Journal of Cell Science
Limitation of individual folding resources in the ER
leads to outcomes distinct from the unfolded protein
Davide Eletto1, Avinash Maganty1,*, Daniela Eletto1,2, Devin Dersh1,3, Catherine Makarewich1,`,
Chhanda Biswas4,§, James C. Paton5, Adrienne W. Paton5, Shirin Doroudgar6, Christopher C. Glembotski6and
1Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, PA 19104, USA
2Department of Pharmaceutical Sciences, University of Salerno, Fisciano (SA), Italy
3Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine at the University of Pennsylvania, and4Department of
Surgery, The University of Pennsylvania, Philadelphia, PA 19104, USA
5Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, Australia
6San Diego State University Heart Institute and the Department of Biology, San Diego State University, San Diego, CA 92182, USA
*Present address: Weill Cornell Medical College, 1300 York Ave., New York, NY 10065, USA
`Present address: Program on Cellular and Molecular Physiology, Temple University School of Medicine, Philadelphia, PA 19140, USA
§Present address: Dept. of Pediatrics, Children’s Hospital of Philadelphia, PA 19104, USA
"Author for correspondence (email@example.com)
Accepted 6 June 2012
Journal of Cell Science 125, 4865–4875
? 2012. Published by The Company of Biologists Ltd
ER stress leads to upregulation of multiple folding and quality control components, known as the unfolded protein response (UPR).
Glucose Regulated Protein 78 (GRP78) (also known as binding immunoglobulin protein, BiP, and HSPA5) and GRP94 are often
upregulated coordinately as part of this homeostatic response. Given that endoplasmic reticulum (ER) chaperones have distinct sets of
clients, we asked how cells respond to ablation of individual chaperones. The cellular responses to silencing BiP, GRP94, HSP47,
PDIA6 and OS-9, were distinct. When BiP was silenced, a widespread UPR was observed, but when GRP94 was either inhibited or
depleted by RNA interference (RNAi), the expression of only some genes was induced, notably those encoding BiP and protein
disulfide isomerase A6 (PDIA6). Silencing of HSP47 or OS-9 did not lead to any compensatory induction of other genes. The
selective response to GRP94 depletion was distinct from a typical ER stress response, both because other UPR target genes were not
affected and because the canonical UPR signaling branches were not activated. The response to silencing of GRP94 did not preclude
further UPR induction when chemical stress was imposed. Importantly, re-expression of wild-type GRP94 in the silenced cells
prevented the upregulation of BiP and PDIA6, whereas re-expression of an ATPase-deficient GRP94 mutant did not, indicating that
cells monitor the activity state of GRP94. These findings suggest that cells are able to distinguish among folding resources and
generate distinct responses.
Folding of secreted and membrane proteins, their post-translational
modifications and their quality control are performed by
endoplasmic reticulum (ER) resident chaperones, enzymes and co-
factors. When these processes are compromised by accumulation of
misfolded substrates, a signaling mechanism initiates the stress
response known as the unfolded protein response (UPR), which
aims to restore ER homeostasis (Ron and Walter, 2007; Walter and
Ron, 2011). The UPR is initiated not only by pathological
circumstances,but also in
differentiation of secretory cells, in preparation for an increased
demand on the ER folding capacity (van Anken et al., 2003).
In metazoa, the UPR comprises three signaling branches
emanating from the transmembrane transducers inositol-requiring
enzyme 1 (IRE1), activated transcription factor 6 (ATF6) and
protein kinase RNA-activated ER kinase (PERK) (Ron and
Walter, 2007). The mode of function of these pathways has been
elucidated mostly by using chemically induced ER stress, such as
with tunicamycin, thapsigargin or dithiothreitol (DTT) (Ron and
Walter, 2007; Walter and Ron, 2011). Other mechanistic insights
have come through the expression in the ER of misfolded
proteins as models for various protein conformation diseases
(Ron, 2002). These substrates are ‘proteotoxic’ because they are
thought to occupy folding resources that in turn leads to the UPR
(Balch et al., 2008). We sought to explore a complementary
approach – limiting individual folding components of the ER by
RNAi in order to assess the consequences to the cell.
In canonicalUPR,hundredsof ERgenesareco-induced,including
many components of the protein folding machinery (Kamauchi et al.,
2005; Murray et al., 2004; Travers et al., 2000). Nonetheless, because
the ER fulfils multiple additional functions, such as calcium
homeostasis and lipid synthesis, different physiological conditions
may require distinct outcomes, characterized by the upregulation of
UPR signaling can cause differential target gene expression
depending on the nature of the stress (Thibault et al., 2011).
Two of the most inducible ER proteins are glucose-regulated
protein 94, GRP94 (gp96 or HSP90B1) and BiP (immunoglobulin
Journal of Cell Science
binding protein or GRP78), which are hallmarks of both
pathological and physiological UPR (Chang et al., 1989; Shiu
et al., 1977; Wiest et al., 1990). BiP functions as the ‘first
encounter’ chaperone of the secretory pathway and interacts with
many newly synthesized secretory proteins (Ma and Hendershot,
2004). BiP is also a negative regulator of the UPR, through its
association with IRE1, ATF6 and PERK (Ron and Walter, 2007):
its depletion induces ER stress signaling through all three UPR
transducers(Paton etal., 2006). Incontrast,less isknown aboutthe
identities of GRP94’s clients and interacting proteins, although for
the few known clients GRP94 is essential (Yang et al., 2007). At
least in some folding pathways, GRP94 acts later than BiP
(Melnick et al., 1992; Melnick et al., 1994; Muresan and Arvan,
1997). Also in contrast to BiP, GRP94 has not been found to bind
directly to the ER stress transducers.
Even though the two chaperones display no obvious genetic
redundancy with each other, Link et al. described compensatory
regulation in C. elegans: loss of either GRP94 or BiP upregulated
expression of the other and activated IRE1 (Kapulkin et al., 2005).
GRP94-deficient murine cells also expressed more BiP and other
ER proteins, but unlike C. elegans, they were less responsive to ER
stress because the level of spliced X-box binding protein 1 (XBP1)
compensatory network among at least some of the ER chaperones,
but the underlying molecular mechanisms remain unclear.
The present study was designed to ask how the ER responds to
limitation of individual folding resources such as BiP or GRP94. We
identified a novel transcriptional network that appears distinct from
the canonical UPR, by which the ER monitors the activity state of
GRP94 and responds to the perturbation of this chaperone’s function.
