Double-Stranded RNADependent Protein Kinase Is Not Required for Double-Stranded RNAInduced Nitric Oxide Synthase Expression or Nuclear Factor-
B Activation by Islets
Libby A. Blair,
Monique R. Heitmeier,
Anna L. Scarim,
Leonard B. Maggi, Jr., and
John A. Corbett
From the Edward A. Doisy Department of Biochemistry and Molecular
Biology, St. Louis University School of Medicine, St. Louis, Missouri.
Address correspondence and reprint requests to Dr. John A. Corbett, St. Louis
University School of Medicine, Department of Biochemistry and Molecular
Biology, 1402 South Grand Blvd., St. Louis, MO 63104. E-mail:
corbettj{at}slu.edu
.
 |
ABSTRACT
|
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Environmental factors, such as viral infection, have been implicated in the
destruction of ß-cells during the development of autoimmune diabetes.
Double-stranded RNA (dsRNA), produced during viral replication, is an active
component of a viral infection that stimulates antiviral responses in infected
cells. Previous studies have shown that treatment of rat islets with dsRNA in
combination with
-interferon (IFN-
) results in a nitric
oxide-dependent inhibition of glucose-stimulated insulin secretion. This study
examines the role of nuclear factor-
B (NF-
B) and the
dsRNA-dependent protein kinase (PKR) in dsRNA + IFN-
-induced nitric
oxide synthase (iNOS) expression and nitric oxide production by rat, mouse,
and human islets. Treatment of rat and human islets with dsRNA in the form of
polyinosinic-polycytidylic acid (poly IC) and IFN-
resulted in iNOS
expression and nitric oxide production. Inhibitors of NF-
B
activationthe proteasome inhibitor MG-132 and the antioxidant
pyrrolidinedithiocarbamate (PDTC)prevented poly IC +
IFN-
-induced iNOS expression and nitric oxide production. Incubation of
rat islets for 3 h or human islets for 2 h with poly IC alone or poly IC +
IFN-
resulted in NF-
B nuclear translocation and degradation of
the NF-
B inhibitor protein, I
B, events that are prevented by
MG-132. PKR has been shown to participate in dsRNA-induced NF-
B
activation in a number of cell types, including mouse embryonic fibroblasts.
However, poly IC stimulated NF-
B nuclear translocation and I
B
degradation to similar levels in islets isolated from mice devoid of PKR
(PKR-/-) and wild-type mice (PKR+/+). Furthermore, the
genetic absence of PKR did not affect dsRNA + IFN-
-induced iNOS
expression, nitric oxide production, or the inhibitory actions of these agents
on glucose-stimulated insulin secretion. These results suggest that
1) NF-
B activation is required for dsRNA + IFN-
-induced
iNOS expression, 2) PKR is not required for either dsRNA-induced
NF-
B activation or dsRNA + IFN-
-induced iNOS expression by
islets, and 3) PKR is not required for dsRNA + IFN-
-induced
inhibition of glucose-stimulated insulin secretion by islets.
 |
INTRODUCTION
|
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Autoimmune diabetes is characterized by a local inflammatory reaction in
and around the pancreatic islets, followed by selective destruction of
insulin-producing ß-cells
(1). Viral infection has been
implicated as one environmental factor that may trigger the initial autoimmune
reaction that targets and destroys ß-cells in genetically susceptible
individuals
(2,3,4,5).
Viruses have been isolated from the pancreata of acutely diabetic deceased
patients, and viral-specific IgM responses have been identified in newly
diagnosed diabetic patients
(2,5).
Autoimmune diabetes can also be induced in genetically susceptible strains of
rats and mice by infection with Kilham rat virus
(6) and encephalomyocarditis
virus (7), respectively. In
these animal models, viral-induced diabetes is associated with increased
cytokine expression and nitric oxide production
(6,7).
