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INTRODUCTION |
The endoplasmic reticulum
(ER)1 is an important
organelle in which newly synthesized secretory and membrane-associated
proteins are correctly folded and assembled. Perturbations in the ER
environment result in a condition known as ER stress, which can
threaten cell survival. ER stress can be induced in cells by a variety
of treatments including agents known to affect calcium homeostasis,
inhibitors of glycosylation, and overloading of the cell with mutant
proteins that cannot be properly folded. Such stress triggers the
activation of a complex response termed the unfolded protein response
(UPR), which in mammalian cells is characterized by coordinate
transcriptional up-regulation of a number of proteins including
molecular chaperones and folding enzymes, global inhibition of protein
synthesis, and activation of apoptotic pathways (1). The first two
components serve to reduce the load of client proteins and alleviate
the stress, whereas the third functions to eliminate severely damaged cells.
At least three separate mechanisms contribute to the transcriptional
response to ER stress in mammalian cells. The first involves IRE1
and IRE1
, transmembrane protein kinases with endoribonuclease function, that sense the presence of unfolded proteins in the ER,
leading to their activation (2-4). In turn, activation of IRE1 results
in the splicing of mRNA encoding the transcription factor XBP-1,
increasing its efficiency of translation, thereby enhancing its
expression (5-7). A second mechanism contributing to the
transcriptional response to ER stresses is mediated through the ATF6
transcription factor, a member of the ATF/cAMP-response element-binding
protein family of basic leucine zipper proteins. ATF6 is a type
II ER transmembrane protein with its NH2-terminal DNA-binding domain facing the cytosol and its COOH terminus in the ER
lumen (8). In response to ER stress, the cytosolic domain of ATF6 is
cleaved off and translocates to the nucleus to activate transcription
of ER stress target genes (7, 8, 10-15). Overexpression of the
active nuclear form of ATF6 is sufficient for transcriptional induction
of GRP78 and transcription factors Gadd153 and XBP-1 (7, 16). The third
mechanism contributing to transcriptional regulation by ER stress
involves the transcription factor ATF4, another ATF/cAMP-response
element-binding protein family member. Its expression is regulated via
the PERK/eIF2
pathway as discussed below. Like ATF6, ATF4 plays an
important role in the activation of CHOP/Gadd153 (17, 18).
Repression of protein synthesis in response to ER stress is mediated
through the increased phosphorylation of eukaryotic initiation factor 2 (eIF2
), a modification that interferes with the formation of an
active 43 S translation-initiation complex (19). Phosphorylation of
eIF2
during ER stress is carried out by the pancreatic eIF2
kinase (PEK or PERK), which is activated as part of the unfolded protein response (20-22). In addition to PERK, three other eukaryotic protein kinases are known to phosphorylate eIF2
, each of which responds to distinct stress signals. These include the heme-regulated inhibitory kinase, which phosphorylates eIF2
in response to
heme depletion (23, 24), the general control non-derepression-2-kinase, activated in response to amino acid starvation (25, 26), and the
interferon-induced protein kinase (PKR), which is activated by dsRNA
produced during viral infection (27, 28). PERK, like IRE1, is a
transmembrane protein, comprised of a cytoplasmic domain possessing
kinase function and a stress-sensing lumenal domain (20). PERK and IRE1
are activated through a similar mechanism involving oligomerization and
autophosphorylation (1, 2, 30). Although PERK-mediated eIF2
phosphorylation leads to a general suppression of translation, it
promotes the preferential translation of certain mRNAs. Most
notable among these is the transcription factor ATF4 (18).
Genome-wide expression analysis using DNA microarrays has revealed that
activation of the UPR in yeast results in the up-regulation of more
than 350 genes. These include genes involved in various aspects of the
secretory pathway, such as protein folding, ER to Golgi vesicular
transport, and ER-associated protein degradation (22). Although many of
the molecular details of the UPR have been conserved in yeast and
mammalian systems, the scope of UPR outputs in the mammalian cell is
more complex and diverse. Accordingly, it is likely to involve a
greater number of proteins and gene expression changes than seen in yeast.
The present study utilized DNA microarray analysis to search for novel
genes induced by ER stress in mouse embryo fibroblasts (MEFs). We
report here the identification of P58IPK, an inhibitor of
the interferon-induced double-stranded RNA-activated protein kinase
(PKR), as a gene whose expression is up-regulated in response to ER
stress. Additional studies provide evidence that ATF6 contributes to
the induction of P58IPK and P58IPK plays a role
in regulating the activity of the PERK/eIF2
/ATF4 pathway.
