From the Steno Diabetes Center and
§ Hagedorn Research Institute, 2820 Gentofte, Denmark,
Vertex Pharmaceuticals Inc., Cambridge, Massachusetts 02139, and the ** University of Colorado Health Sciences Center, Denver,
Colorado 80262
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ABSTRACT |
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Interleukin-1 (IL-1
) is cytotoxic to rat
pancreatic
-cells by inhibiting glucose oxidation, causing DNA
damage and inducing apoptosis. Nitric oxide (NO) is a necessary but not
sufficient mediator of these effects. IL-1
induced kinase activity
toward Elk-1, activation transcription factor 2, c-Jun, and heat shock protein 25 in rat islets. By Western blotting with phosphospecific antibodies and by immunocomplex kinase assay, IL-1
was shown to
activate extracellular signal-regulated kinase (ERK) 1/2 and p38
mitogen-activated protein kinase (p38) in islets and rat insulinoma cells. Specific ERK1/2 and p38 inhibitors individually reduced but in
combination blocked IL-1
-mediated islet NO synthesis, and reverse
transcription-polymerase chain reaction of inducible NO synthase
mRNA showed that ERK1/2 and p38 controlled IL-1
-induced islet
inducible NO synthase expression at the transcriptional level.
Hyperosmolarity caused phosphorylation of Elk-1, activation transcription factor 2, and heat shock protein 25 and activation of
ERK1/2 and p38 in islets comparable to that induced by IL-1
but did
not lead to NO synthesis. Inhibition of p38 but not of ERK1/2
attenuated IL-1
-mediated inhibition of glucose-stimulated insulin
release. We conclude that ERK1/2 and p38 activation is necessary but
not sufficient for IL-1
-mediated
-cell NO synthesis and that p38
is involved in signaling of NO-independent effects of IL-1
in
-cells.
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INTRODUCTION |
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Interleukin-1
(IL-1
)1 is cytotoxic to
rat pancreatic
-cells (1, 2), and IL-1
in combination with tumor
necrosis factor-
and interferon-
is cytotoxic to human
-cells
(3, 4), implicating IL-1
as a central immune mediator of
-cell
destruction leading to insulin-dependent diabetes mellitus
(5). Nitric oxide (NO) produced by IL-1
mediated expression of
-cell cytokine inducible NO synthase (iNOS) (6, 7) has been
suggested as the second messenger for the
-cell cytotoxic effect of
IL-1
(8). However, because inhibition of NO synthesis only partially
protects rat
-cells from IL-1
-induced cytotoxicity (9), other
signals for this effect must be involved. The intracellular signal
transduction pathways leading to induction of iNOS and other genes
contributing to IL-1
-mediated
-cell cytotoxicity are not fully
elucidated. Tyrosine kinase-dependent activation of NF-
B
seems to be central for IL-1
-mediated
-cell iNOS expression (10,
11).
Recently, the activation of c-Jun NH2-terminal kinase (JNK)
1 and the transcription factor activating transcription factor 2 (ATF2)
by IL-1 in an insulin-producing rat insulinoma (RIN) m5F
-cell
line was described (12). JNKs (or stress-activated protein kinases) are
a subgroup of the mitogen-activated protein kinase (MAPK) family of
threonine or serine kinases, which upon activation phosphorylate and
thereby activate transcription factors leading to changes in gene
expression (reviewed in Refs. 13 and 14). Additional subgroups are p38
MAPK (hereafter termed p38) and extracellular signal-regulated kinase
(ERK) 1/2. p38 is activated in many cells by IL-1 and other cellular
stresses, including UV irradiation, hyperosmolarity, tumor necrosis
factor-
, and lipopolysaccharide (15-18), generally in parallel with
JNK, although JNK and p38 may also be activated independently (19). ERK1/2 are generally activated by growth factors (20, 21) and to a
minor extent by cellular stress, illustrated by the fact that IL-1
activates ERK1/2 in some (22, 23) but not all (20, 24) cells
investigated. Recently, specific inhibitors of ERK1/2 (25) and p38 (26)
signaling pathways have been identified, providing effective tools for
investigating the role of ERK1/2 and p38 in cellular signaling.
