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Role of endoplasmic reticulum chaperones GRP78 and GRP94 in regulating unfolded protein response and cell survival

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Role of endoplasmic reticulum chaperones GRP78 and GRP94 in regulating unfolded protein response and cell survival

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ROLE OF ENDOPLASMIC RETICULUM CHAPERONES GRP78 AND GRP94 IN
REGULATING UNFOLDED PROTEIN RESPONSE AND CELL SURVIVAL

by
Miao Wang

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETICS, MOLECULAR AND CELLULAR BIOLOGY)

December 2010

Copyright 2010 Miao Wang
ii
Dedication
To my dearest parents, Hexiao Wang and Zhimei Song, who have continuously given me
pure love and full support; and my husband and soulmate Risheng Ye, who has brought
me happiness and endless love.
iii
Acknowledgements
I would like to express my sincere appreciation for my mentor, Dr. Amy S. Lee. She
demonstrates to me the characters of being a successful scientist. With the excellent and
enthusiastic guidance from her, I become a confident and competent scientist today. I
also would like to thank the former Lee lab members, Shengzhan Luo, Changhui Mao,
Jianze, Li, Brenda Lee, Peter Baumeister, Yong Fu and Min Ni, Dezheng Dong as well as
my current labmates, Hui Zhou, Yi Zhang, Kyle Pfaffenbach, Risheng Ye, Shiuan Wey,
Biquan Luo, Wan-Ting Chen and Genyuan Zhu, for their scientific contributions to my
projects and personal assistance.
I am thankful to my dissertation committee members, Drs. Pradip Roy-Burman and Louis
Dubeau, for their suggestions and instructions on my research projects. I am also grateful
to the numerous collaborators that have made my projects possible. Drs. Louis Dubeau
and David Hinton, as well as Ernesto Barron have contributed to the content in Chapter 2,
which has been published in the journal Cell Death and Differentiation 17: 488–98, 2010.
Robin Wesselschmidt has contributed to the work in Chaper 3, which has been published
in the journal PLoS One 5:e10852. Dr. Siyi Chen and his previous postdoc Sunghyung
Lee have contributed to the study in Chapter 4.
My parents and other family members have always supported me with all their love
through the whole journey, which I have truly appreciated. In addition, I am really
iv
thankful to all the friends I have made in this city. I really enjoyed those times being
together with them.
Last but not least, I would like to express my greatest gratitude to my dearest husband,
Risheng Ye. He has shown and taught me the remarkable courage and exceptional ability
to achieve the career goal and brought the sunshine and happiness into my life.
v
Table of Contents
Dedication ii
Acknowledgements iii
List of Figures vii
Abstract x
Chapter 1: Introduction 1
1.1 Endoplasmic reticulum (ER) 1
1.2 ER stress and unfolded protein response (UPR) 1
1.3 GRP78 in development and human diseases 4
1.4 GRP94 in development and human diseases 11

Chapter 2: Role of GRP78 in neuronal cell protection
18
2.1 Introduction 18
2.2 Materials and methods 19
2.3 Results 24
2.3.1 Generation of the GRP78 conditional knockout mouse
model in Purkinje cells

24
2.3.2 Accelerated Purkinje cell degeneration and cerebellar
atrophy in Grp78 F/-; pc-Cre mice

27
2.3.3 Severe motor function deficiency in Grp78 F/-; pc-Cre
mice
29
2.3.4 GRP94 is not required for the survival of Purkinje cells 30
2.3.5 Modulation of UPR signaling pathway in GRP78 null
PCs
32
2.3.6 Reduction of cytosolic ubiquitin staining in GRP78
null PCs

36
2.3.7 ER expansion in GRP78 null PCs 40
2.3.8 Generation of the GRP78 and CHOP double knockout
mouse model in Purkinje cells
43
2.3.9 CHOP knockout can not rescue GRP78 null Purkinje
cells from apoptosis

44
2.4 Discussion 37

Chapter 3: Role of GRP94 in regulating UPR

52

3.1 Introduction 52
3.2 Materials and methods 55
vi
3.3 Results 57
3.3.1 GRP94 null embryonic stem cells (ESCs) upregulate
specific ER chaperones

57
3.3.2 Modulation of UPR signaling pathways in GRP94 null
ESCs

59
3.3.3 Differential GRP94 expression profiles in adult mouse
tissues

65
3.4 Discussion 68

Chapter 4: Role of GRP94 in the normal mouse mammary gland development
and HER2/Neu induced tumorigenesis

72
4.1 Introduction 72
4.2 Materials and methods 74
4.3 Results 75
4.3.1 Generation of the Grp94 F/-; MMTV-Cre and Grp94
F/-; MMTV-Cre; MMTV-Neu mice

75
4.3.2 The Grp94 F/-; MMTV-Cre mice show normal
morphology and branching of the mammary gland

79
4.3.3 The Grp94 F/-; MMTV-Cre; MMTV-Neu mice show
less branches of the mammary ducts than the Grp94 F/F;
MMTV-Neu mice

82
4.4 Discussion 84

Chapter 5: Conclusions and Perspectives 86

Bibliography 91

vii
List of Figures
Figure 2.1: Generation of Grp78 Purkinje cell-specific knockout mice.

26
Figure 2.2: GRP78 depletion in PCs of F/-; pc-Cre mice.

26
Figure 2.3: Postnatal growth retardation in F/-; pc-Cre mice.

27
Figure 2.4: Body size and food consumption comparation between F/- mice and
their F/-; pc-Cre littermates.

28
Figure 2.5: Progressive PC degeneration and cerebellar atrophy in the F/-; pc-
Cre mice.

28
Figure 2.6: Caspase 3 activation and TUNEL staining in PCs of F/- and F/-; pc-
Cre mice.

29
Figure 2.7: Behavioral abnormalities of the F/-; pc-Cre mice.

30
Figure 2.8: PC-specific knockout of GRP94 caused no morphological or
behavioral defect.

31
Figure 2.9: Modulation of UPR pathways in the GRP78 null PCs.

34
Figure 2.10: Quantitation of the IHC and IF stainings of UPR parameters.

35
Figure 2.11: Knockout of Grp94 has no effect on UPR signaling pathways.

35
Figure 2.12: Reduction of protein ubiquitination and p62 level in GRP78 null
PCs.

37
Figure 2.13: Reduction of protein ubiquitination staining in the GRP78 null
prostate epithelial cells.

39
Figure 2.14: Similar expression levels of calnexin, non-phosphorylated
neurofilament and calbindin in the PCs of control and knockout mice.

39
Figure 2.15: Electron micrograph of PCs from F/- and F/-; pc-Cre mice,
demonstrating the whole PC morphology.

41
Figure 2.16: High magnification of electron micrograph of PCs from F/- and F/-
; pc-Cre mice.

41
viii
Figure 2.17: ER expansion and electron dense aggregates inside the ER in the
PCs of a 3.5 wk old F/-; pc-Cre mouse.

42
Figure 2.18: Altered ER structure in GRP78 null PCs shown by immunogold
labeling.

43
Figure 2.19: Generation of the Grp78 F/-; pc-Cre, Chop -/- mice.

44
Figure 2.20: Knockout of Chop in GRP78 null PCs can not rescue them from
cell death.

46
Figure 2.21: Summary of the alteration in UPR signaling pathways and
pathological phenotypes in GRP78 null PCs.

47
Figure 3.1: Western blots of ER chaperones in ESCs.

58
Figure 3.2: Quantitation of Western blot results on ER chaperones in ESCs.

59
Figure 3.3: Representative Western blot results on the effect of GRP94
depletion on UPR targets.

61
Figure 3.4: Representative Western blot results on eIF2α phosphorylation, total
eIF2α, ATF4 and HSP70.

61
Figure 3.5: Quantitation results of p-PERK and p-eIF2α from Western blots.

62
Figure 3.6: Effect of GRP94 depletion on Xbp-1 mRNA splicing.

63
Figure 3.7: Reduction of the spliced form of XBP-1 protein in GRP94 null
ESCs.

63
Figure 3.8: Representative Western blot results on effect of GRP94 depletion on
cell proliferation and apoptosis.

64
Figure 3.9: GRP94 protects ESCs from ER stress-induced cell death.

64
Figure 3.10: Relative Grp94 mRNA levels in adult mouse tissues and primary
cells.

66
Figure 3.11: GRP94 and GRP78 staining and mRNA expression in adult mouse
tissues.

67
Figure 4.1: Creation of GRP94 mammary gland conditional knockout (KO)
mice with or without Neu transgene.
77
ix

Figure 4.2: Immunofluorescence staining for GRP94 on the mammary glands of
Grp94 F/F, F/- and cF/- mice at 3 months old.

78
Figure 4.3: Wholemount staining of mammary glands from Grp94 F/F, F/- and
cF/- mice at 1, 1.5 and 3 months old.

80
Figure 4.4: H&E staining of mammary glands from Grp94 F/F, F/- and cF/-
mice at 1, 1.5 and 3 months old.

81
Figure 4.5: Wholemount staining of mammary glands from Grp94 F/F; Neu and
cF/-; Neu mice at 3 months old.

82
Figure 4.6: H&E staining of mammary glands from Grp94 F/F; Neu and cF/-;
Neu mice at 3 months old.

83

x
Abstract
Endoplasmic reticulum (ER) is a key cellular organelle responsible for membrane and
secretary protein folding and maturation. When the ER folding capacity is overwhelmed
by protein loading, the unfolded protein response is activated. As the master regulator of
UPR, the role of GRP78 in cell survival has been studied intensively through cell culture
systems. To further understand the in vivo role of GRP78, a novel conditional knockout
mouse model with GRP78 specifically depleted in Purkinje cells (PCs) is created. The
GRP78 null PCs activate UPR signaling pathways including the upregulation of GRP94
and PDI, the induction of CHOP and GADD34, the feedback suppression of eIF2α
phosphorylation and apoptotic cell death. Cytosolic protein ubiquitination level is
strikingly reduced, in contrast to many neurodegenerative mouse models where abnormal
accumulation of ubiquitinated protein is prominent. Ultrastructural evaluation reveals that
the ER shows prominent dilatation with focal accumulation of electron-dense material
surrounded by ER membrane. Growth retardation, severe motor coordination ability
defect by week 5 and cerebellar atrophy by week 13 were observed in the conditional
knockout mice. Therefore GRP78 is required for neuronal cell survival and provides a
novel link between GRP78 depletion and reduction of protein ubiquitination level,
suggesting the important role of GRP78 in ER associated protein degradation. As another
important ER chaperone, GRP94 is discovered to be dispensable for Purkinje cells,
mouse embryonic stem cells (mESCs) and mammary epithelial cells. GRP94 null
Purkinje cells survive and function as well as wildtype control. Under ER stress
conditions, GRP94 depletion in mESCs leads to induction of specific ER chaperones like
xi
GRP78, calnexin (CNX) and calreticulin (CRT), but not PDI and reduction of XBP-1
splicing, a major downstream target of IRE1 signaling pathway. However, PERK
signaling pathway is not affected in GRP94 null cells. In addition, specific knockout of
Grp94 in mouse mammary epithelial cells reveals that GRP94 is not required for the
survival of these cells or the normal development and function of mammary gland.
Conditional depletion of GRP94 in the mammary gland epithelial cells may be able to
inhibit HER2/Neu induced tumorigenesis. Therefore our study provides novel insights for
the role of GRP78 and GRP94 in mouse development, cell survival and UPR activation
by utilizing multiple mouse models.

1
Chapter 1: Introduction
1.1 Endoplasmic Reticulum (ER)
Endoplasmic Reticulum (ER) is one of the most conserved organelles in eukaryotic cells.
ER is originally classified into rough ER (RER) and smooth ER (SER) through distinct
morphology under electron microscope. RER is defined as ribosome bound sheets like
membranes and SER is simply defined by the absence of ribosome bound tubules like
membranes (Shibata, Voeltz et al. 2006). Except for the morphological difference, RER
and SER have distinct functions. RER is responsible for the biosynthesis, folding and
modification of the membrane and secretary proteins. While SER is mainly involved in
several metabolic processes, including synthesis of lipids and steroids, metabolism of
carbohydrates and steroid, regulation of calcium concentration, detoxification of drugs
and attachment of receptors on cell membrane proteins (Maxfield and Wustner 2002). In
addition, ER is also a signal-transducing organelle when cells encounter various stress
conditions which are collectively termed ER stress (Kaufman 1999).
1.2 ER stress and unfolded protein response (UPR)
Various conditions disrupting ER homeostasis can cause ER stress including
physiological, pathological and chemical insults (Lee 2001). Under normal physiological
conditions, ER stress can be activated during the development of professional secretary
cells such as plasma cells and pancreatic β-cells. Genetic mutations such as DNA damage
or mutated secretary proteins can also induce ER stress. ER stress is detected under

2
pathogenesis like viral infection, tumor microenvironment and altered metabolism
(Rutkowski and Kaufman 2004). Numerous chemicals will induce ER stress, including
Thapsigargin (Tg), A23187, Ionomycin and EGTA which can deplete Ca
2+
from the ER,
Tunicamycin(Tu), Glucosamine and 2-Deoxyglucose which can block N-linked
glycosylation, Dithiothreitol (DTT), β-Mercaptoethonal and Homocysteine which can
cause reductive stress in the ER (Lee 2001). Upon ER stress, a coordinated adaptive
program called unfolded protein response (UPR) will be activated to alleviate the stress
(Rutkowski and Kaufman 2004).
Upon ER stress, the master regulator of UPR GRP78 will be released from ER
transmembrane signal transducers including PKR-like ER kinase (PERK), Inositol-
requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), leading to the
activation of these signaling pathways (Lee 2005). PERK dimerizes to promote its
autophosphorylation and activation following the release of GRP78 (Rutkowski and
Kaufman 2004). Eukaryotic translation initiation factor 2α (eIF2α)  is phosphorylated on
Serine 51 by PERK to attenuate the rate of general translation initiation and prevent
further protein synthesis (Shi, Vattem et al. 1998; Harding, Zhang et al. 1999). However,
phosphorylated eIF2α can selectively increase the translation of some mRNAs which
contain inhibitory upstream open reading frames (uORFs) in their 5’ untranslated region.
The best studied example is activating transcription factor 4 (ATF4) (Harding, Novoa et
al. 2000; Lu, Harding et al. 2004). ATF6 is translocated from ER to Golgi to get cleaved.
The cleaved form of ATF6 migrates into the nucleus and acts as an active transcription
factor to upregulate proteins that augment ER folding capacity like ER chaperones such

3
as GRP78 and GRP94 and folding enzymes such as PDI (Haze, Yoshida et al. 1999; Li,
Baumeister et al. 2000; Ye, Rawson et al. 2000; Yoshida, Okada et al. 2000). Activated
IRE1 has endoribonuclease activity and splices a 26-base intron from the mRNA
encoding the X-box binding protein 1 (XBP-1) (Yoshida, Matsui et al. 2001). XBP-1 is a
transcriptional factor with target genes including DnaJ, p58, ERdj4, EDEM and PDI, all
involved in protein folding and ERAD (Calfon, Zeng et al. 2002; Lee, Iwakoshi et al.
2003). Thus, the UPR progresses through transient attenuation of translational and
transcriptional induction of ER chaperones, folding enzymes, proteins involved in ERAD
to alleviate protein aggregation in the ER as an adaptive response.
If the stress is too severe to overcome, the UPR will activate pathways that lead to cell
death through apoptosis (Wu and Kaufman 2006). Upon ER stress, the pro-apoptotic
BH3 protein BAX/BAK in the ER membrane undergoes conformational change and
permits Ca
2+
efflux to the cytosol, which activates m-calpain and subsequently cleaves
and activates procaspase 12 and leads to the activation of the caspase cascade (Nakagawa
and Yuan 2000; Rao, Castro-Obregon et al. 2002; Scorrano, Oakes et al. 2003; Zong, Li
et al. 2003). CHOP, one of the UPR downstream effectors, inhibits B cell
leukemia/lymphoma 2 (Bcl-2), activates growth arrest and DNA damage inducible gene
34 (GADD34) and ER oxidoreductin 1 (ERO1) and thus promotes apoptosis
(McCullough, Martindale et al. 2001; Marciniak, Yun et al. 2004). However, the role of
CHOP in cell death and survival may be context dependent since GADD34 upregulation
by CHOP results in feedback inhibition of eIF2α phosphorylation. This could lead to
recovery of translation which may be beneficial, on the other hand when translation

4
persists under ER stress conditions, build up of abnormal proteins may further erode the
ER folding capacity, leading to cell death.
Activated IRE1 binds to c-Jun-N-terminal inhibitory kinase (JIK) and recruits TRAF2,
which leads to the activation of apoptosis signal-regulating kinase 1 and c-Jun amino
terminal kinase (ASK1/JNK) and also the release of the procaspase 12 from the ER
(Yoneda, Imaizumi et al. 2001; Nishitoh, Matsuzawa et al. 2002). ER stress also activates
the pro-apoptotic p53-upregulated modulator of apoptosis (PUMA) and NOXA, leading
to BAX and BAK activation and apoptosis (Li, Lee et al. 2006). Furthermore, by
monitoring the activation and maintenance of representative UPR pathways in cells
treated with low concentrations of chemical ER stress inducers in tissue culture systems,
it was discovered that survival during mild stress is attained through intrinsic instabilities
of mRNA and proteins that promote apoptosis, and compared to those that facilitate
protein folding and adaptation such as GRP78 (Rutkowski, Arnold et al. 2006).
1.3 GRP78 in development and human diseases
ER homeostasis is maintained by ER chaperones. One of the best characterized ER
chaperones is the 78 kDa glucose-regulated protein (GRP78), which is also referred to as
BiP (immunoglobulin heavy chain binding protein) or HSPA5. GRP78 is a
muiltifunctional protein participating in many cellular processes, including translocating
the newly synthesized polypeptides across the ER membrane, facilitating the folding and
assembly of proteins, targeting misfolded proteins for ER-associated degradation
(ERAD), regulating calcium homeostasis and serving as an ER stress sensor (Hendershot

5
2004; Lee 2005; Li and Lee 2006). Despite extensive investigation on GRP78 function in
regulating the UPR using tissue culture systems, important questions pertaining to the
physiological function of GRP78 in the context of a whole organism remain to be
addressed. To directly investigate the function of GRP78 in vivo, mouse models deficient
in GRP78 through gene targeting have been created (Luo, Mao et al. 2006). Strikingly,
homozygous knockout mice of GRP78 results in lethality by embryonic day 3.5 (E3.5),
with much reduced proliferation rate of embryonic cells and massive apoptotic death of
the inner cell mass (Luo, Mao et al. 2006). In contrast, the heterozygous Grp78+/- mice
are viable and phenotypically normal (Luo, Mao et al. 2006). The Grp78+/- mouse model
serves as a valuable resource to examine the effect of partial reduction of GRP78,
mimicking potential therapeutic intervention outcome, on progression of human diseases.
GRP78 is reported to be involved in many human disease, mainly including cancer and
neurodegenerative diseases. Up to date, the role of GRP78 in cancer can be divided into
two categories: one is the conventional GRP78 localized in the ER, the other is the cell
surface specific GRP78. The ER GRP78 has been discovered for more than twenty years
with multiple significant functions. Extensive studies have shown that ER GRP78 is
involved in cancer progression, metastasis and drug resistance (Lee 2007). The study on
cell surface GRP78 is just emerging but attracts more and more attentions. Cell surface
GRP78 is shown to be able to bind to lots of ligands that can promote cell growth (Wang,
Wey et al. 2009). The signaling pathways and underlying mechanisms regarding the
function of cell surface GRP78 are waiting to be discovered.