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MATERIALS AND METHODS |
Cell Culture, Treatments, Plasmid Construction, and
Transfection--
MEFs, human embryo kidney fibroblasts
(HEK-293), and human cervical carcinoma HeLa cells were cultured in
Dulbecco's modified essential medium (Invitrogen). All media
were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT),
100 units of penicillin/ml, and 100 µg of streptomycin (Invitrogen)
per ml, and were maintained in a humidified atmosphere containing 5%
CO2. Recombinant DNA techniques were performed by standard
procedures. Plasmid pCGN-atf6 was kindly provided by Dr. R. Prywes (Columbia University) and used to construct
pCGN-p50atf6 by inserting a PCR fragment spanning the
NH2-terminal 1-373 amino acids of atf6,
followed by a stop codon into the XbaI and BamHI
sites of the pCGN vector. HEK-293 and HeLa cells were grown
to 60% confluency in 100-mm plates and transfected using Polyfect
reagent (Qiagen, Valencia, CA). Six µg of
pCGN-p50atf6,
pCDNA3-atf4 (kindly provided by Dr. J. Leiden, University of Chicago),
pCDNA1-p58IPK (kindly provided by Dr.
M. G. Katze, University of Washington), pcDNA-perk
(kindly provided by Dr. D. Ron, New York University School of
Medicine), or an empty vector were transfected per plate. Tunicamycin
and thapsigargin were from Sigma.
Western Blot Analysis--
Whole cell lysate protein aliquots
(20-30 µg) were size-fractionated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and then transferred to
nitrocellulose membranes (Schleicher & Schuell) by standard techniques.
Blots were hybridized with the following antibodies: monoclonal
anti-P58IPK (gift from Dr. M. G. Katze, University of
Washington), polyclonal anti-Gadd153 (Santa Cruz Biotechnology Inc.,
Santa Cruz, CA), polyclonal anti-ATF4 (Santa Cruz Biotechnology Inc.),
monoclonal anti-KDEL (Stressgen, Victoria, BC, Canada), polyclonal
anti-eIF2
(Cell Signaling Technology, Beverly, MA), polyclonal
anti-phospho-eIF2
(Cell Signaling Technology, Beverly, MA),
polyclonal anti-phospho-PERK (Cell Signaling Technology), polyclonal
anti-PKR (Cell Signaling Technology), polyclonal anti-phospho-PKR (Cell
Signaling Technology), monoclonal anti-cleaved PARP (Cell Signaling
Technology), polyclonal anti-cleaved caspase-3 (Cell Signaling
Technology), monoclonal anti-myc (9E10) (Santa Cruz Biotechnology
Inc.), and monoclonal anti-GAPDH (Abcam Ltd., Cambridge, UK). Secondary
horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit
antibodies were from Amersham Biosciences. Proteins signals were
detected by using Western Lightning Chemiluminescence Reagent
Plus (PerkinElmer Life Sciences).
RT-PCR and Northern Blot Analysis--
To analyze RNA expression
by reverse transcription-PCR (RT-PCR), total RNA from each sample was
treated with DNase I and used for RT-PCR with SuperScript One-Step
RT-PCR with the Platinum Taq system (Invitrogen). The
primers for gene-specific RT-PCR analysis were as follows: for
P58IPK, GAGGTTTGTGTTGGGATGCAG (5') and
GCTCTTCAGCTGACTCAATCAG (3'); for ATF4, AGGAGTTCGCCTTGGATGCCCTG (5') and
AGTGATATCCACTTCACTGCCCAG (3'); for ATF6, ATCAGTTTACAACCTGCACCCAC (5')
and CTGTCTCCTTAGCACAGCAATATC (3'); for Gadd153, CTGAGTCATTGCCTTTCTCTTCG
(5') and CTCTGACTGGAATCTGGAGAGTG (3'); GAPDH, ACATCAAGAAGGTGGTGAAGCAGG
(5') and CTCTTGCTCTCAGATCCTTGCTGG (3'). Equal aliquots of the PCR
products were electrophoresed through 2% agarose gels. For Northern
blot analysis, 4-µg aliquots of total RNA (harvested using Nucleo
Spin RNAII kit (Clontech, Palo Alto, CA)) were run
on agarose-formaldehyde gels and transferred onto GeneScreen Plus
membranes (PerkinElmer Life Sciences). cDNAs corresponding to
atf4, gadd153, and grp78 (a generous
gift from Amy S. Lee), were labeled by the random primer method and
used to detect corresponding mRNAs on Northern blots. An
end-labeled 24-bp oligonucleotide complementary to 18 S rRNA
(ACGGTATCTGATCGTCTTCGAACC) was used as a probe to verify RNA integrity
and loading differences.
cDNA Array Analysis--
Total RNA was extracted from all
samples using a NucleoSpin RNAII kit (Clontech,
Palo Alto, CA). Atlas Human 1.2 K filters (www.Clontech) each containing 1174 genes were
used. Total RNA (5 µg) was reverse transcribed and labeled with
[
-32P]dATP using the Clontech
cDNA array labeling kit. Hybridizations and washes were performed
as recommended by the manufacturer. The cDNA array membranes were
visualized for analysis by using a PhosphorImager (Amersham
Biosciences), and were quantitated as described (31).