The contribution of p38 and ERK1/2 to IL-1 signal transduction in
insulin-producing
-cells has not been investigated. Therefore, we
examined the involvement of ERK1/2 and p38 in IL-1
signaling in
intact rat islets of Langerhans and in a RIN
-cell line. We report
that both ERK1/2 and p38 are activated by IL-1
in intact islets and
in a RIN
-cell line and that ERK1/2 and p38 are both important steps
in signaling leading to NO synthesis, but p38 is also involved in
signaling of NO-independent effects of IL-1
in intact islets.
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EXPERIMENTAL PROCEDURES |
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Reagents--
All reagents were from Sigma unless otherwise
specified. Recombinant human IL-1 (400 units/ng) was from Novo
Nordisk (Bagsværd, Denmark). Reagents for SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) were all from Bio-Rad.
[
-32P]ATP (3000 Ci/mmol) was from Amersham Pharmacia
Biotech, glutathione S-transferase (GST)-Elk-1 was a gift
from Peter Shaw (Max-Planck-Institut für Immunbiologie, Freiburg,
Germany), GST-ATF2 (1-109) was a gift form Roger J. Davis (Howard
Hughes Medical Institute, University of Massachusetts Medical School,
Worcester, MA), GST-c-Jun was a gift from Peter Angel (Deutsh
Krebsforschungszentrum, Heidelberg, Germany), and recombinant murine
25-kDa heat shock protein (Hsp25) was from Stressgen (Victoria,
Canada). Antibodies against p38, ERK1/2, phospho-p38, or phospho-ERK1/2
were from New England Biolabs (Hitchin Hertfordshire, United Kingdom),
and MAPK-activated protein kinase-2 (MAPKAP-K2) antibody was from
Upstate Biotechnology (Lake Placid, NY).
Inhibitors-- The highly specific inhibitor of p38 (compound VK-19.577, identical to SB203580 and p38i) (26, 27) was from Vertex Pharmaceuticals Inc. (Cambridge, MA). Compound PD 098059 from New England Biolabs is the specific inhibitor of MAPK/ERK kinase activation (hereafter termed MEK inhibitor (MEKi)) (25, 28). p38i and MEKi were both dissolved in DMSO to stock concentrations of 25 and 100 mM, respectively. Inhibitors were added 1 h before islet stimulation, and a final 0.14% (v/v) DMSO was added to all conditions as control for the DMSO used to dissolve MEKi and p38i.
Islet Isolation and Preculture-- Islets of Langerhans from 5-7 day old Wistar Furth rats (Charles River, Sulzfeldt, Germany) were isolated by handpicking after collagenase digestion (Collagenase A from Boehringer Mannheim) of the pancreata (29). Islets were precultured in 5-ml dishes (Nunc, Roskilde, Denmark) for 7 days at 37 °C in atmospheric humidified air in complete RPMI medium (CM)) + 10% FCS (Life Technologies, Inc.): RPMI 1640 medium (11 mM glucose) supplemented with 100,000 IU/liter penicillin, 100 mg/liter streptomycin, 20 mM HEPES buffer, 2 mM L-glutamine, and 0.038% NaHCO3 (all from Life Technologies, Inc.).
Islet Culture--
150 randomly picked islets/300 µl of CM + 0.5% human serum (final osmolarity, 300 mosM) were placed
in 4-well dishes (Nunc) treated with IL-1, with or without
inhibitors, or exposed to hyperosmolarity (addition of hyperosmolar
saline to CM as described previously (17)), as indicated in the figure
legends. For immunoprecipitation and Western blotting experiments, each
condition was made in duplicate, and islets were pooled prior to
lysis.
RIN Cell Preculture and Culture--
RIN-5AH-T2B cells of low
passage number (10-17) were maintained in 80-cm2 tissue
culture flasks (Nunc) in CM supplied with 10% heat-inactivated FCS
(HyClone, Logan, UT) at 37 °C in a 5% CO2/95% air
mixture. When confluent RIN cells were trypsinized, 150,000 cells
seeded in 96-well dishes (Costar, Cambridge, UK) containing 200 µl of CM + 10% heat-inactivated FCS and precultured for 24 h before experimentation, by which time the cell number had doubled. The RIN
cells were then exposed to IL-1 as described in the Fig. 2
legend.
