Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104
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ABSTRACT |
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The
inhibitory actions of 15-deoxy-12,14-prostaglandin
J2 (PGJ2) on inflammatory gene expression have
been attributed to the ability of this prostaglandin to inhibit the
activation of NF-
B. In this study, we have identified an additional
signaling pathway sensitive to inhibition by PGJ2. We show
that PGJ2 inhibits interferon (IFN)-
-stimulated phosphorylation and DNA-binding activity of STAT1. The inhibitory actions on STAT1 phosphorylation are first apparent after a 1- to 2-h
incubation and are maximal after a 6-h incubation with PGJ2, and they correlate with the expression of heat shock
protein (HSP)70 in islets. In previous studies, we have correlated the inhibitory actions of PGJ2 on inducible nitric oxide
synthase (iNOS) expression and NF-
B activation in response to IL-1
with the increased expression of HSP70. Using overexpression and
antisense depletion, we provide evidence that HSP70 does not mediate
the inhibitory actions of PGJ2 on IL-1-induced NF-
B or
IFN-
-induced STAT1 activation or cytokine-stimulated iNOS expression
by
-cells. Last, we show that the inhibitory actions of a short 6-h
pulse with PGJ2 on IL-1 plus IFN-
-stimulated iNOS
expression and NO production by
-cells are persistent for extended
periods (
48 h). These findings suggest that PGJ2 inhibits
multiple cytokine-signaling pathways (IL-1 and IFN-
), that the
inhibitory actions are persistent for extended periods, and that
increased HSP70 expression correlates with, but does not appear to
mediate, the inhibitory actions of PGJ2 on IL-1 and IFN-
signaling in
-cells.
interferon-; signal transducer and activator of transcription-1; peroxisome proliferator-activated receptor-
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INTRODUCTION |
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AUTOIMMUNE DIABETES IS
CHARACTERIZED by islet inflammation followed by the selective
destruction of insulin-secreting -cells found in pancreatic islets
of Langerhans (14). Cytokines released from inflammatory
macrophages and T cells are believed to participate in
-cell damage
during diabetes development (3, 35). Treatment of rat
islets with IL-1 results in a time- and concentration-dependent inhibition of glucose-stimulated insulin secretion that is followed by
islet degeneration (8, 22, 29). The inhibitory and
destructive actions of IL-1 on
-cell function correlate with the
time-dependent expression of inducible nitric oxide synthase (iNOS) and
production of nitric oxide and can be prevented by inhibitors of iNOS
(10, 12, 36). Alone, interferon (IFN)-
does not appear
to modulate
-cell function; however, IFN-
has been shown to both
prime and potentiate the stimulatory actions of IL-1 on iNOS expression by rat islets (19, 21). In addition, a combination
of IL-1 and IFN-
is required to stimulate iNOS expression by mouse
and human
-cells (6, 41). Nitric oxide, produced
following iNOS expression, impairs
-cell function by inhibiting
mitochondrial oxidative metabolism, and this inhibition in
mitochondrial function, coupled with the ability of nitric oxide to
induce DNA damage, appears to mediate islet cell death (12, 18,
24).
In an effort to identify mechanisms to protect -cells from
cytokine-mediated damage, we have shown that IL-1 fails to inhibit glucose-stimulated insulin secretion by rat islets heat shocked before
cytokine stimulation (33). The protective actions of heat
shock on
-cell function are associated with the accumulation of heat
shock protein (HSP)70, the inhibition of IL-1-induced NF-
B
activation, and the inhibition of IL-1-induced iNOS expression (33). Consistent with the protective actions of heat
shock, liposomal delivery of HSP70 has been shown to prevent the
inhibitory actions of IL-1 on glucose-stimulated insulin secretion
(27), and the overexpression of HSP70 in insulinoma cell
lines prevents the damaging actions of nitric oxide, delivered
exogenously, on cell viability (1). These findings suggest
that HSP70 may protect
-cells from the damaging actions of cytokines
by inhibiting signaling pathways activated by cytokine treatment or by
attenuating the damaging actions of free radicals on
-cell function.
