2, Figure 1F). It is unclear why the
expression of ERdj4 varied in ERdj4GT/GT mice; however, it is possible that elevated ER
stress induced by ERdj4 deficiency increased ERdj4 promoter activity leading to
transcription of wildtype ERdj4 (related to splicing around the GT cassette).
154 Unfortunately, the level of ERdj4 protein in ERdj4GT/GT mice remains unknown because
of the lack of useful antibodies, including an antibody generated in our laboratory
against recombinant ERdj4. Erik Snapp et al. have alluded to the generation of a monoclonal antibody against ERdj4, which will be described in an upcoming publication and may help to resolve these unanswered questions7.
Approximately half of the ERdj4GT/GT mice died perinatally (Chapter 2, Tables 1-2)
and we hypothesize that hypomorphic expression of ERdj4 likely prevented fully
penetrant lethality. The surviving ERdj4 hypomorphic mice reached adulthood, but had
defects in glucose metabolism (Chapter 2) and B cell development (Chapter 3). Adult
mice also displayed signs of a vestibular disorder and female mice were infertile,
although these defects were not pursued in the current study. Overall, gene ablation of
ERdj4 using gene trapping instead of homologous recombination likely enabled the
study of ERdj4 in adult mice, revealing several interesting phenotypes that have yet to
be fully explored.
2. What is the function of ERdj4 in perinatal survival?
Perinatal lethality of ERdj4GT/GT mice was associated with growth retardation and hypoglycemia (Chapter 2, Figure 3A-C). After birth, pups endure a starvation period before the mother can provide nutrients from suckling. Survival during this time period depends on self-nourishment, which is mediated through glycogenolysis and the balance of insulin, glucagon and glucocorticoids to regulate metabolism11,12.
Interestingly, neonatal lethality and hypoglycemia were also observed in mice
expressing a nonphosphorylatable mutant of eIF2α, a component of the UPR signaling
155 pathway. These neonatal mice had reduced hepatic glycogen, although whether this
was the result of a defect in glycogen synthesis/storage or increased utilization of
glycogen remains unclear13. Glycogen storage was assessed in E18.5 livers of
ERdj4+/+ and ERdj4GT/GT mice by PAS staining (data not shown), but the result was
inconsistent and revealed no discernable difference between genotypes. A more
sensitive method to evaluate glycogen content will be pursued by using an assay that
quantitates glycogen in biological samples based on the production of glucose by
glucoamylase.
The underlying cause(s) of perinatal growth restriction in ERdj4GT/GT mice
(Chapter 2, Figure 3A-B) remains entirely unknown. However, since ERdj4 is highly
expressed in the placenta8, it is possible that defects in placental development affect nutrient and gas exchange leading to fetal growth restriction14. Further, the IRE1α
signaling pathway is essential for development of the labyrinth layer of the placenta,
which is the site of nutrient and gas exchange between the mother and fetus15. Since the IRE1α signaling pathway regulates expression of ERdj4, future studies will investigate whether ERdj4 deficiency affects placental development. Placental morphology will be analyzed by H&E staining in E18.5 ERdj4+/+ and ERdj4GT/GT mice.
Given that fetal essential fatty acids (EFAs) are obtained only from the mother, nutrient transport will be assessed in fetal ERdj4+/+ and ERdj4GT/GT mice by quantitating EFAs
through gas chromatography15. Interestingly, low birth weight is associated with type 2 diabetes in adulthood due to accelerated growth that alters food intake and glucose metabolism16,17. ERdj4GT/GT mice reached normal weight by 8 weeks of age (data not
156 shown), but were glucose intolerant, suggesting that growth retardation during
embryogenesis affects glucose homeostasis in adult ERdj4GT/GT mice.
Several lines of evidence indicated that ERdj4GT/GT neonates have a delay in
pancreatic development, including altered distribution of α- and β-cells and decreased
pancreatic insulin and glucagon (Chapter 2, Figure 3D-F). Further studies are needed
to confirm the defect in pancreatic development, including enumeration of α- and β- cells, as well as assessment of maturation markers (e.g. MafA and MafB). Plasma glucagon and insulin also need to be quantitated in newborn ERdj4GT/GT mice. Given
that ERdj4GT/GT neonates are hypoglycemic, we expect to observe lower levels of circulating glucagon, thereby reducing glycogenolysis in the liver.
