From the ¶ Edward A. Doisy Department of Biochemistry and
Molecular Biology and the Department of Molecular Microbiology
and Immunology, St. Louis University School of Medicine, St. Louis,
Missouri 63104
Received for publication, November 18, 2002, and in revised form, February 5, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Double-stranded (ds) RNA, which
accumulates during viral replication, activates the antiviral response
of infected cells. In this study, we have identified a requirement for
extracellular signal-regulated kinase (ERK) in the regulation of
interleukin 1 (IL-1) expression by macrophages in response to dsRNA and
viral infection. Treatment of RAW 264.7 cells or mouse macrophages with dsRNA stimulates ERK phosphorylation that is first apparent following a
15-min incubation and persists for up to 60 min, the accumulation of
iNOS and IL-1 mRNA following a 6-h incubation, and the expression of iNOS and IL-1 at the protein level following a 24-h incubation. Inhibitors of ERK activation prevent dsRNA-induced ERK phosphorylation and IL-1 expression by macrophages. The regulation of macrophage activation by ERK appears to be selective for IL-1, as ERK inhibition does not attenuate dsRNA-induced iNOS expression by macrophages. dsRNA
stimulates both ERK activation and IL-1 expression by macrophages isolated from dsRNA-dependent protein kinase (PKR)-deficient
mice, indicating that PKR does not participate in this antiviral
response. These findings support a novel PKR-independent
role for ERK in the regulation of the antiviral response of IL-1
expression and release by macrophages.
Double-stranded (ds)1
RNA, which accumulates at various stages of viral replication, plays a
primary role in the activation of antiviral responses in virally
infected cells (1). Prominent sources of dsRNA include viral RNAs
containing ds secondary structure and dsRNA formed during viral
replication (1). The accumulation of these dsRNAs activates several
antiviral responses including the dsRNA-dependent protein
kinase, PKR (2, 3). PKR has been implicated in both the transcriptional
activation of antiviral genes (4-7), and in the inhibition of protein
translation mediated by the phosphorylation of the initiation factor
eIF2 The molecular mechanisms by which viral infection or dsRNA stimulates
iNOS and IL-1 expression by macrophages are not well-defined. We have
shown that dsRNA-induced iNOS and IL-1 expression by RAW 264.7 cells is
prevented by the expression of dominant negative (dn) PKR, and that
this PKR dependence can be overcome by IFN- Mitogen-activated protein kinase (MAPK) signaling pathways are
activated by dsRNA (24, 27, 28) and viral infection (25), and MAPK have
been implicated in the regulation of iNOS and IL-1 expression (29-31).
The role of PKR in dsRNA-induced MAPK kinase activation is less clear.
It was first shown that dsRNA stimulates c-Jun N-terminal kinase (JNK)
and p38 activation in MEF. In these studies, dsRNA-induced p38
activation was attenuated in PKR In this study, the role of the MAPK pathway, specifically ERK, in
dsRNA-induced macrophage activation has been examined. We show that the
MAP/ERK kinase (MEK) selective inhibitors U0126 and PD98059 prevent
dsRNA- and virus-induced ERK phosphorylation, IL-1 expression, IL-1 Materials and Animals--
Defined fetal bovine serum was
purchased from Hyclone (Logan, UT). Poly IC, myelin basic protein, and
protein A-Sepharose were purchased from Sigma Chemical Co. Poly IC was
prepared as previously described (4). U0126 was from CalBiochem (San
Diego, CA), and PD98059 was purchased from Alexis Biochemicals (San
Diego, CA). The human IL-1 Cell Culture, Peritoneal Macrophage Isolation, and EMCV
Infection--
RAW 264.7 and RINm5F cells were removed from growth
flasks by treatment with 0.05% trypsin, 0.02% EDTA for 5 min at
37 °C. Cells were washed twice with media and plated at the
indicated concentrations. The cells were allowed to adhere for 2 h
under an atmosphere of 5% CO2, 95% air before the
initiation of experiments. Peritoneal exudate cells (PEC) were isolated
from PKR Nitrite and IL-1 Determinations--
Nitrite production was
determined by the Greiss assay, and IL-1 release was measured using the
RINm5F cell bioassay as previously described (38, 39). The RINm5F cell
IL-1 bioassay assay measures cumulative biological activity of both
IL-1 Western Blot Analysis--
Total cellular protein was separated
by SDS-PAGE and transferred to High Bond ECL nitrocellulose membranes
(Amersham Biosciences) under semidry conditions as previously described
(9). Antibody dilutions were: rabbit anti-mouse iNOS, 1:2,000; mouse
anti-pro-IL-1 Northern Blot and RT-PCR Analysis--
Total RNA was isolated
from RAW 264.7 cells using the RNeasy kit (Qiagen, Santa Clarita, CA)
according to the manufacturer's instructions. Northern blot analysis
and RT-PCR were performed using iNOS, IL-1 Transient Transfections and IL-1 Immunocomplex Kinase Assay--
The K71R mutation in human ERK1
was made using the Stratagene (La Jolla, CA) QuickChange PCR-plasmid
mutagenesis kit using 5'-TGGCCATCAGAAAGATCAGC-3' and
5'-GCTGATCTTTCTGATGGCCA-3' (mutated bases in bold) primers
according to the manufacturer's specifications. This mutation, which
was confirmed by sequence analysis, has previously been shown to
inhibit ERK kinase activity (42). RAW 264.7 cells were transiently
transfected with 2 µg of wild-type ERK1 or ERK1(K71R) using Superfect
(Qiagen) according to the manufacturer's instructions. After a 48-h
incubation at 37 °C, poly(IC) was added, and the cells were cultured
for 30 additional min. The cells were then harvested, ERK was
immunoprecipitated, and the ability of immunoprecipitated ERK to
phosphorylate myelin basic protein was examined as previously described
(42). ERK kinase activity was quantitated by densitometry.