6
Elevation of GRP78 is observed in a variety of cancer cell lines, solid tumors and human
cancer biopsies including breast cancer, prostate cancer, hepatocarcinoma, lung cancer
and cervical cancer, associating with malignancy and metastasis (Fernandez, Tabbara et
al. 2000; Shuda, Kondoh et al. 2003; Uramoto, Sugio et al. 2005; Fu and Lee 2006; Lee,
Nichols et al. 2006; Li and Lee 2006; Pootrakul, Datar et al. 2006; Lee 2007). Prostate
cancer patients with strong GRP78 staining in the primary tumor have a higher chance of
clinical recurrence and death compared with those have lower level of GRP78
(Daneshmand, Quek et al. 2007). Overexpression of GRP78 is detected in chemoresistant
human breast cancer patient samples (Dong, Ko et al. 2005). Thus GRP78 expression
level is closely correlated with tumor malignancy, drug resistance, patient survival and
clinical recurrence, demonstrating that the level of GRP78 could provide prognostic
information for cancer patients (Lee 2007).
Up to today, cell surface expression of GRP78 has been identified in multiple cell lines
including prostate cancer cells and vascular endothelium (Delpino, Piselli et al. 1998;
Davidson, Haskell et al. 2005). This is probably due to the high expression level of
GRP78 in those cells. Overexpression of GRP78 in 293T cells enhances the level of
GRP78 on cell surface (Zhang, Liu et al. 2010). It is discovered that cell surface GRP78
can function as receptors mediating the activation of multiple signaling pathways (Wang,
Wey et al. 2009). High-affinity binding interaction between Kringle 5 and surface GRP78
induces apoptosis in the stimulated endothelial cells and stressed cancer cells (Davidson,
Haskell et al. 2005). Surprisingly, as a generally recognized cytosolic and nuclear protein,
Par-4 can be secreted out and induce apoptosis in cancer cells through binding to cell

7
surface GRP78 (Burikhanov, Zhao et al. 2009; Lee 2009). Except for apoptotic pathways,
cell surface GRP78 is also reported to mediate cell survival and proliferation pathways.
Binding between surface GRP78 and α2-macroglobulin in prostate cancer cells can
activate multiple mechanisms that directly promote cellular proliferation like PI3K/AKT
pathway but also block mechanisms triggering programmed cell death (Misra,
Deedwania et al. 2005; Misra, Deedwania et al. 2006). The complex of the multiple
functional protein Cripto and surface GRP78 can enhance the growth of tumor through
inhibition of TGF-β signaling (Shani, Fischer et al. 2008). Surface GRP78 is also
essential for the T-cadherin dependent signal transduction in vascular endothelial cells
(Philippova, Ivanov et al. 2008). Thus GRP78 on cancer cell surface could mediate
cancer specific therapy, without affecting adjacent normal cells.
The activation of UPR signaling pathway may explain the upregulation of GRP78 in
tumor cells. The microenvironment of tumor often associates with lower pH value, high
glucose metabolic rate, less oxygen and nutrition, leading to the onsite of ER stress and
activation of UPR in those cells (Lee 2007). The underlying mechanisms for GRP78
promoting cancer progression can be dissected into three aspects. First of all, GRP78 is
critical for tumor cell growth and proliferation. This is convinced by both cell culture
system and mouse models. Heterozygous Grp78 tumor cells grow slower than their
wildtype control during in vitro culture (Dong, Ni et al. 2008). Knockdown of GRP78 by
siRNA inhibits the growth of HeLa, PC-3 and glioma cells but not other cell lines with
low basel level of GRP78, which is consistent with high level expression of GRP78 in
HeLa, PC-3 and glioma cells (Pyrko, Schonthal et al. 2007; Suzuki, Lu et al. 2007).

8
Grp78 heterozygosity strikingly prolongs the latency and retards the progression of the
oncogene-induced mammary tumors in the well-established MMTV-PyVT mouse model
with mouse growth, organ development and antibody production unaffected (Dong, Ni et
al. 2008). In addition, knockout of Grp78 specifically in mouse prostate epithelium
suppresses PTEN depletion induced prostate tumorigenesis without affecting postnatal
prostate development and growth (Fu, Wey et al. 2008). This corresponds with
suppression of AKT phosphorylation by GRP78 knockdown in prostate cancer cell line
(Fu, Wey et al. 2008). Second, GRP78 protects tumor cells from apoptosis. Knockdown
of GRP78 induced apoptosis in HeLa and PC-3 cells (Suzuki, Lu et al. 2007).
Heterozygous Grp78 tumor cells show enhanced TUNEL staining and induction of
CHOP, indicating the more onsite of apoptosis (Dong, Ni et al. 2008). The anti-apoptotic
properties of GRP78 can be explained through two aspects. One is that GRP78 binds to
inactivated caspase-7 and block the cleavage of it (Reddy, Mao et al. 2003). The other is
that GRP78 can inhibit BIK induced cell apoptosis through interaction with BIK (Fu, Li
et al. 2007). Third, GRP78 promotes angiogenesis in tumors. Grp78 heterozygosity
remarkably reduces vasculature in tumors, but not in normal organ and tissues,
suggesting tumor angiogenesis preferably depends on GRP78 (Dong, Ni et al. 2008).
Knockdown of GRP78 in human umbilical vein endothelial cells (HUVECs) significantly
suppressed VEGF-induced endothelial cell proliferation and VEGF-induced
phosphorylation of extracellular-regulated kinase (ERK) 1/2, phospholipase C-γ, and
VEGF receptor-2 in HUVECs (Katanasaka, Ishii et al. 2010). Other mechanisms

9
regarding GRP78 and cancer such as the role of GRP78 in stress induced autophagy and
Ca
2+
buffering in the ER remain to be elucidated in the future.
Other than cancer, GRP78 is also implicated in neurodegenerative diseases. Onsite of ER
stress and upregulation of GRP78 have been frequently detected in neurodegenerative
diseases such as Alzheimer’s disease, Parkinson’s disease and Marinesco-Sjögren (MS)
syndrome (Nakagawa, Zhu et al. 2000; Imai, Soda et al. 2001; Rao and Bredesen 2004;
Zhao, Longo-Guess et al. 2005). This may be caused by the accumulation and
aggregation of misfolded proteins which can elicit ER stress.
The function of GRP78 in MS syndrome is the most studied case among
neurodegenerative diseases. MS syndrome is a rare autosomal recessive multisystem
disorder mainly characterized by cerebellar ataxia with cerebellar atrophy, early-onsite
cataract, myopathy and mild to severe mental retardation (Anttonen, Mahjneh et al. 2005;
Senderek, Krieger et al. 2005). MS syndrome was described in literature as early as 1931
by Marinesco and co-authors (Georgy, Snow et al. 1998). The underlying mechanisms
remain obscure until recent years. It was discovered at 2005 that loss-of-function
mutations in SIL1 genes which encodes a nucleotide exchange factor for the major ER
chaperone GRP78 are strongly associated with MS syndrome (Anttonen, Mahjneh et al.
2005; Senderek, Krieger et al. 2005). Later on, more and more mutations in SIL1 gene
including deletions, homozygous frameshift insertions and homozygous substitution
caused missense mutations are identified in MS syndrome patients all over the world
(Eriguchi, Mizuta et al. 2008; Riazuddin, Amiri-Kordestani et al. 2009; Takahata,
Yamada et al. 2010). Up to date, SIL1 seems to be the only one gene that directly

10
associated with MS syndrome, although it was also reported that some individuals with
typical MS syndrome do not have SIL1 mutations (Takahata, Yamada et al. 2010). These
findings implicate MS syndrome as an ER dysfunction related disease and suggest ER
protein quality control is involved in the multisystem disorders. SIL1 homozygous mutant
mice develop adult-onset ataxia and progressive Purkinje cell (PC) degeneration (Zhao,
Longo-Guess et al. 2005). Two ER stress markers GRP78 and CHOP are upregulated in
the degenerating PCs, suggesting the activation of UPR pathways (Zhao, Longo-Guess et
al. 2005). The non-lethal phenotype of SIL1 deficiency in both human and mice indicate
the existence of other co-factors for GRP78. It is discovered that GRP170 may function
as the nucleotide exchange factor for GRP78 (Weitzmann, Volkmer et al. 2006).
To further understand the role of GRP78 in neuronal cell development, a knock-in mouse
model expressing a mutant form of GRP78 with deletion of the ER retrieval sequence
KDEL by homologous recombination is created (Mimura, Hamada et al. 2007). The
homozygous mutant neonates die soon after birth because of respiratory failure which is
caused by impaired secretion of pulmonary surfactant by alveolar type II epithelial cells
(Mimura, Hamada et al. 2007). The mutant mice display disordered layer formation in the
cerebral cortex and cerebellum, a neurological phenotype of reeler mutant-like
malformation (Mimura, Yuasa et al. 2008). Besides that, the whole brain size and the
protein level of reelin which is secreted by Cajal-Retzius (CR) cells are remarkably
reduced. It is also observed that the maturation and secretion of reelin in CR cells and
other factors related to neural migration may be enhanced by GRP78 (Mimura, Yuasa et
al. 2008). Furthermore, reelin and its mRNA are significantly reduced in patients with

11
schizophrenia (Impagnatiello, Guidotti et al. 1998) and epigenetic aberration of the
human reelin gene is associated with psychiatric disorders (Tamura, Kunugi et al. 2007).
Collectively these results imply that mutant form of GRP78 and aberrant ER protein
quality control may cause various neurological disorders. Considering the potential
important roles of GRP78 in neuronal development and disorders, the deeper
understanding of the pathological role of GRP78 in neurological disorders and
neurodegenerative diseases warrants further investigation. Most recently, it has been
reported that a small molecule BIX that preferentially induces GRP78 and slight
induction of GRP94 and calreticulin can protect neurons from ER stress (Kudo,
Kanemoto et al. 2008). Thus, there is translational potential for GRP78 induction as
therapy against neurodegenerative diseases and other neurological disorders.
1.4 GRP94 in development and human diseases
Glucose regulated protein 94 (GRP94), is an ER resident member belonging to the Heat
Shock Protein 90 (HSP90) family (Little, Ramakrishnan et al. 1994). It is also named as
gp96, endoplasmin, ERp99, HSP108, CaBP4 and Tra-1 (Yang and Li 2005). All of these
names refer to one specific gene HSP90B1. So far it is the only HSP90-like protein found
located in the ER. GRP94 is ubiquitously expressed in all vertebrates and some
invertebrates like C. elegans and Drosophila and also plants, but not in yeast (Argon and
Simen 1999; Chen, Zhong et al. 2006; Morales, Wu et al. 2009).
GRP94 is a soluble ER chaperone and its structure was determined by many methods
including X-ray crystallography, electron microscopy and small angle X-ray scattering

12
(Immormino, Dollins et al. 2004; Dollins, Warren et al. 2007; Krukenberg, Bottcher et al.
2009). Four domains have been identified in GRP94: an N-terminal domain (NTD), an
acidic linker domain (LD), a middle domain (MD) and a C-terminal domain (CTD). The
ATPase activity of GRP94 is essential for its normal chaperone activity and relies on all
of the NTD, LD and MD. NTD functions as the ATP binding domain and LD and MD
work cooperatively to fulfill the ATP hydrolysis process. The CTD is responsible for the
constitutive dimerization of GRP94. It is reported that ATPase activity is essential for in
vivo chaperone activity of GRP94 and the essential peptide-binding domain of GRP94 is
distinct from the NTD through an in vitro cell survival assay (Ostrovsky, Makarewich et
al. 2009). It remains to be discovered that which sites of GRP94 are responsible for
binding with other proteins such as those GRP94 specific clients and other ER
chaperones. In addition, there are no co-chaperones found to be able to affect the ATPase
activity of GRP94, which awaits further investigation (Eletto, Dersh et al. 2010).
The general function of GRP94 can be divided into three categories, chaperoning client
proteins, buffering Ca
2+
in the ER and involving in ER associated protein degradation
(ERAD) (Eletto, Dersh et al. 2010). The major function of GRP94 is to fold or assembly
membrane or secretary proteins. Unlike GRP78 which folds numerous proteins, GRP94
is pretty selective and has limited number of client proteins including immunoglobulin,
insulin like growth factor I and II (IGF-I and II), Toll-like receptors (TLR) and integrins
(Eletto, Dersh et al. 2010).
Immunoglobulin was the first GRP94 client protein found in early 90s. Compared with
GRP78, which prefers binding an early disulfide intermediate of immunoglobulin light

13
chain (LC) and heavy chain (HC), GRP94 tends to fold advanced folding intermediates,
the fully oxidized substrates (Melnick, Dul et al. 1994). However, the level of serum
immunoglobulin was not affected in B-cell specific GRP94 null mice (Liu and Li 2008).
This indicates that there could be compensatory mechanisms which can facilitate the
assembly and secretion of immunoglobulin without the help of GRP94.
Genetically targeted disruption of Grp94 gene in mice results in embryonic lethality
around embryonic day 7.5 (E7.5) and Grp94 -/- embryos fail to develop mesoderm,
primitive streak, or proamniotic cavity (Wanderling, Simen et al. 2007; Mao, Wang et al.
2010). This implies the essential role of GRP94 in embryonic development. Compared
with Grp94 +/+ embryonic stem cells (ESCs), Grp94 -/- ESCs grow normally in culture
and are able to form embryonic bodies (EB) and differentiate into cells of all three germ
layers (Wanderling, Simen et al. 2007; Mao, Wang et al. 2010). However, Grp94 -/-
ESCs are unable to differentiate into cardiac, smooth or skeletal muscle which is caused
by the deficiency in production of IGF II by GRP94 depleted ESCs (Wanderling, Simen
et al. 2007; Mao, Wang et al. 2010). Exogenous expression of IGF I or II will restore the
ability of Grp94 -/- ESCs differentiating into muscle cells (Wanderling, Simen et al.
2007). Later on, studies further prove that GRP94 is indispensable for the intracellular
processing and secretion of IGF II, thus establishing that IGF II is a client protein strictly
depending on GRP94 (Ostrovsky, Ahmed et al. 2009). This function is elucidated by
tissue culture systems. Utilizing established myogenic cell lines, it is discovered that
GRP94 is not required for the initial steps of myogenesis, but GRP94 is essential for the
expression of contractile proteins and in vitro fusion of myoblasts purcursors into

14
myotubes, downstream of myoD upregulation (Ostrovsky, Eletto et al. 2010). This is
consistent with the known function of IGF I or II signaling (Ostrovsky, Eletto et al.
2010).
It is reported previously that GRP94 is not required for cell survival but responsible for
the normal expression of a small subset of cell surface receptors including TLRs through
a GRP94 deficient B-cell line (Randow and Seed 2001). Later on, a macrophage-specific
knockout mouse model of GRP94 reveal that GRP94 is the master regulator for both cell-
surface and intracellular TLRs including TLR2, TLR4, TLR5, TLR7 and TLR9 (Yang,
Liu et al. 2007). This is achieved by the LysM-Cre mediated specific deletion of the
Grp94 gene in the macrophage of the Grp94 F/- mice (Yang, Liu et al. 2007). TLRs
recognize many microbial ligands such as bacterial lipoproteins, double stranded RNA,
and lipopolysaccharide (Harding 2007). Grp94 F/-; LysM-Cre mice show normal
hematopoiesis with the numbers of macrophages, dendritic cells and other leukocytes
unaffected (Yang, Liu et al. 2007). Although the responsiveness to ligands is impaired in
GRP94 deficient macrophages, the expression level of those TLRs is not affected
revealed by immunoblotting, which is consistent with the working model that GRP94 is
required for the post-translational folding and maturation of TLRs (Harding 2007). In
Drosophila, gp93, which shares 74% homology to mammalian GRP94, can chaperone
multiple clients of GRP94 including TLRs and integrins in GRP94 deficient mouse cells
(Chen, Zhong et al. 2006; Morales, Wu et al. 2009).
In order to examine the role of GRP94 in B cells in vivo, a B cell-specific Grp94
knockout mouse model is created by CD19-Cre mediated homologous recombination

15
(Liu and Li 2008). GRP94 null B cells have no problem in development, with normal
plasma cell differentiation, immunoglobulin assembly, class-switching and
immunoglobulin production, which indicate that GRP94 is dispensable for
immunoglobulin folding and secretion in vivo (Liu and Li 2008). This study further
demonstrates that GRP94 optimizes the function of B-cells through folding limited
number of clients such as TLRs and selective but not all integrins (Liu and Li 2008). The
role of GRP94 in folding integrins is further investigated in hematopoietic systems
utilizing a tamoxifen-inducible Grp94 knockout mouse model (Staron, Yang et al. 2010).
Out of 17 integrin pairs expressed in the hematopoietic system, 14 are dependent on
GRP94 (Staron, Yang et al. 2010). GRP94 null hematopoietic stem cells (HSC) are able
to self-renew and support long-term myelopoiesis (Staron, Yang et al. 2010). However,
the B- and T-cell development is blocked in an early stage, which implies that GRP94
plays a selective but not general role during hematopoiesis (Staron, Yang et al. 2010). It
is still unclear that whether the block of B- and T-cell development is due to the lack of
integrins, or other client proteins of GRP94, which awaits further investigation (Merchant
2010).
Except for folding client proteins, GRP94 is also one of the major Ca
2+
-binding proteins
located in the ER (Eletto, Dersh et al. 2010). Each GRP94 can bind between 16 and 28
Ca
2+
atoms, providing about 30μm Ca
2+
storage capacity (Eletto, Dersh et al. 2010). In
addition, the peptide-binding activity of GRP94 is enhanced at physiological Ca
2+
levels
(Eletto, Dersh et al. 2010).