Islet and RIN Cell Lysis--
Following stimulation, islets and
RIN cells were lysed for 30 min on ice in 25, 50, and 100 µl of lysis
buffer (20 mM Tris acetate, pH 7.0, 0.27 M
sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM
Na3VO4, 50 mM NaF, 1% Triton
X-100, 5 mM sodium pyrophosphate, 10 mM
-glycerophosphate, 1 mM dithiothreitol, 1 mM
benzamidine, and 4 µg/ml leupeptin) for whole cell lysate kinase
assay, Western blotting, and immunoprecipitation, respectively. The
detergent-insoluble material was pelleted by centrifugation at 15,000 rpm for 5 min at 4 °C. The supernatants containing whole cell lysate
were either immediately used for whole cell lysate kinase assay,
immunoprecipitation, or Western blotting or stored at
80 °C.
Whole Cell Lysate Kinase Assay--
The GST-Elk-1, GST-ATF2, and
Hsp25 phosphotransferase reactions were carried out in a final volume
of 25 µl at 30 °C for 30 min after addition of 5 µl of whole
cell lysate, 17 µl of reaction buffer (2 µg of GST-Elk-1, 2 µg of
GST-ATF2, 1 µg of Hsp25, 25 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4,
1 µM cAMP-dependent protein kinase inhibitor
peptide, and 10 mM Mg-acetate), and 3 µl of ATP mixture
(1 mM ATP and 3 µCi [-32P]ATP).
Reactions were terminated by addition of 25 µl of SDS sample buffer
(125 mM Tris-HCl, pH 6.8, 4% SDS, 0.1 M
dithiothreitol, 10% glycerol, and 0.02% bromphenol blue) and boiling
for 5 min. The samples were then subjected to SDS-PAGE as described by
Laemmli (30), using a 4% stacking gel and a 12% separating gel. After electrophoresis, the separating gel was washed for 15 min in a mixture
of 10% acetate and 40% methanol. The gels were dried, and the
proteins were visualized by autoradiography and quantitated by
PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). To asses
the possible contamination of the used GST-ATF2, we performed Coomassie
Blue staining and kinase assay with GST-ATF2 and ATF2 (1-96 and
1-505) from Santa Cruz Biotechnology (Santa Cruz, CA), which confirmed
the substrate specificity and purity of the GST-ATF2.
Immunoprecipitation, Immunocomplex, and JNK Kinase Assay (Solid
Phase Assay)--
100 µl of whole cell lysates from RIN cells or 300 islets, diluted 1:4 in washing buffer (lysis buffer with 0.1% Triton
X-100), were immunoprecipitated by incubation overnight with
anti-MAPKAP-K2 or anti-ERK1/2 antibodies and then with protein
A-Sepharose beads (Amersham Pharmacia Biotech) for 3 h at 4 °C
(MAPKAP-K2 and ERK1/2) or incubated with GST-c-Jun (5 µg) coupled to
glutathione-Sepharose beads for 3 h at 4 °C (JNK kinase assay).
The beads were washed three times in washing buffer and twice in kinase
buffer (20 mM HEPES (pH 7.5), 20 mM
-glycerophosphate, 10 mM MgCl2, 1 mM dithiothreitol, 50 µM
Na3VO4). Kinase reactions were carried out for
30 min at 30 °C in 30 µl of kinase buffer containing 10 µCi
[
-32P]ATP and 1 µg of Hsp25 (MAPKAP-K2) or 5 µg of
GST-Elk-1 (ERK1/2). Reactions were terminated with 30 µl of SDS
sample buffer, and the samples were analyzed as in the whole cell
lysate kinase assay.
Western Blotting-- Pooled duplicate experiments (a total of 300 islets/condition) were lysed in 50 µl of lysis buffer. Twenty µl (~20 µg) of whole cell lysates were added to 20 µl of SDS sample buffer and boiled for 5 min. SDS-PAGE (12%) was performed, and Western blotting was carried out according to standard protocols (31). Anti-total p38, anti-total ERK1/2, anti-phosphospecific p38, or anti-phosphospecific ERK1/2 antibodies were used. Enhanced chemiluminescence was used for detection. Lysate from the same experiment was separated on two gels and probed with either phosphospecific or total antibody as control for sample variation in protein content.