Densitometry and Image Analysis--
Autoradiograms were scanned
into NIH Image version 1.59 using a COHU high performance CCD camera
(Brookfield, WI) and densities determined using NIH Image version 1.59 software.
Effects of MEK Inhibition on dsRNA-induced iNOS Expression, Nitrite
Production, and IL-1 Expression and Release by RAW 264.7 Macrophages--
Macrophage activation in response to dsRNA includes
the expression of iNOS and production of nitric oxide (9, 14). Also, dsRNA and viral infection have been shown to stimulate ERK activation in macrophages (15, 24, 27), and LPS-induced iNOS expression is
sensitive to ERK inhibition (43). Therefore, the potential role of ERK
in dsRNA-induced macrophage activation was examined using selective
inhibitors (U0126 and PD98059) of MEK, the upstream kinase that is
responsible for the activation of ERK. Treatment of RAW 264.7 macrophages with 50 µg/ml poly(IC) results in the accumulation of
iNOS mRNA following a 6-h incubation, the expression of iNOS at the
protein level and a 5-fold increase in nitrite production following a
24-h incubation (Fig. 1,
A-C). The ERK selective inhibitor U0126, at concentrations
of 0.01-5 µM, does not attenuate iNOS mRNA
accumulation, iNOS protein expression, or nitrite production by RAW
264.7 cells. Similar results for nitrite production and iNOS expression
were obtained with the second MEK inhibitor, PD98059 at concentrations
of 0.1-10 µM (data not shown). MEK inhibition slightly
increases the levels of nitrite produced by RAW 264.7 cells treated
with poly(IC); however, this minor increase did not achieve statistical
significance.
A second antiviral response activated by dsRNA in macrophages is the
expression and release of the proinflammatory cytokine, IL-1 (4, 9). A
24-h incubation of RAW 264.7 cells with poly(IC) (50 µg/ml) results
in an ~50-fold increase in IL-1 release and the accumulation of
pro-IL-1
To confirm these findings the effects of a second MEK selective
inhibitor, PD98059 on IL-1 Effects of MEK Inhibition on EMCV-induced pro-IL-1 Expression by
RAW 264.7 Macrophages--
To confirm that poly(IC) recapitulates host
responses to a viral infection; the effects of encephalomyocarditis
virus (EMCV) infection on the expression of IL-1 dsRNA Stimulates ERK Activation and dnERK Inhibits dsRNA-induced
IL-1 Expression by Macrophages--
To confirm that U0126 prevents ERK
activation, the effects of this MEK kinase inhibitor on ERK
phosphorylation were examined by Western blot analysis. Treatment of
RAW 264.7 cells for 30 min with poly(IC) (50 µg/ml) results in the
phosphorylation of ERK, an effect that is prevented by U0126 at
concentrations (5 µM) that inhibit dsRNA-induced IL-1
expression and release (Fig. 5A). In a similar manner
PD98059 (10 µM) prevents poly(IC)-stimulated ERK
phosphorylation (data not shown). Consistent with the stimulatory actions of poly(IC) on ERK phosphorylation, dsRNA activates ERK as
determined by immunocomplex kinase assays. For these experiments, RAW
264.7 cells, transiently transfected with either wild type or dnERK
(ERK1(K71R)), were incubated with or without poly(IC) for 30 min, and
then the phosphorylation of myelin basic protein by immunoprecipitated
ERK was examined. A 30-min incubation with poly(IC) stimulates an
~4.5-fold increase in ERK activity (myelin basic protein
phosphorylation) in RAW 264.7 cells expressing wild-type ERK; however,
poly(IC) fails to stimulate myelin basic protein phosphorylation in RAW
264.7 cells expressing dnERK (Fig. 5B). In this
immunocomplex kinase assay wild type and dnERK are expressed at levels
2-3-fold higher than the levels of endogenous ERK (Fig. 5B,
inset). RT-PCR was used to examine the role of ERK in the regulation of IL-1 MEK Inhibition Does Not Affect dsRNA-induced iNOS Expression and
Nitrite Production but Prevents IL-1 Expression and Release by
Peritoneal Macrophages--
Consistent with the lack of a role for ERK
in dsRNA-induced iNOS expression by RAW 264.7 cells, U0126 (5 µM) does not modulate poly(IC) + IFN-
While ERK does not appear to participate in the regulation of iNOS
expression, it is required for poly(IC) and poly(IC) + IFN-
To confirm that dsRNA stimulates ERK activation, and to determine if
PKR is required for this activation, the effects of poly(IC) on ERK
phosphorylation by PEC isolated from PKR Recently, Magun and co-workers (25) have proposed that dsRNA
triggers two separate antiviral programs, cell suicide (apoptosis), and
a survival pathway associated with proinflammatory cytokine production.