DAPI Staining--
DAPI staining was performed as described
previously (24). In brief, prior to staining, the cells were fixed with
4% paraformaldehyde for 30 min at room temperature, and then washed
with phosphate-buffered saline. DAPI was added to the fixed cells for
30 min, after which they were examined by fluorescence microscopy.
Apoptotic cells were identified by condensation and fragmentation
of nuclei. Percentage of apoptotic cells was calculated as the ratio of
apoptotic cells to total cells counted ×100. A minimum of 400 cells
were counted for each treatment.
Construction of Small Interference RNA (siRNA) Duplexes and
Transfection--
Twenty-one nucleotide double-stranded RNAs were
transcribed in vitro using the SilencerTM siRNA
construction kit according to the manufacturer instructions (Ambion
Inc.). The targeting sequence of human P58IPK (accession
number U28424), corresponding to nucleotide positions 137-157 (coding
region), was AATTACTTGCAGCTGGACAGC. An siRNA targeting the luciferase
mRNA (accession number X65324) served as a control. Cells were
seeded in 6-well plates on the day before transfection at a
concentration of 105 cells per well. Cells were transfected
with OligofectAMINE reagent according to the manufacturer's
instructions (Invitrogen). Briefly, Opti-MEM (165 µl) was mixed with
20 µl of 1 µM siRNA duplex. In a separate tube, 12 µl
of Opti-MEM I was incubated with 3 µl of OligofectAMINE for 5 min at
room temperature. The two mixtures were combined, gently mixed, and
incubated for another 20 min at room temperature. The entire mixture
was added to the cells in 0.8 ml of 10% fetal bovine serum-containing
Dulbecco's modified Eagle's medium without antibiotics. Cells were
assayed at different time intervals after transfection.
[35S]Methionine Metabolic Labeling--
Cells were
seeded into 6-well plates at a density of 105 cells per
well. Twenty-four h following P58IPK siRNA transfection,
cells were placed for 30 min in methionine-free minimal essential
medium (BIOSOURCE, Camarillo, CA), and labeled by
addition of [35S]methionine (20 µCi/ml; 1,000 Ci/mmol;
Amersham Biosciences) to the culture medium for 2 h. Cells were
washed twice with ice-cold phosphate-buffered saline and collected in
lysis buffer (20 mM Hepes, pH 7.4, 2 mM EGTA,
50 mM
-glycerophosphate, 1 mM
Na3VO4, 5 mM NaF, 1% Triton X-100,
10% glycerol, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin). The protein concentration was measured using the Bio-Rad protein DC assay kit. The [35S]methionine incorporation
was measured by cold trichloroacetic acid precipitation and analyzed by
SDS-PAGE. For trichloroacetic acid precipitation, equal amounts of
protein were added to 0.5 ml of 0.1 mg/ml bovine serum albumin
containing 0.02% sodium azide and placed on ice. Ice-cold 20%
trichloroacetic acid (0.5 ml) was added and samples were vortexed
vigorously and incubated for 30 min on ice. Cell suspensions were
filtered through glass microfiber discs (Whatman). Discs were washed
three times with ice-cold 10% trichloroacetic acid, and twice with
100% ethanol, after which they were air-dried, and the radioactivity
was measured by scintillation counting.
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RESULTS |
Identification of P58IPK as an ER Stress-induced
Gene--
We sought to investigate the UPR stress response in
mammalian cells by assessing changes in gene expression profiles after ER stress using the Atlas cDNA Gene Array
(Clontech). This array contains cDNAs for 1174 genes involved in apoptosis, cell cycle control, stress responses,
transcription, and signaling. Total RNA was isolated from MEFs that
were either left untreated or treated for 6 h with 2 µg/ml
tunicamycin, an agent that causes ER stress by inhibiting protein
N-glycosylation. Analysis of the resulting signals on
cDNA arrays (carried out as described under "Materials and
Methods") revealed 19 candidate genes whose expression was increased
in response to tunicamycin treatment. Among these were a number of
genes previously shown to play a role in the UPR response, including
grp78, gadd153, and erp72. One novel
gene whose expression was significantly elevated in MEFs following ER
stress was p58IPK, an established inhibitor of the
eIF2
kinase PKR, a kinase related to the ER-specific kinase, PERK
(Fig. 1A). Hence, we further
investigated the regulation of p58IPK by ER stress
and examined its potential role during the UPR response.

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Fig. 1.