16
The role of GRP94 in ERAD is suggested by the impairment of degradation of a model
ERAD substrate in the GRP94 depleted cells (Christianson, Shaler et al. 2008). This
could be due to the physiological interaction between GRP94 and OS-9, which is
essential for the proper degradation of several ERAD substrates. Studies on this specific
function of GRP94 just start and further investigations are needed to uncover the
underlying mechanisms.
GRP94 has been implicated in cancer, autoimmune disease and prion diseases (Ni and
Lee 2007). Induction of GRP94 is often associated with cellular transformation,
tumorigenicity and reduced sensitivity to radiation therapy, whereas suppression of
GRP94 by antisense RNA sensitizes tumor cells to etoposide treatment (Reddy, Lu et al.
1999; Lee 2001; Banerjea, Ahmed et al. 2004; Kubota, Suzuki et al. 2005). GRP94 in
tumor cells may be selectively targeted since GRP94 has less monosaccharides in
aggressive cancer tissues than normal tissues (Suriano, Ghosh et al. 2005; Fu and Lee
2006). It is also suggested that GRP94 may function as a cell-surface receptor for signal
transduction (Cabanes, Sousa et al. 2005). In addition, anti-GRP94 vaccines have been
widely studied in cancer therapy based on the discoveries that GRP94 is associated with
diverse peptides including tumor specific antigens (Srivastava 2006). Currently, vaccines
anti-GRP94 is being tested in cancer patients of various clinical trials (Pilla, Patuzzo et al.
2006). The important function of GRP94 in chaperoning TLRs and integrins leads to the
impression that GRP94 could play important roles in immune system and autoimmune
disease. Despite no apparent gross abnormality, the mice with enforced overexpression of
cell-surface GRP94 develop spontaneous autoimmune diseases at 20 weeks old (Liu, Dai

17
et al. 2003). Prion diseases, also known as transmissible spongiform encephalopathies
(TSEs) are infectious neurological disorders characterized by accumulation of PrP
sc

(scrapie-associated PrP, the abnormal form of PrP), and consequent neudegeneration
(Lindholm, Wootz et al. 2006). Compared with the normal soluble prion protein, the
PrP
sc
is protease-resistant and insoluble, which is believed to cause the neurotoxicity and
cell death (Ni and Lee 2007). The upregulation of GRP94 has been observed in PrP
sc
-
infected neuroblastoma cells and samples of the cortex of TSEs patients (Hetz,
Russelakis-Carneiro et al. 2003; Lindholm, Wootz et al. 2006). The onset of ER stress
has been detected in PrPsc aggregated neuronal cells, which could induce ER stress
mediated apoptotic pathways (Hetz, Russelakis-Carneiro et al. 2003).
In summary, the study on the role of GRP94 in development and human diseases just
begins; numerous efforts are needed in the future to elucidate the underlying mechanisms.
Up to date, all the information we know about GRP94 suggests that it is a unique ER
chaperone with specific function in different tissues and diseases. Future studies on
GRP94 will enhance our understanding more deeply how GRP94 interacts with client
proteins, whether there are cochaperones acts with GRP94 and how its action cycle is
regulated, etc.

18
Chapter 2: Role of GRP78 in neuronal cell protection
2.1 Introduction
A common feature shared by neurodegenerative diseases is the accumulation and
aggregation of misfolded proteins (Rubinsztein 2006). The presence of misfolded
proteins either within or outside of the ER will elicit cellular stress response including ER
stress response (Rao and Bredesen 2004). Disruption of ER homeostasis will trigger a
serious signal transduction termed as unfolded protein response (UPR) (Wu and Kaufman
2006). When the protein load exceeds the folding capacity of ER, the UPR signaling
pathways will be activated (Lee 2001). ER stress has been implicated in the pathogenesis
of neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases (Nakagawa,
Zhu et al. 2000; Imai, Soda et al. 2001). ER protein quality control system is critical for
cerebellar Purkinje cell survival (Zhao, Longo-Guess et al. 2005; Hara, Nakamura et al.
2006; Lee, Beebe et al. 2006) and is linked to Marinesco-Sjögren (MS) syndrome in
human (Anttonen, Mahjneh et al. 2005; Senderek, Krieger et al. 2005). One of the best
characterized ER chaperones GRP78 is crucial in maintaining ER homeostasis and
regulating UPR (Wang, Wey et al. 2009). The role of GRP78 in promoting cell survival
and anti-apoptosis has been studied intensively using tissue culture systems, however, the
direct role of GRP78 in regulating UPR in vivo and neurodegeneration remains unclear,
partially due to the early embryonic lethality of Grp78-/- embryos (Luo, Mao et al. 2006).
Purkinje cells (PCs) are the principal neurons and the sole output of the computational
circuitry of the cerebellar cortex, providing signals required for balance, motor

19
coordination and cognitive learning (Sillitoe and Joyner 2007). It is reported that
mutations in SIL1, a nucleotide exchange factor for GRP78, cause MS syndrome in
human and protein accumulation and PC degeneration in mice (Anttonen, Mahjneh et al.
2005; Senderek, Krieger et al. 2005; Zhao, Longo-Guess et al. 2005). Interestingly,
induction of GRP78 and CHOP is detected in SIL1 null PCs, suggesting the onsite of ER
stress (Zhao, Longo-Guess et al. 2005).
To investigate the role of GRP78 in regulating UPR signaling pathways in vivo and
neuronal cell survival, a Purkinje cell (PC)-specific knockout mouse of Grp78 was
created. The PC-specific knockout mice developed severe motor coordination defect
caused by progressive PC degeneration, associating with cerebellar atrophy. GRP78
depletion in PCs leads to specific modulation of UPR signaling pathways and reduction
in cytosolic ubiquitination level. It is also revealed by ultrastructural evaluations that
GRP78 is essential in maintaining ER integrity and morphology. Thus our study shows
GRP78 is required for Purkinje cell survival and normal function of cerebellum and
establishes a novel mouse model of accelerated cerebellar degeneration with basic and
clinical applications.
2.2 Materials and methods
Mouse models The detailed information on generation of the Grp78 F/F, F/- mice and
the prostate epithelial cell-specific Grp78 conditional knockout mice were described
previously (Fu, Wey et al. 2008). In brief, Grp78 T/+ mice (Luo, Mao et al. 2006)
carrying the targeted allele (T) were mated with the EIIA-Cre transgenic mice, and

20
generated offspring carrying the floxed (F) or knockout (-) allele. The Purkinje cell (PC)
specific Cre (pc-Cre) transgenic mice (line L7Cre-2) were obtained from the Jackson
Laboratory (Barski, Dethleffsen et al. 2000). The Grp78 F/-; pc-Cre mice were generated
through breeding between Grp78 F/- mice and the PC specific Cre transgenic mice.
The generation of the Grp94 F/F and F/- mice was described previously (Mao, Wang et al.
2010). The Grp94 F/F; pc-Cre mice were generated through breeding between Grp94 F/F
mice and the PC specific Cre transgenic mice.
The Chop-/- mice were obtained from the Jackson Laboratory (Marciniak, Yun et al.
2004). The Grp78, Chop double knockout mice in PCs were generated from the mating
between Grp78 F/-; pc-Cre mice and Chop -/- mice.
All protocols for animal use and euthanasia were reviewed and approved by the
University of Southern California Institutional Animal Care and Use Committee.
Primary antibodies Primary antibodies used include: rabbit anti-GRP78 (H129) and
anti-PERK (H-300) (Santa Cruz Biotechnology), anti-cleaved caspase-3 (Asp175) and
phospho-eIF2α (Ser51) (Cell Signaling), anti-GRP94 (SPA-851), PDI (SPA890) and
calnexin (SPA-865) (Stressgen) and anti-p62 (SQSTM1) (BIOMOL), mouse anti-
calbindin and anti-β-actin (Sigma-Aldrich), anti-CHOP (GADD153 B-3) and GADD34
(C-19) (Santa Cruz Biotechnology), anti-eIF2α (L57A5) and ubiquitin (P4D1) (Cell
Signaling) and anti-neurofilament (non-phosphorylated) (SMI-32) (Sternberger
Monoclonals), rat anti-GRP94 (Stressgen).

21
Histological, immunohistochemical and immunofluorescence staining analyses
Mouse brains were exposed by removing the cranial bones and fixed in situ overnight in
10% buffered formalin. The entire brains were then removed from the skull and cut
midsagittally before being embedded in paraffin using standard protocols.
Immunohistochemical and immunofluorescence staining were carried out as described
previously (Dong, Ni et al. 2008). In brief, Vectastain Elite avidin-biotin complex kit
(Vector Laboratories) was used for immunohistochemistry. After antigen retrieval with
retrivagen A (pH 6.0) (BD Pharmingen), paraffin sections were incubated with primary
antibodies in blocking solution (1.5% serum in PBS) at 4°C overnight with antibodies
against GRP78 (1:100), calbindin (1:100), cleaved caspase-3 (1:50), GRP94 (1:200), PDI
(1:300), CHOP (1:50), eIF2α (1:50), phospho-eIF2α (Ser51) (1:50), ubiquitin (1:200)
and p62 (1:1000). For immunofluorescence staining, paraffin sections were incubated
with primary antibodies including calbindin (1:100), GADD34 (1:200), ubiquitin (1:100),
GRP78 (1:100), calnexin (1:200) and neurofilament (1:100) at 4°C overnight. For
fluorescent detection of calbindin, GADD34, ubiquitin and neurofilament, Alexta Fluor
488-conjugated goat anti-mouse IgG (1:200) (Invitrogen) was used. For fluorescent
detection of GRP78 and calnexin, Alexta Fluor 594-conjugated goat anti-rabbit IgG
(1:200) (Invitrogen) were used. TUNEL assay was performed with Peroxidase In Situ
Apoptosis Detection Kit (Chemicon International).
Motor coordination tests Rota rod test and footprint test were performed as previously
described (Hara, Nakamura et al. 2006). In brief, for the rota rod test, mice were placed
on a rod rotating at 30 rpm, and the time taken for them to fall from the rod was measured.

22
If a mouse stayed on the rod longer than the 2 min trial, a time of 120 sec was recorded.
For the footprint test, the forelimbs and hindlimbs of mice were painted as blue and red
with non-toxic ink respectively. Then their paw placement was recorded using a narrow
tunnel (10 cm wide, 40 cm long and 10 cm high) with white paper on the bottom.
Electron microscopy (EM) and immunoEM analysis For regular EM, mouse brain
tissues were dissected from the skull and immersion fixed in half strength Karnovsky’s
fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer, pH 7.4) for 24 h at 4°C. Tissues were postfixed in 1% osmium tetroxide for 2 h on
ice. Samples were dehydrated in graded alcohol and infiltrated with Eponate (Ted Pella
Inc., Redding CA). Ultrathin sections were cut at 70 nm and stained with uranyl acetate
and lead citrate. For immunoEM labeling, mouse brain tissues were dissected from the
skull and immersion fixed in fixative (2% paraformaldehyde and 0.1% glutaraldehyde in
0.1M sodium cacodylate buffer) for 24 h at 4°C. Tissues were infiltrated with LR White
acrylic resin (Ted Pella Inc.). After polymerization, ultrathin sections were cut at 70 nm.
After incubation with blocking solution (1% BSA and 0.1% Triton-X100 in PBS) for 30
min, sections were stained with anti-calnexin antibody (1:40) at 4
o
C for overnight
followed by 1 h incubation with 15 nm gold-conjugated goat anti-rabbit antibody (1:40)
(Ted Pella Inc.). Sections were counter stained with uranyl acetate. All sections were
examined on a JEOL JEM 2100 electron microscope (Peabody, MA) and photographed
with the Orius SC1000B Gatan digital camera (Pleasanton, CA).

23
Body weight and food consumption measurements Fasting body weights of the mice
were measured weekly from 3- to 10-wk old. The food consumption was measured for
seven successively days at 3- and 9-wk old.
Western blotting Embryonic stem cells (ESCs) derived from E3.5 Grp94 +/+ and -/-
mouse embryos were maintained on feeders in ESC specific media with leukemia
inhibitory factor (LIF) added. Western blots were performed as previously described (Ye,
Jung et al. 2010). In brief, lysates from the untreated or 6 h 300nM ER stress inducer
thapsigargin (Tg) treated cells were extracted in ice-cold RIPA buffer (50 mM Tris-Cl,
150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing cocktails
of proteinase inhibitors and phosphatase inhibitors (Roche), by centrifugation (13000g,
15 min) following 3 freeze-thaw cycles. Proteins were separated by 6%, 8% or 10% SDS-
PAGE, and transferred to nitrocellulose membrane (Pall). The primary antibodies used
include the following: rat anti-GRP94 (1:5000) from Stressgen; rabbit anti-PERK (H-
300) (1:500), rabbit anti-GADD34 (1:1,000) from Santa Cruz Biotechnology; rabbit anti-
phosphorylated PERK (Thr980) (1:500), rabbit anti-phosphorylated eIF2α (1:1000) and
rabbit anti-eIF2 α (1:1,000) from Cell Signaling; mouse anti-β-actin (1:5000) from
Sigma-Aldrich. The experiments were repeated two to three times.
RT-PCR analysis of Xbp-1 mRNA splicing The cells were either untreated or treated
with 300 nM ER stress inducer thapsigargin (Tg) for 6 h. After that, total RNA was
extracted using TRI reagent (Sigma-Aldrich) following the manufacturer’s instructions.
First-strand cDNA was synthesized with SuperScript II (Invitrogen). To detect both

24
unspliced and spliced Xbp-1 mRNA, PCR was performed as described (Mao, Dong et al.
2004). β-actin was used as internal control. These experiments were repeated three times.
2.3 Results
2.3.1 Generation of the GRP78 conditional knockout mouse model in
Purkinje cells
ER quality control system has been implicated in the pathogenesis of neurodegenerative
diseases such as Alzhermer’s disease and Parkinson’s disease (Nakagawa, Zhu et al. 2000;
Imai, Soda et al. 2001; Mao, Dong et al. 2004). As one of the major ER chaperones and
the master regulator of UPR, the role of GRP78 in cytoprotection and regulating UPR has
been studied intensively in in vitro systems, however, the in vivo role of GRP78 in cell
survival and ER stress signaling remains obscure until recent years. Homozygous
knockout of Grp78 in mice results in embryonic lethality around embryonic day 3.5
(E3.5) (Luo, Mao et al. 2006). In order to investigate the function of GRP78 in adult
neuronal cells, creation of specific knockout mouse models in neuronal cells are required.
Purkinje cells (PCs) are the principal neurons and the sole output of the computational
circuitry of the cerebellar cortex, providing signals required for balance, motor
coordination and cognitive learning (Sillitoe and Joyner 2007). Thus a PC-specific Grp78
knockout can be used as a valuable tool to answer those questions.
The PC-specific Grp78 knockout mice were created by crossing Grp78 F/F or F/- mice
with PC-specific promoter driven Cre recombinase transgenic mice (L7-Cre) (Barski,

25
Dethleffsen et al. 2000). Grp78 F/F or F/- mice were generated from Grp78 T/+ mice.
The Grp78 T/+ mice (Luo, Mao et al. 2006) carrying the targeted allele (T) were mated
with the EIIA-Cre transgenic mice, and offsprings carrying the floxed (F) or knockout (-)
allele were selected by genotyping. The illustration of the Grp78 wildtype (WT), F and –
alleles is shown in Figure 2.1A. The Grp78 PC-specific mice were abbreviated below as
F/-; pc-Cre. The successful generation of mice bearing various Grp78 alleles has been
confirmed by genotyping (Figure 2.1B). Cohorts of mice bearing the following
genotypes: (1) Grp78F/F, (2) Grp78 F/- and (3) Grp78 F/-; pc-Cre were breed for growth
and development. No obvious phenotypes or developmental defects were observed in
Grp78 F/- mice in agreement with previous report that Grp78 heterozygosity affects
neither mouse development nor normal organ function (Luo, Mao et al. 2006). PCs in
Grp78 F/- mice are intact and similar GRP78 level was detected when compared with
Grp78 F/F mice by immunohistochemical staining (Figure 2.1C and D).
In the F/-; pc-Cre mice, Cre recombinase was expressed specifically in the PCs after
postnatal day 6, causing deletion of exon 5 to exon 7 of Grp78 which encodes both the
ATPase and peptide-binding domain, both of them are essential for the normal function
of GRP78. At 2 week of age, GRP78 staining was partially reduced in about 10% of the
PCs of the F/-; pc-Cre mice compared to the F/- control; and by week 3, GRP78 was
undetectable in about 90% of the PCs of the F/-; pc-Cre mice by immunohistochemical
staining (Figure 2.2A and B).