Peroxisome proliferator-activated receptor- (PPAR
)
agonists are known insulin sensitizers that have been shown to
stimulate the expression of HSP70 in a number of cell types, including
RINm5F cells and islets (5, 11, 26). We have shown
that a 6-h pretreatment of RINm5F cells or rat islets with the
naturally occurring PPAR
agonist
15-deoxy-
12,14-prostaglandin J2
(PGJ2) and the synthetic agonist troglitazone stimulates HSP70 expression and that HSP70 expression correlates with
an impairment of IL-1-induced NF-
B activation and JNK
phosphorylation (26). In contrast, a 30-min preincubation
of islets or RINm5F cells with either agonist does not stimulate HSP70
expression and also does not inhibit the ability of IL-1 to stimulate
JNK phosphorylation or NF-
B activation (26).
Importantly, NF-
B activation is required for
-cell expression of
iNOS in response to IL-1 treatment (16, 17, 25). Although
previous studies have shown that PGJ2 attenuates NF-
B
activation by directly inhibiting I
B kinase (IKK) as well as
inhibiting NF-
B DNA binding activity (30, 31, 38), this
direct inhibition of IKK or NF-
B DNA binding activity does not
appear to account for the inhibitory actions of PGJ2 on
NF-
B activation in RINm5F cells or rat islets, as a 30-min
pretreatment with PGJ2 does not affect the ability of IL-1
to stimulate the activation of NF-
B (26).
In this study, the potential role of PGJ2 as an inhibitor
of IFN--stimulated signaling pathways and whether the inhibitory actions of PGJ2 are mediated by the increased expression of
HSP70 in isolated rat islets as well as insulinoma RINm5F cells have been examined. Evidence is presented indicating that pretreatment of
RINm5F cells and rat islets for 6 h with PGJ2 results
in the attenuation of IFN-
-induced STAT1 phosphorylation, nuclear
localization, and DNA binding. Although the inhibitory actions of
PGJ2 on IFN-
-stimulated STAT1 activation correlate with
the time-dependent expression of HSP70, antisense depletion of HSP70
does not influence the ability of PGJ2 to inhibit
IFN-
-stimulated STAT1 activation. In addition, overexpression of
HSP70 in RINm5F cells does not modulate the stimulatory actions of IL-1
on NF-
B or IFN-
on STAT1 activation. These findings suggest that
the inhibitory actions of PGJ2 on proinflammatory cytokine
signaling are not limited to the inhibition of NF-
B activation in
response to IL-1, as PGJ2 appears to be a potent inhibitor
of IFN-
-induced STAT1 activation in islets and RINm5F cells. In
addition, these studies indicate that, although HSP70 expression
correlates with an inhibition of cytokine signaling, HSP70 does not
appear to mediate the inhibitory actions of PGJ2 on
cytokine signaling in RINm5F cells.
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EXPERIMENTAL PROCEDURES |
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Materials and animals.
Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN). RPMI
1640 containing 1× L-glutamine, CMRL-1066 tissue culture medium, L-glutamine, penicillin, streptomycin, and rat
recombinant IFN- were from GIBCO-BRL (Grand Island, NY). Fetal calf
serum was obtained from Hyclone Laboratories (Logan, UT). Superfect reagent was obtained from Qiagen (Valencia, CA). Human recombinant IL-1
was obtained from Cistron Biotechnology (Pine Brook, NJ). PGJ2 was from Cayman Chemicals (Ann Arbor, MI).