At the core of this proposal is the ability of PKR to regulate each of
these antiviral responses. The first antiviral pathway is that of
apoptosis, a process of self-elimination that removes virally infected
cells. This pathway of programmed cell death is activated by dsRNA, is
dependent on PKR (45, 46), and appears to be a widely used antiviral
response as evidenced by the multiple strategies used by viruses to
evade apoptosis (Ref. 8 and references therein). The second antiviral
pathway activated by dsRNA or viral infection is the expression and
release of proinflammatory cytokines such as interferons and
interleukins. The role of PKR in the regulation of cytokine expression
has not been clearly defined, in part because of results that support or refute a role for PKR in the regulation of NF- In this study, we present evidence to further support the presence of a
PKR-independent antiviral response pathway that is activated by viral
infection and dsRNA and that regulates the expression and release of
the proinflammatory cytokine IL-1. We show that dsRNA treatment or EMCV
infection stimulates IL-1 expression and release in an
ERK-dependent fashion. Selective inhibition of MEK, the
upstream ERK kinase, prevents dsRNA and EMCV-induced IL-1 expression
and release. The role of ERK in regulating the antiviral response
appears to be selective for IL-1 expression as MEK inhibition does not
modulate dsRNA-induced iNOS expression or nitric oxide production by
macrophages. PKR does not appear to be required for this response, as
dsRNA stimulates ERK activation and ERK-dependent IL-1
expression by macrophages isolated from PKR In summary, these studies have identified a novel mechanism by which
viral infection and dsRNA stimulate the expression and release of IL-1
by macrophages. dsRNA or EMCV infection stimulates ERK activation and
ERK participates in the transcriptional regulation of IL-1 expression
by macrophages. PKR does not appear to be required for dsRNA-induced
ERK activation or the transcriptional activation of IL-1 expression.
The downstream targets of ERK that regulate IL-1 expression in response
to dsRNA and virus infection have yet to be defined. One likely
candidate is PU.1, a member of the ets family of transcription factors,
and a transcription factor known to participate in the activation of
IL-1 expression in response to LPS. In support of a role for PU.1 in
the regulation of virus-induced IL-1 expression, we have recently shown
by gel shift analysis that poly(IC) stimulates the DNA binding activity
of PU.1 in macrophages in an U0126-sensitive
manner.2 Additional
transcription factors also participate in the regulation of IL-1
expression in response to dsRNA. Inhibitors of NF-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(8). In macrophages, dsRNA and viral infection stimulate the
expression of proinflammatory cytokines such as IL-1
, IL-1
, tumor
necrosis factor, and IL-6 as well as the inducible isoform of
nitric-oxide synthase (4, 9-12). Nitric oxide appears to play a
primary role in host defense against a viral infection as the
inhibitory actions of IFN-
-activated macrophages on viral
replication are mediated by nitric oxide (13-16). Also, iNOS-deficient
mice have higher mortality rates and reduced viral clearance as
compared with wild-type mice following virus infection (17-19). These
findings support a role for nitric oxide, produced following iNOS
expression, in the antiviral response.
(4). In addition, dsRNA + IFN-
stimulate iNOS expression to similar levels in peritoneal
macrophages (PEC) isolated from PKR
/
and
PKR+/+ mice (4). These findings suggest that PKR may
participate in, but is not essential for, dsRNA-induced iNOS
expression. In contrast, nuclear factor
B (NF-
B) activation
appears to be required for iNOS and IL-1 expression by macrophages.
Inhibition of NF-
B activation prevents iNOS and IL-1 expression
stimulated by dsRNA in RAW 264.7 cells and dsRNA + IFN-
in primary
macrophages (9). NF-
B is comprised of heterodimer or homodimers of
the p50/NF-
B1 and p65/RelA subunits and is sequestered in the
cytoplasm of unstimulated cells in complex with inhibitor protein
B
(I
B, Ref. 20). Pathways that stimulate the activation of NF-
B are
characterized by I
B phosphorylation, polyubiquitination, and
degradation by the 26 S proteasome complex (21). This allows for the
release of NF-
B and its translocation to the nucleus. PKR has been
implicated in the activation of NF-
B, either by directly stimulating
I
B phosphorylation or by activating I
B kinase (IKK) through
physical interaction (22-24). Recent studies have identified a
PKR-independent pathway that results in the activation of NF-
B by
dsRNA. Magun and co-workers (25) have shown that dsRNA and
encephalomyocarditis virus (EMCV) infection stimulates NF-
B
activation to similar levels in mouse embryonic fibroblasts (MEF)
isolated from PKR
/
and PKR+/+ mice. In
addition, dsRNA stimulates I
B degradation and NF-
B nuclear
localization to similar levels in macrophages and islets of Langerhans
isolated from PKR
/
and PKR+/+ mice (4,
26).
/
MEF while JNK
appeared to be activated by a PKR-independent mechanism (24). More
recently, dsRNA and viral infection have been shown to stimulate p38
and JNK activation in MEF isolated from PKR
/
mice.
While JNK activation appears to require a PKR-dependent inhibition of protein synthesis (25), p38 activation by dsRNA is not
dependent on PKR or PKR-mediated inhibition of protein synthesis (25).
While these findings indicate that MAPK are activated by dsRNA, the
role of PKR and the mechanisms by which PKR may mediate MAPK activation
have yet to be fully elucidated.
reporter activity, and IL-1 release by macrophages. The role of ERK in
the regulation of inflammatory gene expression in response to virus
infection appears to be selective for the IL-1 pathway, as ERK
inhibition does not affect the ability of dsRNA to stimulate iNOS
expression or nitrite production by macrophages. Using peritoneal
macrophages isolated from PKR+/+ and PKR
/
mice we show that the genetic absence of PKR does not modulate the
ability of dsRNA to induce IL-1 expression and release or to stimulate
ERK phosphorylation. These findings support a role for ERK activation
in the PKR-independent regulation of the antiviral response of IL-1
expression and release by macrophages.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter luciferase reporter plasmid
(XL-Luc) has been previously described (32) and was provided by Dr.
Matthew Fenton (Boston University, Boston, MA). Rabbit anti-phospho-ERK was purchased from Promega (Madison, WI). Rabbit anti-ERK1 was a gift
from Dr. John C. Lawrence, Jr. (University of Virginia, Charlottesville, VA). Rabbit anti-mouse macrophage iNOS was a gift from
Dr. Tom Misko (Amersham Biosciences). 3ZD monoclonal mouse anti-IL-1
was from Biological Resources Branch, DCTD at the NCI.