26

Figure 2.1. Generation of Grp78 Purkinje cell-specific knockout mice. (A) Schematic
drawing for the Grp78 wild-type allele (+), floxed allele (F) and knockout allele (-). The
loxP sites were indicated by black arrowhead. The size and position of the expected PCR
products were shown with bars and numbers on top. (B) Representative genotyping
results of Grp78 +, F, and - alleles, and Cre transgene with expected PCR product size.
(C) Representative immunohistochemical (IHC) staining for GRP78 in the Purkinje cells
(PCs) of Grp78 F/F mice and their F/- littermates at 3 week of age. The staining was
performed on the sagittal sections of the cerebellum of mice. (D) Representative
immunofluorescence (IF) staining for calbindin shows there is no PC degeneration in
Grp78 F/- mice compared with their F/F littermates at 4.5 week of age. Green: Calbindin.
Blue: DAPI. Scale bars: 50 μm (C and D).

Figure 2.2. GRP78 depletion in PCs of F/-; pc-Cre mice. (A) Immunostaining for
GRP78 in the PCs of F/-; pc-Cre mice and their F/- littermated at 2 week of age. (B) The
same as in (A) with the mice at 3 week of age. n=3 mice per group. Scale bars: 50 μm
(upper panel A and B), 25 μm (lower panel A and B).
A B
A B
C D

27
2.3.2 Accelerated Purkinje cell degeneration and cerebellar atrophy in F/-;
pc-Cre mice

The F/-; pc-Cre mice were born with the expected Mendelian ratio and there was no
difference in survival between them and their Grp78 F/F or F/- siblings. However, both
male and female mice exhibited significant lower bodyweight comparing with the F/-
mice (Figure 2.3A and B). Accordingly, their overall body size was also smaller than
their Grp78 F/- littermates (Figure 2.4A). Food consumption of the F/-; pc-Cre mice was
also significantly less than their F/- littermates, which might in part explaining the lower
body weight (Figure 2.4B).

Figure 2.3. Postnatal growth retardation in F/-; pc-Cre mice. (A) Body weights of
Grp78 F/- and their F/-; pc-Cre littermates were measured weekly from 3 week old to 10
week old. The male F/-; pc-Cre exhibited significantly lower body weights than the F/-
mice. (B) The same as in (A) with the body weights of female mice. n=10 per group.
Data are presented as mean±s.e.m. ∗ p≤0.05, ∗∗p ≤0.01, ∗∗∗p ≤0.001 (Student’s t-test).

Staining with PC specific marker calbindin showed partial loss of the PCs at week 4.5
(Figure 2.5A). Extensive PC degeneration was observed in all ten lobules of the
cerebellum with almost no PC survived at 13 week of age (Figure 2.5B). Correlating with
PC degeneration, cerebellar atrophy was evident in F/-; pc-Cre mice by week 13 and
A B

28
more severe by week 27 (Figure 2.5C). Collectively, these results establish that GRP78 is
required for PC survival and cerebellum integrity.

Figure 2.4. Body size and food consumption comparation between F/- mice and their
F/-; pc-Cre littermates. (A) A representative male F/-; pc-Cre mice and its F/- littermate
at 5.5 week of age. (B) Food consumption of male F/- mice and their F/-; pc-Cre
littermates at both 3 and 9 week of age. n=4 mice per group. Data are presented as
mean±s.e.m. ∗∗p≤0.01, ∗∗∗p≤0.001 (Student’s t-test).

Figure 2.5. Progressive PC degeneration and cerebellar atrophy in the F/-; pc-Cre
mice. (A) Representative IF staining for calbindin in F/- and F/-; pc-Cre mice at 4.5 week
of age. (B) IHC staining for calbindin on the cerebellum sections of F/- and F/-; pc-Cre
mice at 13 week old. Cerebellar lobules are indicated by Roman numbers. (C)
Hematoxylin and eosin (H&E) staining for the whole cerebellum of F/- and F/-; pc-Cre
mice, demonstrating cerebellar atrophy. Scale bars: 50 μm (A), 1.5 mm (B and C). n=3
mice per group for all the panels.
B
A
B A
C

29
To investigate whether apoptosis is the underlying mechanism account for PC
degeneration, cleaved caspase-3 (C-3) staining and TUNEL assay were performed. The
activation of C-3 and positive TUNEL staining were detected in about 10% of the PCs at
week 3.5 (Figure 2.6A and B), suggesting onset of apoptosis in the PCs of F/-; pc-Cre
mice.

Figure 2.6. Caspase 3 activation and TUNEL staining in PCs of F/- and F/-; pc-Cre
mice. (A) IHC staining for cleaved caspase-3 on PCs of F/- and F/-; pc-Cre mice at 3.5
week old. (B) TUNEL staining on PCs of F/- and F/-; pc-Cre mice at 3.5 week old. n=3
mice per group. Scale bars: 25 μm (A and B).
2.3.3 Severe motor function deficiency in F/-; pc-Cre mice
By week 5.5, both male and female Grp78 F/-; pc-Cre mice exhibited severe motor
coordination defects, consistent with partial degeneration of PCs. It was difficulty for
them to walk in a straight line. Even when they stand still, they could not maintain
balance and appeared “tipsy”. All these phenotypes suggest the onset of cerebellar ataxia
in those mice. In order to further characterize their ataxic phenotype, neurological tests
such as rota rod test and footprint record were performed. Rota rod test were performed
in both male and female cohorts, and the results show that F/-; pc-Cre mice stayed on the
rod for a much shorter time than F/- mice, consistently over multiple trials (Figure 2.7A).
Footprint recording further revealed ataxic walking pattern (Figure 2.7B), with reduced
stride/width ratio compared to the F/- mice, which is a normal indicator for ataxia (Figure
A B

30
2.7C). Thus severely impaired motor coordination ability of the F/-; pc-Cre mice was
confirmed by the results from both tests.

Figure 2.7. Behavioral abnormalities of the F/-; pc-Cre mice. (A) Rota rod test results
of F/- mice (open symbols) and their F/-; pc-Cre littermates (closed symbols) at 5.5 week
old. Each time the mice fell off the rod rotating at 30pm was recorded. n=4 to 5 mice per
group. p=0.016 (male mice) and p=0.008 (female mice) (randomization test). (B)
Footprint of a typical F/- and F/-; pc-Cre mice at 5.5 week old. The blue and red prints
were from the painted forelimbs and hindlimbs, respectively. (C) Quantitation results of
panel (B). The ratio of stride length divided by paw-based widths was shown here. n=4
mice per group. Data are presented as mean±s.e.m. for panel (A) and (C). ∗∗∗p≤0.001
(Student’s t-test).
2.3.4 GRP94 is not required for the survival of Purkinje cells
Loss function of GRP78 in PCs results in apoptosis; we wonder whether loss function of
another important ER chaperone GRP94 will also cause neurodegeneration in PCs. The
Grp94 PC-specific knockout mice were generated by mating between Grp94 F/F, F/-
B
A
C

31
mice and PC-specific promoter driven Cre recombinase transgenic mice (L7-Cre) (Barski,
Dethleffsen et al. 2000). The creation and characterization of the Grp94 +/- and F/F mice
were described previously (Mao, Wang et al. 2010). The Grp94 PC-specific mice were
abbreviated below as Grp94 F/-; pc-Cre.

Figure 2.8. PC-specific knockout of GRP94 caused no morphological or behavioral
defect. (A) IHC staining for GRP94 on the sagittal sections of Grp94 F/F mice and their
Grp94 F/-; pc-Cre littermates cerebella at 5 month of age. (B) H&E staining on the
cerebellum sections of Grp94 F/F mice and their Grp94 F/-; pc-Cre littermates at 5 month
old. (C) Results of rotor rod test given to the Grp94 F/F male mice and their Grp94 F/-;
pc-Cre male littermates for 5 trials at 5 month old, n=5 for each group. The time of 120
sec denotes that the mice stayed on the rod for the duration of the test and none fell off
the rod. Scale bars: 50 μm (A) and 100 μm (B).
A
B
C

32
As Grp78 F/- mice, Grp94 F/- mice also show no obvious phenotypes or developmental
defects. Interestingly, we did not observe any PC degeneration in Grp94 F/-; pc-Cre mice
even when they are already 5 month old (Figure 2.8A and B). Neither did we observe any
behavioral defects in those mice. They can walk as well as their Grp94 F/F or F/-
littermates. Rota rod test also reveals that they can stay on the rod as long as Grp94 F/F
mice, which clearly showing that there is no motor coordination defects in those Grp94
F/-; pc-Cre mice. The distinct phenotypes of Grp78 and Grp94 F/-; pc-Cre mice indicate
the unique role of GRP78 in neuroprotection and cerebellar integrity, which can not be
compensated by other chaperones in the ER.
2.3.5 Modulation of UPR signaling pathways in GRP78 null PCs
To investigate whether GRP78 knockout will activate UPR in PCs, we performed
immunostaining experiments with variety of UPR parameters on both 2 and 3 week old
mice cerebellum. At 2 week old, about 90% PCs in F/-; pc-Cre mice still have similar
amount of GRP78 protein as their F/- littermates; however, GRP78 are almost completely
depleted at nearly 90% PCs of F/-; pc-Cre mice when they are 3 week old. Thus through
direct compare of the staining results between 2 and 3 week old mice, the role of GRP78
in regulating UPR can be clearly exhibited. One of the pro-survival branches of UPR is to
upregulate ER chaperones such as GRP78, GRP94 and PDI to enhance ER folding
capacity. We observed that the level of both GRP94 and PDI were upreguated in PCs of 3
week old F/-; pc-Cre mice when compared to their F/- littermates (Figure 2.9A). As a
major down stream target of PERK signaling pathway, CHOP is an important ER stress
specific marker. The results show that CHOP was induced in about 90% of PCs at 3 week

33
old, while only about 5% PCs were CHOP positive at 2 week old, consistent with the loss
of GRP78 in those cells (Figure 2.9B). The upregulation of CHOP downstream target
GADD34 was also detected at 3 week old PCs but not at 2 week old (Figure 2.9C). The
phosphorylation of eIF2α, which was observed at 2 week old, was dramatically reduced
in 3 week old PCs. However, the total levels of eIF2α remain the same at both 2 and 3
weeks old (Figure 2.9D and E). The changes in UPR parameters were summarized in
Figure 2.10. Depletion of GRP78 in PCs results in upregulation of GRP94, PDI, CHOP
and GADD34 and suppression of phosphorylation of eIF2α without affecting the total
level of eIF2α.
In contrast to GRP78, knockout of Grp94 did not cause the activation of UPR signaling
pathways by utilizing our lab established Grp94 +/+ and -/- mouse embryonic stem cells
(mESCs). Compared with Grp94 +/+, depletion of GRP94 in mESCs confirmed by
Western blot will not activate PERK and IRE1 pathways. Neither induction of PERK and
eIF2α phosphorylation nor GADD34 upregulation was detected in Grp94 -/- mESCs
(Figure 2.11A). Under thapsigargin-induced ER stress, Grp94 -/- exhibited the same level
of induction of PERK and eIF2α phosphorylation, GADD34, and splicing of XBP-1,
further supporting that GRP94 did not regulate UPR signaling pathways (Figure 2.11A
and B).
Collectively, these data indicate that GRP94 is not required for the survival of either the
terminally differentiated PCs or the totipotent mESCs which can differentiate into cells of
all three germ layers. Different from GRP78 depleted cells, GRP94 null cells will not

34
activate UPR, consistent with the in vitro data that GRP78 not GRP94 is the master
regulator of UPR signaling pathways.

Figure 2.9. Modulation of UPR pathways in the GRP78 null PCs. (A-B) IHC staining
for GRP94 and PDI (A) and CHOP (B) on PCs of Grp78 F/- mice and their F/-; pc-Cre
littermates at indicated ages. (C) IF staining for GADD34 on the PCs of F/- mice and
their F/-; pc-Cre littermates at 2 and 3 wk of age. Green: GADD34. Blue: DAPI. (D-E)
IHC staining for p-eIF2α (Ser51) and eIF2α on PCs of F/- mice and their F/-; pc-Cre
littermates at indicated ages. n=3 mice per group for all panels. Scale bars: 25 μm.
A B
D E
C

35

Figure 2.10. Quantitation of the IHC and IF stainings of UPR parameters. The
relative protein levels were quantitated by NIH software ImageJ. The mean level of
staining in each group was determined by 40 randomly selected areas within PCs on the
sections. The level of p-eIF2α was normalized to eIF2α. For GRP94 and PDI, the
staining of PCs in F/- mice at 3 wk old was set as 1. For GADD34, p-eIF2α and eIF2α,
the staining of PCs in F/- mice at 2 wk old was set as 1. Data are presented as
mean±s.e.m. ***p ≤0.001 (Student’s t-test).

Figure 2.11. Knockout of Grp94 has no effect on UPR signaling pathways. (A)
Grp94+/+ and -/- mouse embryonic stem cells (mESCs) were either untreated or treated
with 300 nM ER stress inducer thapsigargin (Tg) for 6 h. The cell lysates were subjected
to Western blotting. a) The numbers on the left indicate the size of the proteins (in kD).
(B) XBP-1 splicing is not affected by Grp94 knockout. The same treatment as in panel
(A) with total RNA subjected to RT-PCR for detection of the unspliced (u) or spliced (s)
form of XBP-1. The numbers on the left indicate the size of the PCR products (in bp).
For all panels, the experiments were repeated two to three times.
B A

36
2.3.6 Reduction of cytosolic ubiquitin staining in GRP78 null PCs
In the mammalian ER associated protein degradation (ERAD) system, misfolded protein
released from calnexin will be captured by a recognition complex including EDEM,
passed on to GRP78 and PDI to get unfolded, retrotranslocated into the cytosol to
become polyubiquitinated by the E1-E2-E3 system and finally degraded by the
proteasome (Yoshida 2007). The malfunction of ERAD has been reported to be involved
in many neurological disorders including Alzheimer’s disease, Huntington’s disease and
Parkinson’s disease (Yoshida 2007). Upon ER stress, the proteins related to ERAD such
as EDEM, ERdj5 are upregulated to promote the malfolded protein degradation process.
Aggregated ubiquitinated proteins were often observed in cells with dysfunctional ER
chaperones or mutant proteins (Zhao, Longo-Guess et al. 2005). Strikingly, we did not
observe aggregated ubiquitinated proteins in the GRP78 null PCs. At 2 week old, the
protein ubiquitination level was detectable in the PCs of F/- mice and their F/-; pc-Cre
littermates. However, the level was greatly reduced at 2.5 week old, and almost
undetectable at 3 week old by both IHC and IF staining (Figure 2.12A and C). The
quantitation from multiple samples further confirmed that the protein ubiquitination level
decrease about 50% and 90% at 2.5 and 3 week old, respectively (Figure 2.12B and D).
p62/SQSTM1 is a cytosolic protein which binds to polyubiquitinated proteins and targets
them to autophagy machinery for degradation. p62 itself is stabilized by the
polyubiquitinated protein aggregates (Bjorkoy, Lamark et al. 2005; Seibenhener, Geetha
et al. 2007). Consistent with the level of protein ubiquitination, the level of p62 was also
reduced in PCs of F/-; pc-Cre mice at 3 week old, but not in PCs of 2 week old mice

37
(Figure 2.12E). Quantitation of the p62 staining levels shows that the p62 protein level
was significantly reduced at 3 week old (Figure 2.12F).