[
-32P]dATP and enhanced chemiluminescence (ECL)
reagents were purchased from Amersham Pharmacia Biotech (Piscataway,
NJ). The HSP70 gene, cloned into the pBluescript plasmid, was a
generous gift from Dr. Ken Polonsky (Washington University School of
Medicine, St. Louis, MO). Horseradish peroxidase-conjugated donkey
anti-rabbit and donkey anti-mouse IgG were obtained from Jackson
ImmunoResearch Laboratories (West Grove, PA). Mouse anti-rabbit HSP70
was from StressGen (Victoria, BC, Canada). Rabbit anti-phospho-STAT1
was from Upstate Biotechnology (Lake Placid, NY). Rabbit anti-STAT1 and
rabbit anti-I
B were from Santa Cruz Biotechnology (Santa Cruz, CA).
Rabbit anti-iNOS antiserum was a gift from Dr. Pam Manning (Parmacia,
St. Louis, MO). All other reagents were from commercially available sources.
Islet isolation. Islets were isolated from 250- to 300-g male Sprague-Dawley rats by collagenase digestion as previously described (28). Islets were cultured overnight in complete CMRL-1066 (containing 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin) at 37°C under an atmosphere of 95% air-5% CO2 before experimentation.
Nitrite determination. Nitrite formation was measured by mixing 50 µl of culture media with 50 µl of the Griess reagent (15) as previously described. Absorbance was measured at 540 nm, and nitrite concentrations were calculated from a sodium nitrite standard curve.
Electrophoresis and Western blotting.
Cellular proteins were separated by SDS-polyacrylamide gel
electrophoresis and transferred to Highbond ECL nitrocellulose membranes (Amersham) as previously described (33).
Antibody dilutions were as follows: rabbit anti-phospho-STAT1, 1:800;
rabbit anti-STAT1, 1:1,000; mouse anti-HSP70, 1:1,000; rabbit
anti-IB
, 1:1,000; rabbit anti-iNOS, 1:1,000; horseradish
peroxidase-conjugated donkey anti-mouse, 1:5,000; and horseradish
peroxidase-conjugated donkey anti-rabbit, 1:7,000. Antigens were
detected by ECL (Amersham Pharmacia Biotech).
Gel shift analysis.
Nuclear proteins were isolated, and gel shift analysis was performed as
previously described (20) using a 32P
end-labeled probe containing the consensus sequence for STAT1 binding (5'-CATGTTATGCATATTCCTGTAAGTG-3') or NF-B binding
(5'-AGTTGAGGGGACTTTCCCAGGC-3'). Binding reactions were run on a 4%
nondenaturing gel in a Tris-glycine buffer system.
Transfections.
RINm5F cells were transiently transfected using Superfect reagent
according to the manufacturer's specifications. Briefly, RINm5F cells
were plated at 4 × 105 cells/2 ml complete CMRL-1066
and grown to ~50% confluence (~24 h at 37°C under 95% air-5%
CO2). Plasmid DNA (2 µg) was incubated in 100 µl of
CMRL-1066 (containing 2 mM L-glutamine) for 5 min at room
temperature, 6 µl of Superfect was added, and the mixture was
incubated for an additional 15 min at room temperature followed by the
addition of 600 µl of complete CMRL-1066. The cells were washed once
with sterile PBS, and then the DNA-Superfect mixture was added dropwise
onto cells. After a 4- to 6-h incubation at 37°C, 1.3 ml of complete
CMRL-1066 was added to the cells followed by an overnight incubation at
37°C. Transfection efficiencies of 70-80%, as determined by
cotransfection of -galactosidase, were routinely obtained using this protocol.
Expression plasmids. Antisense or sense HSP70 cDNA was subcloned into the pDEST12.2 expression plasmid under control of the CMV promoter using the Gateway Expression recombination system according to the manufacturer's guidelines (Life Technologies, Rockville, MD).
Densitometry and statistical analysis. Autoradiograms were scanned using a COHU high-performance charge-coupled device camera, and densities were determined using NIH Image version 1.59 software (Brookefield, WI). Statistical comparisons were made between groups using a one-way analysis of variance (ANOVA). Significant differences between groups (P < 0.05) were determined by Bonferroni post hoc analysis.