PKR
/
mice (C57BL/6(J)×SV129 background) were the
generous gift of Dr. Randal Kaufman (University of Michigan, Ann Arbor,
MI) and have been previously described (33-35). C57BL/6(J)×SV129
(PKR+/+) mice were obtained from Jackson Laboratories (Bar
Harbor, ME). iNOS and cyclophilin cDNA were the generous gift of
Dr. Charles Rodi (Amersham Biosciences) and Dr. Steve Carroll
(University of Alabama-Birmingham, Birmingham, AL), respectively.
IL-1
and IL-1
cDNAs were gifts from Dr. Cliff Bellone (Saint
Louis University, St. Louis, MO) and have been previously described
(36). The wild-type ERK1 plasmid was the gift of Dr. Joseph Baldassare
(Saint Louis University). The B-strain of encephalomyocarditis virus (EMCV) was the generous gift of Dr. J. W. Yoon (University of Calgary,
Calgary, Alberta, Canada). All other reagents were obtained from
commercially available sources.
/
and PKR+/+ mice by lavage as
previously described (37). After isolation, 4 × 105
cells/condition were incubated in 400 µl of complete CMRL-1066 (CMRL-1066 containing 10% heat-inactivated fetal bovine serum, 1×
L-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin) for 2 h under an atmosphere of 5%
CO2, 95% air. The cells were washed three times with 400 µl of complete CMRL-1066 to remove non-adherent cells before
initiation of experiments. RAW 264.7 cells (4 × 105/400 µl of DME) were infected with the
indicated amounts of EMCV virus in 100 µl of DME for 30 min, 300 µl
of DME was added, and the cells were incubated for 24 additional hours.
EMCV was propagated as previously described (15).
and IL-1
.
, 1:2,000; rabbit anti-phospho-ERK, 1:1,000; rabbit
anti-phospho-p38, 1:1,000; rabbit anti-phospho-JNK, 1:1,000; rabbit
anti-ERK1, 1:1,000; rabbit anti-JNK, 1:1,000; horseradish
peroxidase-conjugated donkey anti-mouse, 1:5,000; and horseradish
peroxidase-conjugated donkey anti-rabbit, 1:7,000. Antigen detection
was by ECL (Amersham Biosciences) according to the manufacturer's specifications.
, IL-1
, cyclophilin,
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes as
previously described (9, 40). Cyclophilin was used as an internal RNA
loading control for Northern blots, and GAPDH used as a control for the
RT reaction.
Reporter Assays--
RAW
264.7 cells were transiently transfected with 1 µg of the human
IL-1
luciferase reporter (positions
3757 to +11; XL-LUC) and 1 µg of the pCMV-SPORT-
-galactosidase (Invitrogen) control plasmid
using the Qiagen Superfect reagent according to the manufacturer's instructions. After an 8-h incubation at 37 °C experiments were initiated by addition of poly(IC) or poly(IC) and U0126 as indicated in
the figure legends. Twenty-four hours after stimulation, the cells were
harvested and lysed in Reporter Lysis Buffer, and luciferase activity
was determined using the luciferase assay system according to the
manufacturer's specifications (Promega).
-galactosidase assays were
performed as previously described (41). Luciferase activities are
reported relative to
-galactosidase activity to control for
transfection efficiencies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
Effects of MEK inhibition on dsRNA-induced
iNOS expression and nitrite production by RAW 264.7 macrophages.
RAW 264.7 cells (4 × 105/400 µl of DME) were
pretreated for 30 min with the indicated concentrations of U0126,
poly(IC) (50 µg/ml) was added, and the cells were cultured for 24 additional hours. Nitrite production was determined on culture
supernatants (A) and the expression of iNOS at the protein
level was determined by Western blot analysis of the isolated cells
(B). C, RAW 264.7 cells (5 × 106/2 ml of DME) were pretreated with U0126 or
Me2SO (vehicle control, DMSO) for 30 min at
37 °C, poly(IC) (50 µg/ml) was added, and the cells were incubated
for 6 additional hours. Total RNA was isolated and used to determine
iNOS mRNA accumulation by Northern blot analysis. Cyclophilin
mRNA accumulation is shown as a control for RNA loading. Results
for nitrite production are the average ± S.E. of three
independent experiments, and iNOS protein expression and mRNA
accumulation are representative of three independent experiments.
protein (Fig. 2, A
and B, respectively). Consistent with IL-1 release,
treatment of RAW 264.7 cells with poly(IC) stimulates the accumulation
of both IL-1
and IL-1
mRNA as determined by Northern blot
analysis following a 6-h incubation, and IL-1
luciferase reporter
activity following a 24-h incubation (Fig. 2, C and
D, respectively). The MEK selective inhibitor U0126 attenuates poly(IC)-induced IL-1
and IL-1
mRNA accumulation, IL-1
promoter activity, pro-IL-1
protein expression, and IL-1 release by RAW 264.7 cells, with maximal inhibition at 5 µM.
View larger version (27K):
[in a new window]
Fig. 2.
MEK inhibition prevents dsRNA-induced IL-1
expression and release by RAW 264.7 macrophages. RAW 264.7 cells
(4 × 105/400 µl of DME) were pretreated with the
indicated concentrations of U0126 for 30 min, poly(IC) (50 µg/ml) was
added, and the cells were cultured for 24 additional hours. IL-1
released into the culture supernatant was determined using the RINm5F
cell bioassay (A), and pro-IL-1 protein expression was
determined by Western blot analysis of the isolated cells
(B). RAW 264.7 cells (5 × 106/2 ml DME)
were pretreated with U0126 or Me2SO (vehicle control,
DMSO) for 30 min, poly(IC) was added, and the cells were
incubated for 6 additional hours at 37 °C. Total RNA was isolated
and used to determine IL-1
and IL-1
mRNA accumulation by
Northern blot analysis (C). Cyclophilin mRNA
accumulation is shown as a control for RNA loading. RAW 264.7 cells
(4 × 106/2 ml DME), transiently transfected with the
human IL-1
reporter construct XL-Luc, were pretreated for 30 min
with U0126, poly(IC) was added, and the cells were cultured for 24 additional hours. Total cellular protein was isolated and luciferase
activity determined as described under "Experimental Procedures."