This response does not preclude responses to other stress conditions,
suggesting that the ER is able to distinguish among various metabolic
stresses and respond to each adaptively.
Silencing GRP94 produces a response distinct from the
To determine the consequences of depleting individual ER
chaperones we used RNAi knockdown (KD) approach and
monitored the expression of individual proteins via western blot
analysis. An example of the primary data is shown in Fig. 1A and
the data are summarized in the subsequent panels. The effects of
RNAi KDs were compared to the effects of tunicamycin (Tun) or
thapsigargin (TG) treatments as reference points, since it is well
known that when cells are treated with these agents they respond
by upregulating a large battery of ER chaperones, enzymes and
components of the quality control system. We measured the
changes in protein levelsof eight UPR target proteins, representing
a few different classes of luminal ER stress response genes. In
response to Tun, seven of the eight are upregulated (Fig. 1B) and
in response to TG, six of eight were upregulated (supplementary
material Fig. S1), as expected from coordinate UPR regulation.
BiP could generally be depleted only up to 60%, but even this
partial depletion was sufficient to upregulate a similar pattern of
Fig. 1. Silencing GRP94 produces a response distinct from a general
UPR. (A) shRNA-mediated silencing of either BiP or GRP94 leads to
upregulation of other ER components. In this representative experiment,
murine 10T1/2 cells stably expressing shRNA against GRP94, BiP or an
irrelevant shRNA (Ctrl) were lysed and analyzed by immunoblotting with
antibodies to the proteins indicated on the right. Signals were quantified using
a Li-Cor imager, because of its superior dynamic range. Levels of expression
of the indicated proteins were determined by quantification of exposures at
the linear range and normalized for protein load by comparison to the levels
of tubulin and 14-3-3 in the same sample. The density of each band in the
shCtrl lane is defined as 1.0. The relative fold-increase is indicated below
each band. Similar results were obtained in various other cells, of either
human of mouse origin, where the pattern was always consistent even if the
amplitude of the effect differed. (B) Quantification of the response of the
indicated eight ER proteins to tunicamycin treatment (2 mg/ml for 24 hours),
as a typical chemical inducer of ER stress. Shown are means6s.d. of four
independent experiments, performed and quantified as in A. (C) The protein
levels of the same eight ER components as in B were determined by
immunoblotting of lysates of cells stably expressing lentivirus shRNA against
BiP. Shown are means6s.d. of four independent experiments. (D) The level
of expression of the same protein set in cells silenced by shRNA against
GRP94. Shown are means6s.d. of .4 independent experiments. (E) The
level of expression of the same protein set in cells silenced by shRNA
targeting HSP47. Shown are means6s.d. of three independent experiments.
ND, not determined.
Journal of Cell Science 125 (20) 4866
Journal of Cell Science
target proteins as that caused by the chemical stress inducers
(Fig. 1A–C). Thus, partial silencing of BiP is sufficient to cause
UPR, as also noted by others (Ye et al., 2010). A more complete
ablation of BiP is provided by using subtilase AB (subAB), a
bacterial endopeptidase which cleaves BiP specifically at a di-
leucine motif (L416–L417), rendering it nonfunctional (Paton
et al., 2006). SubAB treatment depleted BiP acutely and
completely, causing a wide spread ER stress response (Fig. 4E
and Paton et al., 2006).
In contrast to the broad consequences of BiP silencing, when
other chaperones are depleted by RNAi the cellular response is
much more selective. For example, silencing GRP94 expression
by lentiviral infection of short hairpin RNAs (shRNAs) triggered
overexpression of BiP and of another KDEL-containing protein
with an apparent molecular mass of 50 kDa (Fig. 1D), which we
identified as PDIA6 (supplementary material Fig. S2). In
contrast, calreticulin, HSP47, OS-9 and GRP170, other ER
proteins that have been reported to be physically or functionally
related to GRP94 (Christianson et al., 2008; Meunier et al.,
2002), are marginally or not at all affected by silencing of GRP94
(Fig. 1D). These genes are not responsive to GRP94 depletion
even though they are upregulated by chemical stimulation of ER
stress (Fig. 1B) or in some cases by complete BiP ablation by
subAB (data not shown). Particularly informative is the lack
of response by GRP170 to GRP94 depletion, since GRP170
is highly induced by chemical ER stress (Fig. 1A). This
underscores the selective nature of the response and argues
against the possibility that all highly responsive genes are
induced by GRP94 depletion. Therefore, the response to the
silencing of GRP94 is different from the response to chemically
induced ER stress. It is termed ‘the GRP94-specific response’
throughout this work.
The GRP94-specific response is unique also because it is not
the common response to the depletion of any resident ER protein.
Silencing of HSP47, a collagen chaperone in fibroblasts, or of
OS-9, a component of ER-associated degradation, leads to little if
any upregulation of the proteins in our test set (Fig. 1E and data
not shown), and silencing of PDIA6 induces GRP94 and BiP only
marginally (supplementary material Fig. S1B, Fig. S2D). Thus,
the consequences of depleting individual ER chaperones are
distinct rather than common, from a broad UPR through selective
upregulation to very little response. On the basis of these data, we
conclude that cells are able to distinguish between the ablation of
particular chaperones in the ER, generating distinct outcomes.
The GRP94-specific response is also obtained by means
other than RNAi
The upregulation of BiP and PDIA6 is not a peculiarity of the
method of lentiviral delivery and is not due to the shRNA method
of silencing, since (1) infection with a lentivirus expressing a non-
targeting sequence (shRNA-Ctrl) or green fluorescence protein
(GFP) does notinducethetwogenes(Fig. 2A); (2) BiPandPDIA6
are upregulated also when the effective shRNAs are introduced by
transfection (data not shown); (3) the GRP94-specific response is
observed in multiple cell lines from different species and tissue
types(twoshowninFig. 2A);and(4) theextentofBiPinductionis
inversely proportional to the level of GRP94 expression [we used
observed that the lesser the expression of GRP94, the higher the
induction of BiP (Fig. 2B)].