Although other possible causes of neonatal lethality, including abnormal feeding and respiration, appeared normal in ERdj4GT/GT neonates (data not shown), it remains
unclear whether hypoglycemia directly caused their demise. To address this question,
glucose will be administered to neonatal mice during the starvation period, a procedure
that has previously been shown to rescue lethal hypoglycemia18. We hypothesize that
ERdj4GT/GT neonates that receive glucose will survive, thus confirming the link between
hypoglycemia and neonatal lethality.
3. ER stress and Type 2 Diabetes
Type 2 diabetes is a heterogeneous group of metabolic illnesses characterized
by high blood glucose levels due to insulin resistance in the periphery and/or impaired
insulin secretion by pancreatic β-cells. Although obesity and insulin resistance are
linked to type 2 diabetes, insulin-resistant individuals only develop the disease with the
157 onset of β-cell failure19. Current lifestyles, with increased consumption of energy-rich foods and reduced physical activity, have contributed to the prevalence of diabetes, affecting approximately 285 million adults worldwide. Further, global estimates predict a
54% increase in diabetes by 2030 due to population growth, ageing and urbanization associated with a sedentary lifestyle20. Although improved survival has been reported for other chronic, noninfectious diseases21, mortality attributed to diabetes increased
29% from 2007 to 2010, a frequency expected to rise as a result of increased
prevalence22. Unfortunately, the pathogenesis of diabetes is not well understood and
further insight is necessary for the prevention and treatment of disease.
Several studies have linked ER stress to β-cell dysfunction and type 2 diabetes23-
25. Chapter 2 highlights the importance of ERdj4 in glucose metabolism in vivo.
However, several questions remain including, whether ERdj4GT/GT mice develop diabetes with metabolic stress, how the loss of ERdj4 causes β-cell failure and whether
rescue of ERdj4 expression in pancreatic β-cells will correct the defects in glucose
metabolism. Further, the role of ERdj4 in pancreatic α-cells remains entirely unclear.
These questions and issues will each be addressed in turn.
3a. Do physiological stressors cause diabetes in ERdj4GT/GT mice?
Although fasting blood glucose levels were normal in young adult ERdj4GT/GT mice, they
were considered pre-diabetic based on glucose intolerance, hypoinsulinemia and an
increased proinsulin to insulin ratio (Chapter 2, Figure 6). It remains unknown whether
increased physiological β-cell stress, including pregnancy26, age27 or high-fat diet
(HFD)24,28, would promote diabetes in ERdj4GT/GT mice. Pregnancy cannot be used as
158 model of metabolic stress because female ERdj4GT/GT mice are infertile; however, aging
and HFD studies are concurrently underway. We hypothesize that age and/or HFD will
result in hyperglycemia in ERdj4GT/GT mice, given the β-cell dysfunction and prediabetic
phenotype already observed in these animals. Since insulin stimulates lipogenesis in
peripheral tissues and ERdj4GT/GT mice are hypoinsulinemic, it is possible that a HFD
will result in increased levels of circulating FFAs, which could promote cardiovascular
disease in ERdj4GT/GT mice.
If ERdj4GT/GT mice do not develop diabetes with physiological stressors, an
alterative approach would be to cross ERdj4GT/GT mice to the Akita mouse model of type
2 diabetes. These mice carry a C96Y mutation in the insulin 2 (insulin2C96Y) gene, resulting in disruption of disulfide bond formation and accumulation of misfolded protein.
Akita mice develop hyperglycemia and hypoinsulinemia at approximately 4 weeks of
age29. Our laboratory previously showed that ERdj4 facilitates the removal of insulin2C96Y from the ER1. Thus, we hypothesize that the loss of ERdj4 in Akita mice
would further increase accumulation of insulin2C96Y in the ER, resulting in an
exacerbated diabetic phenotype.
3b. What causes β-cell failure in ERdj4GT/GT mice?