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RESULTS |
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PGJ2 inhibits IFN--induced STAT1 activation in
RINm5F cells and rat islets.
Previous studies have shown that pretreatment of rat islets and RINm5F
cells with PGJ2 and troglitazone for 6 h results in an
inhibition of IL-1-induced NF-
B activation and JNK phosphorylation, and these inhibitory actions correlate with the expression of HSP70
(26). To determine whether signaling pathways in addition to IL-1 are sensitive to inhibition by PGJ2, the effects of
this PPAR
agonist on IFN-
-induced STAT1 phosphorylation and
nuclear localization were examined in rat islets and RINm5F cells.
Treatment of RINm5F cells (Fig.
1A) or isolated rat islets
(Fig. 1B) with IFN-
for 30 min results in STAT1
phosphorylation. Alone, a 6-h pretreatment with PGJ2 does
not stimulate STAT1 phosphorylation; however, this PGJ2
pretreatment attenuates the stimulatory actions of IFN-
on STAT1
phosphorylation in both RINm5F cells and rat islets. Similar to the
attenuation of IL-1 signaling (26), the inhibitory actions
of a 6-h pretreatment with PGJ2 on IFN-
-induced STAT1
phosphorylation correlate with the increased expression of HSP70 by rat
islets and RINm5F cells (Fig, 1, A and B). In addition, a 6-h preincubation of RINm5F cells with PGJ2
results in an inhibition of IFN-
-induced STAT1 nuclear localization
and DNA binding to
-activated sequences as determined by gel shift analysis (Fig. 1C). To confirm the presence of STAT1 in
these DNA-protein complexes, inclusion of STAT1 antiserum in the DNA binding reaction results in a reduction in the mobility of the STAT1-DNA protein complex, and excess cold oligonucleotide probe inhibits the formation of the STAT1-DNA probe complex (data not shown).
These findings indicate that PGJ2 inhibits IFN-
-induced STAT1 phosphorylation, nuclear localization, and DNA binding and that
these inhibitory actions of PGJ2 correlate with an increase in HSP70 expression.
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Role of HSP70 in mediating the inhibitory actions of PPAR
agonists on IFN-
signaling.
Because the time-dependent inhibition of IFN-
signaling in
PGJ2-treated RINm5F cells parallels the induction of HSP70,
and HSP70 overexpression has been shown to protect islets from
cytokine-induced damage (27, 33), we examined whether
HSP70 depletion would modulate the inhibitory actions of
PGJ2 on IFN-
signaling in RINm5F cells. For these
experiments, RINm5F cells were transiently transfected with antisense
HSP70 or the pDEST 12.2 empty vector for 48 h at 37°C. The cells
were then incubated with PGJ2 for 6 h followed by a
30-min stimulation with IFN-
. The cells were isolated, and HSP70 and
STAT1 expression and STAT1 phosphorylation were evaluated by Western
blot analysis. As shown in Fig.
2A, expression of antisense
HSP70 reduces PGJ2-stimulated HSP70 expression by >90% at
all time points examined (1-6 h). Importantly, the attenuation of
HSP70 accumulation does not modulate the ability of PGJ2 to
inhibit STAT1 phosphorylation. The initial inhibitory actions of
PGJ2 on IFN-
-stimulated STAT1 phosphorylation are first
observed after a 2-h pretreatment and are maximal after a 5- to 6-h
pretreatment with this PPAR
agonist (Fig. 2A). Figure 2B shows that, to control for nonspecific actions of the
expression vector, transfection with the pDEST 12.2 empty vector does
not modulate 1) the ability of IFN-
to stimulate STAT1
phosphorylation following a 30-min incubation, 2) the
inhibitory actions of a 6-h PGJ2 pretreatment on
IFN-
-induced STAT1 phosphorylation, or 3) the stimulatory
actions of a 6-h PGJ2 pretreatment on HSP70 expression.