Silencing of GRP94 expression in fibroblastic cell lines is
long-lived; it persists for months, and when it subsides, BiP
expression returns to the basal level of expression (data not
shown). This is in contrast to the tolerance of lymphoid or
myogenic cells, where silencing of GRP94 expression is shorter-
lived (Kropp et al., 2010; Ostrovsky et al., 2010)
To ask whether the induction is responsive to levels of GRP94
transcripts, protein or activity, we took advantage of 17-N-
allylamino-17-demethoxygeldanamycin (17AAG), which inhibits
the ATPase activity of GRP94 (and HSP90). The treatment with
17AAG induces a response comparable to the RNAi, in dose-
dependent fashion: BiP and PDIA6 were induced but GRP170
expression increased only slightly and OS-9 expression did not
change (Fig. 2C,D). Interestingly, the level of expression of
GRP94 itself increases, suggesting that cells respond to limit of
the activity, rather than of the amount of GRP94 mRNA or
consistently in a variety of cell lines, we asked if it was also
evident in vivo. To test this, we examined a conditional GRP94
knockout mouse, where GRP94 is deleted postnatally in skeletal
muscle, due to Cre recombinase driven by the skeletal muscle-
specific promoter of muscle creatine kinase (Barton et al.,
manuscript submitted). As shown in Fig. 2E, GRP94-deleted
muscle displays similar induction of BiP and PDIA6 in murine
Importantly, distinct regulation of GRP94 and BiP is also observed
in normal physiology: during aging, GRP94 expression, but not
BiP expression in skeletal muscles declines (Fig. 2F). The
selective reduction in GRP94 during aging is correlated with the
reduced expression of insulin-like growth factors in aging muscle
(Musaro ` et al., 2001), and are thought to be contributing factors to
aging-related sarcopenia (Bartke, 2009). We showed that the
activity of GRP94 is essential for production of IGF (Barton et al.,
2012; Ostrovsky et al., 2010; Wanderling et al., 2007).
Another example of divergent chaperone regulation is provided
by the androgen-responsive prostate carcinoma cell line LnCap
(supplementary material Fig. S3). When treated with the androgen
analog 5a-dehydroepiandrosterone (DHEA), there is a selective
increase in GRP94 expression, without a concomitant increase in
BiP in these cells. This increase appears to be cell-type specific,
since PC-3, another prostate cancer cell line that is androgen-
insensitive, as well as the embryonic kidney fibroblast 293T, do
not respond in this fashion. These data indicate that GRP94
expression can be increased considerably even without a general
The GRP94-specific response is transcriptional
Besides an increase in steady state levels of BiP protein in
GRP94-depleted cells, BiP mRNA levels are also elevated.
(Fig. 3A). Moreover, the induction of BiP is abolished when cells
are pre-treated with actinomycin D, to arrest transcription
(Fig. 3B). In this experiment, we used inhibition of activity
rather than RNAi, because under this condition the upregulation
is fast and the time course of the experiment is compatible with
cell viability. Yet a third line of evidence that the GRP94 specific
response is transcriptional is provided by a luciferase reporter
analysis. Using a construct where luciferase expression is driven
by regulatory regions of the BiP gene, compared to controls,
there is ,3.5-fold induction of luciferase in 293T cells in which
GRP94 was knocked down (Fig. 3C). The three ER stress
Distinct responses to chaperone ablation4867
Journal of Cell Science
elements (ERSEs) (Yoshida et al., 1998) of the BiP promoter are
essential for the response to GRP94 silencing, since luciferase
activity is abolished when all three ERSEs are mutated (Fig. 3C).
Thus, ablation of GRP94 initiates a transcription response at
the BiP promoter. While we did not check the transcription of
PDIA6 explicitly, its promoter has the same organization of three
ERSE elements as the BiP promoter and ERSE1 shows
excellent correspondence to the canonical sequence (Fig. 3D;
supplementary material Fig. S6). Not all ER stress-responsive
promoters are induced by silencing of GRP94. One example is the
promoter of PDIA1, which has only one ERSE and its sequence
diverges from the canonical sequence (supplementary material
Fig. S6). Furthermore, we tested another reporter, where luciferase
is driven by the promoter of the Herpes Simplex Virus ICP0 gene
(Burnett et al., 2012). This reporter is responsive to chemically
induced ER stress (Burnett et al., 2012) and is also responsive to
silencing of BiP (Fig. 3C). However, the ICP0-luciferase reporter
is not induced by silencing of GRP94, supporting the conclusion
Fig. 2. The GRP94-specific response occurs when GRP94 is ablated by several means in vitro and in vivo. (A) The GRP94-specific RNAi response is
observed in several cell lines. Murine myoblasts (C2C12) and embryonic fibroblasts (10T1/2) were infected with a lentivirus encoding shRNA targeting GRP94 or
with a control, non-targeting, shRNA (Ctrl) or a GFP-expressing lentivirus (GFP). Lysates were analyzed by immunoblotting with anti-KDEL antibody to detect
GRP94, BiP and PDIA6 simultaneously; a-tubulin served as the loading control. (B) BiP induction is proportional to the degree of silencing of GRP94. GRP94
expression in C2C12 was knocked down to different extents with the indicated shRNAs. Levels of expression of GRP94 (white) and BiP (gray) were determined
by densitometry of immunoblots. The means6s.d. of three independent experiments are shown. The numbers indicate the relative expression of BiP or GRP94
with each of the indicated shRNAs. (C) Pharmacological inhibition of GRP94 also induces BiP and PDIA6 expression. 10T1/2 cells were treated with 17AAG at
the indicated concentrations for 24 hours [D, 0.1% (v/v) DMSO]. Lysates were analyzed by immunoblotting. Data are from a representative experiment out of
three. (D) Pharmacological inhibition of GRP94 does not induce upregulation of calreticulin (CRT) or either OS-9 isoform, and only marginally induces GRP170
expression (in contrast to tunicamycin treatment, see Fig. 1B). 10T1/2 cells were treated with 17AAG at the indicated concentrations for 24 hours. Lysates were
analyzed as in C. Note that unlike the shRNA treatment, 17AAG causes destabilization of AKT, indicating that the compound affects cytosolic HSP90 as well as
GRP94. The numbers below the bands are the relative protein levels, determined by densitometry and normalized to the 14-3-3 loading control. (E) BiP and
PDIA6, but not calreticulin (CRT) are induced in GRP94 knocked-out muscle. Gastrocnemius muscles were obtained from transgenic floxed-GRP94 mice
expressing MCK-Cre recombinase (Mut) or from littermate controls (WT). 40 mg of muscle protein extracts were analyzed by immunoblotting as in A. 14-3-3
served as loading control. Note that the comparisons are normalized within each pair with the value of each protein in the WT mouse defined as 1.0. (F) Selective
decline in GRP94 expression occurs during aging. WT (C57Bl/6) mice were killed and dissected at the indicated ages. Protein extracts from dissected tibialis
anterior muscles were subjected to immunoblotting with anti-KDEL antibody to determine simultaneously the levels of GRP94 (black circles) and BiP (white
squares). Means of relative expression levels (6s.d.) of 2–4 animals are shown, except the 25-month-timepoint, which is based on only one mouse. *timepoints
when GRP94 expression values are significantly different from those of BiP in the same samples; P,0.002; two-tailed Student’s t-test.