The results for IL-1 release and IL-1
promoter activity are the
average ± S.E. of three independent experiments, pro-IL-1
protein expression and IL-1 mRNA accumulation are representative of
three independent experiments.
and IL-1
mRNA, and protein
expression in response to poly(IC) treatment was examined. PD98059 at
10 µM inhibits poly(IC)-induced IL-1
and IL-1
mRNA accumulation and pro-IL-1
protein expression (Fig.
3, A and B). To
confirm the selectivity of the ERK inhibitors, the effects of PD98509 and U0126 on dsRNA-induced p38 and JNK phosphorylation were examined. At concentrations that prevent dsRNA-induced IL-1
and IL-1
expression, PD98059 (10 µM, Fig. 3C) and U0126
(5 µM, data not shown) do not attenuate
poly(IC)-stimulated p38 or JNK phosphorylation. These findings suggest
that dsRNA selectively regulates iNOS and IL-1 expression by
macrophages, in that ERK appears to participate in the transcriptional
activation of IL-1 but does not regulate iNOS expression in response to
dsRNA.
View larger version (51K):
[in a new window]
Fig. 3.
Effects of PD98059 on dsRNA-induced IL-1
expression by RAW 264.7 macrophages. RAW 264.7 cells (5 × 106/2 ml DME) were pretreated with PD98059 for 30 min,
poly(IC) was added, and the cells were incubated for 6 additional hours
at 37 °C. Total RNA was isolated and used to determine IL-1 and
IL-1
mRNA accumulation by Northern blot analysis (A).
Cyclophilin mRNA accumulation is shown as a control for RNA
loading. RAW 264.7 cells (4 × 105/400 µl of DME)
were pretreated with the indicated concentrations of PD98059 for 30 min, poly(IC) (50 µg/ml) was added, and the cells were cultured for
24 additional hours. The cells were then isolated and pro-IL-1
protein expression was determined by Western blot analysis
(B). The concentration-dependent effects of PD98059
on dsRNA-induced p38 and JNK phosphorylation following a 30-min
incubation were examined by Western blot analysis using phosphospecific
(p38-P, JNK-P) antisera (C). Total JNK is shown as a loading
control. The results IL-1 mRNA accumulation, pro-IL-1
protein
expression, and p38 and JNK phosphorylation are representative of three
independent experiments.
by RAW 264.7 cells
were evaluated. As shown in Fig.
4A, infection of RAW 264.7 cells with EMCV stimulates the accumulation of IL-1
following a 24-h
incubation. Importantly, EMCV-induced IL-1
expression is attenuated
by U0126 (5 µM, Fig. 4B) at a concentration
that prevents poly(IC)-induced IL-1 expression by RAW 264.7 cells (Fig.
2). These findings indicate that EMCV and poly(IC) elicit similar
antiviral responses in RAW 264.7 cells and provide additional evidence
to support a role for ERK in the regulation of IL-1
expression by
macrophages in response to a viral infection.
View larger version (28K):
[in a new window]
Fig. 4.
Effects of MEK inhibition on EMCV-induced
pro-IL-1 expression by RAW 264.7 cells.
A, RAW 264.7 cells (4 × 105/400 µl DME)
were infected with the indicated multiplicity of infection (MOI) of
EMCV for 24 h, the cells were isolated and pro-IL-1
expression
was determined by Western blot analysis. B, RAW 264.7 cells,
pretreated with U0126 for 30 min, were infected for 24 h with EMCV
at an MOI of 0.25. The cells were then isolated, and pro-IL-1
expression was determined by Western blot analysis. Results are
representative of three independent experiments.
and IL-1
mRNA accumulation by macrophages in response to dsRNA and EMCV infection. A 6-h incubation with poly(IC)
results in the accumulation of both IL-1
and IL-1
mRNA in RAW
264.7 cells transfected with wild-type ERK (Fig. 5C). In contrast, IL-1
and IL-1
mRNA accumulation is attenuated in
RAW 264.7 cells transfected with dnERK. In a similar fashion,
EMCV-induced IL-1
and IL-1
mRNA accumulation is attenuated in
RAW 264.7 cells transfected with dnERK (Fig. 5D). The
transfection efficiency in these experiments was greater then 70% (as
determined by
-galactosidase staining, and the levels of wild type
and dnERK expression were comparable to those shown in Fig.
5B, inset). These findings indicated that dsRNA
stimulates ERK activation and provide further evidence to support a
role for ERK in the regulation of dsRNA- and EMCV-induced IL-1
expression by macrophages.
View larger version (42K):
[in a new window]
Fig. 5.
dsRNA stimulates ERK activation and dnERK
inhibits dsRNA-induced IL-1 expression by macrophages.
A, RAW 264.7 cells (4 × 105/400 µl DME)
were pretreated with 5 µM U0126 for 30 min, poly(IC) (50 µg/ml) was added, and the cells were cultured for an additional 30 min. The cells were isolated and phosphorylated ERK (p44-P and p42-P)
and p44 ERK (loading control) were determined by Western blot analysis.