Cytoplasmic vacuolation, ER dilation and upregulation of the UPR (Chapter 2, Figure 4),
indicated that ERdj4 deficiency caused ER stress in pancreatic β-cells. What remains
unclear, however, is whether increased ER stress results in apoptosis or
dedifferentiation of β-cells in ERdj4GT/GT mice. The UPR activates cell death pathways in response to protein accumulation in the ER that cannot be resolved through
159 attenuation of protein translation or upregulation of protein folding/degradation
machinery (Chapter 1, Section 2cii)24. β-cell death, apparent in several mouse models with genetic alterations in the UPR30,31, will be assessed in pancreatic tissue sections from ERdj4GT/GT mice by using the TUNEL assaya. However, a recent study by Talchai
et al. indicated that enhanced ER stress did not result in apoptosis, but instead caused
β-cell dedifferentiation to a progenitor phenotype, expressing Neurogenin3, Oct4,
Nanog and L-Myc32, that no longer produced insulin. Importantly, β-cell dedifferentiation also led to an increased number of α-cells, resulting in hyperglucagonemia. Based on the α-cell hyperplasia and hyperglucagonemia observed in ERdj4GT/GT mice, we hypothesize that increased ER stress causes β-cell dedifferentiation and not apoptosis in ERdj4GT/GT mice. Thus, we expect to observe an increase in the number of progenitor cells in islets of ERdj4GT/GT mice compared to
controls. These findings will provide a mechanistic understanding of how increased ER
stress causes β-cell failure in ERdj4GT/GT mice.
3c. Does rescue of ERdj4 expression in pancreatic β-cells result in normal insulin
secretion and glucose metabolism in ERdj4GT/GT mice?
Pancreatic β-cells activate the UPR in order to maintain ER homeostasis during
increased insulin biosynthesis. The UPR increases protein folding in the ER through
upregulation of foldases and chaperones24. Our data suggest that the chaperone activity of ERdj4 plays an important role in insulin biosynthesis. ERdj4 associated with insulin, as well as several insulin processing enzymes, including Pcsk1, Pcsk2 and CPE
a The terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay detects DNA fragmentation associated with apoptosis.
160 (Chapter 2, Figure 5C). Loss of ERdj4 led to accumulation of proinsulin in the ER,
contributing to increased β-cell stress (Chapter 2, Figures 4 and 5). Reduced insulin
biogenesis resulted in hypoinsulinemia and glucose intolerance in ERdj4GT/GT mice
(Chapter 2, Figure 6). To confirm that ERdj4 deficiency impairs insulin biosynthesis,
proinsulin processing will be assessed by pulse chase analyses using a pancreatic β- cell line transfected with small interfering RNA (siRNA) against ERdj4. We hypothesize that ERdj4 deficiency in β-cells will disrupt proinsulin processing leading to reduced production and secretion of mature insulin.
Since ERdj4 is important for β-cell homeostasis and function, we hypothesize
that rescuing ERdj4 expression in β-cells will enhance insulin secretion and ameliorate
glucose intolerance in ERdj4GT/GT mice. These studies will be performed in vitro by
transfecting ERdj4 into pancreatic islets isolated from ERdj4GT/GT mice33 and analyzing
insulin biosynthesis by pulse chase analyses. We expect that rescue of ERdj4
expression will enhance proinsulin processing and, as a result, increase secretion of
mature insulin. This hypothesis could be further tested in vivo by rescuing ERdj4
expression in pancreatic β-cells of ERdj4GT/GT mice. ERdj4 cDNA would be subcloned
into a plasmid containing the porcine insulin promoter, which directs expression in
mouse pancreatic β-cells34. Expression of the ERdj4 transgene would be confirmed in
vitro by transfecting a pancreatic β-cell line with the recombinant plasmid and assessing
ERdj4 expression by qRT-PCR. The DNA fragment would then be microinjected into
ERdj4+/GT oocytes and fertilized embryos would be transferred into pseudopregnant mice. Founder mice would be assessed for the presence of the ERdj4 transgene by
Southern blot analysis and multiple transgenic lines would be generated to exclude
161 artifacts related to random transgenic integration. ERdj4 mRNA expression would be
analyzed in pancreatic β-cells isolated from transgenic mice by qRT-PCR. We hypothesize that rescuing ERdj4 expression in pancreatic β-cells would reduce ER stress and increase insulin biosynthesis leading to normal glucose tolerance. If
ERdj4GT/GT mice develop diabetes with age and/or HFD (as discussed in section 3a), we
predict that rescuing ERdj4 in pancreatic β-cells would prevent the onset of diabetes.