Although this evidence supports a role for viral infection in the development
of diabetes, the mechanisms by which viruses initiate ß-cell damage have
been difficult to determine because multiple viruses from both RNA and DNA
viral families have been implicated in this disease. One common feature of a
viral infection is the formation of double-stranded RNA (dsRNA), which
accumulates during replication. dsRNA is an active component of a viral
infection that stimulates host antiviral responses
(8). The synthetic dsRNA
molecule polyinosinic-polycytidylic acid (poly IC) also activates the
antiviral response (8), and has
been shown to stimulate the development of diabetes in diabetes-resistant BB
rats and accelerate disease development in diabetes-prone BB rats
(9,10).
One cellular target activated in response to dsRNA is the transcriptional
regulator nuclear factor-
B (NF-
B)
(11). dsRNA has been shown to
stimulate NF-
B nuclear translocation in endothelial cells
(12) and murine macrophages
(13). NF-
B is a
heterodimer comprised of p50 and p65 (RelA) subunits, and is found sequestered
in the cytoplasm of unstimulated cells as an inactive complex with the
NF-
B inhibitory protein (I
B). Upon stimulation, I
B is
phosphorylated and degraded in a ubiquitin-dependent manner. Free of
I
B, the NF-
B heterodimer translocates to the nucleus and
activates mRNA transcription
(14). NF-
B is activated
by several cytokines, including interleukin-1 (IL-1) and tumor necrosis
factor-
(15), and
appears to play a primary role in the regulation of inducible nitric oxide
synthase (iNOS) gene expression
(16,17,18).
NF-
B activation is required for IL-1ß and IL-1ß +
IFN-
-induced iNOS expression by rat and human islets, respectively
(19,20).
The dsRNA-dependent protein kinase (PKR) is an important regulator of the
antiviral response. NF-
B activation in response to dsRNA appears to be
dependent on functional PKR. dsRNA-induced NF-
B activation is
attenuated in mouse embryonic fibroblasts isolated from PKR-deficient mice
(21,22).
PKR has been shown to phosphorylate I
B in vitro
(11), although recent evidence
suggests that PKR may mediate dsRNA-induced NF-
B nuclear localization
by activating the I
B kinase (IKK)
(23,24).
PKR is a 65-68 kDa serine/threonine kinase whose expression is induced by
interferons (IFNs) (25).
Binding to dsRNA, an event that leads to dimerization and autophosphorylation,
activates PKR (26). Once
activated, PKR participates in the antiviral response by inhibiting
translation through phosphorylation of eukaryotic initiation factor-2
(25). However, PKR is also
thought to participate in other cellular and antiviral responses, such as
transcription factor activation, cell cycle control, and apoptosis
(21,27,28).
In this study, we examined the roles of NF-
B and PKR in dsRNA +
IFN-
-induced iNOS expression and nitric oxide production by rat, mouse,
and human islets. We show that dsRNA (in the form of poly IC) stimulates the
activation of NF-
B in rat and human islets. Pyrrolidinedithiocarbamate
(PDTC) and MG-132, inhibitors of NF-
B, prevent dsRNA and dsRNA +
IFN-
-induced NF-
B nuclear localization and I
B degradation
as well as dsRNA + IFN-
-induced iNOS expression and nitric oxide
production by islets. Furthermore, the genetic absence of PKR does not
adversely affect dsRNA-induced NF-
B activation or dsRNA +
IFN-
-induced iNOS expression by mouse islets. These findings suggest
that 1) NF-
B activation is required for dsRNA +
IFN-
-induced iNOS expression by islets, and 2) dsRNA-induced
NF-
B activation and dsRNA + IFN-
-induced iNOS expression by
islets occur by PKR-independent mechanisms.