RAW 264.7 cells (4 × 105/2 ml of DME), transiently
transfected with ERK or dnERK, were treated with 50 µg/ml poly(IC)
for 30 min, and ERK activity was determined by immunocomplex kinase
assay (B). The expression levels of ERK and dnERK were
determined by Western blot analysis and are shown in the
inset (B). RAW 264.7 cells (4 × 105/2 ml DME), transiently transfected with ERK or dnERK,
were treated with 50 µg/ml poly(IC) (C) or EMCV (0.25 MOI,
D) for 6 h, total RNA was isolated and used to examine
IL-1 and IL-1
mRNA accumulation by RT-PCR. GAPDH mRNA
accumulation was used as an internal control for the RT and PCR
reactions. The results for ERK phosphorylation and IL-1 mRNA
accumulation are representative of three independent experiments. The
results for ERK kinase activity are the average of two independent
experiments.
-induced nitrite
production (Fig. 6A) or iNOS expression (Fig. 6B) by PEC isolated from
PKR
/
and PKR+/+ mice. In contrast to RAW
264.7 cells, naive mouse peritoneal macrophages require two
proinflammatory signals for iNOS expression and nitrite production
(44), and we have previously shown that neither poly(IC) nor IFN-
alone stimulate iNOS expression or nitric oxide production by PEC (4,
9). Also, we have shown that PKR is not required for poly(IC) + IFN-
-induced iNOS expression and nitric oxide production by mouse
PEC (4).
View larger version (30K):
[in a new window]
Fig. 6.
MEK inhibition does not affect dsRNA + IFN- -induced iNOS expression or nitrite
production by peritoneal macrophages. Peritoneal macrophages
isolated from PKR+/+ and PKR
/
mice (4 × 105 cells/400 µl of complete CMRL-1066) were
preincubated for 30 min with U0126, poly(IC) was added, and the cells
were cultured for 24 additional hours. Nitrite production was
determined on the culture supernatants (A), and iNOS protein
expression determined by Western blot analysis of the isolated cells
(B). The results for nitrite production are the average ± S.E. of three independent experiments, and iNOS protein expression
is representative of three independent experiments.
-induced
IL-1 release by mouse macrophages. Treatment of PEC isolated from
PKR
/
and PKR+/+ mice for 24 h with
either poly(IC) or poly(IC) + IFN-
results in the release of IL-1 to
similar levels, and this IL-1 release is prevented by U0126 (Fig.
7A). U0126 also prevents
poly(IC) (data not shown) and poly(IC) + IFN-
-induced IL-1
protein accumulation in PEC isolated from PKR
/
and
PKR+/+ mice (Fig. 7B). These findings, which are
consistent with the inhibitory actions of U0126 on IL-1 expression and
release by RAW 264.7 cells, support a PKR-independent role for ERK in
dsRNA-induced IL-1 expression and release by primary mouse
macrophages.
View larger version (35K):
[in a new window]
Fig. 7.
MEK inhibition prevents dsRNA and dsRNA + IFN- -induced IL-1 expression and release by
PEC isolated from PKR
/
and
PKR+/+ mice. Peritoneal macrophages
isolated from PKR+/+ and PKR
/
mice (4 × 105/400 µl of complete CMRL-1066) were pretreated with
U0126 or the vehicle control Me2SO (DMSO) for 30 min, poly(IC) (50 µg/ml), and IFN-
(150 units/ml) were added, and
the cells were cultured for 24 additional hours. IL-1 released into the
culture supernatants was determined using the RINm5F cell bioassay
(A), and pro-IL-1
protein expression determined by
Western blot analysis of the isolated cells (B). The
time-dependent effects of poly(IC) on ERK activation
(C), and the inhibitory actions of U0126 on
poly(IC)-stimulated ERK activation following a 30-min incubation
(D) were examined in PEC isolated from PKR
/
and PKR+/+ mice by Western blot analysis using antisera
specific for phospho-ERK (p44-P, p42-P) and total
ERK (p44). Results for IL-1 release are the average ± S.E. of
three independent experiments, and the results for IL-1
protein
expression and ERK phosphorylation are representative of three
independent experiments.
/
and
PKR+/+ mice were examined. As shown in Fig. 7C,
poly(IC) stimulates the time-dependent phosphorylation of
ERK that is first apparent following a 15-min incubation and that
persists for up to 60 min. PKR does not appear to participate in ERK
activation, as poly(IC) stimulates ERK phosphorylation to similar
levels in PEC isolated form PKR
/
and PKR+/+
mice. In addition, the stimulatory actions of a 30-min incubation with
poly(IC) on ERK phosphorylation are prevented by U0126 (5 µM, Fig. 7D). These findings indicate that the
presence of PKR is not required for the activation of ERK by dsRNA in
PEC.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and MAPK
activation in response to dsRNA. PKR was originally believed to be
required for dsRNA-induced NF-
B activation and p38 phosphorylation
based on studies in MEF isolated from PKR
/
mice (24,
35). More recently, dsRNA and EMCV infection have been shown to
stimulate NF-
B and p38 activation in MEF isolated from
PKR
/
mice to levels similar to MEF isolated from
wild-type mice (25), suggesting that PKR-independent pathways also
participate in the antiviral response.
/
mice.
These findings suggest that PKR is not required for the activation of
ERK or the expression and release of IL-1 by macrophages in response to
dsRNA or viral infection. However, this conclusion must be tempered by
the possibility that other "PKR-like" molecules may compensate for
the absence of PKR (in PKR-deficient macrophages), and the recent
evidence that the PKR
/
mice used in this study may be
incomplete knockouts (47).
B have been shown
to prevent dsRNA-induced IL-1 expression and release by mouse
macrophages (9). In addition, interferon regulatory factor-4 in
combination with PU.1 has been shown to synergistically activate the
human IL-1 reporter by interacting at the distal PU.1 consensus binding
element (32, 48). Importantly, this transcriptional regulation of
antiviral response genes following viral infection or treatment with
dsRNA appears to be selective. We show that dsRNA-stimulated iNOS
expression is not sensitive to inhibitors of ERK, while ERK inhibition
prevents virus and dsRNA-stimulated IL-1 expression and release.