Journal of Cell Science 125 (20)4868
Journal of Cell Science
that depletion of the two major ER chaperones is not equivalent
and the GRP94-specific transcriptional response is mechanistically
distinct from UPR.
The response to GRP94 ablation is not mediated by UPR
Transcriptional responses of ER quality control genes are usually
initiated by the UPR signaling pathways, through activation of
IRE1, PERK or ATF6. Therefore, we investigated whether
depletion of GRP94 triggers these three transducers, using two
approaches: (1) assaying the response to GRP94 deletion in cells
where thesignalpathways aregeneticallyablated (loss-of-function
approach) and (2) measuring the activity of each transducer in
The GRP94-specific response does not require IRE1, because
it occurs in IRE1-deficient mouse embryonic fibroblasts (MEF)
just as it does in wild-type MEFs (Fig. 4A). Similarly, the
GRP94-specific response is normal in PERK-deficient and in
ATF6a-deficient MEF (Fig. 4B,C). Therefore, none of the three
transducers is needed individually for the response.
To test the possibility that in the absence of one transducer,
signaling could occur via another UPR branch, we tested whether
Fig. 3. The GRP94-specific response is
transcriptional. (A) Total RNA derived from 293T
cells stably expressing shRNAs against GRP94, BiP
or an irrelevant shRNA (Ctrl) was subjected to RT-
PCR. Amplicons of BiP and b-actin were then
resolved on agarose gels. The gel shown is
representative of two independent experiments.
(B) 10T1/2 cells were treated with either 5 mM
17AAG, 0.5 mg/ml actinomycin D (ActD) or both.
After 24 hours, cells were lysed and the indicated
proteins resolved and quantified by immunoblotting.
(C) 293T cells in which an irrelevant gene (white
bars, shCtrl), GRP94 (black bars), or BiP (gray bars)
were silenced, were transiently transfected with
reporter plasmids carrying luciferase driven by: the
minimal promoter of BiP; a mutant version of the BiP
promoter where the ERSEs were disrupted; or the
minimal promoter of HSV ICP0, a UPR-responsive
viral gene. All cells were also co-transfected with a
plasmid carrying Renilla luciferase. Luciferase
activity was assayed 24 hours post-transfection and
relative luciferase activity was calculated as a
percentage after normalization against Renilla values.
Results are mean6s.e.m.; n56 transfections for the
wild-type and mutant BiP promoters; n52 for the
ICP0 promoter. Left: schematic representation of
intact or mutated promoter-luciferase constructs.
Numbers indicate the nucleotide position from the
transcription start site. (D) Schematic representation
of the promoters of select murine UPR targets, as well
as HSV ICP0 genes. The ERSEs in each promoter,
are as defined previously (Yoshida et al., 1998), and
are shown as by shaded boxes. The solid gray boxes
are identical in sequence, as shown in supplementary
material Fig. S6. The numbers below the ERSEs
define the bp position relative to the transcription
start site (TSS).
Distinct responses to chaperone ablation4869
Journal of Cell Science
the main substrates mediating the signaling are activated. No
splicing of XBP1 was observed at one, two or three days after
infection with shRNA-GRP94-encoding virus, during the time
frame needed to silence GRP94, and also not after 27 days, long
after silencing was established (Fig. 4D). In contrast, tunicamycin
and DTT treatments induce XBP1 splicing within a few hours, as
expected, serving as positive controls for the assay. Since RNAi
silencing requires days, we asked if there was an earlier XBP1
splicing event when GRP94 was inhibited by 17AAG, under
conditions that induce BiP upregulation (Fig. 3). As shown in
Fig. 4E, there was no detectable XBP1 splicing within 4 hours of
17AAG treatment. In contrast, efficient XBP1 splicing was observed
already at the earliest time point of BiP ablation by subAB (Fig. 4E),
was not because of low sensitivity of detection; as shown in Fig. 4F,
splicing is easily detectable in our hands even at a dose of 100 ng/ml
tunicamycin, 10-206lower than the dose commonly used to induce
UPR. Finally, the GRP94-specific response was intact in XBP1-
deficient MEF (supplementary material Fig. S4A), showing that it is
independent of either IRE1 or XBP1.