Induction of P58IPK by ER
stress-inducing agents. A, Atlas array analysis of gene
expression profiles following treatment with tunicamycin. MEF cells
were treated for 6 h with 2 µg/ml tunicamycin. Reverse
transcribed, [ -32P]dATP-labeled cDNA probes
synthesized from total cellular RNA present in either untreated
(left) or tunicamycin-treated (right) populations
were hybridized to Atlas Mouse 1.2 Array membranes, as described under
"Materials and Methods." Induction of P58IPK expression
is indicated by the arrow. B, HEK-293 and MEF
cells were treated with either 2 µg/ml tunicamycin or 1 µM thapsigargin for the times shown, whereupon
p58IPK and gapdh mRNA levels
following exposure to ER stress agents were detected by RT-PCR as
indicated under "Materials and Methods." Assessment of
gapdh levels served to control for RNA integrity and
loading. C, immunoblot analysis of P58IPK and
GAPDH protein levels in HEK-293 and MEF cells treated with tunicamycin
(2 µg/ml) for the times indicated.
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To validate the findings obtained by microarray analysis,
p58IPK expression levels in MEFs and HEK-293 cells
were examined using RT-PCR after treatment with either tunicamycin or
thapsigargin (another ER stress agent that inhibits ER
Ca2+-ATPase, thereby causing ER stress through a different
mechanism). As shown, p58IPK mRNA levels
increased in both cell lines in response to each ER stress agent in a
time-dependent manner (Fig. 1B). Further characterization of the response was carried out through kinetic analysis of P58IPK protein expression by Western blot
analysis. As depicted in Fig. 1C, P58IPK protein
levels similarly increased following treatment with tunicamycin in both
cell lines (Fig. 1C).
Because ATF4 and ATF6 are known to play a role in the transcriptional
activation of ER stress-inducible genes, we examined their contribution
to the induction of p58IPK expression. To
investigate the potential role of ATF6, we transfected HEK-293 cells
with a plasmid that expresses p50 ATF6 (the soluble form of ATF6,
capable of translocating into the nucleus). Cells were harvested
48 h after transfection and p58IPK mRNA was
examined using RT-PCR (Fig. 2).
Overexpression of p50 ATF6 alone in HEK-293 cells resulted in
up-regulation of the p58IPK mRNA, even in the
absence of ER stress. In contrast, overexpression of full-length ATF4
alone did not alter p58IPK mRNA expression. To
ensure that the ATF4 construct was functional in this assay, we also
analyzed expression of gadd153, an established transcriptional target of both ATF4 and ATF6. gadd153
mRNA was induced similarly by ectopically overexpressed p50 ATF6
and ATF4, indicating that the ATF4 construct was functional. These
observations indicate that p58IPK is a novel target
of the ATF6, but not ATF4 pathway.

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Fig. 2.
Overexpression of ATF6 induces
p58IPK expression. A, HEK-293
cells were transfected with an empty vector or vectors expressing
either atf4 or atf6. Cells were harvested 48 h after transfection and total RNA prepared for assessment of the
expression levels of mRNAs encoding p58IPK and
gapdh (to control for RNA integrity and loading) by RT-PCR.
B, cells were transfected as described in the legend of
panel A and total RNA was prepared to assess the expression
levels of gadd153, atf4, atf6, and
gapdh by RT-PCR.
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Overexpression of P58IPK Inhibits eIF2
Phosphorylation and ATF4 and Gadd153 Induction--
To gain a better
understanding of the biological role of P58IPK in the UPR,
we tried to generate stable cell lines overexpressing P58IPK using both HEK-293 and HeLa cells. Despite our best
efforts, no stable P58IPK-overexpressing clones could be
obtained (data not shown). As an alternative strategy, we transiently
transfected a plasmid expressing P58IPK into HeLa cells,
where high transfection efficiencies could be achieved. ER stress is
known to induce translational repression, which is mediated by
phosphorylation of eIF2
. Phosphorylation of eIF2
leads to
down-regulation of translation initiation through a well characterized
mechanism involving inhibition of eIF2
activity (32).
P58IPK is a known inhibitor of the eIF2
kinase PKR, and
it has been shown that overexpression of P58IPK can inhibit
dsRNA-induced phosphorylation of eIF2
by PKR. Because ER stress
leads to elevated P58IPK expression, we hypothesized that
P58IPK might affect the eIF2
phosphorylation during the
UPR. To address this possibility HeLa cells were transiently
transfected with a P58IPK expression vector or empty vector
(Fig. 3A). Forty-eight hours post-transfection, cells were treated with tunicamycin, and protein lysates were analyzed for both eIF2
phosphorylation and total eIF2
protein levels by Western blotting. As shown in Fig.
3B, overexpression of P58IPK significantly
attenuated tunicamycin-induced eIF2
phosphorylation.

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Fig. 3.