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Effects of PGJ2 and HSP70 overexpression on IL-1
signaling in RINm5F cells.
Because the inhibitory actions of PGJ2 on IL-1 signaling
correlate with HSP70 expression (26), and induction of
HSP70 by heat stress results in impairment in IL-1 signaling
(33), the effects of HSP70 overexpression on IL-1-induced
IB degradation and NF-
B nuclear localization were examined.
Treatment of RINm5F cells for 30 min with IL-1 results in I
B
degradation and NF-
B nuclear localization as determined by
Western blot and gel shift analysis, respectively (Fig.
3). Consistent with our previous findings, a 6-h pretreatment with PGJ2 results in the
inhibition of IL-1-induced I
B degradation, and these inhibitory
actions correlate with the increased expression of HSP70 (Fig.
3A). To determine whether HSP70 alone is sufficient to
attenuate IL-1-stimulated I
B degradation and NF-
B nuclear
localization, RINm5F cells were transiently transfected with HSP70 and
then stimulated with IL-1 for 30 min. As shown in Fig.
3, HSP70 overexpression does not attenuate IL-1-induced I
B degradation or NF-
B nuclear
localization and DNA binding activity. The levels of HSP70 expressed by
transient transfection are similar in magnitude to the levels induced
by a 6-h treatment with PGJ2, indicating that the lack of
an inhibitory effect of HSP70 overexpression on cytokine signaling is
not due to differences in expression of this stress response protein. Also, transfection of RINm5F cells with the pDEST 12.2 empty vector does not attenuate IL-1-induced I
B degradation or NF-
B nuclear localization in RINm5F cells. Taken together, these findings suggest that the increased expression of HSP70 does not inhibit cytokine signaling and that HSP70 does not appear to mediate the inhibitory actions of PGJ2 on cytokine signaling in
-cells.
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Effects of HSP70 overexpression on IL-1-induced iNOS expression.
Although HSP70 overexpression does not attenuate cytokine signaling,
increased HSP70 expression has been associated with the inhibition of
iNOS expression by islets and RINm5F cells (33). Therefore, the effects of HSP70 overexpression on IL-1-induced iNOS
expression and nitrite production by RINm5F cells were examined. As
shown in Fig. 4, IL-1 stimulates the expression of iNOS and the
production of nitrite to similar levels in RINm5F cells and RINm5F
cells overexpressing HSP70. In addition, HSP70 overexpression does not
modulate the stimulatory actions of IL-1 plus IFN- on iNOS
expression and nitric oxide production by RINm5F cells (data not
shown). These findings are consistent with the lack of an inhibitory
action of HSP70 overexpression on the intracellular signal transduction
pathways activated by these cytokines and suggest that HSP70 does not
mediate the inhibitory actions of heat shock on cytokine-induced iNOS
expression by islets.
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PPAR agonists inhibit IL-1 plus IFN-
-stimulated iNOS
expression and nitric oxide accumulation.
Although HSP70 does not appear to mediate the inhibitory actions of
PGJ2 on cytokine signaling and cytokine-stimulated iNOS expression, we present evidence in Fig. 5
to confirm that, under conditions in which cytokine signaling is
impaired, cytokine-induced iNOS expression and nitric oxide production
by rat islets and RINm5F cells are also inhibited. As shown in Fig. 5,
the stimulatory actions of IL-1 plus IFN-
on iNOS expression
(inset) and nitrite production by RINm5F cells (Fig.
5A) and isolated rat islets (Fig. 5B) are
prevented by a 6-h pretreatment with PGJ2 before cytokine stimulation. These findings show that, under conditions in which PGJ2 inhibits cytokine signaling, cytokine-induced iNOS
expression is also impaired.
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Persistence of the inhibitory actions of PGJ2 on
cytokine-induced iNOS expression by islets.