Figure 2.12. Reduction of protein ubiquitination and p62 level in GRP78 null PCs.
(A) IHC staining for ubiquitin in F/- and F/-; pc-Cre mice at indicated ages. (B)
Quantitation of ubiquitin staining, with the level in the PCs of F/- mice at 2 wk old set as
1. (C) IF staining for ubiquitin in F/- and F/-; pc-Cre mice at indicated ages. (D)
Quantitation of ubiquitin fluorescence staining with the level in the PCs of F/- mice at 2.5
wk old set as 1. (E) IHC staining for p62 in PCs of F/- and F/-; pc-Cre mice at 2 and 3 wk
of age. (F) Quantitation of p62 staining with the level in the PCs of F/- mice at 2 wk old
set as 1. All relative levels shown were quantitated by NIH software ImageJ. The mean
level of staining in each group was determined by 40 randomly selected areas within the
PCs on the IHC or IF stained sections. For all panels, n=3 mice per group. Data are
presented as mean±s.e.m. ***p ≤0.001 (Student’s t-test). All scale bars: 25 μm.
A B
C D
E F

38
In the woozy mutant mouse model which was caused by homozygous mutation of a
GRP78 cochaperone SIL1, the aggregation of ubiquitinated proteins were detected inside
the PCs. While in our GRP78 depleted PCs, we observed significant reduction in protein
ubiquitination. In order to further investigate the role of GRP78 in protein degradation,
another Grp78 conditional knockout mouse model was used. PB-Cre is the abbreviation
form of probasin-Cre, which can only be expressed in the prostate epithelial cells. IHC
and IF stainings for GRP78 clearly show the depletion of GRP78 protein in the ventral
prostate epithelial cells (Figure 2.13A). In a high magnification, the reduction of protein
ubiquitin level was also observed, correlating with the depletion of the GRP78 protein
(Figure 2.13B). It was reported that conditional knockout of Grp78 in prostate epithelial
cells does not affect the gross morphology or the normal function of the prostate and
those cells do not undergo cell death (Fu, Wey et al. 2008). Thus the reduction of protein
ubiquitination is unlikely due to those cells were undergoing cell death process. One
possible reason that we observed significant lower level of protein ubiquitination is that
those PCs did not make enough proteins since they were undergoing apoptosis. To show
the specificity of the reduction of protein ubiquitination, we performed immunostaining
with calnexin, which is an ER protein, neurofilament and calbindin, which are
cytoskeletal and cytosolic proteins, respectively. Similar level of calnexin, neurofilament
and calbindin was detected in the PCs of F/-; pc-Cre and F/- mice (Figure 2.14A).
Quantitation of the staining also revealed the same results (Figure 2.14B).

39

Figure 2.13. Reduction of protein ubiquitination staining in the GRP78 null prostate
epithelial cells. (A) IHC (upper panel) and IF (lower panel) staining for GRP78 on the
ventral prostate of the Grp78 F/F and their Grp78 F/F; PB-Cre littermates at 5 month old.
PB-Cre refers to cre-recombinase driven by the probasin promoter. (B) IF staining for
GRP78 (Red) and ubiquitin (Green) on the ventral prostate of Grp78 F/F mice and their
Grp78 F/F; PB-Cre littermates at 5 month old. Blue: DAPI. Scale bars: 200 μm (A) and
50 μm (B).

Figure 2.14. Similar expression levels of calnexin, non-phosphorylated
neurofilament and calbindin in the PCs of control and knockout mice. (A) IF
staining for calnexin and neurofilament and IHC staining for calbindin in PCs of Grp78
F/- mice and their F/-; pc-Cre littermates at 3 wk of age. (B) Quantitation of the IF and
IHC stainings shown in panel (A). The relative protein levels were quantitated by NIH
software ImageJ. The mean level of staining in each group (n=3 mice per group) was
determined by 40 randomly selected areas within the PCs on the sections. For all the
quantitation, the staining of PCs in F/- mice at 3 wk old was set as 1. Data are presented
as mean±s.e.m. Scale bars: 25 μm.
A B
A B

40
2.3.7 ER expansion in GRP78 null PCs
As one of the major ER chaperones, it is well established that GRP78 plays an important
role in maintaining ER homeostasis. We wonder what will happen to the ER structure
when GRP78 is completely depleted. The ER morphology of PCs from 3.5 week old F/-
mice and their F/-; pc-Cre littermates was examined under transmission electron
microscope. Compared with the normal typical linear structure of ER in the PCs of F/-
mice, the ER exhibited prominently dilatated morphology with accumulation of
flocculent material, and in some of them, varying degrees of electron dense material
aggregation (Figure 2.15, 2.16 and 2.17). In tissue culture studies, tunicamycin-induced
ER stress will activate the formation of double membrane bound autophagosome
structures inside the cell. However, in our case, we did not observe any double membrane
bound inclusions that would suggestive of autophagosome formation, consistent with the
previously report that GRP78 is required for the formation of autophagosome under ER
stress conditions (Li, Ni et al. 2008).

41

Figure 2.15. Electron micrograph of PCs from F/- and F/-; pc-Cre mice,
demonstrating the whole PC morphology. (A) Representative electron micrograph
(EM) of a 3.5 wk old F/- mouse cerebellum shows normal PC ultrastructural morphology.
The white arrowheads indicate normal ER structure. (B) EM of a 3.5 wk old F/-; pc-Cre
mouse cerebellum shows prominent expansion of the ER in the perikaryon and proximal
apical dendrite. Scale bars: 5 μm.

Figure 2.16. High magnification of electron micrograph of PCs from F/- and F/-; pc-
Cre mice. (A) Higher magnification of the proximal dendrite area. Yellow arrowheads
indicate electron dense materials inside ER. (B) Varying degrees of electron dense
aggregates inside the expanded ER of the GRP78 null PCs as indicated by yellow
arrowheads. I to III indicate increasing amount of electron dense materials inside the ER.
Scale bars: 3μm (A) and 0.5μm (B).
A B
A B

42

Figure 2.17. ER expansion and electron dense aggregates inside the ER in the PCs of
a 3.5 wk old F/-; pc-Cre mouse. White arrowheads indicate normal ER structure
undergoing expansion. Black arrowheads indicate electron dense aggregates inside the
ER. Scale bar: 0.5 μm.
To further prove those expanded structures are ER not other cell organelles,
immunoelectron microcopy examination was performed with ER specific antibody,
calnexin. The ER structure was clearly seen by the immunogold labeling. In the F/- mice,
the typical tubular structure of ER was labeled by the gold particle; whereas in those
GRP78 null PCs, expanded structures enclosing electron dense or flocculent materials
were labeled (Figure 2.18). The ER identity of those expanded structures was further
confirmed by the results.

43

Figure 2.18. Altered ER structure in GRP78 null PCs shown by immunogold
labeling. Representative electron micrographs showing immunogold labeled ER
structures. PCs from 4.5 wk old F/- mice and their F/-; pc-Cre littermates were labeled
with the ER marker calnexin. The 15 nm immunogold particles were indicated by open
arrows. Scale bars: 0.2 μm.

2.3.8 Generation of the GRP78 and CHOP double knockout mouse model in
Purkinje cells
It was published that CHOP promotes cell death through activation of GADD34 and
ERO1α under ER stress conditions (Marciniak, Yun et al. 2004). In the GRP78 null PCs,
the induction of CHOP and GADD34 may partially contribute to the cell death. We
wonder if we simultaneously inactivate both GRP78 and CHOP, whether PCs can be
rescued from apoptosis and survive longer. Since Chop -/- mice are viable, fertile and
have no obvious developmental defects, they can be directly crossed with Grp78 F/-; pc-
Cre mice to get the Grp78 F/-; pc-Cre; Chop -/- mice. After several generations of
breeding, we have successfully generated the Grp78 and Chop double knockout mice in

44
PCs. The genotyping results with four cohorts of mice (1) Grp78 F/-, (2) Grp78 F/-; pc-
Cre, (3) Grp78 F/-; Chop -/- and (4) Grp78 F/-; pc-Cre; Chop -/- were presented in Figure
2.19. Grp78 F/-; Chop -/- mice were phenotypically normal, with no behavioral
abnormalities. Both Grp78 F/-; Chop -/- and Grp78 F/-; pc-Cre; Chop -/- mice were
fertile and survive as well as Grp78 F/- mice.

Figure 2.19. Generation of the Grp78 F/-;pc-Cre, Chop -/- mice. Representative
genotyping results from four cohorts of mice, Grp78 F/-, Grp78 F/-; pc-Cre, Grp78 F/-;
Chop -/- and Grp78 F/-; pc-Cre; Chop -/-. The left two lanes serve as positive and
negative control for the PCR reaction.
2.3.9 CHOP knockout can not rescue GRP78 null Purkinje cells from cell
death
After we obtained the Grp78 F/-; Chop -/- and Grp78 F/-; pc-Cre; Chop -/- mice, the next
question was whether the PCs can be rescued. There was no PC degeneration in Grp78

45
F/-; Chop -/- mice, which have similar number of PCs as Grp78 F/F or F/- mice. We
examined the number of PCs in the double knockout mice by H&E staining and
compared the cell number with Grp78 F/-; Chop -/- mice. The results show that PCs in
the Grp78 F/-; pc-Cre; Chop -/- mice still undergo age-dependent degeneration, with the
same rate as the Grp78 F/-; pc-Cre mice (Figure 2.20A and B). The ataxia walking
pattern was also detected in the Grp78 F/-; pc-Cre; Chop -/- mice by week 5.5. These
preliminary data that CHOP depletion can not rescue the GRP78 mutation caused cell
death in PCs suggest that CHOP does not play an essential role in this process. However,
whether UPR signaling pathways play an important role in this process still awaits further
investigation. In order to better understand all the signaling pathway modulations and
pathological phenotypes we observed in the Grp78 conditional knockout mouse, a
summary is presented in Figure 2.21.

46

Figure 2.20. Knockout of Chop in GRP78 null PCs can not rescue them from cell
death. (A) Age-dependent PC degeneration in the Grp78 F/-; pc-Cre mice compared with
their F/- littermates. The number of PCs determined by H&E staining was quantitated
within the same areas of cerebellar sections and plotted against age of the mice. The
number of PCs in 3 wk old F/- mouse cerebellum was set as 100%. (B) The same as in
panel (A) with the comparation between Grp78 F/-; Chop -/- mice and their Grp78 F/-;
pc-Cre; Chop -/- littermates. The number of PCs in 6.5 wk old Grp78 F/-; Chop -/- mouse
cerebellum was set as 100%. Data are presented as mean±s.e.m. **p ≤0.01, ***p ≤0.001
(Student’s t-test). For all panels, n=3 mice per group were examined.
A
B

47

Figure 2.21. Summary of the alteration in UPR signaling pathways and pathological
phenotypes in GRP78 null PCs. Grp78 knockout in PCs leads to ER protein
accumulation, UPR activation, ER expansion, protein ubiquitination reduction and
apoptosis.
2.4 Discussion
Although the role of GRP78 in regulating UPR has been studied intensively in tissue
culture systems in the past decades (Wu and Kaufman 2006), in this study, we investigate
the role of GRP78 in adult neuronal cell survival and UPR signaling pathway activation
in mouse models. According to previous studies, homozygous knockout of Grp78 in
mouse results in embryonic lethality at E3.5 (Luo, Mao et al. 2006). The Grp78 null
embryos show extensive TUNEL positive cells compared with negligible level of
staining in the wildtype control, suggesting the onsite of apoptosis in the embryonic stem
cells (ESCs). Thus GRP78 is required for the survival of mESCs. However, tissue
specific knockout of Grp78 in prostate epithelial cells does not affect their viability,
indicating that GRP78 is dispensable for the survival of those cells (Fu, Wey et al. 2008).
Knockdown of Grp78 by siRNA in tissue culture cell lines only induce apoptosis in two
out of seven human cell lines tested (Suzuki, Lu et al. 2007). The seven cell lines include
HeLa cells, Rsa cells, two pancreatic cancer cell lines, KP4 and PANC-1, two prostate

48
cancer cell lines PC-3 and DU 145, and a melanoma cell line, SK-MEL-28. Only HeLa
and PC-3 cells with high basal level of GRP78 show obvious upregulation of GRP94 and
apoptosis activation (Suzuki, Lu et al. 2007). Thus GRP78 may be only essential for the
survival of some cells, like ESCs, neuronal cells and certain type of cancer cells.
Compared with SIL1 deficient “woozy” mice, the Grp78 PC knockout mice show
accelerated Purkinje cell degeneration rate and cerebellar atrophy. The PC degeneration
begins at 4.5 wk and all ten lobules degenerate simultaneously and completely by 3
month. In contrast, PC degeneration in woozy mice starts around 3 month and lobule X
and caudal lobule IX still have intact PC even at more than 1 year old (Zhao, Longo-
Guess et al. 2005). Thus the function of GRP78 can not be substitute by any other
chaperones in all the lobules. A recent study reveals that overexpression of GRP170,
another nucleotide exchange factor for GRP78 (Weitzmann, Volkmer et al. 2006;
Weitzmann, Baldes et al. 2007), can suppress ER stress and rescue PC degeneration in
woozy mice (Zhao, Rosales et al. 2010). Additionally, Grp170+/- exacerbates those
phenotypes in woozy mice, and except PCs, other neurons in the brain did not show any
degeneration (Zhao, Rosales et al. 2010). However, p58
IPK
-/- ameliorates both the onsite
of ER stress and PC degeneration in SIL1 deficient mice (Zhao, Rosales et al. 2010).
Normally, p58
IPK
works as a co-chaperone associating with GRP78 in the ER, promoting
the ATP hydrolysis process of GRP78 (Rutkowski, Kang et al. 2007). These results
indicate that GRP170 and SIL1 are two alternative nucleotide exchange factors for
GRP78 with interchangeable functions and they may work under different scenetios. Not
all tissues are affected equally in either woozy mice or MS syndrome patients, implying

49
that relative amount of GRP170 and SIL1 varies in different tissues. Multiple DNAJ
domain proteins have been identified to function as co-chaperones to regulate the
different functions of GRP78 (Otero, Lizak et al. 2010). Future investigations are needed
to elucidate the special requirement of co-chaperones for GRP78 to fulfill different
functions.
Although it is not possible to perform biochemical analysis with the whole cerebellum
tissue since PCs only constitute a small portion, the large size of PCs with distinct nuclear
and cytosolic compartments allows us to use immunostaining techniques to detect the
UPR activation kinetically. It took about two weeks for GRP78 to be completely depleted
from PCs after the activation of Cre recombinase around one week after birth (Barski,
Dethleffsen et al. 2000). GRP78 null PCs upregulated GRP94 and PDI, which is also
observed by previous reports that both Grp78 heterozygous MEFs and knockdown of
GRP78 in vitro by siRNA showed the same compensatory response (Luo, Mao et al.
2006; Suzuki, Lu et al. 2007). We also detected the induction of CHOP and GADD34 in
GRP78 depleted PCs at 3 weeks old. Instead of upregulation, the phosphorylation of
eIF2α was substantially reduced in GRP78 null PCs. This could be due to the
upregulation of GADD34 which can recruit a catalytic subunit from one of several
related protein phosphatase I (PPP1) isoforms to dephosphorylate phosphorylated eIF2α
(Harding, Zhang et al. 2009). We did not observe activation of UPR in GRP94 null ESCs,
consistent with no PC degeneration and no ataxic phenotype in Grp94 PC knockout mice.
It is quite unexpected that the level of cytosolic ubiquitin staining was greatly reduced in
GRP78 null PCs. Downregulation of Grp78 should lead to more misfolded proteins in the

50
ER and those malfolded proteins will be ubiquitinated and degraded by proteasome or
autophagy. There are two possible explanations for this observation. The first one is that
GRP78 is required for ER protein retrotranslocation to be ubiquitinated in the cytosol.
Evidence has been shown in both yeast and mammalian cells. GRP78 facilitates the
export of mutant proteins to be degraded in the cytosol in yeast (Plemper, Bohmler et al.
1997). In mammalian cells, GRP78 stably binds to unfolded proteins and delivers them to
ERAD (Skowronek, Hendershot et al. 1998). The involvement of GRP78 in ERAD is
further clarified through the recent discovery that EDEM, ERdj5 and GRP78 form a
functionally important ERAD complex (Ushioda, Hoseki et al. 2008). ERdj5 can function
as a disulfide reductase which cleaves the disulfide bonds of misfolded proteins. ERdj5
binds to the ATP-bound form of GRP78 and activates ATP hydrolysis of GRP78,
resulting in dissociation of GRP78 from ERdj5. In turn the ADP-bound form of GRP78
binds the protein substrates and holds them in a dislocation-competent state until they are
transferred to the retrotranslocation channel in the ER membrane, for export to the
cytosol and subsequent ubiquitination and degradation. The second explanation is that ER
integrity is required for the retrotranslocation and degradation of proteins. GRP78
knockout will destroy the integrity and normal function of ER. This is shown by
ultrastructural examination of the GRP78 null PCs. Instead of typical tubular ER
structures in the F/- mice, we observed greatly expanded ER structures inclosing electron
dense or flocculent materials in GRP78 null PCs. Plus, the accumulation of electron
dense materials was only detected inside the expanded ER, not outside the ER. Thus
those electron dense materials could be aggregation of misfolded proteins. Nonetheless,

51
the tipsy mouse model does not allow us to test this mechanism biochemically. Future
studies are required to understand the role of GRP78 in protein ubiquitination.
Autophagy represents a catabolic process for the degradation and recycling of cytosolic,
long-lived or aggregated proteins and excess or defective organelles. ER stress induces
autophagy and promotes cell survival by enabling the use of intracellular resources under
starvation conditions (Ogata, Hino et al. 2006). GRP78, as a critical component of the ER,
is required for ER integrity and ER stress induced autophagy. In cells where GRP78 is
knocked down by siRNA, the ER structure is disrupted and autophagosome formation
under both ER stress and nutrient starvation conditions are suppressed (Li, Ni et al. 2008).
Consistent with this observation, we did not detect any autophagosome in the GRP78
depleted PCs, further confirming the role of ER and GRP78 in autophagosome formation.
In summary, our studies establish the Grp78 floxed mice as valuable tools for studying
the physiological role of GRP78 and the UPR in specific organs, as exemplified for the
creation of the tipsy mice. Considering that PCs are part of the core circuitry in the motor
learning process, the tipsy mice provide a unique model for behavioral studies of Purkinje
cell specific function. Further, the accelerated cerebellar degeneration in these mice will
facilitate the development of therapeutic agents combating ER dysfunction in
neurodegeneration and other diseases.