 |
RESEARCH DESIGN AND METHODS
|
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Materials and animals. CMRL-1066 tissue culture medium, L-glutamine,
penicillin, streptomycin, and rat recombinant IFN-
were obtained from
Gibco BRL-Life Technologies (Grand Island, NY). Fetal calf serum was obtained
from Hyclone (Logan, UT); human recombinant IL-1ß from Cistron
Biotechnology (Pine Brook, NJ); human IFN-
from Boehringer Mannheim
(Indianapolis, IN); and mouse IFN-
from R & D Systems (Minneapolis,
MN). Poly IC, PDTC, MG-132, and collagenase type XI were obtained from Sigma
(St. Louis, MO). [
-32P]ATP and enhanced chemiluminescence
reagents were obtained from Amersham Pharmacia Biotech (Arlington Heights,
IL). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG was
obtained from Jackson ImmunoResearch Laboratories (West Grove, PA), and rabbit
antiserum specific for the C-terminal 27 amino acids of mouse macrophage iNOS
was a generous gift from Dr. Thomas Misko (G.D. Searle, St. Louis, MO).
NF-
B consensus oligonucleotide, rabbit anti-human I
B
, and
rabbit anti-human nitric oxide synthase-2 (NOS-2) were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Male SD rats (250-300 g) and male
C57BL/6J mice (7-8 wks) were purchased from Harlan (Indianapolis, IN).
PKR-/- mice in a C57BL/6 x 129 background
(22) were a generous gift from
Dr. Randal J. Kaufman (University of Michigan Medical Center, Ann Arbor, MI).
C57BL/6 x 129 mice were obtained from Jackson Laboratories (Bar Harbor,
ME). Human islets were obtained from the Juvenile Diabetes Federation
International Islet Isolation Centers at Washington University School of
Medicine (St. Louis, MO), the Diabetes Research Institute at the University of
Miami (Miami, FL), and the Diabetes Institute for Immunology and
Transplantation (University of Minnesota). All other reagents were from
commercially available sources.
Islet isolation. Islets were isolated from male SD rats, male or
female PKR-/- mice, male C57BL/6J or male C57BL/6 x 129
(PKR+/+) mice by collagenase digestion as previously described
(29). After isolation, islets
were cultured overnight in complete CMRL-1066 (containing 2 mmol/l
L-glutamine, 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and
10 µg/ml streptomycin) under an atmosphere of 95% air and 5% CO2
at 37°C. Human islets were incubated for 48 h at 37°C in complete
CMRL-1066 before the initiation of experiments. Before each experiment, islets
were cleaned by hand picking, washed three times in complete CMRL-1066,
counted, and cultured for an additional 2 h at 37°C. No differences in
islet function or cytokine signaling were observed between male and female
PKR-/- mice. Data presented in Figs.
5 and
6 were obtained using islets
isolated from C57BL/6J mice. To control for potential strain differences, the
effects of dsRNA + IFN-
on iNOS expression, nitric oxide production,
and NF-
B activation were compared among islets isolated from C57BL/6
x 129 and C57BL/6J mice; no differences were observed (data not
shown).
Nitrite determination. Nitrite formation was determined by mixing 50
µl of culture medium with 50 µl of Griess reagent
(30). Absorbance was measured
at 540 nm, and nitrite concentrations were calculated from a sodium nitrite
standard curve.
Western blot analysis. Western blot analysis of iNOS and I
B
was performed as previously described
(31), using the following
antibody dilutions: rabbit anti-mouse iNOS (1:2,000 dilution), rabbit
anti-human I
B
(1:1,500 dilution), rabbit anti-human NOS-2
(1:1,000 dilution), and HRP-conjugated donkey anti-rabbit IgG (1:7,000
dilution). Antigen was detected by enhanced chemiluminescence according to
manufacturer's specifications.
Insulin secretion. Glucose-stimulated insulin secretion was
performed as previously described
(29). In brief, islets
isolated from PKR-/- and PKR+/+ mice (180/ml of complete
CMRL-1066) were cultured for 40 h with the indicated concentrations of poly
IC, IFN-
, IL-1, and aminoguanidine (AG). The islets were washed three
times in Krebs-Ringer bicarbonate buffer (KRBB; 25 mmol/l HEPES, 115 mmol/l
NaCl, 24 mmol/l NaHCO3, 5 mmol/l KCl, 1 mmol/l MgCl2,
2.5 mmol/l CaCl2, and 0.1% bovine serum albumin, pH 7.4) containing
3 mmol/l D-glucose, and then preincubated for 30 min in 200 µl KRBB
containing 3 mmol/l glucose. The preincubation buffer was removed and the
islets were incubated for 30 min in 200 µl of KRBB containing either 3 or
20 mmol/l glucose. The incubation buffer was removed and insulin content was
determined by radioimmunoassay
(32).