Recently, we have identified a role for the calcium-independent
phospholipase A2 (iPLA2) in the regulation of
dsRNA and EMCV-induced iNOS expression, and the role of
iPLA2 in this antiviral response appears to be selective
for iNOS as inhibitors of this phospholipase fail to modulate IL-1
expression in response to dsRNA (49). In this study we have not
examined the upstream molecules that activate ERK; however, it is
likely that the recently described receptor for dsRNA, TLR3 mediates
ERK activation (50). These findings indicate that activation of the
antiviral responses is cell type selective and that the pathways that
regulate specific antiviral responses may be selective for individual
target genes or cellular responses.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Colleen Bratcher for expert
technical assistance and Dr. Joseph Baldassare for advice concerning
the immunocomplex kinase assays. We also thank Dr. Matthew Fenton for
the human IL-1 luciferase reporter construct and Dr. J. W. Yoon for
providing EMC virus.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK-52194 and AI-44458 (to J. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Internal Medicine, Division of Molecular
Oncology, Washington University School of Medicine, St. Louis, MO 63110.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8165; Fax: 314-577-8156; E-mail: corbettj@slu.edu.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M211744200
2 L. B. Maggi and J. A. Corbett, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ds, double-stranded;
IL, interleukin;
INF-, interferon-
;
iNOS, inducible nitric-oxide
synthase;
poly(IC), polyinosinic-polycytidylic acid;
ERK, extracellular
signal-regulated kinase;
PKR, dsRNA-dependent protein
kinase;
MEF, mouse embryonic fibroblasts;
MAPK, mitogen-activated
protein kinase;
PEC, peritoneal exudate cells;
JNK, Jun N-terminal
kinase;
DME, Dulbecco's modified Eagle's medium.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Jacobs, B. L., and Langland, J. O. (1996) Virology 219, 339-349[CrossRef][Medline] [Order article via Infotrieve] |
2. | Hovanessian, A. G. (1993) in Translational regulation of gene expression (Ilan, J., ed) , pp. 163-185, Plenum Press, New York |
3. | Samuel, C. E., Kuhen, K. L., George, C. X., Ortega, L. G., Rende-Fournier, R., and Tanaka, H. (1997) Int. Journal Hematol. 65, 227-237[CrossRef] |
4. |
Maggi, L. B., Jr.,
Heitmeier, M. R.,
Scheuner, D.,
Kaufman, R. J.,
Buller, R. M.,
and Corbett, J. A.
(2000)
EMBO J.
19,
3630-3638 |
5. |
Koromilas, A. E.,
Cantin, C.,
Craig, A. W.,
Jagus, R.,
Hiscott, J.,
and Sonenberg, N.
(1995)
J. Biol. Chem.
270,
25426-25434 |
6. |
Bandyopadhyay, S. K.,
de La Motte, C. A.,
and Williams, B. R.
(2000)
J. Immunol.
164,
2077-2083 |
7. | Offermann, M. K., Zimring, J., Mellits, K. H., Hagan, M. K., Shaw, R., Medford, R. M., Mathews, M. B., Goodbourn, S., and Jagus, R. (1995) Eur. J. Biochem. 232, 28-36[Abstract] |
8. | Gale, M., Jr., and Katze, M., G. (1998) Pharmacol. Therap. 78, 29-46[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Heitmeier, M. R.,
Scarim, A. L.,
and Corbett, J. A.
(1998)
J. Biol. Chem.
273,
15301-15307 |
10. |
Eliopoulos, A. G.,
Gallagher, N. J.,
Blake, S. M.,
Dawson, C. W.,
and Young, L. S.
(1999)
J. Biol. Chem.
274,
16085-16096 |
11. | Milhaud, P. G., Machy, P., Colote, S., Lebleu, B., and Leserman, L. (1991) J. Interfer. Res. 11, 261-265[Medline] [Order article via Infotrieve] |
12. |
Nath, A.,
Conant, K.,
Chen, P.,
Scott, C.,
and Major, E. O.
(1999)
J. Biol. Chem.
274,
17098-17102 |
13. | Bukrinsky, M. I., Nottet, H. S., Schmidtmayerova, H., Dubrovsky, L., Flanagan, C. R., Mullins, M. E., Lipton, S. A., and Gendelman, H. E. (1995) J. Exp. Med. 181, 735-745[Abstract] |
14. | Kreil, T. R., and Eibl, M. M. (1996) Virology 219, 304-306[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Hirasawa, K.,
Jun, H. S.,
Han, H. S.,
Zhang, M. L.,
Hollenberg, M. D.,
and Yoon, J. W.
(1999)
J. Virol.
73,
8541-8548 |
16. | Karupiah, G., Xie, Q. W., Buller, R. M., Nathan, C., Duarte, C., and MacMicking, J. D. (1993) Science 261, 1445-1448[Medline] [Order article via Infotrieve] |
17. | Flodstrom, M., Horwitz, M. S., Maday, A., Balakrishna, D., Rodriguez, E., and Sarvetnick, N. (2001) Virology 281, 205-215[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Reiss, C. S.,
and Komatsu, T.
(1998)
J. Virol.
72,
4547-4551 |
19. |
Zaragoza, C.,
Ocampo, C. J.,
Saura, M.,
Bao, C.,
Leppo, M.,
Lafond-Walker, A.,
Thiemann, D. R.,
Hruban, R.,
and Lowenstein, C. J.
(1999)
J. Immunol.
163,
5497-5504 |
20. | Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-179[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Zandi, E.,
and Karin, M.
(1999)
Mol. Cell. Biol.