A second well-studied branch of the UPR machinery, ATF6, is
activated by relocation to the Golgi complex, where proteolytic
cleavage frees the N-terminal domain to become a transcription
factor (Haze et al., 1999). We tested the activation of ATF6 by
using 293T cells stably expressing an HA-tagged version of the
protein (Wang et al., 2000). As shown in Fig. 4G, in GRP94-
silenced cells, the basal level of the p50 active fragment of ATF6
is equal to that seen in control cells and there is no detectable
accumulation of active ATF6. Note that the p50 fragment is
unstable and is best detected when proteasome degradation is
Fig. 4. The response to GRP94 ablation is not mediated by UPR signaling. (A–C) The GRP94-specific response is observed in MEFs deficient for each of the
three UPR transducers. (A) GRP94 and BiP were silenced individually, using lentiviral infections, in IRE1-deficient MEFs. Cell lysates were analyzed by western
blotting. Relative band intensities, normalized to the loading control (14-3-3) are indicated. (B) Response of PERK-deficient MEFs to lentiviral-mediated
silencing of GRP94. (C) Response of ATF6a-deficient MEFs to lentiviral-mediated silencing of GRP94. (D) XBP1 splicing is not triggered in GRP94-depleted
cells. Total RNA was extracted from 293T cells that were either untransduced (U), transduced with a lentivirus with shRNA to GRP94 (GRP94) or with an
irrelevant shRNA (Ctrl). Cells were harvested at the indicated times after viral transduction and XBP1 was amplified by RT-PCR. The unspliced (u, 473 bp) and
spliced (s, 447 bp) forms of XBP1 differ by 26 nucleotides. Only the unspliced form is seen in all samples, whereas the positive controls for cells mounting active
UPR show either efficient splicing (DTT treatment, 1 mM for 6 hours) or partial splicing (Tun, 10 mg/ml for 8 hours). (E) Inhibition of GRP94 does not trigger
XBP1 splicing, whereas ablation of BiP does. XBP1 splicing in 10T1/2 cells that were treated with 100 ng/ml subAB, a toxin that cleaves BiP selectively and
induces global UPR, or exposed to 10 mM 17AAG overnight. Cells were harvested at the indicated time points. RNA extracts were assayed as in panel D, with b-
actin amplification serving as a loading control. (F) Sensitivity of detection of XBP1 splicing. 293T cells were subjected to the indicated doses of tunicamycin
overnight and splicing was detected as shown in Fig. 4D,E. The percentage splicing is the ratio of the spliced band over the total amplicon [s/(u + s)] in each gel
lane. Even though there is a higher level of basal activity in our 3T3 cells compared with that of the 293T cells or MEFs, splicing in response to as little as 0.1 mg/
ml tunicamcyin was already detectable. Doses up to 2.0 mg/ml were used in the other figures. (G) ATF6 endo-proteolysis is induced in BiP-, but not in GRP94-
depleted cells. Nuclear extracts of 293T cells stably co-expressing HA-tagged ATF6 with shRNA against BiP, GRP94 or Ctrl genes were analyzed by
immunoblotting. The nuclear ATF6 fragment was detected with anti-HA antibody. Consistent with previous results (Haze et al., 1999), the nuclear ATF6 fragment
was detected when cells were treated with tunicamycin (Tun; 2 mg/ml for 3 hours) to induce ER stress. The same fragment is induced in BiP-depleted cells,
whereas the pattern of GRP94-depleted is similar to the control cells. Enhanced levels of the ATF6 fragment were detected when cells were treated with MG132
(10 mM for 3 hours) to prevent degradation. A longer exposure is presented in supplementary material Fig. S4C. (H) Depletion of BiP, but not of GRP94, triggers
PERK activity. BiP, GRP94 or an irrelevant gene (Ctrl) were silenced with the appropriate shRNA-encoding lentivirus and then assayed by western blotting with
the indicated antibodies. The anti-phospho-eIF2a antibody measures the phosphorylation site most indicative of PERK activation. The asterisk indicates a non-
specific band. The data shown in all panels of this figure are representative of at least three independent experiments.
Journal of Cell Science 125 (20) 4870
Journal of Cell Science
inhibited by MG-132 (Fig. 4G). In contrast, both tunicamycin
treatment and BiP depletion are associated with accumulation of
The main substrate of the third UPR sensor, PERK, is the
translation initiation factor 2 alpha (eIF2a) (Harding et al., 2000).
that in turn mediates the expression of ER proteins. Therefore, we
assessed the phosphorylation of eIF2a in GRP94-silenced cells
(Fig. 4H). Phospho-eIF2a levels do not appear significantly
different in GRP94-depleted cells; in contrast, phosphorylation of
eIF2aisinducedbydepletion ofBiP(Fig. 4HandseealsoWolfson
et al., 2008). Further evidence against the involvement of PERK is
that the GRP94-specific response occurs in ATF4-deficient MEF
(supplementary material Fig. S4B).
On the basis of these combined data, we conclude that the
GRP94-specific response does not cause activation of either IRE1,
PERK or ATF6, while depletion of BiP by the same methods does,
supporting that the GRP94-specific response is not a canonical
UPR, but rather is mediated by a different mechanism.
The responsiveness to ER stress is not diminished by
ablation of GRP94
If the GRP94-specific response involves upregulation of two
proteins but not the activation of UPR sensors, is the UPR still
functional under these conditions? To address this question we
compared the responses to tunicamycin or thapsigargin of cells
that are either GRP94-deficient or -sufficient.
In terms of viability, GRP94-deficient cells survived in
increasing concentrations of Tun or TG less well than GRP94-
sufficient cells, but the differences were not dramatic (Fig. 5A,B)
when compared to the effect of BiP-deficient cells (see figure 5 in
Morinaga et al., 2008) or to the GRP94-null ES cells (see figure 6
in Biswas et al., 2007). These observations indicate that despite the
ubiquitous upregulation of GRP94 under ER stress conditions, this
chaperone contributes to, but is not essential for coping with
ER stress. A different conclusion was reached by Biswas and
colleagues, and Ostrovsky and co-workers (Biswas et al., 2007;
Ostrovsky et al., 2009), showing that GRP94 knockout (KO) ES
cells are hypersensitive to stress, suggesting a pro-survival role for
GRP94. The difference is likely due to the upregulation of BiP and
PDIA6 seen in the KD cells but not in grp942/2ES cells, arguing
that the upregulation of the two compensates for some function of
GRP94. Long-term loss of GRP94 function leads to adaptation at a
cost of increased sensitivity to ER stress.
As a second readout for UPR responsiveness we measured the
extent of BiP induction by Tun or TG stress. As shown in Fig. 5C,
GRP94-silenced cells can overexpress BiP when challenged with
Tun or TG beyond the level of the GRP94-specific response,
demonstrating that the stable induction of BiP when GRP94 is
silenced is not maximal, and further increase of BiP is possible.