Nuclear extraction and gel shift analysis. After treatment with poly
IC and cytokines, islets were dispersed into single cells by trypsin treatment
(33) and nuclear proteins were
extracted as previously described
(13). The probe consisted of a
double-stranded oligonucleotide containing the consensus binding sequence for
NF-
B (5'-AGTTGAGGGGAC TTTCCCAGGC-3') end-labeled with
[
-32P]ATP using T4 polynucleotide kinase (Promega; Madison,
WI). Binding reactions consisted of 10 µg of nuclear protein extract, 0.5
ng of DNA probe, and 1 µg/ml of poly (dI-dC) in a buffer containing 10
mmol/l HEPES (pH 7.8), 50 mmol/l NaCl, 1 mmol/l EDTA, 10% glycerol, and 1
mmol/l dithiothreitol. Reactions were incubated at 30°C for 30 min, and
the NF-
B/oligonucleotide complex was separated on 4.5% Tris-glycine
nondenaturing polyacrylamide gels in a 2x Tris-glycine (50 mmol/l
Tris-HCl [pH 8.3], 0.38 mol/l glycine, and 2 mmol/l EDTA) buffer system
(34). Gels were dried on
Whatman paper and subjected to autoradiography.
Polymerase chain reaction. Total RNA was isolated from islets using
the RNeasy RNA isolation kit (QIAGEN, Santa Clarita, CA) and was used to
prepare first-strand cDNA by reverse transcription using the SuperScript
Preamplification System (Life Technologies, Grand Island, NY) as described
(35). iNOS,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and PKR primers were designed
according to these published sequences: 1) iNOS forward primer
5'-CCAACCGGAGAAGGGGACGAACT-3', reverse primer
5'-GGAGGGTGGTGCGGCTGGAC-3' (PCR product size = 297 bp);
2) GAPDH forward primer 5'-GCTGGGGCTCACCTGAAGGG-3',
reverse primer 5'-GGATGACCTTGCCACGCC-3' (PCR product size = 343
bp); and 3) murine PKR forward primer
5'-GCCAGATGCACGGAGTAGCC-3', reverse primer
5'-GAAAACTTGGCCAAATCCACC-3' (PCR product size = 722 bp). A
standard 25-µl PCR reaction of 30 cycles was performed
(35), and PCR products were
separated on 1.5% agarose gels containing 0.5 µg/ml ethidium bromide and
visualized by ultraviolet light exposure.
Statistical analysis. Statistical comparisons were made between
groups using a one-way analysis of variance. Significant differences between
groups (P < 0.05) were determined by Scheffe's F test
post-hoc analysis.
 |
RESULTS
|
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Effects of PDTC and MG-132 on poly IC + IFN-
-induced nitrite
formation and iNOS expression by rat islets. Recently we showed that dsRNA
+ IFN-
stimulates iNOS expression and nitrite production by rat islets,
and that nitric oxide mediates the inhibitory actions of these agents on
glucose-stimulated insulin secretion
(36). The role of NF-
B
in dsRNA + IFN-
-stimulated iNOS expression and nitric oxide production
was evaluated using PDTC and MG-132, which are inhibitors of NF-
B
activation
(37,38).
As shown in Fig. 1A,
the combination of 50 µg/ml poly IC and 150 U/ml IFN-
stimulated an
approximately fivefold increase in nitrite formation by rat islets. Alone,
neither poly IC nor IFN-
stimulated nitrite production by rat islets.