We next evaluated the duration of the protective response induced by
PGJ2 that renders islets inert to cytokine signaling. In
this experiment, rat islets were pulsed with 10 µg/ml
PGJ2 for 6 h, washed, and then incubated in fresh
media at 37°C for increasing time intervals before a 24-h incubation
with IL-1. As shown in Fig. 6, IL-1 fails
to stimulate iNOS expression or nitrite production by rat islets
pretreated for 6 h with PGJ2 before cytokine
stimulation. In a similar fashion, a 6-h pulse with PGJ2
prevents the subsequent stimulation of iNOS expression in response to
IL-1 plus IFN- for periods as long as 48 h (data not shown).
These findings suggest that the inhibitory actions of a short 6-h pulse
with PGJ2 on IL-1 and IFN-
-induced iNOS expression are
persistent for extended periods of time up to 48 h.
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DISCUSSION |
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Agonists of PPAR such as PGJ2 and synthetic
thiazolidinediones (TZD; e.g, troglitazone) have been shown to
attenuate proinflammatory cytokine, iNOS, and inducible cyclooxygenase
2 expression by LPS-treated macrophages (4, 5, 23, 38),
and we have shown that these agonists attenuate IL-1-induced iNOS
expression by RINm5F cells and rat islets (26). The
specific mechanisms by which these agonists inhibit inflammatory gene
expression are associated with an inhibition of transcription factor
activation, as these cyclopentonone prostaglandins and TZD drugs have
been shown to directly inhibit IKK activation, NF-
B-DNA-binding
activity (31, 37), and the activation of AP-1
(39). Activation of PPAR
does not appear to be required
for the inhibitory actions of PGJ2 on NF-
B activation, as PGJ2 has been shown to inhibit LPS-induced NF-
B
activation in RAW 264.7 cells deficient in PPAR
. However,
PPAR
may participate directly in the inhibitory actions of
PGJ2 on inflammatory gene expression, as transfection of
PPAR
increases the sensitivity of RAW 264.7 cells to the inhibitory
actions of PGJ2 on TNF-induced iNOS reporter gene
activation (38). In the present study, we have identified
the IFN-
-signaling pathway as an additional signaling pathway that
is inhibited by PPAR
agonists. Treatment of isolated rat islets or
RINm5F cells with IFN-
for 30 min results in STAT1 phosphorylation
and nuclear localization. In a time-dependent manner, PGJ2
prevents IFN-
-induced STAT1 phosphorylation and nuclear localization
with the initial inhibitory actions observed following a 1- to 2-h
pretreatment and maximal actions after a 6-h pretreatment before
cytokine stimulation. The inhibitory actions of PGJ2 on
IFN-
-induced STAT1 activation also correlate with induction of HSP70
expression by rat islets and RINm5F cells.
Previous studies have shown that, similar to the inhibition of IFN-
signaling, PGJ2 and troglitazone attenuate IL-1-induced I
B degradation and JNK phosphorylation in isolated rat islets and
RINm5F cells (26). In these studies, we showed that a
30-min pretreatment with PGJ2 or troglitazone does not
modulate the ability of IL-1 to stimulate NF-
B activation or JNK
phosphorylation; however, after a 6-h incubation, IL-1-induced I
B
degradation and JNK phosphorylation are significantly attenuated
(26). The inhibitory actions of these agonists on NF-
B
and JNK activation are associated with the induction of HSP70
expression or induction of a stress response. These findings suggest
that PGJ2 and troglitazone may attenuate cytokine-mediated
cell signaling and cytokine-induced inflammatory gene expression by an
additional mechanism that is associated with the induction of HSP70 expression.