GRP94-silenced cells responded by increasing the level of BiP in a
dose-dependent fashion, similarly to the control cells, but BiP
induction appeared saturated at lower concentration of Tun in
Fig. 5. The responsiveness to ER stress is not diminished by ablation of
GRP94. (A,B) HeLa cells stably expressing shRNA against GRP94
(shGRP94, black squares) or shRNA-Ctrl (white squares) were exposed to
various doses of thapsigargin (TG) (A) or tunicamycin (Tun) (B). After
48 hours, cell viability was assayed by XTT and plotted as percentage relative
to DMSO control. Results are the average 6 s.d. of three (TG) and two (Tun)
independent experiments performed in quadruplicate. (C) 293T cells stably
expressing shRNA-CTRL or against GRP94 were treated with 10 mM TG,
10 mg/ml Tun or vehicle control (DMSO) for 18 hours. The protein levels
were quantified by densitometry of immunoblots probed with anti-KDEL
antibody to detect BiP and GRP94. The levels of expression of GRP94 (white)
and BiP (black) were normalized against the DMSO levels and plotted in the
bar graph. Values are means+s.d. of three experiments. (D) BiP response to
titration of Tun doses. GRP94-silenced or control 293T cells were treated
with Tun at the indicated concentrations for 18 hours. Cell lysates were
analyzed as in C. The response values were renormalized to the response seen
in each of cells without Tun, to separate the response to Tun from the inherent
upregulation of BiP upon GRP94 KD. Note that BiP induction at the lower
drug concentration is similar in the two cell lines, whereas they differ at
higher concentrations of Tun. White bars, fold changes of GRP94 expression
versus no Tun. Black bars, fold changes of BiP expression versus no Tun.
Values are means+s.d. of three experiments. (E) CHO-tet cells were induced
with 50 ng/ml doxycycline to express a Flag-tagged wild-type GRP94 cDNA.
After 2 days, doxycycline or untreated cells were exposed to 10 ng/ml subAB
to trigger ER stress. At the indicated time points after subAB addition, protein
extracts were collected and analyzed as in A. White, level of GPR94; black,
BiP; gray, FLAG–GRP94. Dox, doxycycline. The means6s.d. of four
independent experiments are shown.
Distinct responses to chaperone ablation4871
Journal of Cell Science
comparison to the control cells (Fig. 5D). Interestingly, GRP94-
silenced cells responded with similar amplitude to the control cells
at the lowest concentration of Tun, indicating that the trigger is not
compromised by the lack of GRP94. A third readout for UPR
responsiveness was the XBP1-Venus reporter, where YFP
fluorescence is induced by splicing of the upstream XBP1
sequence (Iwawaki and Akai, 2006). While GRP94-silenced
cells displayed only background reporter levels, when treated
with Tun, the reporter in these cells was activated (Eletto and
Argon, data not shown). Together, these readouts show that the
GRP94-specific response does not obviate the ability of cells to
mount the UPR transcriptional response.
If depletion of GRP94 does not impair the ER stress response
and since GRP94 is normally induced under such stress conditions,
we also testedthe effect of overexpressing GRP94 on the ER stress
response. Taking advantage of doxycycline-inducible CHO cells,
we expressed a controlled amount of Flag-tagged GRP94 in cells
that were simultaneously treated with subAB (the time course of
BiP ablation by this method is compatible with the time course of
doxycycline induction). Depleting cells of BiP resulted in 3–4-fold
induction of the endogenous GRP94, even in cells that already
overexpressed Flag-tagged GRP94 (Fig. 5E). This indicates that
the ER has a selective mechanism of sensing levels of BiP that
fails to monitor the abundance of GRP94. Surprisingly, the
overexpressionof GRP94isnotcounter-balanced bya reductionin
the level of BiP, as mirrored by the loss-of-function experiment.
Altogether, these results suggest that: (a) the relative abundance of
GRP94 is monitored only below a minimal threshold; (b) cells
employ a distinct mechanism to sense the relative abundance of
GRP94; (c) although GPR94 is one of the major UPR targets, it is
not required for mounting the response to chemical stresses.
Cells monitor the activity, not the protein level of GRP94 in
Since RNAi was not the only treatment that induced BiP and
PDIA6 and chemical inhibition led to the same response, we asked
if cells sense the expression of GRP94 or its activity. To this end,
we determined whether WT GRP94 or an ATPase-deficient
(E82A) mutant can substitute for the endogenous protein when the
latter is silenced in doxycycline-inducible CHO cells. The
exogenous GRP94 genes were constructed to be shRNA-resistant
(supplementary material Fig. S5). When an active version of
GRP94 (Flag-taggedWT GRP94),
endogenous GRP94, the induction of BiP and PDIA6 was
abolished. On the other hand, expression of E82A GRP94 failed
to prevent the induction of the two genes (Fig. 6). Together with
the effect of geldanamycin, these results strongly suggest that the
mechanism that monitors the amount of GRP94 in the ER is
dependent on the activity of the chaperone. Since GRP94 activity
is much more client-selective than BiP activity, loss of this
chaperone may necessitate a more restricted and mechanistically
distinct response than UPR.
replaced the silenced
The present work shows that limitation of folding resources in the
ER has distinct outcomes, depending on which chaperone or
enzyme activity is limited. When GRP94 is either inhibited or
depleted by RNAi, a selective transcriptional response and de
novo synthesis are initiated, resulting in upregulation of BiP and
PDIA6. This response is different from the UPR that is induced
by loss of BiP and is characterized by several functional and
mechanistic features. Silencing of PDIA6, HSP74 or OS-9 does
not cause compensatory protein upregulation, while depleting
BiP causes a more global UPR (Wolfson et al., 2008; Ye et al.,
2010 and this work). Such distinct ER stress responses illustrate
the adaptability of the ER to various types of stress.
The response to GRP94 ablation is much more selective than
standard UPR and does not include many UPR target genes, even
those that are known to interact with GRP94 (such as OS-9,
Christianson et al., 2008). Among all the genes we tested, only
BiP and PDIA6 are very responsive to ablation of GRP94. This
contrasts with the typical ER stress response, which is
characterized by transcriptional activation of hundreds of ER
proteins (Kamauchi et al., 2005; Murray et al., 2004). Consistent
with this selectivity, not every UPR target promoter is activated
by silencing of GRP94, even if it contains ER stress responsive
elements. The second distinguishing feature of the transcriptional
response to silencing of GRP94 is that it does not rely on either
Fig. 6. Cells monitor the level of active GRP94 in the
ER. CHO-tet cells were induced with 50 ng/ml
doxycycline to express Flag-tagged wild-type (WT)
(A,B) or E82A (C,D) GRP94 cDNA, which is resistant to
shRNA. Cells were then infected with shRNA against
GRP94 or CTRL lentivirus and selected in presence of
2 mg/ml puromycin, to silence the endogenous GRP94.
After 4 days, cells were harvested and subjected to
immunoblotting to determine the level of expression of
endogenous GRP94, Flag–GRP94, BiP and PDIA6. A and
C show the level of expression of endogenous GRP94
(endo. 94, white bars) and of exogenous Flag–GRP94 (exo.