Pretreatment of rat islets for 2 h with 100 µmol/l PDTC or for 90 min with
50 µmol/l MG-132 inhibited dsRNA + IFN-
-induced nitrite formation by
90%. At these concentrations, PDTC
(19) and MG-132 (39; data not
shown) have been shown to maximally inhibit IL-1-induced NF-
B
activation by rat islets without adversely affecting islet total protein
synthesis or oxidative metabolism
(19,39).
As a control, the inhibitory effects of PDTC and MG-132 on IL-1-induced
nitrite production are also shown. In addition, neither PDTC nor MG-132 alone
stimulated nitrite production by rat islets, and DMSO, the vehicle control for
both inhibitors, neither stimulated nor inhibited nitrite production in
response to dsRNA + IFN-
(data not shown).
We previously reported that poly IC + IFN-
stimulates maximal iNOS
mRNA accumulation in rat islets after an 18-h incubation
(36). As shown in
Fig. 1B, pretreatment
of rat islets for 90 min with MG-132 (50 µmol/l) prevented poly IC- and
poly IC + IFN-
-induced iNOS mRNA accumulation, as determined by reverse
transcriptase-polymerase chain reaction (RT-PCR). Similar to iNOS mRNA
accumulation, MG-132 (50 µmol/l; data not shown) and PDTC (100 µmol/l)
also prevented poly IC + IFN-
-induced iNOS expression at the protein
level, as determined by Western blot analysis
(Fig. 1C). These
findings indicate that NF-
B activation may be required for dsRNA +
IFN-
-induced iNOS expression by rat islets. It is also clear from these
experiments that poly IC alone stimulates iNOS mRNA accumulation, but does not
induce iNOS protein expression or nitrite formation by rat islets. Previously,
we were unable to detect poly IC-induced iNOS mRNA accumulation in rat islets
by Northern blot analysis
(36). The detection of iNOS
mRNA accumulation in response to dsRNA in these studies appears to reflect the
increased sensitivity of PCR over Northern blot analysis in the evaluation of
mRNA expression.
Effects of poly IC on NF-
B activation in rat islets. To
determine whether dsRNA stimulates NF-
B activation in rat islets,
NF-
B nuclear localization was examined by gel shift analysis and
I
B degradation was determined by Western blot analysis. Treatment of
rat islets for 3 h with 50 µg/ml poly IC alone or in combination with 150
U/ml IFN-
resulted in the maximal nuclear translocation of NF-
B,
as demonstrated by the reduced mobility of the radio-labeled DNA probe
containing the NF-
B consensus sequence
(Fig. 2A; data not
shown for time-dependence of NF-
B nuclear translocation). As controls,
inclusion of antiserum specific for the p65 subunit of NF-
B in the
binding reaction further reduced the NF-
B/DNA complex migration
(supershift), and the addition of excess unlabeled NF-
B oligonucleotide
prevented the NF-
B/DNA complex formation (data not shown). Consistent
with NF-
B nuclear localization, treatment of rat islets for 3 h with 50
µg/ml poly IC and poly IC + 150 U/ml IFN-
resulted in the
degradation of I
B
. Pretreatment of islets for 90 min with MG-132
(50 µmol/l) attenuated poly IC- and poly IC + IFN-
-induced
NF-
B nuclear localization and I
B
degradation
(Fig. 2). Alone, neither MG-132
nor IFN-
stimulated NF-
B nuclear translocation or
I
B
degradation (data not shown). As a positive control, the
stimulatory effects of IL-1 on NF-
B nuclear translocation and
I
B
degradation in rat islets are also shown
(Fig. 2).