Overexpression of P58IPK reduces
eIF2 phosphorylation and expression of ATF4
and Gadd153. A, 48 h after transfection with
either an empty plasmid or a vector expressing
p58IPK, HeLa cells were treated with 2 µg/ml
tunicamycin for the indicated times and P58IPK and GAPDH
protein levels were assessed by immunoblot. B, cells were
transfected as described in panel A, whereupon they were
treated with 1 µM thapsigargin (Tg) for 1 h and phospho-eIF2 and total eIF2 levels were assessed by
immunoblotting. C, cells were treated as described in
panel A, then treated with 2 µg/ml tunicamycin
(Tn) for the times indicated. Expression of ATF4, Gadd153,
and GAPDH proteins was assessed by immunoblot analysis. The values
between the panels represent the -fold induction of ATF4 and Gadd153
levels compared with control levels after normalization to GAPDH
levels, as determined by densitometric analysis. D, control
and P58IPK-overexpressing HeLa cells were harvested at the
indicated times, and PARP cleavage was assessed by Western blot
analysis using a monoclonal antibody that recognizes cleaved PARP.
E, HeLa cells that were treated with tunicamycin for the
times indicated were fixed with 4% paraformaldehyde, stained with
DAPI, and subjected to counting of apoptotic nuclei by fluorescence
microscopy. Data represent the mean ± S.E.
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Given that elevated P58IPK expression decreased the
phosphorylation levels of eIF2
, we sought to analyze the levels of
ATF4, a downstream target of this pathway, and Gadd153, whose
expression is in turn regulated (at least in part) by ATF4.
P58IPK overexpression profoundly inhibited ATF4 protein
accumulation in tunicamycin-treated HeLa cells (Fig. 3C),
consistent with previous reports showing that the production of ATF4
protein requires eIF2
phosphorylation (18). Gadd153 protein
induction in the P58IPK overexpressed cells was ~70% of
that seen in control cells after 4-6 h treatment with tunicamycin
(Fig. 3C).
Previous reports have shown that P58IPK can protect cells
from dsRNA or tumor necrosis factor-
-induced apoptosis. We were
therefore interested in determining whether P58IPK could
protect cells from ER stress-induced apoptosis. HeLa cells transiently
transfected with either a p58IPK-containing
expression vector or an empty vector were treated with tunicamycin for
different time periods and then analyzed for apoptosis. A hallmark of
apoptosis is cleavage of the nuclear 116-kDa PARP (poly(ADP-ribose)
polymerase) protein to an 85-kDa inactive polypeptide. Inactivation of
PARP through proteolytic cleavage facilitates chromosomal DNA
fragmentation as part of the cellular apoptotic program (33). Our
results show that the tunicamycin-induced PARP proteolysis was similar
in vector and P58IPK-transfected HeLa cells (Fig.
3D). DAPI staining revealed a similar pattern of condensed
and fragmented nuclei for both the control and
P58IPK-transfected cells (Fig. 3E). Taken
together, these results suggest that under the conditions utilized
here, overexpression of P58IPK does not alter the
apoptotic response to ER stress.
Silencing of P58IPK by siRNA Induces
Apoptosis--
Whereas elevated P58IPK expression failed
to alter the cellular outcome following ER stress, it remained possible
that a reduction in P58IPK could influence the response. We
decided to employ the RNA interference technique to address this
possibility. A small inhibitory double-stranded RNA homologous to a
21-nucleotide sequence unique to the human P58IPK was used
to reduce P58IPK expression. As shown in Fig.
4A, transfection of HeLa or
HEK-293 cells with P58IPK siRNA resulted in a reduction of
P58IPK protein levels, although control transfections with
siRNA specific for luciferase, carried out in parallel, showed no
effect on P58IPK expression. These observations indicate
that the P58IPK siRNA treatment specifically reduced the
abundance of P58IPK protein.

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Fig. 4.
Reduction of P58IPK protein
expression by small interference RNA (siRNA) causes apoptosis.
A, reduction of P58IPK protein expression by RNA
interference. An siRNA duplex specific for human P58IPK as
well as a duplex specific for the firefly gene luciferase (described
under "Materials and Methods") were transfected into HEK-293 and
HeLa cells. Whole cell extracts were prepared 20 h after
transfection and were used to detect P58IPK and GAPDH by
immunoblotting. B, induction of cell death by
P58IPK siRNA. HEK-293 and HeLa cells were transfected with
siRNA control duplexes and duplexes specific for human
P58IPK. At different time intervals after transfection,
cells were counted and assessed for viability by trypan blue dye
exclusion. C, induction of caspase 3 and PARP cleavage by
P58IPK siRNA. HeLa cells were transfected with siRNA for
human P58IPK and harvested at the indicated times. Whole
cell lysates were subjected to immunoblot analysis to assess the levels
of cleaved caspase-3 by using a polyclonal cleaved caspase-3
(Asp175) antibody that detects endogenous levels of the
large fragment (17/19 kDa) of activated caspase-3 resulting from
cleavage adjacent to (Asp175). PARP cleavage was assessed
by using a monoclonal antibody that recognizes cleaved PARP.
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Approximately 28 h after transfection with the P58IPK
siRNA duplex, we observed that many cells showed reduced ability to
adhere to the plate and floated in the medium. To determine whether
silencing P58IPK causes cell death, we examined the cell
growth and viability of P58IPK siRNA-transfected cells.