94, gray shaded bars) with and without doxycyclin (DOX).
B and D show the level of BiP (striped bars) and of PDIA6
(spotted bars) before and after replacement of GRP94
expression. The means6s.d. of four independent
experiments are shown. *P#0.05; **P#0.01.
Journal of Cell Science 125 (20)4872
Journal of Cell Science
the IRE1, PERK or ATF6 pathways of ER stress signaling. At no
time after inhibition or depletion of GRP94 was there activation
of the proximal mediators of each of the UPR pathways – spliced
XBP-1, phospho-eIF2a or cleaved ATF6. Furthermore, the
transcriptional response to loss of GRP94 still occurs in MEFs
deficient for IRE1, XBP-1, PERK, ATF4 or ATF6a.
The use of two luciferase reporter constructs show that the
GRP94-specific transcriptional response is mediated by some
promoter elements, and not only by the ERSE cis elements that
are normally activated by ER stress signaling. We notice that
there are genes that are strong responders to canonical ER stress
(e.g. GRP170), but are not responsive to GRP94 depletion in
spite of the presence of one or more ERSEs. Furthermore, the
number of predicted ERSE does not correlate with the amplitude
of either the GRP94-specific response or the general UPR.
We speculate that there is a different transcription factor,
perhaps analogous to OASIS (Kondo et al., 2005), which binds to
the promoters and mediates the activation of BiP and PDIA6.
While the functional ERSEs in BiP and GRP94 promoters have
been extensively studied (Yoshida et al., 1998), little is known
about the regulation of PDIA6. It is regulated during ER stress by
XBP1 (Lee et al., 2003; and our data) and we have found that the
PDIA6 promoter has a perfectly homologous ERSE. The same
motif is either poorly conserved or not present at all in other
PDIAs, explaining why they are not induced and PDIA6 is highly
responsive to ER stress.
Despite GRP94 being one of the main hallmarks of the ER
stress response, its loss or inhibition do not impair the cells’
ability to mount a stress response and cope with ER stress.
GRP94-depleted cells, which have increased levels of BiP, can
still further upregulate BiP when ‘insulted’ with chemical stress
inducers. In contrast, a GRP94-null clone that does not show BiP
induction, is dramatically more susceptible to ER stress (Biswas
et al., 2007). We propose that ER stress responsiveness is not
impaired without GRP94 as long as there is sufficient activity of
BiP and PDIA6. This explanation fits with the cytoprotective
effect of BiP overexpression (Dorner et al., 1992). Also
consistent with this explanation is the inability of cells to
survive simultaneous ablation of GRP94 and BiP (D.E.,
unpublished data). Furthermore, KD of GRP94 in myeloma
lines, which are specialized secretory cells, is barely achievable
and only lasts for few days (Kropp et al., 2010). In contrast,
GRP94 gene remained silenced for indefinite period of time in
non-professionally secretory cells, as reported here. Thus, GRP94
may have a central role in general UPR only when there is high
demand on folding resources, when even mild accumulation of
folding intermediates may be harmful.
It is unclear why BiP and PDIA6 in particular are overexpressed
when GRP94 activity is lost. There is no obvious functional reason
why upregulation of either one would compensate for the loss of
another component. BiP and GRP94 do work in concert on the
folding of some client proteins, such as immunoglobulins or
thyroglobulin, but they are not redundant and work sequentially,
apparently recognizing distinct folding intermediates (Melnick
et al., 1994). We hypothesize that when GRP94 is ablated, BiP,
which recognizes the less advanced folding intermediates, must be
induced to deal with accumulation of GRP94 clients. PDIA6 is a
BiP partner that binds to BiP clients (Jessop et al., 2009), and its
level is co-regulated with BiP under chemically induced ER stress
and in response to the accumulation of misfolded proteins (this
work and Hartley et al., 2010). GRP94 and BiP may also be
affectedby each other due toother shared commonfunctions. Both
are calcium-binding proteins (Biswas et al., 2007; Lie `vremont
et al., 1997) and both are involved in the degradation of soluble
substrates (Christianson et al., 2008; Kabani et al., 2002). In
conclusion, the expression network formed by BiP, GRP94 and
PDIA6canberationalized byseveralknownfunctional parameters
that can be tested in the future.
Cells must have developed mechanisms to monitor the
abundance of chaperones in order to keep their levels within a
certain range that is compatible with their cooperative mode of
upregulated, due to the cooperation of these two chaperones in the
folding pathways of many kinases and transcription factors
(Bagatell et al., 2000; Zou et al., 1998). Such compensatory
mechanisms are likely based on sensing the loss of chaperone
activity. We provide evidence that the presence of GRP94 is also
detected based on its activity and not simply the abundance of the
protein. First, the transcriptional response of BiP and PDIA6 is
activated not only when GRP94 is depleted, but also when it is
inhibited pharmacologically. Second, the upregulation of BiP and
PDIA6 occurs even if an inactive GRP94 mutant is overexpressed,
but is prevented when active GRP94 is supplied. Interestingly, the
detection mechanism works only when GRP94 activity is reduced;
overexpression of GRP94 does not perturb the normal functioning
of the ER stress response machinery.
These results suggest the following model: when GRP94
activity is insufficient, either because of a high demand of
obligate clients that accumulate in the ER or because of another
need, a dedicated sensor is activated, which reports on the limiting
GRP94 through a signaling pathway, culminating in activation of
transcription of BiP, PDIA6, and perhaps other genes.
Our results show that ER proteins are not necessarily
upregulated as a cohort. Rather, multiple signal transduction
pathways may be evocated in response to distinct stimuli or
stresses. The plasticity of ER dynamics may be due to its ability to
initiate such distinct stress responses, leading to distinct outcomes
and different kinetics.
Materials and Methods
All experiments were approved by the CHOP and the University of Pennsylvania
animal care committees.
Chemicals and plasmids
Actinomycin D, doxycycline, tunicamycin and thapsigargin were purchased from
Sigma Chemicals (St Louis, MO). MG-132 was from Calbiochem (San Diego, CA).
Lipofectamine 2000 transfection reagent was from Invitrogen (Carlsbad, CA). 17-
Allylamino-17-demethoxygeldanamycin (17AAG), puromycin and G418 were from
InvivoGen (San Diego, CA); the XTT cell proliferation kit from Biotium, Inc.