Effects of MG-132 on poly IC + IFN-
-induced nitrite formation and
iNOS expression by human islets. Treatment of human islets with dsRNA +
IFN-
resulted in the concentration-dependent expression of iNOS and
production of nitric oxide that was maximal at 400 µg/ml poly IC and 750
U/ml IFN-
(M.R.H. and J.A.C., unpublished observation). To determine if
NF-
B activation is required for dsRNA + IFN-
-induced nitric
oxide formation, human islets were pretreated for 90 min with 50 µmol/l
MG-132, followed by incubation with poly IC + IFN-
for 48 h. Alone,
neither poly IC nor IFN-
stimulated nitrite production by human islets
(Fig. 3A)
(40); however, in combination,
poly IC + IFN-
stimulated an approximately threefold increase in
nitrite formation. Similar to rat islets, MG-132 (50 µmol/l) prevented
nitrite formation by human islets in response to poly IC + IFN-
. As a
positive control, the inhibitory actions of MG-132 on IL-1 +
IFN-
-induced nitrite formation by human islets are also shown
(Fig. 3A)
(20,40).
Consistent with its inhibitory actions on nitrite formation, MG-132
prevented poly IC + IFN-
-induced iNOS expression by human islets
(Fig. 3B). Treatment
of human islets for 48 h with poly IC + IFN-
resulted in the expression
of iNOS to levels similar in magnitude to that induced by IL-1 + IFN-
.
MG-132 prevented both poly IC + IFN-
- and IL-1 + IFN-
-induced
iNOS expression by human islets. Alone, neither poly IC
(Fig. 3B) nor
IFN-
(39; data not shown) stimulated iNOS protein expression by human
islets.
Poly IC stimulates NF-
B activation in human islets. To
determine whether poly IC alone or in combination with IFN-
activates
NF-
B in human islets, NF-
B nuclear translocation was examined by
gel shift analysis. Treatment of human islets for 2 h with 400 µg/ml poly
IC or poly IC + 750 U/ml IFN-
stimulated maximal NF-
B nuclear
translocation (Fig.
4A; data not shown for time-dependence of NF-
B
nuclear translocation). MG-132 attenuated NF-
B nuclear translocation
induced by poly IC and poly IC + IFN-
. Alone, neither IFN-
(20) nor MG-132 (data not
shown) stimulated NF-
B nuclear localization in human islets. Consistent
with the effect of dsRNA on NF-
B nuclear localization, poly IC- and
poly IC + IFN-
-induced I
B
degradation was maximal after a
2-h incubation (Fig.
4B; data not shown for time-dependence of
I
B
degradation); the addition of MG-132 (50 µmol/l)
attenuated I
B
degradation under these conditions.
The genetic absence of PKR does not affect islet expression of iNOS or
production of nitrite in response to poly IC + IFN-
. PKR is
believed to regulate dsRNA-induced NF-
B activation by either directly
phosphorylating I
B or by activating the upstream I
B kinase, IKK
(11,22).
As shown in Figs.
1,2,3,4
NF-
B activation appears to be required for dsRNA-induced iNOS
expression by islets. Therefore, the effects of dsRNA + IFN-
on iNOS
expression and nitric oxide production were examined in islets isolated from
both PKR+/+ and PKR-/- mice. Islets isolated from either
PKR-/- or PKR+/+ mice were treated with poly IC +
IFN-
, which resulted in the production of similar levels of nitrite
after a 40-h incubation (Fig.
5A). Poly IC + IFN-
also stimulated iNOS
expression to similar levels in islets isolated from both PKR-/-
and PKR+/+ mice (Fig.
5B). Similar to human islets, a concentration of 400
µg/ml poly IC + 150 U/ml IFN-
was required to stimulate maximal iNOS
expression and nitrite production by mouse islets. Also, similar to rat and
human islets, neither poly IC nor IFN-
alone stimulated nitrite
production by islets isolated from PKR-/- and PKR+/+
mice. The level of nitrite produced by mouse islets in response to poly IC +
IFN-
was similar to the levels induced in response to 15 U/ml IL-1 +
150 U/ml IFN-
. RT-PCR was used to confirm the lack of PKR mRNA
accumulation in islets isolated from PKR-/- mice
(Fig. 5C).