52
Chapter 3: Role of GRP94 in regulating UPR
3.1 Introduction
Glucose regulated protein 94 (GRP94), also known as gp96, endoplasmin, ERp99,
HSP108, CaBP4 and Tra-1, is the most abundant ER resident glycoprotein belonging to
the HSP90 family (Little, Ramakrishnan et al. 1994; Argon and Simen 1999). It shares
50% amino acid homology with HSP90 (Little, Ramakrishnan et al. 1994). It is
ubiquitously expressed in all vertebrates, some invertebrates like C. elegans and
Drosophila and also plants, but not in yeast (Argon and Simen 1999; Chen, Zhong et al.
2006; Morales, Wu et al. 2009).
GRP94 is a soluble ER chaperone and its structure has been determined by many
methods (Immormino, Dollins et al. 2004; Dollins, Warren et al. 2007; Krukenberg,
Bottcher et al. 2009). Four domains have been identified in GRP94: an N-terminal
domain (NTD), an acidic linker domain (LD), a middle domain (MD) and a C-terminal
domain (CTD). The ATPase activity of GRP94 is essential for its normal chaperone
activity and relies on all of the NTD, LD and MD (Ostrovsky, Makarewich et al. 2009).
NTD functions as the ATP binding domain and LD and MD work cooperatively to fulfill
the ATP hydrolysis process. The CTD is responsible for the constitutive dimerization of
GRP94 (Eletto, Dersh et al. 2010).
The general function of GRP94 contains three categories, chaperoning client proteins,
buffering Ca
2+
in the ER and involving in ER associated protein degradation (ERAD)

53
(Eletto, Dersh et al. 2010). The major function of GRP94 is to fold or assembly
membrane or secretary proteins. Unlike GRP78 which folds numerous proteins, GRP94
is pretty selective and has limited number of client proteins including immunoglobulin,
insulin like growth factor I and II (IGF-I and II), Toll-like receptors (TLR) and integrins
(Eletto, Dersh et al. 2010).
Genetically targeted disruption of Grp94 gene in mice results in embryonic lethality
around embryonic day 7.5 (E7.5) and Grp94-/- embryos fail to develop mesoderm,
primitive streak, or proamniotic cavity (Wanderling, Simen et al. 2007; Mao, Wang et al.
2010). This implies the essential role of GRP94 in embryonic development. Compared
with Grp94 +/+ embryonic stem cells (ESCs), Grp94 -/- ESCs grow normally in culture
and are able to form embryonic bodies (EB) and differentiate into cells of all three germ
layers (Wanderling, Simen et al. 2007; Mao, Wang et al. 2010). However, Grp94 -/-
ESCs are unable to differentiate into cardiac, smooth or skeletal muscle which is caused
by the deficiency in production of IGF II by GRP94 depleted ESCs (Wanderling, Simen
et al. 2007; Mao, Wang et al. 2010). Exogenous expression of IGF I or II will restore the
ability of Grp94 -/- ESCs differentiating into muscle cells (Wanderling, Simen et al.
2007). Later on, studies further prove that GRP94 is indispensable for the intracellular
processing and secretion of IGF II (Ostrovsky, Ahmed et al. 2009). A macrophage-
specific knockout mouse model of GRP94 reveal that GRP94 is the master regulator for
both cell-surface and intracellular TLRs including TLR2, TLR4, TLR5, TLR7 and TLR9
(Yang, Liu et al. 2007). In addition, B-cell specific knockout of Grp94 demonstrates that
GRP94 optimizes the function of B-cells through folding limited number of clients such

54
as TLRs and selective but not all integrins (Liu and Li 2008). This was further elucidated
by utilizing a tamoxifen-inducible Grp94 knockout mouse model (Staron, Yang et al.
2010). Out of 17 integrin pairs expressed in the hematopoietic system, 14 are dependent
on GRP94 (Staron, Yang et al. 2010). GRP94 null hematopoietic stem cells (HSC) are
able to self-renew and support long-term myelopoiesis (Staron, Yang et al. 2010).
However, the B- and T-cell development is blocked in an early stage, which implies that
GRP94 plays a selective but not general role during hematopoiesis (Staron, Yang et al.
2010).
Except for folding client proteins, GRP94 is also an important ER stress inducible protein
(Lee 2007). Upon ER stress, GRP94 is coordinately regulated with GRP78, the master
regulator of UPR signaling pathways (Li and Lee 2006). GRP78, GRP94, PDI and other
co-chaperones form a complex chaperone network process the unfolded protein
substrates (Ni and Lee 2007). In addition, GRP94 has similar promoter regulatory
sequences as GRP78 and is inducible by a variety of ER stress conditions (Ni and Lee
2007).
To better understand the in vivo role of GRP94, mouse models that allow both the
conventional and conditional knockout of Grp94 allele were created in our lab (Mao,
Wang et al. 2010). Through numerous efforts made by our collages Robin Wesselschmidt
and Changhui Mao, one Grp94-/- and multiple Grp94+/+ and +/- ESC lines were
established. Thus we were able to examine the role of GRP94 in regulating UPR
pathways and cell survival by utilizing the Grp94+/+ and -/- ESCs. We discovered that
under ER stress conditions, GRP94 depletion leads to induction of specific ER

55
chaperones like GRP78, calnexin (CNX) and calreticulin (CRT), but not PDI and
reduction of XBP-1 splicing, a major downstream target of IRE1 signaling pathway.
However, PERK signaling pathway is not affected in GRP94 null cells. Furthermore,
from analysis of microarray database of relative Grp94 mRNA level in adult mouse
tissues, we present predictions where GRP94 may play important roles in specific organ
homeostasis and normal function. This was further confirmed by immunostaining results
for GRP94 in some important organs such as lung, pancreas and kidney.
3.2 Materials and methods
Generation of ESCs Grp94+/+ and -/- mESCs were derived from interbred Grp94+/-
mice (backcrossed to C57BL/6 background for 3 or 4 generations). The blastocysts from
E3.5 pregnant female mice were recovered and cultured on MEFs. One Grp94-/- and
multiple +/+ and +/- ESC lines were established.
Western Blotting Grp94+/+ and -/- mESCs were homogenized in RIPA buffer with a
Dounce homogenizer, followed by centrifugation at 13,000g at 4°C for 15 min after 3
freeze-thaw cycles. The detailed information about Western blots was described
previously (Ye, Jung et al. 2010). The primary antibodies used included the following: rat
anti-GRP94 (1:2500), rabbit anti-calnexin (1:2000), and rabbit anti-calreticulin (1:2000)
from Stressgen; goat anti-GRP78(C20) (1:5000), rabbit anti-PERK (H-300) (1:500),
rabbit anti-ATF4 (CREB-2) (1:1000), goat anti-HSP70 (K-20) (1:1000), mouse anti-
PCNA (PC10) (1:1000), mouse anti-CHOP (1:1000), goat anti-EDEM (1:500) and rabbit
anti-XBP1 (1:500) from Santa Cruz Biotechnology; rabbit anti-phospho-PERK (Thr980)

56
(1:500), mouse anti-cleaved-caspase-7 (Asp198), rabbit anti-phospho-eIF2α (1:1000),
rabbit anti- eIF2α (1:1000) and rabbit anti-cleaved caspase-3 (Asp175) (1:500) from Cell
Signaling; rabbit anti-PDI (1:2000) from Assay Designs; mouse anti-β-actin (1:5000)
from Sigma-Aldrich; and rabbit anti-p62 (SQSTM1) (1:1000) from BIOMOL. The
experiments were repeated 2 to 5 times. Protein levels were visualized by Western blot
films and then quantitated using Quantity One software. Phospho-PERK was quantitated
against total PERK, and phospho-eIF2α was quantitated against total eIF2α. Both spliced
and unspliced XBP-1 were quantitated against β-actin. All the other proteins were
quantitated against β-actin.
RT-PCR Analysis of Xbp-1 mRNA Splicing The cells were either untreated or treated
with 300 nM ER stress inducer thapsigargin (Tg) for 4 hr or 16 hr. After that, total RNA
was extracted using TRI reagent (Sigma-Aldrich) following the manufacturer’s
instructions. First-strand cDNA was synthesized with SuperScript II (Invitrogen). To
detect both unspliced and spliced Xbp-1 mRNA, PCR was performed as previously (Mao,
Dong et al. 2004). The primers for PCR of Xbp-1 and β-actin were described previously
(Wang, Ye et al. 2010). These experiments were repeated two to three times.
Immunohistochemical Staining Mouse tissues were fixed overnight in 10% buffered
formalin and embedded in paraffin using standard protocols. Immunohistochemical
staining was carried out as described previously (Dong, Ni et al. 2008). In brief,
Vectastain Elite avidin-biotin complex kit (Vector Laboratories) was used for staining.
Paraffin sections were incubated with rat anti-GRP94 antibody (1:1000) (Stressgen),
mouse anti-BiP/GRP78 antibody (1:200) (BD Biosciences) or without primary antibody

57
(control staining) in blocking solution (1.5% serum in PBS) at 4°C overnight after
antigen retrieval with retrivagen A (pH 6.0) (BD Biosciences).
Microarray Analysis The raw data of relative Grp94 and Grp78 mRNA expression level
was downloaded from the Amazonia microarray database, which can be accessed freely
on the website: http://amazonia.transcriptome.eu/expression.php?geneId=Hs.192374#
(Grp94) and http://amazonia.transcriptome.eu/expression.php?geneId=Hs.716396
(Grp78). The data we presented in Figure 10 were from studies performed with
embryonic and adult mouse normal tissues (U133A) chip for HSP90B1(Grp94) and
HSPA5 (Grp78).
3.3 Results
3.3.1 GRP94 null embryonic stem cells (ESCs) upregulate specific ER
chaperones
While Grp94+/- mice were born in the expected Mendelian ratio, Grp94-/- embryos were
not viable. GRP94 null embryos die around embryonic day 7.5 (E7.5) (Mao, Wang et al.
2010), which enables us to isolate Grp94-/- ESCs from the blastocysts. Through
numerous efforts made by our collages Robin Wesselschmidt and Changhui Mao, one
Grp94-/- and multiple Grp94+/+ and +/- ESC lines were established in our lab.
By utilizing Grp94+/+ and -/- ESCs, we were able to examine the role of GRP94 in
regulating UPR. Grp94+/+ and -/- ESCs were treated with 300nM Thapsigargin (Tg) for
0, 1, 2, 4, 6 and 6 hours (hr), then whole cell lysates were collected for either RT-PCR or

58
Western blots. First of all, we confirmed by Western blot that the Grp94-/- ESCs are
devoid of GRP94 (Figure 3.1). Later on, the level of ER chaperones including GRP78,
PDI, calnexin (CNX) and calreticulin (CRT) were examined. We discovered that the
compensatory upregulation was evident for GRP78, CNX and CRT in GRP94 null ESCs
both the basal level and Tg-induced level (Figure 3.1). However, the level of PDI did not
change between Grp94+/+ and -/- ESCs (Figure 3.1).

Figure 3.1. Western blots of ER chaperones in ESCs. ER chaperones GRP78, CNX
and CRT but not PDI were upregulated in Grp94-/- ESCs. Grp94+/+ and -/- ESCs were
treated with 300 nM ER stress inducer thapsigargin (Tg) for the indicated time period
(hrs). The whole cell lysates were subjected to Western blot.
The quantitation results from multiple blots show that while the level of GRP78
significantly increased during all the time courses, CNX and CRT only increased
significantly at some time points in the GRP94 null ESCs (Figure 3.2). For PDI, there is

59
no significant change at all time points (Figure 3.2). These results implicate that specific
elimination of GRP94 may trigger a regulatory circuit to enhance the expression of
GRP78, CNX and CRT, but not PDI.

Figure 3.2. Quantitation of Western blot results on ER chaperones in ESCs.
Summary of the Western blot results representing 2 to 3 independent experiments.
Quantitation was performed using Quantity One software with the level of each
chaperone in Grp94+/+ ESCs at 0 hr set as 1. The data are presented as mean±s.e.m.
*p≤0.05, **p≤0.01 (Student’s t-test).

3.3.2 Modulation of UPR signaling pathways in GRP94 null ESCs
There are three important ER stress transducers in the cells: PERK, IRE1 and ATF6. It is
well accepted that GRP78 is the master regulator for those three signaling molecules.
Since GRP94 and GRP78 form a chaperone complex in the ER, we wonder whether

60
elimination of GRP94 will affect the UPR pathways. Grp94+/+ and -/- ESCs were
subjected to Tg-induced ER stress for different time courses and the important UPR
targets were measured. Overall, the phosphorylation of PERK does not change at most
time points, with slightly induction at 16hr (Figure 3.3). The quantitaion of p-PERK
against total PERK from several independent experiments also shows no significant
change between the two genotypes (Figure 3.5). The downstream effectors of PERK
signaling pathways including eIF2α phosphorylation, ATF4 and CHOP induction are not
affected in GRP94 null ESCs compared with wildtype (Figure 3.3 and 3.4). There were
substantial variations on Tg-induced eIF2α phosphorylation level in the Grp94-/- ESCs
however on average there was no significant change (Figure 3.4 and 3.5). The level of
cytosolic protein p62 which binds to polyubiquitinated proteins and targets them for
autophagy mediated protein degradation was not affected (Figure 3.3). The 16 hr Tg-
induced level of EDEM was reduced in Grp94-/- ESCs (Figure 3.3). Besides, the level of
HSP70, a major heat shock protein related to GRP78, was not affected either (Figure 3.4).

61

Figure 3.3. Representative Western blot results on the effect of GRP94 depletion on
UPR targets. Grp94+/+ and -/- ESCs were treated with 300 nM Tg for the indicated time
(hr). The whole cell lysates were subjected to Western blot to detect the level of PERK
phosphorylation, total PERK, CHOP, EDEM and p62.

Figure 3.4. Representative Western blot results on eIF2α phosphorylation, total
eIF2α, ATF4 and HSP70. Grp94+/+ and -/- ESCs were treated with 300 nM Tg for 0, 4
and 16 hr. These experiments were repeated two to four times.

62

Figure 3.5. Quantitation results of p-PERK and p-eIF2α from Western blots. The
protein levels of p-PERK and p-eIF2α were quantitated and normalized against PERK
and eIF2a, respectively. The data are presented as mean±s.e.m. n=2 for p-PERK and n=4
for p-eIF2α.
Due to the technical difficulty, we were unable to detect the level of IRE1
phosphorylation and cleaved ATF6. Since XBP-1 splicing is the major downstream target
of activated IRE1, the IRE1 pathway is measured through detection of spliced XBP-1 at
both mRNA and protein level. 4 hr Tg treatment induced a high level of Xbp-1 splicing in
Grp94+/+ ESCs, while the level was reduced about 30% in GRP94 null ESCs (Figure
3.6). The level of spliced Xbp-1 was similar at 16 hr Tg treatment in those two genotypes
(Figure 3.6). At the protein level, while Grp94+/+ ESCs showed robust induction of
spliced XBP-1 at 4 hr treatment and residual amount at 16 hr, Grp94-/- ESCs exhibited
much reduced amount of spliced XBP-1 at 4 hr and non-detectable amount at 16 hr
(Figure 3.7). Quantiation of the Western blot results showed 75% reduction of spliced
XBP-1 at 4 hr Tg treatment (Figure 3.7). The protein level of unspliced XBP-1 was stable
through all the Tg treatment period, with a relatively lower level in GRP94 null ESCs
compared to wildtype (Figure 3.7).

63

Figure 3.6. Effect of GRP94 depletion on Xbp-1 mRNA splicing. The RNA extracts of
Grp94+/+ and -/- ESCs treated with Tg for indicated hrs were subjected to RT-PCR. The
positions of the PCR products of both the unspliced (u) and spliced (s) Xbp-1 are
indicated by arrows. The Xbp-1(s) mRNA level was quantitated using Quantity One
software and was normalized against β-actin. The level of Xbp-1(s) in Grp94+/+ ESCs
treated with Tg for 16 hr was set as 1. These experiments were repeated three times.

Figure 3.7. Reduction of the spliced form of XBP-1 protein in GRP94 null ESCs.
Whole cell lysates of Grp94+/+ and -/- ESCs treated as indicated were subjected to
Western blot. The protein level was quantitated and normalized against β-actin. The level
of XBP-1(s) in Grp94+/+ ESCs treated with Tg for 16 hr was set as 1. These experiments
were repeated three times.
Under Tg-induced ER stress condition, Grp94+/+ and -/- ESCs showed similar level of
cell proliferation, indicated by the comparable level of proliferating cell nuclear antigen
(PCNA) (Figure 3.8). However, the level of activated caspase-7 (C-7) was substantially
higher in Grp94-/- ESCs, implying the protective role of GRP94 in ER stress induced cell
apoptosis (Figure 3.8). This result was further confirmed by cleavage of caspase-3 (C-3).

64
Consistent with C-7, the level of activated C-3 was also elevated in GRP94 null ESCs,
with the treatment of another ER stress inducer tunicamycin (Tu) (Figure 3.9).