(Hayward, CA). DMEM was from Mediatech, Inc. (Manassas, VA), fetal bovine
serum was from Gemini (West Sacramento, CA). Glutamine, penicillin/streptomycin
supplement was from Gibco-Invitrogen (Grand Island, NY). Subtilase AB toxin was a
kind gift of J. C. Paton (Univ. of Adelaide, Australia). A Flag-tagged GRP94
expressed into the pTRE Vector (Clontech, Mountain View, CA) was subjected to
site-directed mutagenesis using the QuickChange Kit (Agilent, Santa Clara, CA) to
generate the E82A GRP94 ATPase deficient mutant.
Cell culture and GRP94 conditional KO mice
C2C12, 10T1/2,NIH-3T3, 293T, andHeLa cells were fromthe ATCC. CHO-K1 Tet-
On were from Clontech. 293T cells stably expressing a HA-tagged ATF6 were a kind
gift of Drs H. Steiner and Haass (Ludwig Maximilians University, Germany). These
cell lines weregrown in DMEM in the presence of10% FBS andGln/Pen/Strept, and,
when needed, the proper eukaryotic selection agent (puromycin or G418).
IRE1 and XBP1 KO, PERK and ATF4 KO MEFs were generous gifts from Dr D
Ron (Univ. of Cambridge, UK). ATF6alpha KO MEFs from Dr K. Mori (Univ. of
Kyoto, Japan), Mice containing a floxed allele of GRP94 (grp94flox) (Yang et al.,
2007) were crossed with muscle creatine kinase (MCK)-Cre transgenic mice (on a
Distinct responses to chaperone ablation4873
Journal of Cell Science
C57Bl/6 background; Jackson Labs, stock 006475). Double heterozygous progeny
were then bred to grp94flox/floxmice, in order to deplete GRP94 within skeletal and
cardiac muscle. Tissue samples were collected and processed for immunoblotting as
reported in Barton et al. (Barton et al., 2010).
Cells were lysed with a 0.5% NP-40/Igepal detergent solution as described in
Ostrovsky et al. (Ostrovsky et al., 2009). Images were recorded using an Alpha
Innotech (Santa Clara, CA) or Odyssey (Li-Cor, Lincoln, NE) imagers. Band
intensities were normalized for total protein loads using house-keeping proteins
(a–tubulin or 14-3-3). Fold changes were calculated relative to internal references
(wt or control samples), as indicated in the figure legends.
Antibodies: rabbit anti-14-3-3 (C16) and mAb anti-myogenin (F5D) were
purchased from Santa Cruz, Biotechnology, Santa Cruz, CA. mAb anti-
desmin(D33) was from Imgenex (San Diego CA); mAb anti-KDEL was from
StressGen (Vancouver, BC); anti-HSP90 was from BD Transduction Laboratories
(San Jose, CA) and anti-caspase 3 and anti-cleaved caspase 3 (Asp175) were from
Cell Signaling; the anti-GRP94 monoclonal antibody 9G10 (SPA-850) from
Stressgen Biotechnologies (Victoria, BC). The monoclonal anti-HA antibody
HA.11 (clone 16B12) was obtained from Covance (Princeton, NJ). Secondary
antibodies conjugated to HRP were from Jackson ImmunoResearch Laboratories
(West Grove, PA), secondary antibodies conjugated to near-infrared fluorophores
were from Li-Cor.
GRP94, BiP, PDIA6, HSP47 or OS-9 were knocked-down, using the following
shRNAs from Sigma Life Science (S Louis, MO): SHCLNG-NM_011631 (a set of
five vectorsreferredto in this paper as shRNA23 to 27;
TRCN0000008455, SHCLNG-NM_027959 (a set of five vectors, supplementary
material Fig. S2D), TRCN0000008534, TRCN0000175937, respectively. Briefly,
cells were transduced with lentiviral particles encoding the shRNA sequences, as
in Ostrovsky et al. (Ostrovsky et al., 2010). The efficiency of knockdown was
consistently .90% for GRP94, PDIA6, HSP47 and OS-9. BiP expression typically
could only be reduced to 40% of control level.
Analysis of XBP1 mRNA splicing and ATF6 endoproteolysis
XBP1 and b-actin were PCR amplified from total RNA as in Calfon et al. (Calfon
et al., 2002). 293T cells stably expressing HA-tagged ATF6 were analyzed by
immunoblotting with anti HA-antibody HA.11 after the cells were stressed with Tun
and treated with MG132, to block degradation of the ATF6 fragment. The levels of
the ATF6 fragment nuclear fractions were enriched as describedin Li et al. (Li et al.,
Luciferase reporter assay
Constructs composed of nucleotides 2284 to +221 of the human GRP78 promoter
driving luciferase were described in Doroudgar et al. (Doroudgar et al., 2009). The
construct composed of 1024 nucleotides upstream of the coding sequence of the
herpes simplex virus ICP0 gene fused to luciferase was from Dr Liu (Burnett et al.,
2012). 293T stably expressing shRNA targeting either GRP94, BiP or a non-relevant
sequence were transfected andanalyzed forluciferase activity as in (Doroudgar et al.,
Statistical analysis was performed using a one-way analysis of variance followed
by Student’s Newman-Keul’s post hoc analysis of variance (*, P,0.05, unless
otherwise stated in the figure legends).
We are grateful to K. Mori (University of Kyoto, Japan), D. Ron
(Cambridge University, UK), J. C. Paton (University of Adelaide), H.
Steiner and C. Haass (Ludwig-Maximilians-University, Germany), R.
Prywes (Columbia University) and R. Lu (University of Guelph) for
generous gifts of cell lines, plasmids andother reagents. We also thank
Yina Dong and Erikka Carr for technical support and T. Gidalevitz,
M. Marzec, M. Chou and A. Gentilella for their comments and
This work was funded by the National Institutes of Health [grant
numbers GM077480, AG18001 to Y.A., HL085577, HL75573,
HL104535, EB011698 to C.G.G.]. D.D. was supported by a National
Institutes of Health training grant [grant number GM008275]. S.D.
was supported by the Rees-Stealy Research Foundation, the San
Diego Chapter of the Achievement Rewards for College Scientists
(ARCS) Foundation, an American Heart Association Predoctoral
Fellowship and an Inamori Foundation Fellowship. The funders had
no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript. Deposited in PMC for
release after 12 months.
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