Poly IC stimulates NF-
B nuclear translocation and I
B
degradation in islets isolated from PKR-/- and PKR+/+
mice. The effects of dsRNA on I
B degradation and NF-
B
nuclear translocation in islets isolated from PKR-/- and
PKR+/+ mice were examined by Western blot and gel shift analyses,
respectively. As shown in Fig. 6A
and B, a 1-h incubation with poly IC or poly IC +
IFN-
stimulated I
B degradation and NF-
B nuclear
localization in islets isolated from PKR-/- mice. The effects of
poly IC and poly IC + IFN-
on I
B degradation and NF-
B
nuclear localization were similar to those observed in islets isolated from
PKR+/+ mice. As a positive control, we also showed that a 30-min
incubation with IL-1 + IFN-
stimulated I
B degradation and
NF-
B nuclear translocation in islets isolated from both
PKR-/- and PKR+/+ mice.
Poly IC + IFN-
inhibits glucose-stimulated insulin secretion by
islets isolated from PKR-/- mice. We have previously shown that
dsRNA + IFN-
inhibits glucose-stimulated insulin secretion by rat
islets in a nitric oxidedependent manner
(36). To determine if PKR is
required for the inhibitory actions of poly IC + IFN-
on insulin
secretion, the effects of these agents on glucose-stimulated insulin secretion
by islets isolated from PKR-/- mice were examined. Treatment of
islets isolated from PKR-/- mice for 40 h with 400 µg/ml poly IC
+ 150 U/ml IFN-
resulted in
75% inhibition of glucose-stimulated
insulin secretion (Fig. 7). AG,
the iNOS selective inhibitor, prevented poly IC + IFN-
-induced
inhibition of insulin secretion by islets isolated from PKR-/-
mice, indicating that nitric oxide mediates the inhibitory actions of these
agents on islet function. Alone, neither poly IC nor IFN-
inhibited
glucose-stimulated insulin secretion by PKR-/- mouse islets
(Fig. 7). The inhibitory
actions of poly IC + IFN-
were similar in magnitude to the inhibitory
effects of 15 U/ml IL-1 + 150 U/ml IFN-
on glucose-stimulated insulin
secretion by islets isolated from PKR-/- mice. Similar results were
obtained in islets isolated from PKR+/+ mice (data not shown).
These results suggest that PKR is not required for the inhibitory actions of
dsRNA + IFN-
on glucose-stimulated insulin secretion by mouse
islets.
 |
DISCUSSION
|
---|
Viral infection has been implicated as one environmental factor that may
trigger ß-cell destruction during the development of autoimmune diabetes.
However, the mechanism by which a viral infection induces ß-cell damage
has been difficult to examine because multiple viruses from both DNA and RNA
genome families have been associated with the development of diabetes
(2,3,4,5).
dsRNA is an active component of a viral infection that stimulates antiviral
responses in infected cells
(8). Recently we showed that
treatment of rat islets and primary ß-cells with dsRNA + IFN-
results in a potent inhibition of glucose-stimulated insulin secretion and
islet degeneration, events that require ß-cell production of nitric oxide
(36). In this study, the role
of NF-
B in dsRNA + INF-
-induced iNOS expression and nitric oxide
production by islets was examined. We showed that dsRNA (in the form of poly
IC), in the presence or absence of IFN-
, stimulated NF-
B nuclear
translocation and I
B degradation, and that inhibition of NF-
B
activation prevented dsRNA + IFN-
-induced iNOS expression and nitric
oxide production by rat and human islets. These findings indicate that
NF-
B activation is required for iNOS expression by islets in response
to dsRNA, and provide further evidence supporting a key role for NF-
B
activation in the regulation of iNOS expression by rat and human islets
(19,20).
One mechanism by which dsRNA may activate NF-
B is by PKR-dependent
phosphorylation of I
B, an event that results in I
B degradation
by the proteasome complex
(11). In addition, PKR has
been shown to activate IKK in mouse embryonic fibroblasts, and this activation
results in I
B degradation and NF-
B nuclear localization
(23,24).