Overexpression of HSP70 has been shown to protect islets or insulinoma
cells against cytokine-mediated damage at two levels: 1) the
inhibition of IL-1-induced NF-B activation and the subsequent inhibition of iNOS expression (33), and 2) the
inhibition of nitric oxide donor compound-mediated damage (1,
2). Because PPAR
agonists are known to stimulate HSP70
expression (5, 26) and under conditions in which
-cells
express HSP70 these cells are protected from cytokine-mediated damage
(33), we examined the role of HSP70 in mediating the
protective actions of PPAR
agonists on cytokine signaling and
cytokine-induced iNOS expression by rat islets and RINm5F cells. Using
an antisense approach, we show that, in the absence of HSP70
accumulation, PGJ2 is still capable of impairing
IFN-
-induced STAT1 activation. In addition, antisense depletion of
HSP70 does not affect the ability of PGJ2 to attenuate
IL-1-induced I
B degradation in RINm5F cells (data not shown).
Conversely, overexpression of HSP70 in RINm5F cells to levels similar
in magnitude to those induced by PGJ2 does not result in an
impairment of IL-1-induced NF-
B activation or IFN-
-induced STAT1
activation. In addition, IL-1 and IL-1 plus IFN-
stimulate iNOS
expression and nitric oxide production to similar levels in RINm5F
cells and RINm5F cells overexpressing HSP70. These findings suggest that the inhibitory effects of PGJ2 on cytokine
signaling in rat islets and RINm5F cells are not mediated by HSP70
expression, although HSP70 expression does correlate with the
impairment in cytokine signaling.
Cytokines such as IL-1 and IFN- have been shown to impair
-cell function by stimulating iNOS expression and nitric oxide production by
-cells (18). Evidence to support a role
for iNOS and nitric oxide in cytokine-mediated
-cell damage includes
the following: 1) the ability of iNOS inhibitors to prevent
the inhibitory actions of these cytokines on insulin secretion, islet
oxidative metabolism, and islet degeneration (7, 9, 36);
2) the inability of cytokines to impair glucose-induced
insulin secretion by islets isolated from iNOS-deficient mice
(13); and 3) the induction of diabetes in
transgenic mice expressing iNOS under control of the rat insulin
promoter (40). In the current study, we show that
PGJ2 is a potent inhibitor of IL-1- and IFN-
-stimulated signaling pathways as well as the downstream activation of iNOS expression in islets and RINm5F cells. Importantly, the inhibitory actions of PGJ2 on cytokine-stimulated iNOS expression are
persistent for extended periods. A short 6-h pretreatment of islets
with PGJ2 renders these islets nonresponsive to IL-1 or
IL-1 plus IFN-
for periods as long as 48 h. These new findings
may have important implications for the attenuation of islet graft
rejection in the transplantation setting. The recent success by the
Edmonton group (32, 34) has provided new enthusiasm for
the transplantation of islets, isolated from cadaver donors, as a
method to treat brittle insulin-dependent diabetic patients.
Notwithstanding this recent success, the lack of sufficient supply of
islets and the need to transplant islets isolated from two donor organs
are issues that will need to be addressed if islet transplantation is
to be routinely used for the treatment of insulin-dependent
diabetics. One potential method to reduce the islet mass
required for a successful transplant may be to promote survival or
protect islets from immune-mediated injury. Our findings that
PGJ2 pretreatment provides an extended protection of islets
from cytokine-stimulated iNOS expression suggest that the use of
PGJ2 (or other PPAR
agonists) to attenuate proinflammatory cytokine signaling and induction of inflammatory gene
expression may provide a new strategy to promote islet graft survival
and thereby reduce the islet mass required for a successful transplantation.
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ACKNOWLEDGEMENTS |
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We thank Colleen Kelly Bratcher for expert technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-52194 and AI-44458.
Address for reprint requests and other correspondence: J. A. Corbett, Edward A. Doisy Dept. of Biochemistry and Molecular Biology, Saint Louis Univ. School of Medicine, St. Louis, MO 63104 (E-mail: corbettj{at}slu.edu).
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.
First published January 7, 2003;10.1152/ajpendo.00515.2002
Received 22 November 2002; accepted in final form 14 December 2002.
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