Figure 3.8. Representative Western blot results on effect of GRP94 depletion on cell
proliferation and apoptosis. Grp94+/+ and -/- ESCs were treated with 300 nM Tg for
the indicated time (hr). The whole cell lysates were subjected to Western blot with
antibodies against PCNA, caspase-7 (C-7) and β-actin. Both pro C-7 (the uncleaved
form) and cleaved C-7 (active form) were detected by the anti-C-7 antibody. The cleaved
C-7 protein level was quantitated and normalized against β-actin. The level of cleaved C-
7 in Grp94+/+ ESCs treated with Tg for 4 hr was set as 1. These experiments were
repeated two times.

Figure 3.9. GRP94 protects ESCs from ER stress-induced cell death. The cells were
treated with 1.5μg/ml Tunicamycin (Tu) for 0, 4 and 16 hr. The Western blots were
performed with antibodies against cleaved caspase-3 (C-3) and β-actin. The cleaved C-3
protein level was quantitated and normalized against β-actin. The level of cleaved C-3 in
Grp94+/+ ESCs without Tu treatment was set as 1. These experiments were repeated two
times.

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3.3.3 Differential GRP94 expression profiles in adult mouse tissues
According to the microarray database, in most of the adult mouse tissues, the level of
Grp94 mRNA is relatively low, however, the level is about 20-fold higher in dendritic
cells, 12-fold higher in smooth muscle and lung bronchial epithelium and 10-fold higher
in pancreatic islet, with the level of Grp94 mRNA in whole blood setting as 1 (Figure
3.10). This suggests that GRP94 may play important roles in the homeostasis of specific
cell types or organs. To correlate the mRNA data with the protein level of GRP94, we
examined the level of GRP94 in mouse adult organs such as lung, pancreas and kidney
utilizing immunostaining. We discovered that GRP94 is highly expressed in the lung
bronchial epithelium, pancreatic islets of Langerhans and kidney cuboidal epithelium
with relatively low level expression in the whole lung, pancreas and kidney (Figure
3.11A). In order to show the specificity of GRP94 staining, we also performed
immunohistochemical staining with GRP78 antibody. The results showed that the level of
GRP78 was also relatively high in the lung bronchial epithelium and kidney cuboidal
epithelium, but not in the pancreatic islets of Langerhans (Figure 3.11A). The staining
results for GRP94 and GRP78 were consistent with the relative mRNA level from the
microarray database in the specific tissues being examined (Figure 3.11B)

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Figure 3.10. Relative Grp94 mRNA levels in adult mouse tissues and primary cells.
The data were from Amazonia microarray database. All the data are presented as
mean±s.e.m, with the level of Grp94 mRNA in whole blood set as 1.

67

Figure 3.11. GRP94 and GRP78 staining and mRNA expression in adult mouse
tissues. (A) Immunohistochemical staining for GRP94 and GRP78 in adult mouse lung,
pancreas and kidney tissues. The GRP78 staining and control staining (without primary
antibody) were presented to show the specificity of the GRP94 antibody. The sections
were counterstained with hematoxylin. Brown color depicts GRP94 and GRP78 staining.
White arrowheads indicate lung bronchi epithelium. Black arrowheads indicate
pancreatic islet. Black arrows indicate renal cuboidal epithelium. Scale bars represent 50
μm. (B) Relative levels of Grp94 and Grp78 mRNA in adult mouse lung, broncho
epithelium, pancreas, islet and kidney, with the level of Grp94 and Grp78 mRNA in
whole blood set as 1.

A
B

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3.4 Discussion
During the past decades, ER stress and UPR signaling pathways have been implicated to
play important roles in mammalian development and human diseases. To facilitate the
understanding about the molecular pathways that help cells maintain ER homeostasis
under various stress condition, we focus on ER chaperones which are critical not only for
quality control of proteins processed in the ER, but may also directly or indirectly
regulate ER signaling in response to ER stress and maintenance of calcium homeostasis
(Ni and Lee 2007). GRP94 is ubiquitously expressed in all vertebrates and some
invertebrates like C. elegans and Drosophila and also plants, but not in yeast, suggesting
that it is required for specific functions in vertebrates not shared by yeast. As documented
in earlier reports (Wanderling, Simen et al. 2007; Yang, Liu et al. 2007), creation of
model organisms with targeted mutation of GRP94 allows comprehensive analysis of its
in vivo function.
We discovered that deficiency of GRP94 is distinct in many different aspects from
deficiency in GRP78, which has an established role in regulating ER signaling in addition
to being a major chaperone. While complete knockout of Grp78 results in embryonic
lethality around peri-implantation (E3.5) stage (Luo, Mao et al. 2006), GRP94 null
embryos die around E7.5 and fail to develop mesoderm, primitive streak, or proamniotic
cavity (Wanderling, Simen et al. 2007). This implies that GRP78 plays more essential
roles in mouse embryonic development than GRP94, which is consistent with the limited
number of client proteins of GRP94 compared with GRP78. The inner cell mass in the
E3.5 Grp78-/- blastocysts are undergoing apoptosis, indicating GRP78 is required for the

69
survival of ESCs (Luo, Mao et al. 2006). However, Grp94-/- ESCs growth as well as
their wildtype controls, which shows that GRP94 is not essential for the survival and
proliferation of ESCs (Mao, Wang et al. 2010). Furthermore, we discovered cells were
able to adjust ER chaperone balance according to different conditions. While Grp78+/-
mouse embryonic fibroblasts (MEFs) and liver cells upregulate GRP94 and PDI, but not
CNX and CRT, the Grp94 heterozygous MEFs, embryonic brain and liver cells did not
change ER chaperone balance (Luo, Mao et al. 2006; Mao, Wang et al. 2010). In GRP78
null Purkinje cells, upregulation of GRP94 and PDI was also detected (Wang, Ye et al.
2010). However, in GRP94 null ESCs, we observed that not only is GRP78 upregulated,
but also CNX and CRT. Interestingly, PDI appears to be substantially less affected or
unaffected. Thus, it is possible that feedback mechanisms exist to compensate the
complete loss of GRP94 by increasing GRP78, a partner protein of GRP94 in the
GRP78/GRP94 chaperone system, and also of the CNX/CRT chaperone system, but
apparently not the thiol oxidoreductases such as PDI. Collectively, these results imply
that cells have abilities to adjust the ER chaperone balance in response to depletion of
key chaperones such as GRP94 and GRP78.
The GRP94 null ESCs provide us with a unique opportunity to study whether GRP94, as
a partner protein of the GRP78 chaperone complex, contributes to ER stress signaling. In
the case of GRP78 knockdown or knockout, UPR signaling downstream targets including
phosphorylation of eIF2α, induction of CHOP and GADD34, splicing of XBP-1 are
observed without the treatment of ER stress-inducers, clearly showing GRP78 suppresses
their activation under non-stress conditions (Li, Ni et al. 2008; Wang, Ye et al. 2010).

70
However, none of these was observed in GRP94 null ESCs compared with wildtype in
the absence of ER stress. Under Tg or Tu induced ER stress conditions, we discovered
that the spliced XBP-1 protein level was substantially reduced following Tg treatment in
Grp94 null ESCs compared to wild-type control. Interestingly, for both Grp94+/+ and -/-
ESCs, PERK phosphorylation persisted up to 16 hr after Tg treatment, whereas in most
other cell lines, ER stress-induced PERK phosphorylation was transient and subsided
within a few hours of ER stress. All of these data suggest that while GRP94 may act in
concert with GRP78 in folding protein intermediates, its depletion results in de-regulation
of the specific UPR targets quite distinct from those affected by GRP78 deficiency.
What is the possible underlying mechanism that GRP94 depletion suppresses ER-stress
induced XBP-1 splicing? One possible answer is that GRP94 regulates XBP-1 through
TLRs. It is well established that GRP94 is the master chaperone for TLRs in variety of
cells including macrophages, B-cells and ESCs (Yang, Liu et al. 2007; Liu and Li 2008;
Lee, Hong et al. 2009). Using mouse models of specific knockout of Grp94 in
macrophages, it was demonstrated that GRP94 is the master chaperone for TLRs and is
important in the innate function of macrophages (Yang, Liu et al. 2007). Analysis of B-
cell specific GRP94 null mice further revealed that GRP94 optimizes B-cell function
serving as chaperones for integrin and TLRs but not immunoglobulins (Liu and Li 2008).
In addition, functional TLRs are identified in the mouse ESCs and TLR ligands can
stimulate the proliferation of ESCs and promote their hematopoietic differentiation (Lee,
Hong et al. 2009). Recently, it was discovered that TLR4 and TLR2 specifically activated
the UPR sensor IRE1α and its downstream target XBP-1 in macrophages (Martinon,

71
Chen et al. 2010). Since GRP94 is required for maturation of TLRs, including TLR4 and
TLR2, it may regulate XBP-1 activation through the TLRs. Future studies will be
required to test this and other mechanisms. The differential effect of GRP94 depletion on
XBP-1 splicing but not other UPR signaling pathways also supports the notion that UPR
sensors can be dissociated and selectively regulated.
Through analysis of the amazonia microarray database, we discovered that GRP94
expression is strikingly high in specific cell types such as dendritic cells, smooth muscle
and lung bronchi epithelium among multiple adult mouse tissues and cells. The
microarray data, which is further supported by tissue staining results, predicts potential
role of GRP94 in immunity and function/survival of specific cell types in the lung,
pancreas and kidney. Future studies with conditional KO of Grp94 in these cell types will
address these important issues. Furthermore, the role of GRP94 is human diseases such as
cancer, metabolic diseases and neurodegeneration can be also achieved through creation
of mutant mouse models.

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Chapter 4: Role of GRP94 in normal mouse mammary gland
development and HER2/Neu induced tumorigenesis
4.1 Introduction
HER2, also known as ERBB2 or Neu, is a 185-kD transmembrane protein belonging to
epidermal growth factor receptor family (Guy, Webster et al. 1992). Amplification and
overexpression of human ERBB2 gene has been observed in about 20% of human breast
cancer patients and is always correlated with poor patient prognosis, resistance to therapy
and decreased survival (Fantozzi and Christofori 2006). It was well established that
overexpression of the unactivated Neu oncogene in mice results in the formation of focal
mammary tumors with high incidence of metastasis to lung (Guy, Webster et al. 1992).
Through biochemical analysis, it was suggested that HER2/Neu may be one of the client
proteins of an important ER chaperone, Glucose regulated protein 94 (GRP94) (Chavany,
Mimnaugh et al. 1996; Yang and Li 2005).
GRP94, also named as gp96, endoplasmin, ERp99, HSP108, CaBP4 or Tra-1, is the most
abundant ER resident glycoprotein belonging to the HSP90 family (Little, Ramakrishnan
et al. 1994; Argon and Simen 1999). It shares 50% amino acid homology with HSP90
(Little, Ramakrishnan et al. 1994). It is ubiquitously expressed in all vertebrates, some
invertebrates like C. elegans and Drosophila and also plants, but not in yeast (Argon and
Simen 1999; Chen, Zhong et al. 2006; Morales, Wu et al. 2009).

73
The major function of GRP94 is to fold or assembly membrane or secretary proteins.
Unlike GRP78 which folds numerous proteins, GRP94 is pretty selective and has limited
number of client proteins (Yang and Li 2005; Eletto, Dersh et al. 2010). Among them,
only insulin like growth factor II (IGF-II), Toll-like receptors (TLRs) and integrins have
been demonstrated through creation of knockout mouse models (Wanderling, Simen et al.
2007; Yang, Liu et al. 2007; Liu and Li 2008; Staron, Yang et al. 2010).
To address the question that whether GRP94 is required for the correct folding and
maturation of HER2/Neu and depletion of GRP94 can inhibit HER2/Neu induced
tumorigenesis and metastasis, a Grp94 conditional knockout mouse together with
overexpression of Neu specifically in mammary gland epithelial cells (Grp94 F/-;
MMTV-Cre; MMTV-Neu) was created (Guy, Webster et al. 1992; Wagner, Wall et al.
1997; Mao, Wang et al. 2010). Since some client proteins of GRP94 are involved in the
normal developmental process of mouse mammary gland, we first examined whether
there will be any developmental or functional defects caused by GRP94 depletion.
Through generation of Grp94 specific knockout mice in mammary epithelial cells, we
find that GRP94 is not required for the survival of mammary epithelial cells or the
normal development and function of mammary gland. Future studies are required to
clarify the function of GRP94 in HER2/Neu induced mammary tumor development and
metastasis.

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4.2 Materials and methods
Mouse models The generation and characterization of Grp94 F/F mice were described
previously (Mao and Lee, 2010, PLoS ONE). One male Grp94 F/-; MMTV-Cre mouse
was obtained from Dr. Siyi Chen’s lab. The MMTV-neu transgenic mouse was purchased
from Jackson Laboratory (Muller WJ 1992 PNAS). The Grp94 F/-; MMTV-Cre;
MMTV-Neu (Grp94 cF/-; Neu), Grp94 F/-; MMTV-Cre (Grp94 cF/-), Grp94 F/F;
MMTV-Neu (Grp94 F/F; Neu) and Grp94 F/F mice were generated through the mating
between Grp94 cF/- male mice and Grp94 F/F; Neu female mice. All protocols for
animal use and euthanasia were reviewed and approved by the University of Southern
California Institutional Animal Care and Use Committee.
Wholemount staining of mouse mammary glands Mouse mammary glands were
dissected from the back sides of female virgin mice at 1, 1.5, 3 and 8 month old. The
detailed protocol about wholemount hematoxylin staining of mammary glands was
described previously (Brantly DM 2001 Mol Biol Cell). In brief, glands were fixed in
10% neutral buffered formalin for 24hrs, followed by 48hr actone treatment. Then the
tissues were washed by 100% and 95% ethanol before the overnight staining with the
hematoxylin staining solution (1.225ml stock heamatoxylin (5g hematoxylin in 50ml
95% ethanol), 87.5ml 95% ethanol, 0.14g FeCl
3
in 37.5ml distilled H2O, 25ml 1N HCl,
250ml 95% ethanol). After that, the tissues were washed with distilled H2O, 70%, 95%
and 100% ethanol (three times), and xylene (three times) for 1 hr sequentially. Finally the
stained tissues were placed in glass vials with methyl salicylate for permanent storage.

75
Immunofluorescence staining Mouse mammary glands were dissected and fixed
overnight in 10% buffered formalin followed by embedding in paraffin using standard
protocols. The immunofluorescence staining was carried out as described previously
(Wang M CDD 2010). In brief, after antigen retrieval with retrivagen A (pH 6.0) (BD
Pharmingen), paraffin sections were incubated with primary antibodies in blocking
solution (1.5% serum in PBS) at 4°C overnight with antibodies against GRP94 (1:100),
Alexta Fluor 488-conjugated goat anti-mouse IgG (1:200) (Invitrogen) was used as the
secondary antibody.
4.3 Results
4.3.1 Generation of the Grp94 F/-; MMTV-Cre and Grp94 F/-; MMTV-Cre;
MMTV-Neu mice
We have previously shown the successful generation of Grp94 heterozygous and floxed
mice in our lab (Mao, Wang et al. 2010). Various alleles of the Grp94 gene including the
wildtype (WT) (+) allele, the floxed (F) allele and the knockout (KO) (-) allele are
presented in Figure 4.1A. In the Grp94 KO allele, exon 2 encoding the ATP binding
domain which is critical for the normal function of GRP94 is deleted.
To investigate the role of GRP94 in mammary gland development and HER2/Neu
induced tumor formation, two mouse models were created. One is the Grp94 conditional
knockout mice in mammary epithelial cells, which was generated through the mating
between Grp94 F/- mice (Mao, Wang et al. 2010) and MMTV-Cre transgenic mice

76
(Wagner, Wall et al. 1997). The Grp94 F/-; MMTV-Cre mice are abbreviated below as
Grp94 cF/- mice. The other one is the Grp94 cF/- mice with overexpression of Neu
transgene specifically in mammary epithelial cells. The Grp94 F/-; MMTV-Cre; MMTV-
Neu mice are abbreviated below as Grp94 cF/-; Neu mice. The genotypes of mice
harboring the various Grp94 alleles, the MMTV-Cre allele or the MMTV-Neu allele
were determined by PCR (Figure 4.1B). The cohorts of mice with the following
genotypes (1) Grp94 F/F, (2) Grp94 cF/- (3) Grp94 F/F; Neu and (4) Grp94 cF/-; Neu
were monitored for growth and experiments.