19,
4547-4551 |
22. | Kumar, A., Haque, J., Lacoste, J., Hiscott, J., and Williams, B. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6288-6292[Abstract] |
23. | Ishii, T., Kwon, H., Hiscott, J., Mosialos, G., and Koromilas, A. E. (2001) Oncogene 20, 1900-1912[CrossRef][Medline] [Order article via Infotrieve] |
24. | Chu, W. M., Ostertag, D., Li, Z. W., Chang, L., Chen, Y., Hu, Y., Williams, B., Perrault, J., and Karin, M. (1999) Immunity 11, 721-731[Medline] [Order article via Infotrieve] |
25. |
Iordanov, M. S.,
Wong, J.,
Bell, J. C.,
and Magun, B. E.
(2001)
Mol. Cell. Biol.
21,
61-72 |
26. |
Blair, L. A.,
Heitmeier, M. R.,
Scarim, A. L.,
Maggi, L. B., Jr.,
and Corbett, J. A.
(2001)
Diabetes
50,
283-290 |
27. |
Harcourt, J. L.,
and Offermann, M. K.
(2001)
Eur. J. Biochem.
268,
1373-1381 |
28. | Williams, B. R. (1999) Oncogene 18, 6112-6120[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Pahan, K.,
Sheikh, F. G.,
Khan, M.,
Namboodiri, A. M.,
and Singh, I.
(1998)
J. Biol. Chem.
273,
2591-2600 |
30. |
Da Silva, J.,
Pierrat, B.,
Mary, J. L.,
and Lesslauer, W.
(1997)
J. Biol. Chem.
272,
28373-28380 |
31. |
Chan, E. D.,
Winston, B. W.,
Uh, S. T.,
Wynes, M. W.,
Rose, D. M.,
and Riches, D. W.
(1999)
J. Immunol.
162,
415-422 |
32. |
Marecki, S.,
Riendeau, C. J.,
Liang, M. D.,
and Fenton, M. J.
(2001)
J. Immunol.
166,
6829-6838 |
33. |
Der, S. D.,
Yang, Y. L.,
Weissmann, C.,
and Williams, B. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3279-3283 |
34. |
Kumar, A.,
Yang, Y. L.,
Flati, V.,
Der, S.,
Kadereit, S.,
Deb, A.,
Haque, J.,
Reis, L.,
Weissmann, C.,
and Williams, B. R.
(1997)
EMBO J.
16,
406-416 |
35. | Yang, Y. L., Reis, L. F., Pavlovic, J., Aguzzi, A., Schafer, R., Kumar, A., Williams, B. R., Aguet, M., and Weissmann, C. (1995) EMBO J. 14, 6095-6106[Abstract] |
36. | Godambe, S. A., Chaplin, D. D., and Bellone, C. J. (1993) Cytokine 5, 327-335[Medline] [Order article via Infotrieve] |
37. |
Beckerman, K. P.,
Rogers, H. W.,
Corbett, J. A.,
Schreiber, R. D.,
McDaniel, M. L.,
and Unanue, E. R.
(1993)
J. Immunol.
150,
888-895 |
38. | Hill, J. R., Corbett, J. A., Baldwin, A. C., and McDaniel, M. L. (1996) Anal. Biochem. 236, 14-19[CrossRef][Medline] [Order article via Infotrieve] |
39. | Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., and Tannenbaum, S. R. (1982) Anal. Biochem. 126, 131-138[Medline] [Order article via Infotrieve] |
40. |
Arnush, M.,
Scarim, A. L.,
Heitmeier, M. R.,
Kelly, C. B.,
and Corbett, J. A.
(1998)
J. Immunol.
160,
2684-2691 |
41. |
Baldassare, J. J.,
Bi, Y.,
and Bellone, C. J.
(1999)
J. Immunol.
162,
5367-5373 |
42. |
Weber, J. D.,
Hu, W.,
Jefcoat, S. C.,
Raben, D. M.,
and Baldassare, J. J.
(1997)
J. Biol. Chem.
272,
32966-32971 |
43. |
Chan, E. D.,
Morris, K. R.,
Belisle, J. T.,
Hill, P.,
Remigio, L. K.,
Brennan, P. J.,
and Riches, D. W.
(2001)
Inf. Immun.
69,
2001-2010 |
44. | Hibbs, J. B. J., Taintor, R. R., Vavrin, Z., Granger, D. L., Draiper, J.-C., Amber, I. J., and Lancaster, J. R. J. (1990) in Nitric oxide from L-Arginine: A Bioregulatory System (Moncada, S. , and Higgs, E. A., eds) , pp. 189-223, Elsevier Science Publishing Co., New York, NY |
45. | Barber, G. N. (2001) Cell Death Differ. 8, 113-126[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Scarim, A. L.,
Arnush, M.,
Blair, L. A.,
Concepcion, J.,
Heitmeier, M. R.,
Scheuner, D.,
Kaufman, R. J.,
Ryerse, J.,
Buller, R. M.,
and Corbett, J. A.
(2001)
Am. J. Pathol.
159,
273-283 |
47. |
Baltzis, D.,
Li, S.,
and Koromilas, A. E.
(2002)
J. Biol. Chem.
277,
38364-38372 |
48. | Marecki, S., and Fenton, M. J. (2000) Cell Biochem. Biophys. 33, 127-148[Medline] [Order article via Infotrieve] |
49. |
Maggi, L. B.,
Moran, J. M.,
Scarim, A. L.,
Ford, D. A.,
Yoon, J. W.,
Howat, J. M.,
Buller, R. M. L.,
and Corbett, J. A.
(2002)
J. Biol. Chem.
277,
38449-38455 |
50. | Alexopoulou, L., Holt, A. C., Medzhitov, R., and Flavell, R. A. (2001) Nature 413, 732-738[CrossRef][Medline] [Order article via Infotrieve] |