Alternatively, if rescue of ERdj4 in pancreatic β-cells fails to correct the metabolic
defects in ERdj4GT/GT mice, it is possible that aberrant expression/activity of Pcsk1 indirectly affects insulin secretion. Pcsk1 also processes proglucagon in intestinal L cells to generate glucagon-like peptide (GLP)-135,36, which stimulates glucose- dependent insulin secretion37. Thus, lower levels of GLP-1 in ERdj4GT/GT mice may
cause hypoinsulinemia and glucose intolerance. Overall, these studies would provide
insight into how the absence of ERdj4 causes abnormalities in glucose metabolism.
Since ER stress is associated with obesity, insulin resistance and type 2 diabetes, alleviating ER stress by increasing protein folding capacity may be a novel therapeutic strategy in the treatment of disease. Ozcan et al. tested this hypothesis
using the pharmaceutical chaperones 4-phenyl butyric acid (PBA) and
tauroursodeoxycholic acid (TUDCA) in leptin-deficient mice (ob/ob), a model of obesity
and insulin resistance. PBA and TUDCA are chemical chaperones, approved by the
Federal Drug Administration for use in humans, that function to increase protein folding
38 and degradation in ER . Treatment with either PBA or TUDCA reduced ER stress in peripheral tissues, resulting in normoglycemia with increased insulin sensitivity and resolution of fatty liver disease28. It remains unknown whether PBA or TUDCA are
162 capable of alleviating β-cell stress in other mouse models of type 2 diabetes (e.g. Akita mice). However, since ER stress is tightly associated with disease pathogenesis, restoring ER function by chemical chaperones is a promising therapeutic approach for type 2 diabetes38.
3d. Is the chaperone activity of ERdj4 important in α-cells?
3di. Does loss of ERdj4 affect glucagon biosynthesis?
Glucagon is released from pancreatic α-cells in response to low levels of blood glucose in order to stimulate gluconeogenesis and glycogenolysis. Accumulation of glucagon was associated with increased ER stress in pancreatic α-cells of ERdj4GT/GT mice
(Chapter 2, Figure S2B), suggesting that ERdj4 may be required for glucagon
maturation in the ER. However, co-immunoprecipitation experiments revealed that,
unlike insulin, ERdj4 did not directly associate with glucagon but rather, interacted with
Pcsk2 and CPE, glucagon-processing enzymes (Chapter 2, Figure 5C). Mice deficient
in either Pcsk2 or CPE had defects in proglucagon processing and glucose
metabolism39,40.
Since ERdj4 associated with Pcsk2 and CPE, but not glucagon, we hypothesize
that ERdj4 indirectly affects proglucagon processing in α-cells by influencing Pcsk2 and
CPE maturation. Several experiments are needed to test this hypothesis. First,
glucagon processing and secretion will be assessed by pulse chase analyses using a
pancreatic α-cell lineb transfected with ERdj4 siRNA. We expect that proglucagon will accumulate in α-cells deficient in ERdj4, thus explaining the increase in glucagon
b Islets are not used to assess glucagon processing by pulse chase analysis due to α-cell hyperplasia in ERdj4GT/GT mice.
163 granules observed in pancreatic α-cells of ERdj4GT/GT mice (Chapter 2, Figure S2B).
Second, proglucagon levels will be assessed in the pancreas and serum of ERdj4GT/GT
mice by immunoassay. We hypothesize that proglucagon will be increased in the
pancreas and serum, if expression and/or enzymatic activity of Pcsk2/CPE is altered in
the absence of ERdj4. Third, the maturation of Pcsk2/CPE will be analyzed by pulse
chase analyses in an α-cell line transfected with ERdj4 siRNA. Since ERdj4 interacts
with Pcsk2/CPE, we hypothesize Pcsk2/CPE processing will be reduced in the absence
of ERdj4. These studies will determine if ERdj4 indirectly disrupts glucagon
biosynthesis by affecting the expression and/or maturation of glucagon-processing
enzymes.
ERdj4 also associated with Pcsk1, which processes proglucagon in intestinal L
cells to generate GLP-1 and GLP-235,36 (Chapter 2, Figure 5C). GLP-1 and GLP-2 regulate energy homeostasis by influencing insulin/glucagon secretion and intestinal nutrient absorption, respectively37. The levels of GLP-1 and GLP-2 have not been
assessed in ERdj4GT/GT mice, but it is possible that they are affected by ERdj4 deficiency, thereby contributing to the defects in glucose metabolism (as discussed in section 3c).