Cells transfected with the control siRNA grew very well. By contrast,
cells transfected with P58IPK siRNA showed a marked
reduction in viability (Fig. 4B). Caspase-3 is one of the
key executioners of apoptosis, being responsible either partially or
totally for the proteolytic cleavage of many key proteins such as the
nuclear enzyme PARP. Activation of caspase-3 requires the proteolytic
processing of its inactive zymogen into activated p17 and p12 subunits.
Cleavage of caspase-3 and PARP can be detected by Western blot analysis
and was apparent 20 h after transfection of P58IPK
siRNA in HeLa cells, whereas no caspase-3 or PARP cleavage were detected in the control cells (Fig. 4C). These results
indicate that silencing P58IPK decreases viability by
causing apoptotic cell death.
siRNA Silencing of P58IPK Induces eIF2
Phosphorylation and Inhibits Protein Synthesis--
Because
overexpression of P58IPK can inhibit eIF2
phosphorylation, we postulated that silencing of P58IPK
would induce eIF2
phosphorylation and inhibit protein translation. To determine the effect of P58IPK siRNA on eIF2
phosphorylation, HeLa and HEK-293 cells were transfected with the
control siRNA or P58IPK siRNA and 20 h
post-transfection, protein lysates were prepared and analyzed by
Western blotting (Fig. 5A).
The results show that P58IPK siRNA treatment markedly
increased the level of eIF2
phosphorylation compared with control
transfected cells.

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Fig. 5.
Silencing of P58IPK by siRNA
leads to eIF2 phosphorylation and inhibition
of protein synthesis. A, 20 h after transfection
of HEK-293 and HeLa cells with P58IPK siRNA or control
siRNA (described in the legend of Fig. 4), whole cell protein extracts
were subjected to immunoblot analysis to assess the levels of total
eIF2 and phosphorylated eIF2 . B, whole cell extracts
from HEK-293 and HeLa cells that were transfected with either the
control siRNA duplex or the P58IPK siRNA duplex were
subjected to pulse-labeling of total proteins using
[35S]methionine (described under "Materials and
Methods"). Proteins were resolved by 4-12% gradient SDS-PAGE and
stained with Coomassie Blue R-250 (right panel), or
visualized for analysis using a PhosphorImager.
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To further establish that P58IPK siRNA inhibits overall
protein synthesis, [35S]methionine incorporation was used
to quantify the rate of protein synthesis after P58IPK
silencing. HeLa and HEK-293 cells were transfected with the
P58IPK siRNA duplex and metabolically labeled with
[35S]methionine at 20 h post-transfection. Analysis
by SDS-PAGE and trichloroacetic acid precipitation demonstrated that
general protein synthesis was reduced to 16.5 and 51% for HeLa and
HEK-293, respectively, following P58IPK silencing (Fig.
5B). Taken together these results strongly suggest that
silencing P58IPK activates the eIF2
stress-signaling
pathway resulting in the inhibition of overall protein synthesis.
siRNA Silencing of P58IPK Induces PERK
Phosphorylation--
Having determined that P58IPK siRNA
induces eIF2
phosphorylation we next wished to determine whether any
of the known eIF2
kinases might be implicated in this process.
P58IPK was originally discovered as an inhibitor of PKR, an
interferon-induced, double-stranded RNA-activated kinase that is
activated during virus infection (34). Activation of PKR by
double-stranded RNA results in PKR dimerization and autophosphorylation
at positions Thr446 and Thr451 in the
activation loop (35). To determine whether PKR is activated by
P58IPK siRNA, we examined its phosphorylation state.
P58IPK siRNA did not markedly induce PKR phosphorylation,
as determined by Western blotting with an antiserum reactive to
phospho-Thr446/451 (Fig.
6A). It was interesting to
note that total PKR protein levels were slightly increased by
P58IPK siRNA. Although moderate, such increases in PKR
expression were seen reproducibly.

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Fig. 6.
Silencing of P58IPK by siRNA
leads to PERK phosphorylation. A, 20 h after
transfection of HEK-293 cells with P58IPK siRNA or control
siRNA (described in the legend of Fig. 4) whole cell protein extracts
were subjected to immunoblot analysis to assess the levels of total PKR
and phosphorylated PKR. B, HEK-293 cells were transfected
with PERK and 24 h later split into 6-well plates. Twenty-four h
later, HEK-293 cells were transfected with P58IPK siRNA or
control siRNA (described in the legend of Fig. 4) and after 20 h,
whole cell protein extracts were subjected to immunoblot analysis to
assess the levels of total PERK and phospho-PERK. For the total PERK,
the membrane was probed with the 9E10 antibody, which recognizes a
c-myc epitope tag expressed at the COOH terminus of PERK.