In this study, the role of PKR in both dsRNA + IFN-
-induced iNOS
expression and NF-
B activation was examined using islets isolated from
PKR-deficient mice. In contrast to previous studies
(21,22),
the genetic absence of PKR did not prevent dsRNA or dsRNA +
IFN-
-induced NF-
B nuclear translocation or I
B degradation
in islets isolated from PKR-/- mice. In addition, dsRNA +
IFN-
stimulated iNOS expression and nitric oxide production to similar
levels in islets isolated from PKR-/- and PKR+/+ mice.
Consistent with iNOS expression and nitric oxide production, treatment of
islets isolated from PKR-/- mice with poly IC + IFN-
resulted in a nitric oxidedependent inhibition of glucose-stimulated
insulin secretion. These findings indicate that PKR is not required for
dsRNA-induced NF-
B activation or dsRNA + IFN-
-induced iNOS
expression by isolated mouse islets. The mechanisms responsible for the
differences in dsRNA responsiveness of mouse embryonic fibroblasts as compared
to islets are unknown. However, in support of our findings in islets, we
recently showed that dsRNA + IFN-
-induced iNOS expression and
NF-
B activation in mouse peritoneal macrophages occur by
PKR-independent mechanisms
(41).
Although our findings indicate that PKR is not required for dsRNA-induced
NF-
B activation, the signaling pathways activated by dsRNA in islets
have yet to be identified. The mitogen-activated protein kinases (MAPKs) p38
and c-Jun NH2-terminal kinase (JNK), along with their upstream MAPK
kinases (MKK3, MKK4, and MKK6), are activated by dsRNA and viral infection in
mouse embryonic or immortalized 3T3 fibroblasts, HeLa, and Rat-1 cell lines
(23,42).
Importantly, dsRNA-induced p38 and JNK activation appears to be
PKR-independent; however, these stress kinases do not appear to directly
activate NF-
B in response to dsRNA
(23,42).
It is also possible that dsRNA may interact with other cellular proteins,
distinct from PKR, that contain dsRNA-binding motifs
(43), and that this
interaction may result in the activation of NF-
B. A number of novel
candidate dsRNA-binding proteins have been identified; however, a protein that
contains dsRNA-dependent kinase activity has yet to be described
(44,45,46,47).
Although we have not identified the kinase(s) that regulate dsRNA-induced
NF-
B activation in islets, our findings provide novel evidence that
dsRNA + IFN-
-induced iNOS expression and nitric oxide production
require NF-
B activation and occur by mechanisms that are independent of
PKR.
 |
ACKNOWLEDGMENTS
|
---|
This work was supported by National Institutes of Health Grants DK-52194
and AI-44458, and a Career Development Award from the Juvenile Diabetes
Foundation International (J.A.C.).
We thank Jessica Gorman, Joseph Martin, and Tracey Baird for expert
technical assistance and Dr. Randal Kaufman for providing the PKR-deficient
mice. We also thank the Juvenile Diabetes Federation International Islet
Isolation Centers at Washington University School of Medicine, the Diabetes
Research Institute at the University of Miami, and the Diabetes Institute for
Immunology and Transplantation (University of Minnesota) for providing human
islets used in these studies.
 |
FOOTNOTES
|
---|
AG, aminoguanidine; dsRNA, double-stranded RNA; HRP, horseradish
peroxidase; IFN, interferon; I
B, NF-
B inhibitor protein; IKK,
I
B kinase; IL-1, interleukin-1; iNOS, inducible nitric oxide synthase;
JNK, c-Jun NH2-terminal kinase; KRBB, Krebs-Ringer bicarbonate
buffer; MAPK, mitogen-activated protein kinase; NF-
B, nuclear
factor-
B; NOS-2, nitric oxide synthase-2; PDTC,
pyrrolidinedithiocarbamate; PKR, dsRNA-dependent protein kinase; poly IC,
polyinosinic-polycytidylic acid; RT-PCR, reverse transcriptase-polymerase
chain reaction.
Received for publication January 25, 2000
and accepted in revised form October 4, 2000
 |
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