3dii. What causes α-cell hyperplasia in ERdj4GT/GT mice?
Defective proglucagon processing is associated with α-cell hyperplasia40, a phenotype
observed in pancreatic islets of ERdj4GT/GT mice (Chapter 2, Figure 4D). Since the
mature form of glucagon negatively inhibits α-cell proliferation, decreased mature
glucagon in ERdj4GT/GT mice may promote an increase in α-cell mass. Thus, assessing
164 proglucagon processing (as described above) will elucidate the mechanisms underlying
α-cell hyperplasia. Insulin also regulates glucagon41, another factor likely contributing to the increased number of α-cells and hyperglucagonemia in hypoinsulinemic ERdj4GT/GT
mice (Chapter 2, Figure 6). Several diabetic mouse models30,31,42, as well as humans
with type 2 diabetes43 report α-cell hyperplasia and hyperglucagonemia as a result of insulin deficiency. Thus, it is possible that dysregulation of glucagon and/or insulin in
ERdj4GT/GT mice leads to α-cell hyperplasia.
4. The role of ERdj4 in the bone marrow microenvironment
4a. Does the loss of ERdj4 in osteogenic cells impair B cell development in ERdj4GT/GT
mice?
4ai. Do ERdj4GT/GT osteogenic cells support B cell maturation in vitro?
Although osteogenic cells are reduced in ERdj4GT/GT mice in association with increased
ER stress (Chapter 3, Figure 1), direct evidence linking ERdj4 deficiency in osteogenic cells to the defect in B cell development is lacking. To address this issue, HSCs will be co-cultured with osteogenic cells isolated from either ERdj4+/+ or ERdj4GT/GT mice. Zhu
et al. demonstrated that osteogenic cells isolated from neonatal calvariae supported B
cell development from HSCs through expression of VCAM-1, CXCL12 and IL-76.
However, their isolation technique resulted in a heterogeneous cell population with the
possibility of fibroblast contamination. Thus, a different approach will be used to obtain
primary osteogenic cells; mesenchymal stem cells (MSCs, Lin-CD45-CD31-Sca1+),
isolated from mouse compact bone by collagenase digestion and fluorescence activated
cell sorting (FACS), will be differentiated into osteogenic cells by treatment with
165 BMP244,45. The ability of ERdj4+/+ and ERdj4GT/GT MSCs to undergo osteogenesis will
be assessed by ALP activity and qRT-PCR of Osterix, osteocalcin and type 1 collagen
mRNAs. Further, B cell adhesion molecules and cytokines, including VCAM-1, CXCL12
and IL-7, will be analyzed by flow cytometry and/or immunoassay. Wild-type HSCs will
then be cultured on osteogenic cells for 7 days before assessing B cell development by
flow cytometry6. We expect that ERdj4 deficiency will impair osteogenesis by affecting
maturation and/or survival of osteogenic cells, thereby affecting B cell development. It
is possible that MSCs from ERdj4GT/GT mice could differentiate into osteogenic cells in vitro, resulting in normal B cell development. These findings would suggest that osteogenic cells from ERdj4GT/GT mice are capable of supporting B cell development, but
ERdj4 deficiency may affect growth factors necessary for osteogenesis in vivo.
4aii. Does administration of PTH rescue the defect in B cell development in ERdj4GT/GT
mice?
PTH promotes osteoblast differentiation, function and survival6,46,47. Importantly, PTH
induces the expression of several B cell growth factors, including CXCL12 and IL-7,
thereby enhancing B cell development in vitro6. Defects in the PTH signaling pathway in osteogenic cells reduced B cell development in the bone marrow beginning at the pro-B cell stage, similar to ERdj4GT/GT mice. We expect that exogenous PTH will
stimulate osteogenesis in ERdj4GT/GT mice, thereby rescuing the defect in B cell
development. A potential pitfall to this experiment is the expression of PTH receptor on
B cells; PTH receptor signaling inhibited B cell proliferation by altering intracellular
calcium metabolism48-50. Thus, it is possible that inhibition of B cell proliferation by PTH
166 will prevent rescue of B cell development via the osteogenic pathway. To address this issue, B cell development will be assessed in the bone marrow of wildtype mice upon treatment with PTH. If PTH disrupts B cell development under normal conditions, alternative approaches would be pursued to identify whether impaired osteogenesis reduces B cell development in ERdj4GT/GT mice (as discussed below).