77

Figure 4.1. Creation of GRP94 mammary gland conditional knockout (KO) mice
with or without Neu transgene. (A) Schematic drawings of the Grp94 exon map, the
wild-type (WT) (+) allele, the loxP-FRT targeting vector, the Floxed (F) allele and the
KO (-) allele. The ATP-binding domain of GRP94, the insertion positions of neo cassette,
the loxP (closed triangle) and FRT (open triangle) sites, and the pgk-TK expression
cassette are indicated. The BamHI restriction digestion sites, the position of the 5’-probe
for Southern blotting, the expected location and size for both the Southern blot products
and the genotyping products are also indicated. (B) Representative genotyping results of
various Grp94 alleles (F, + and – alleles), the Cre and Neu transgene. The sizes of the
PCR products are indicated.
A
B

78

Figure 4.2. Immunofluorescence staining for GRP94 on the mammary glands of
Grp94 F/F, F/- and cF/- mice at 3 months old. The results show the partial reduction of
GRP94 in the F/- mice and complete depletion of GRP94 in the cF/- mice mammary
epithelial cells upon 3 months old. The green and blue colors depict GRP94 and DAPI
staining, respectively.
Both the Grp94 cF/- and the Grp94 cF/-; Neu mice were born with the expected
Mendelian ratio and survive as well as their siblings. They are phenotypically normal
compared with their Grp94 F/F or F/- littermates.
Since the MMTV promoter is upregulated by steroid and peptide hormones during estrus
and pregnancy, the Cre recombinase usually begins to express after puberty age (around 3
weeks old). The immunofluorescence staining results for GRP94 indicate that GRP94 has
been completely depleted in most of the mammary epithelial cells in the Grp94 cF/- mice
at 3 months old, compared with the Grp94 F/F mice (Figure 4.2). The expression level of

79
GRP94 in the Grp94 F/- mice is lower than the Grp94 F/F mice and higher than the
Grp94 cF/- mice, consistent with the mouse genotypes (Figure 4.2).
4.3.2 The Grp94 F/-; MMTV-Cre mice show normal morphology and
branching of the mammary gland
The development of mouse mammary gland begins around embryonic day 10 (E10) and
most of the growth happens after puberty (around 3 weeks old) (Moraes, Chang et al.
2009). The rapid elongation of the mammary ducts is driven by the growth of termical
end buds (TEBs) under the regulation of systematic hormones (Moraes, Chang et al.
2009).
To test whether GRP94 is required for the normal development, the mammary glands of
the control group (Grp94 F/F or F/-) and the Grp94 cF/- virgin mice at 1, 1.5 and 3
months old were examined. By the wholemount staining analysis, both groups of mice
exhibit the normal branching of mammary ducts at all three ages (Figure 4.3). Similar
number of TEBs is observed in both groups at 1 and 1.5 months old (Figure 4.3). This is
further confirmed by H&E staining of the mammary glands from those two groups at the
same age (Figure 4.4). The histoarchitecture of TEBs and mammary ducts is identical
between the control group and the Grp94 cF/- mice. Thus GRP94 is not required for the
survival of epithelial cells or the normal development of mammary gland.

80

Figure 4.3. Wholemount staining of mammary glands from Grp94 F/F, F/- and cF/-
mice at 1, 1.5 and 3 months old. The wholemount staining results show that the number
and structure of terminal end buds (TEBs) and branches are normal in Grp94 cF/- mice
compared with F/F or F/- mice at different developmental stages of mammary glands.
In addition, compared with the Grp94 F/F female mice, there was no breeding defect in
the Grp94 cF/- female mice. They were able to get pregnant, give birth to normal number
of pups and feed their pups properly. This implies that the normal function of mammary

81
gland is not disrupted by Grp94 knockout in mammary epithelial cells. Therefore GRP94
is not required for the normal function of mammary gland.

Figure 4.4. H&E staining of mammary glands from Grp94 F/F, F/- and cF/- mice at
1, 1.5 and 3 months old. The H&E staining results show the normal morphology of
TEBs and epithelial ducts in Grp94 cF/- mice compared with F/F or F/- mice at different
developmental stages of mammary glands.

82
4.3.3 The Grp94 F/-; MMTV-Cre; MMTV-Neu mice show less branches of
the mammary ducts than the Grp94 F/F; MMTV-Neu mice
At 3 months old, the TEBs disappear, the ductal growth enters a quiescent stage and the
mammary gland is fully developed. To investigate whether GRP94 depletion can inhibit
the HER2/Neu induced tumorigenesis, we first examined the structure of the mammary
glands from Grp94 F/F; Neu and cF/-; Neu mice using whole mount staining (Figure 4.5).

Figure 4.5. Wholemount staining of mammary glands from Grp94 F/F; Neu and cF/-
; Neu mice at 3 months old. The staining results show the excessive side-branching in
the F/F; Neu mice and less branches in cF/-; Neu mice compared with F/F; Neu mice at 3
months old. Images on the right panel are higher magnification pictures from the boxed
area of the pictures on the left panel.

83
The preliminary results show that there are excessive side branches in the Grp94 F/F;
Neu mice compared with the cF/-; Neu mice at 3 months old (Figure 4.5). H&E staining
results also confirmed the same phenotype (Figure 4.5). We observed more ductal
structures in the Grp94 F/F; Neu mice than the cF/-; Neu mice (Figure 4.6). It takes about
8 months for 50% MMTV-Neu mice in pure FVB background to develop mammary
tumor and 1 year to develop lung metastasis (Brantley-Sieders, Zhuang et al. 2008). In
the future, we will compare the incidence of tumor onset and lung metastasis between the
Grp94 F/F; Neu mice and the cF/-; Neu mice at later time points like 8 months or 1 year
old. Future studies are required to validate these preliminary results.

Figure 4.6. H&E staining of mammary glands from Grp94 F/F; Neu and cF/-; Neu
mice at 3 months old. The staining results show less ductal structures in the Grp94 cF/-;
Neu mice compared with F/F; Neu mice at 3 months old.

84
4.4 Discussion
In the Grp94 cF/- mice, no developmental or functional defects are detected in the
mammary gland compared with the Grp94 F/F or F/- mice. Therefore GRP94 is not
required for the survival of mammary epithelial cells or the normal developmental
process of the mammary gland. Our next step is to examine whether the HER2/Neu
induced tumor growth will be impaired by GRP94 depletion in the epithelial cells. The
preliminary data from the Grp94 cF/-; Neu mice show that the number of branches in the
mammary gland is less than the Grp94 F/F; Neu mice at 3 months old. Therefore GRP94
depletion specifically in the mammary epithelial cells could be able to inhibit HER2/Neu
induced tumorigenesis. According to the literature, it takes about 8 months for 50%
MMTV-Neu mice in pure FVB background to develop mammary tumor and 1 year to
develop lung metastasis (Brantley-Sieders, Zhuang et al. 2008). The genetic background
of the Grp94 F/F; Neu and cF/-; Neu mice is mixed background of C57/BL6, 129Sv and
FVB. There may be some variations in the tumor development due to the background
difference. In the future, we will compare the incidence of tumor onset and lung
metastasis between the Grp94 F/F; Neu mice and the cF/-; Neu mice at later time points
like 8 months or 1 year old. Future studies are required to draw the conclusions.
The mammary stroma is composed of multiple components including pre-adipocytes,
adipocytes, fibroblast, blood vessels, inflammatory cells (macrophages and mast cells)
and extracellular matrix (ECM) (Wiseman and Werb 2002). Although there is no
significant morphological change in mammary gland development, we do observe

85
increased level of stromal cells in the Grp94 cF/- mice according to our preliminary
results. There are two possible explanations for this phenotype at current stage. One is
that there are inflammations going on in the GRP94 depleted mammary gland, thus
attracts more inflammatory cells in the stroma. The other possibility is that the increased
stromal cells are temporary due to the female estrous cycle. In order to exclude this
possibility, we have to synchronize the mice before the dissection and histological
examination. There are two methods to synchronize the mice. One is to intraperitoneally
inject pregnant mare's serum gonadotrophin (PMSG) into mice 48 hrs before the
dissection, which is a well-established approach (Hong, Yen et al. 2010). The other one is
to expose the female mice to the bedding containing male urine prior to the start of the
experiment (Jemiolo, Harvey et al. 1986; Armaiz-Pena, Mangala et al. 2009). To make
sure the estrous cycle of the female mice are synchronized, the stages of the estrous cycle
of each mouse have to be determined by cytological evaluation of vaginal smears for at
least 3 weeks (Hong, Yen et al. 2010). Future studies are needed to validate these
preliminary results.

86
Chapter 5: Conclusions and Perspectives
ER chaperones have been implicated extensively in a variety of diseases, such as
neurodegenerative disorders, inflammation, cancer and diabetes (Ni and Lee 2007; Wang,
Wey et al. 2009). In this the sis, new insights into the role of GRP78 and GRP94 in
regulating UPR and cell survival are provided by studies utilizing multiple mouse models.
GRP78 is discovered to be essential for the survival and function of Purkinje cells (PCs)
(Wang, Ye et al. 2010). In contrast, GRP94 is dispensable for PCs. GRP78 depletion in
PCs leads to the activation of UPR, reduction of ubiquitinated proteins, disruption of ER
structure and accelerated PC degeneration (Wang, Ye et al. 2010). In the other hand, UPR
signaling pathways are not induced by GRP94 depletion in the mouse embryonic stem
cells (mESCs). Under ER stress conditions, the level of spliced XBP1 is substantially
reduced in the Grp94 -/- ESCs compared with Grp94 +/+ ESCs (Mao, Wang et al. 2010).
GRP94 is also discovered to be dispensable for the normal development and function of
mouse mammary gland.
Although the function of GRP78 and GRP94 has been studied intensively in in vitro
systems (Wu and Kaufman 2006), the in vivo functional studies are still in lacking. Here
we investigate the role of GRP78 and GRP94 in development utilizing multiple mouse
models. According to literature, GRP78 is required for the survival of mouse embryonic
stem cells (ESCs), Purkinje cells, several cancer cell lines like HeLa and PC-3 cells, but
not essential for prostate epithelial cells, pancreatic cancer cell lines and a melanoma cell
line, etc. (Luo, Mao et al. 2006; Suzuki, Lu et al. 2007; Fu, Wey et al. 2008). Thus

87
GRP78 may be only essential for the survival of cells with high basal level of GRP78
expression. Our Grp78 F/-; pc-Cre mice provide a valuble tool to study the function of
Purkinje cells in learning and memory. In Addition, the Grp78 F/F mice can be used to
breed with numerous neurons specific Cre transgenic mice to determine the function of
neuronal cells located in different region of the brain. We can also study the role of
GRP78 in neuron stem cell/progenitor cell development in both embryonic and adult
stage using inducible Cre transgenic mice.
There are multiple important ER co-chaperones working together with GRP78. Recent
studies reveal that overexpression of GRP170, another nucleotide exchange factor for
GRP78 (Weitzmann, Volkmer et al. 2006; Weitzmann, Baldes et al. 2007), can suppress
ER stress and rescue PC degeneration in woozy mice (Zhao, Rosales et al. 2010). Besides,
p58
IPK
-/- can also ameliorate both the onsite of ER stress and PC degeneration in SIL1
deficient mice (Zhao, Rosales et al. 2010). Normally, p58
IPK
works as a co-chaperone
associating with GRP78 in the ER, promoting the ATP hydrolysis process of GRP78
(Rutkowski, Kang et al. 2007). These results indicate that GRP170 and SIL1 are two
alternative nucleotide exchange factors for GRP78 with interchangeable functions and
they may work under different scenetios. Not all tissues are affected equally in either
woozy mice or MS syndrome patients, implying that relative amount of GRP170 and
SIL1 varies in different tissues. In addition, multiple DNAJ domain proteins have been
identified to function as co-chaperones to regulate the different functions of GRP78
(Otero, Lizak et al. 2010). Future investigations are needed to elucidate how those co-
chaperones function with GRP78 to fulfill different functions and how the complex ER

88
chaperone network is regulated. As for the role of GRP78 in regulating protein
ubiquitination, there are two possible explanations. The first one is that GRP78 is
required for ER protein retrotranslocation to be ubiquitinated in the cytosol. Evidence has
been shown in both yeast and mammalian cells. GRP78 facilitates the export of mutant
proteins to be degraded in the cytosol in both yeast and mammalian cells (Plemper,
Bohmler et al. 1997; Skowronek, Hendershot et al. 1998). The involvement of GRP78 in
ERAD is further clarified through the recent discovery that EDEM, ERdj5 and GRP78
form a functionally important ERAD complex (Ushioda, Hoseki et al. 2008). The second
possible explanation is that ER integrity is required for the retrotranslocation and
degradation of proteins. GRP78 knockout will destroy the integrity and normal function
of ER as was shown in the GRP78 null PCs. Similar results were observed in in vitro
cultured HEK293T cells with siRNA knockdown of GRP78 (Li, Ni et al. 2008).
We also found that GRP94 can regulate XBP-1 splicing under ER stress conditions. What
are the possible underlying mechanisms? One possible answer is that GRP94 regulates
XBP-1 through TLRs. It is well established that GRP94 is the master chaperone for TLRs
in variety of cells including macrophages, B-cells and ESCs (Yang, Liu et al. 2007; Liu
and Li 2008; Lee, Hong et al. 2009). Using mouse models of specific knockout of Grp94
in macrophages, it was demonstrated that GRP94 is the master chaperone for TLRs and is
important in the innate function of macrophages (Yang, Liu et al. 2007). Analysis of B-
cell specific GRP94 null mice further revealed that GRP94 optimizes B-cell function
serving as chaperones for integrin and TLRs but not immunoglobulins (Liu and Li 2008).
In addition, functional TLRs are identified in the mouse ESCs and TLR ligands can

89
stimulate the proliferation of ESCs and promote their hematopoietic differentiation (Lee,
Hong et al. 2009). Recently, it was discovered that TLR4 and TLR2 specifically activated
the UPR sensor IRE1α and its downstream target XBP-1 in macrophages (Martinon,
Chen et al. 2010). Since GRP94 is required for maturation of TLRs, including TLR4 and
TLR2, it may regulate XBP-1 activation through the TLRs. Future studies will be
required to test this and other mechanisms. The differential effect of GRP94 depletion on
XBP-1 splicing but not other UPR signaling pathways also supports the notion that UPR
sensors can be dissociated and selectively regulated.
GRP94 is also discovered to be dispensible for the survival of mammary epithelial cells
and the normal developmental process of the mammary gland. The next step is to
examine whether the HER2/Neu induced tumor growth and metastasis can be impaired
by GRP94 depletion in the epithelial cells. The preliminary data show that the number of
branches in the mammary gland of Grp94 cF/-; Neu mice is less than the Grp94 F/F; Neu
mice at 3 months old. Therefore GRP94 depletion specifically in the mammary epithelial
cells could be able to inhibit HER2/Neu induced tumorigenesis. In the future, we will
compare the incidence of tumor onset and lung metastasis between the Grp94 F/F; Neu
mice and the cF/-; Neu mice at later time points like 8 months or 1 year old. Future
studies are required to draw the conclusions.
During the past decades, there are hundreds of publications on the role of GRP78 and
GRP94 in the development of normal organ and diseases such as cancer,
neurodegeneration and diabetes; however, the underlying mechanisms are not well
understood. Further investigations will help to define the distinct functions of GRP78 and

90
GRP94 in many different new aspects. Like how GRP78 and GRP94 function in the ER
associated protein degradation (ERAD) (Eletto, Dersh et al. 2010; Wang, Ye et al. 2010),
how GRP78 and GRP94 regulate the cell proliferation and apoptotic signaling pathways
such as the AKT phosphorylation (Fu, Wey et al. 2008; Mao, Wang et al. 2010), how
GRP78 and GRP94 interact with other ER co-chaperones (Otero, Lizak et al. 2010) and
how their action cycle is regulated (Eletto, Dersh et al. 2010), etc. In summary, current
study on the role of ER chaperones GRP78 and GRP94 in development and human
diseases is just a start, a lot of significant and interesting findings are waiting to be
discovered in the future.

91
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Abstract (if available)

Abstract Endoplasmic reticulum (ER) is a key cellular organelle responsible for membrane and secretary protein folding and maturation. When the ER folding capacity is overwhelmed by protein loading, the unfolded protein response is activated. As the master regulator of UPR, the role of GRP78 in cell survival has been studied intensively through cell culture systems. To further understand the in vivo role of GRP78, a novel conditional knockout mouse model with GRP78 specifically depleted in Purkinje cells (PCs) is created. The GRP78 null PCs activate UPR signaling pathways including the upregulation of GRP94 and PDI, the induction of CHOP and GADD34, the feedback suppression of eIF2&alpha phosphorylation and apoptotic cell death. Cytosolic protein ubiquitination level is strikingly reduced, in contrast to many neurodegenerative mouse models where abnormal accumulation of ubiquitinated protein is prominent. Ultrastructural evaluation reveals that the ER shows prominent dilatation with focal accumulation of electron-dense material surrounded by ER membrane. Growth retardation, severe motor coordination ability defect by week 5 and cerebellar atrophy by week 13 were observed in the conditional knockout mice. Therefore GRP78 is required for neuronal cell survival and provides a novel link between GRP78 depletion and reduction of protein ubiquitination level, suggesting the important role of GRP78 in ER associated protein degradation. As another important ER chaperone, GRP94 is discovered to be dispensable for Purkinje cells, mouse embryonic stem cells (mESCs) and mammary epithelial cells. GRP94 null Purkinje cells survive and function as well as wildtype control. Under ER stress conditions, GRP94 depletion in mESCs leads to induction of specific ER chaperones like GRP78, calnexin (CNX) and calreticulin (CRT), but not PDI and reduction of XBP-1 splicing, a major downstream target of IRE1 signaling pathway. However, PERK signaling pathway is not affected in GRP94 null cells.

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Asset Metadata

Creator Wang, Miao (author)

Core Title Role of endoplasmic reticulum chaperones GRP78 and GRP94 in regulating unfolded protein response and cell survival

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Degree Conferral Date 2010-12

Publication Date 10/05/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag ER stress,GRP78,GRP94,HER2/Neu,OAI-PMH Harvest,Purkinje cell,UPR

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Dubeau, Louis (committee chair), Lee, Amy S. (committee member), Roy-Burman, Pradip (committee member)

Creator Email miaoer100@gmail.com,miaoer100@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3492

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Document Type Dissertation

Rights Wang, Miao

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