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PERK, a kinase found in the lumen of the ER, also phosphorylates
eIF2
in response to various stimuli that induce ER stress. A very
recent paper from Yan et al. (36) demonstrates that
P58IPK is associated with the ER and represses PERK
activity. Unfortunately, the low expression levels of endogenous PERK
and the present unavailability of good anti-PERK antibodies did not
allow a clear analysis of the effects of P58IPK siRNA on
PERK phosphorylation. Thus, to gain insight into this potential
regulation, we transiently transfected HEK-293 cells using a plasmid
that expressed wild-type mouse PERK. PERK activation can occur through
autophosphorylation of its cytoplasmic kinase domain, and can be
monitored by immunoblotting with an antibody that recognizes PERK only
when it is phosphorylated on Thr980. HEK-293 cells
transiently overexpressing PERK were transfected with the control siRNA
or P58IPK siRNA and, 20 h after transfection, lysates
were prepared and subjected to Western blot analysis (Fig.
6B). As shown, P58IPK siRNA treatment
markedly increased the level of PERK phosphorylation compared with
control transfected cells.
P58IPK Silencing by siRNA Induces ATF4 and
Gadd153--
Gadd153 is expressed at low or undetectable levels under
normal growth conditions (37), but is highly induced following thapsigargin or tunicamycin treatments, and has been implicated in ER
stress-induced apoptosis (38). Gadd153 transcription is induced by
eIF2
phosphorylation through preferential translation of ATF4, as
previously described (18). Because P58IPK siRNA
increased the level of eIF2
phosphorylation, we predicted that it
would likely induce ATF4 and Gadd153 expression. To determine the
effect of P58IPK siRNA on ATF4 and Gadd153 expression,
Northern and immunoblot analyses were performed. HEK-293 cells were
transfected with control or P58IPK siRNA and 20 h
later were treated with tunicamycin for different time intervals,
whereupon total RNA and protein were prepared for analysis (Fig.
7, A and B). ATF4
protein was low in untreated cells, but increased in response to
tunicamycin treatment. Transfection to decrease P58IPK
levels in HEK-293 cells, did not significantly affect atf4
mRNA levels, but markedly increased ATF4 protein levels, even in
untreated cells. These results are consistent with earlier reports
showing that ATF4 expression is regulated via its preferential
translation during ER stress in an eIF2
-dependent manner
(18). Gadd153, which was barely detectable in the control transfected
cells, was likewise induced after P58IPK siRNA
transfection. This was evident at both the mRNA and protein levels.

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Fig. 7.
Silencing of P58IPK induces ATF4
and Gadd153. Western blot and Northern blot analyses of ATF4,
Gadd153, and GRP78 expression in cells silenced with P58IPK
siRNA. HEK-293 cells were transfected with P58IPK siRNA and
20 h later treated with 2 µg/ml tunicamycin for different time
periods. A, immunoblot analysis of protein lysates from
control transfected populations and from P58IPK
siRNA-transfected cells to monitor ATF4, Gadd153, Grp78, and GAPDH
protein levels. B, abundance of mRNAs encoding
atf4, gadd153, grp78, and
gapdh. Total RNA was prepared from transfected HEK-293 cells
and analyzed by Northern blotting.
|
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Transcriptional induction of gadd153 has been shown to
closely parallel that of grp78 under many ER
stress-triggering conditions and indeed like gadd153,
grp78 mRNA levels were increased at 4 h following
tunicamycin treatment. However, unlike gadd153, expression of grp78 was not increased by P58IPK siRNA.
These findings suggest that silencing P58IPK specifically
affects eIF2
stress signaling pathways and that the
P58IPK protein plays a critical role in regulating ATF4 and
Gadd153 protein levels.
 |
DISCUSSION |
The UPR, initiated in response to perturbations in the ER
environment, is characterized by the activation of signaling pathways that transduce stress signals generated and sensed in the ER to other
cellular compartments, thereby effecting changes in gene transcription
and transiently suppressing translation. The biological objective of
the UPR is to regain homeostasis in the ER by reducing demands on the
organelle and increasing its capacity to carry out its protein folding
and/or modifying functions. So far, three distinct pathways,
IRE1/XBP-1, PERK/eIF2
/ATF4, and ATF6, have been shown to contribute
to the transcriptional response, but there is increasing evidence for
both cross-talk and redundancy in the pathways. For example,
transcriptional up-regulation of XBP-1 through the ATF6 pathway
provides more substrate for IRE1, thus potentiating IRE1 pathway
signaling. In addition, some genes, such as gadd153, appear
to be targets of more than one pathway (i.e. both ATF4 and
ATF6 contribute to its transcription) (15, 17, 40).
In this report, we have identified P58IPK, an inhibitor of
the interferon-induced protein kinase PKR, as a target for
transcriptional up-regulation by the UPR. This finding is consistent
with a previous report by Kaufman and colleagues (41) in which
p58IPK was listed as one of 67 genes induced upon
tunicamycin treatment in MEFs using a different cDNA array.