4aiii. Would rescue of ERdj4 expression in osteogenic cells result in normal B cell development in ERdj4GT/GT mice?
An alternative approach to PTH administration would be to rescue ERdj4 expression in
osteogenic cells of ERdj4GT/GT mice. Mouse ERdj4 cDNA would be subcloned into a
vector containing the rat collagen α1 type I promoter, which directs expression in
osteoblasts, as well as bone-lining cells and osteocytes51,52. The plasmid would be
transfected into an osteoblast cell line to verify expression of the ERdj4 transgene by
qRT-PCR. The DNA fragment would be microinjected into oocytes from ERdj4+/GT mice
and fertilized embryos would be transferred into pseudopregnant mice. Founder mice
would be tested for the presence of the transgene by Southern blot analyses and
multiple transgenic lines would be generated to exclude artifacts related to random
transgenic integration. ERdj4 mRNA expression would be analyzed in osteogenic cells
isolated from transgenic mice by qRT-PCR. Osteogenic cells and B cell development
would then be assessed in transgenic mice by immunohistochemistry and flow
cytometry, respectively. We expect that rescuing ERdj4 expression in osteogenic cells
would increase the number of postosteoblasts resulting in normal B cell development in
167 ERdj4GT/GT mice. These studies would confirm that the loss of ERdj4 in osteogenic cells
causes the defect in B cell development in ERdj4GT/GT mice.
4b. Which cell(s) of the osteogenic lineage affects B cell development?
Osteoblasts are reported to influence hematopoiesis5,53,54. However, this conclusion is based on approaches that affect cells of the osteogenic lineage, not osteoblasts per se.
Conditional depletion of osteoblasts in vivo by expressing herpesvirus thymidine kinase
under control of the rat collagen α1 type I promoter also depleted postosteoblastic cells,
including bone-lining cells and osteocytes55. Ablation of these osteogenic cell types
resulted in a reduction of HSCs, B cells and erythrocytes in the bone marrow5. Further,
Zhu et al. demonstrated that osteoblasts isolated from neonatal calvariae supported B
cell development through VCAM-1, CXCL12 and IL-76. Although these calvarial cells expressed osteogenic-specific markers, the cultures contained multiple cell types within the osteogenic lineage, including MSCs, osteoblasts and osteocytes.
Osteoblasts are active at low frequencies during adulthood; in humans, only 0.1-
7.3% of endosteal surfaces are forming new bone and after bone formation, osteoblasts either undergo cell death or become quiescent bone-lining cells or osteocytes. Thus, if osteoblasts are the only cell in the osteogenic lineage to support hematopoiesis, the hematopoietic niche would be transient, relocating to new areas of bone formation, an unlikely possibility. It is more reasonable to postulate that other cells of the osteogenic lineage, including MSCs, bone-lining cells and osteocytes, influence hematopoiesis56.
MSCs are scattered throughout the bone marrow and a play a pivotal role in
hematopoiesis by stimulating adipogenesis and osteogenesis, as well as the
168 development of bone marrow sinusoids56. Bone-lining cells and osteocytes are the most abundant osteogenic cell types, covering approximately 90% of all bone surfaces57. Osteocytes also express high levels of RANKL, which influences B cell
development58,59. Bone-lining cells were reduced in association with impaired B
lymphopoiesis in ERdj4GT/GT mice (Chapter 3, Figures 1 and 3), although it is possible that these phenotypes are unrelated (addressed experimentally in section 4a).
Unfortunately, the role of MSCs and postosteoblasts in B cell development remains unknown, challenged by the lack of markers defining specific stages of osteogenesis and the plasticity of phenotypes within the osteogenic lineage56.
4c. How does ERdj4 deficiency affect peripheral B cells?
Numbers of follicular and B1b cells were reduced in the spleen and peritoneal cavity of
ERdj4GT/GT mice, respectively. However, not all mature B cell compartments were
compromised; marginal zone and B1a B cells, which function similarly but differ in
anatomical location, were preserved. Similarly, transitional B cells, representing the
most recent bone marrow emigrants, were also unaffected. Bone marrow chimeras
rescued the defect in mature B cell development, supporting a microenvironmental
defect in ERdj4GT/GT mice (Chapter 3, Figure 5).
Follicular B cells continuously circulate throughout the body, visiting the spleen,
lymph nodes, Peyer’s Patches and bone marrow60,61. In the bone marrow, mature B cells reside in the sinusoidal niche, receiving survival signals from dendritic cells through MIF62. Interestingly, the bone marrow sinusoidal niche is formed by osteoclasts, which are regulated by osteocytes56. Thus, it is possible that osteocyte
169 deficiency in ERdj4GT/GT mice affects the sinusoidal niche, thereby impairing the survival of mature recirculating B cells. Experiments addressing whether the loss of ERdj4 in osteogenic cells caused the defect in B cell development are addressed above (section
4a).
Mechanisms for the identified defects in mature peripheral B cells include decreased production, survival and/or proliferation. These possibilities will be explored by using BrdU incorporation to assess cell turnover, as well as cell fate63. BrdU will be
administered to mice through their drinking water for 14 days. Mice will be sacrificed on
days 7 and 14 of the pulse or days 42 or 84 of the chase and mature B cell populations
will be analyzed in the spleen and peritoneal cavity by flow cytometry64. Given that a microenvironmental defect in the bone marrow increased pro-B cell death in ERdj4GT/GT
mice, we expect that BrdU+ mature B cells will decrease more quickly overtime,
indicating a shorter lifespan.
4d. How does the loss of ERdj4 affect postosteoblasts and skeletal development?
4di. Quantitation of osteogenic cells from ERdj4GT/GT mice
Although histological analyses of femur tissue sections revealed a loss of bone-lining
cells in ERdj4GT/GT mice, these cells have yet to be quantitated. To address this issue,
osteoblasts, bone-lining cells and osteocytes will be quantitated using
histomorphometric analyses. Bone sections will be stained with toluidine blue and the
OsteoMeasure system (Osteometrics, Inc.) will be used to count the number of
osteogenic cells per tissue volume65. Anatomical location will distinguish between
osteoblasts, bone-lining cells and osteocytes.
170 4dii. Why are postosteoblasts reduced in ERdj4GT/GT mice?
After bone formation, a majority of osteoblasts undergo apoptosis, while some
differentiate into bone-lining cells and osteocytes57. Bone-lining cells and osteocytes
were reduced in femur tissue sections and primary osteogenic cultures from ERdj4GT/GT
mice, respectively (Chapter 3, Figures 1 and 2). The increased ER stress observed in osteogenic cells in vivo may enhance osteoblast apoptosis, thereby reducing the pool of
cells that become bone-lining cells or osteocytes. Alternatively, postosteoblasts may
have an increased susceptibility to apoptosis. This question will be addressed by using
the TUNEL assay to stain for DNA fragmentation, a marker of apoptosis, on femur
tissue sections from ERdj4GT/GT mice. Osteoblasts, bone-lining cells and osteocytes will be identified by anatomical location. If ERdj4 deficiency does not increase osteoblast
and/or postosteoblast apoptosis, it is possible that osteoblasts fail to differentiate into
bone-lining cells and osteocytes. A block in development would likely result in
accumulation of MSCs and osteoblasts in ERdj4GT/GT mice, which could be assessed by
colony forming assays in vitro.
4diii. Does the loss of postosteoblasts affect skeletal development in ERdj4GT/GT mice?
Postosteoblasts regulate bone remodeling by stimulating osteoclastogenesis through
expression of RANKL. Deficiency of RANKL on osteocytes did not cause any gross
skeletal defects, but resulted in an osteopetrotic phenotype due to reduced
osteoclastogenesis59. Osteogenic cells isolated from ERdj4GT/GT mice expressed significantly lower levels of RANKL (Chapter 3, Figure 2C), suggesting that either osteocytes were reduced within compact bone or expressed lower levels of RANKL.
171 Thus, we hypothesize that reduced osteocytes and/or RANKL expression in ERdj4GT/GT
mice influences osteoclast differentiation and function. To test this possibility,
osteoclasts will be evaluated in femur tissue sections from ERdj4+/+ and ERdj4GT/GT mice
by staining for tartrate-resistant acid phosphatase (TRAP), an osteoclast marker.