However, the physiologic relevance of p58IPK
induction was not addressed in that study, nor was its up-regulation verified by other methods in that report. In the current studies we
have provided evidence suggesting that p58IPK
induction is mediated via ATF6 and that P58IPK plays a role
in down-regulating the activity of the PERK/eIF2
/ATF4 pathway. These
findings identify a novel mechanism contributing to the regulation of
the UPR and define a new avenue for cross-talk between ER
stress-activated pathways.
That ATF6 contributes to the induction of p58IPK
during the UPR is suggested by the finding that overexpression of its
activated nuclear form (p50 ATF6) leads to elevated
p58IPK expression. A 19-bp ER stress responsive
element consisting of the consensus sequence CCAAT(N9)CCACG
has been identified in the promoter region of the
p58IPK gene (36). ER stress-responsive element
sequences have been implicated in the transcriptional activation of
downstream genes after treatment with agents that activate the
mammalian UPR pathway (36), and ATF6 has been shown to interact with
the ER stress-responsive element (16). Whereas definitive proof of its
role in regulating p58IPK must await further
investigation, a role for ATF6 in regulating p58IPK
expression is consistent with studies of Okada and co-workers (42), in
which microarray analysis revealed genes encoding molecular chaperones
and folding enzymes as primary transcriptional targets of the ATF6
pathway. As a member of the DnaJ molecular chaperone family,
P58IPK would fit into this category (34).
P58IPK was first recognized for its ability to inhibit the
double-stranded RNA-activated protein kinase, PKR (34). PKR is part of
the interferon-induced host defense against viral infection, and
functions to repress translation initiation via phosphorylation of
eIF2
(43, 44). The proposed inhibitory mechanism of
P58IPK is as follows. Influenza virus activates the
P58IPK pathway by promoting the dissociation of
P58IPK from its own inhibitor, hsp40. The free
P58IPK represses PKR-mediated eIF2
phosphorylation
through direct protein-protein interaction, and thereby relieves the
PKR-imposed block on mRNA translation (45). Importantly, unlike the
situation seen here for ER stress, P58IPK expression is not
altered by viral infection. Available evidence indicates that PKR is
not involved in the ER stress response, as PKR knockout cells are
unimpaired in their ability to respond to ER stress by attenuating
translation rates (20). Because PERK shares many properties with PKR,
we investigated the possibility that P58IPK could modify
this pathway. A very recent report by Yan and colleagues (36) provides
evidence that P58IPK interacts with PERK and inhibits its
activity. Our data showing that modulation of P58IPK levels
alters the phosphorylation status of PERK and eIF2
is in agreement
with this report. We found that overexpression of P58IPK
inhibited eIF2
phosphorylation and reduced ATF4 protein
accumulation, whereas silencing of P58IPK expression
enhanced PERK and eIF2
phosphorylation and increased ATF4
accumulation. The current model for regulation of ATF4 protein levels
proposes that, under normal conditions, ATF4 mRNA is expressed, but
is translated poorly. However, under ER stress conditions, ATF4 protein
is rapidly synthesized in a manner that is dependent on eIF2
phosphorylation. This then leads to enhanced expression of its target
genes. Previous studies implicate both ATF4 and ATF6 in the
transcriptional activation of gadd153 (15, 17, 40).
The more modest effect of P58IPK overexpression on
gadd153 expression (relative to its effect on ATF4) likely
reflects the contribution of ATF6 to gadd153 induction.
P58IPK has been demonstrated to possess an anti-apoptotic
function, protecting cells against tumor necrosis factor-
- and
dsRNA-induced cell death (39). In our study systems,
overexpression of P58IPK did not alter cell death following
ER stress. However, silencing of p58IPK was
associated with a significant increase in cell sensitivity to
tunicamycin treatment. Higher levels of Gadd153/CHOP, resulting from
increased activity of the PERK/eIF2
/ATF4 pathway, might contribute
to the enhanced sensitivity to undergo apoptosis. Indeed, overexpression of gadd153 has been shown to lead to cell
cycle arrest and apoptosis through mechanisms that likely involve
down-regulation of Bcl-2 (9).
In summary, our findings are consistent with a model in which
P58IPK functions to regulate PERK and eIF2
phosphorylation during normal physiologic conditions and under
conditions of ER stress. Phosphorylation of PERK and eIF2
is
required for translation attenuation, transcriptional induction, and
cellular survival in response to ER stress. However, PERK and eIF2
phosphorylation and inhibition of protein translation is transient in
stressed cells. We propose that enhanced expression of
P58IPK during the UPR serves as an important component of a
negative feedback loop used by the cell to attenuate eIF2
signaling.
This model is reminiscent of that proposed for Gadd34, another gene product induced during the UPR, which has been shown to play an important feedback role in regulating eIF2
phosphorylation, through promotion of its dephosphorylation (29).