Further, microcomputed tomography (µCT) scanning will be performed to assess bone
volume, trabecular thickness/separation and bone mineral content. We expect that a
decrease in osteocytes and/or RANKL in ERdj4GT/GT mice will reduce osteoclastogenesis, leading to a slower rate of bone resorption.
5. Molecular chaperones in the treatment of ER stress-related disease
Several diseases are associated with ER stress, including neurodegenerative diseases, atherosclerosis, interstitial lung disease and diabetes. ER stress results from perturbations that alter redox balance, calcium levels or posttranslational modifications, leading to accumulation of unfolded/misfolded proteins within the ER lumen. The UPR attempts to restore ER homeostasis by attenuating protein translation and upregulating protein folding/degradation machinery. However, a failure to alleviate ER stress results in activation of cell death pathways, thereby contributing to disease pathology.
Molecular chaperones perform a wide variety of functions in the ER including
maintenance of ER calcium stores66, regulation of the translocon pore67, protein folding and degradation68-71, and activation of the UPR72,73. The importance of molecular chaperone activity in cellular homeostasis and function was demonstrated in the current study; ERdj4 deficiency impaired survival, glucose metabolism, osteogenesis and B cell development in vivo.
172 The ability of molecular chaperones to maintain/restore ER homeostasis has
promising therapeutic potential in the treatment of diseases associated with ER stress.
Upregulation of molecular chaperones reduced neurotoxicity in protein folding diseases
such as Alzheimer’s, Huntington’s and Parkinson disease74,75 and therapeutics that induce expression of molecular chaperones are currently being evaluated in clinical trials75-77. This study, clarifying the role of ERdj4 in cellular homeostasis and function,
provides rationale for therapeutic targeting of molecular chaperones in ER stress-
related disease.
6. ERdj4 in cellular function and homeostasis
ERdj4 maintains ER homeostasis by assisting in the removal of unfolded/misfolded
substrates from the ER lumen for proteasomal degradation. ERdj4 deficiency increased
ER stress in a variety of non-secretory and secretory cell types (Chapter 2, Figure S1).
Collectively, these data indicate that the chaperone activity of ERdj4 is important for
cellular homeostasis, but it also suggests that ERdj4 associates with a broad range of
substrates. Our laboratory has identified the only known substrates for ERdj4, which
include unfolded (insulin2WT, Pcsk1, Pcsk2 and CPE) and misfolded (insulin2C96Y and
SP-CΔexon4) proteins that are either non-glycosylated (insulin2 and SP-C) or glycosylated
(Pcsk1, Pcsk2 and CPE) (Chapter 1, Figure 5C)1. Pcsk1, Pcsk2 and CPE play an
important role in glucose metabolism by influencing insulin and glucagon biosynthesis,
but also process many other hormones and neuropeptides in the neuroendocrine
system. Deficiency of Pcsk1, Pcsk2 or CPE in mice resulted in a wide range of neural
and endocrine abnormalities35,36,78. It is possible that ERdj4 deficiency affects the
173 maturation of Pcsk1, Pcsk2 and/or CPE, causing multiple systemic defects in
ERdj4GT/GT mice. Further studies are needed to examine the maturation of these enzymes in the absence of ERdj4 using pulse chase analyses. If Pcsk1, Pcsk2 and/or
CPE fail to achieve native conformation due to the loss of ERdj4, the hormones and neuropeptides processed by these enzymes will be assessed in ERdj4GT/GT mice.
Although several lines of evidence indicate that ERdj4 is a component of ERAD,
it is possible that ERdj4 also promotes the folding and maturation of nascent proteins.
Other ERdjs are known to facilitate protein folding by directly associating with unfolded
substrates and/or the translocon pore, where nascent proteins enter the ER lumen79.
Examining the maturation of Pcsk1, Pcsk2 and CPE will provide evidence as to whether
ERdj4 assists in protein folding, as well as degradation. This work highlights the
importance of ERdj4 chaperone activity in maintaining ER homeostasis under normal
conditions.
Collectively, a great deal has been learned about ERdj4 biology throughout the
course of these studies, revealing novel insights into the role of ERdj4 in survival,
glucose metabolism and B cell development. Future studies hope to address the questions raised in this discussion, as well as explore the other interesting phenotypes resulting from ERdj4 deficiency.
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