Nitric Oxide Synthesis and Insulin Release-- Islet NO production was measured as nitrite accumulation in conditioned media determined by the Griess reaction (32). In brief, 150 µl of medium were mixed with an equal volume of the Griess reagent (one part 0.1% naphtylethylene diamine dihydrochloride and one part 1% sulfanilamide in 5% H3PO4 (Merck, Darmstadt, Germany)) and incubated for 10 min at room temperature. The absorbance at 550 nm was measured on an immunoreader (Nippon Inter Med., Tokyo, Japan). The detection limit was 1 µM, equal to 2 pmol/islet in our conditions. Values below the detection limit were assigned the value of 2 pmol/islet. Intra- and interassay coefficients of variation calculated from three points on the standard curve were as follows: 1 µM, 2.3 and 16.8%; 10 µM, 1.8 and 8.1%; and 25 µM, 1.9 and 11.2%. Accumulated insulin release in the conditioned media was measured by radioimmunoassay (33). The detection limit was 35 fmol/ml. Intra- and interassay coefficients of variation between three known controls were as follows: A, 6.1 and 12.2%; B, 4.3 and 11.4%; and C, 3.1 and 8.2%.
Reverse Transcription-PCR--
Total RNA from snap frozen islets
was extracted and cDNA was prepared with cDNA Cycle® kit
(Invitrogen, Leek, The Netherlands) as described previously (34).
Reverse transcription-PCR was performed using
[-32P]dCTP (Amersham Pharmacia Biotech) and a fixed
volume (5 µl) of cDNA dilution. Each analysis was performed with
a set of iNOS primers in combination with a set of primers for
TATA-binding protein (35) as an internal standard. The PCR products
were separated on a 6% polyarylamide gel (Life Technologies, Inc.), visualized by autoradiography, and quantitated by PhosphorImager. Expression data are given as ratios to the co-amplified internal standard (TATA-binding protein).
Statistical Analysis-- Results are presented as mean ± S.E. (n > 2) or as mean ± range (n = 2). Wilcoxon's matched-pair test was used, and p < 0.05 was chosen as the level of significance.
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RESULTS |
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IL-1 Activates p38 and ERK1/2 in Rat Islets--
To investigate
whether p38 and ERK1/2 were activated by IL-1
in rat islets, we
first assayed the kinase activities toward Elk-1, ATF2, and Hsp25 in
whole cell lysates of IL-1
stimulated islets. Detectable kinase
activity using Elk-1, ATF2, and Hsp25 as substrates was present in
control islets (0', baseline) (Fig. 1A). IL-1
-enhanced
phosphorylation of Elk-1 was evident within 20 min, peaked at 90 min
with a 4.1-fold increase over baseline, and was sustained at 12 h.
Enhanced ATF2 phosphorylation was found within 1 min, with a maximum
3.8-fold activation of phosphotransferase activity at 20 min, and was
sustained at 12 h. Increased Hsp25 phosphorylation was apparent
within 1 min, with a 3.1-fold peak activity at 20 min, and was
sustained at 12 h.
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Detection of ERK1/2 and p38 Activity in RIN Cells--
To
investigate whether the ERK1/2 and p38 activities found in intact
islets were due to the presence of these kinases in -cells, the RIN
-cell line was assayed for ERK1/2 and p38 activities. The RIN
-cell line is comparable to primary
-cells in terms of
IL-1
-mediated NO production, iNOS expression, and cytotoxicity, albeit at a higher concentration than is needed in islets (36, 37).
Based on the IL-1
time course from intact islets (Fig. 1A), an IL-1
exposure period of 20 min was used. IL-1
stimulated ATF2 and Hsp25 kinase activities in a
dose-dependent manner, whereas IL-1
-induced Elk-1 kinase
was not further activated by an increased concentration of IL-1
above 150 pg/ml (Fig. 2A). The
binding of phosphospecific antibodies (pERK1/2 and pp38) showed that
both ERK1/2 and p38 were phosphorylated in RIN cells after a 20-min exposure to 1500 pg/ml IL-1
(Fig. 2B). p38 and ERK1/2
activation by IL-1
was further demonstrated by the immunocomplex
kinase assay that showed a 3.9- and 8.1-fold increase in ERK1/2 and
MAPKAP-K2 activity in IL-1
-exposed RIN cells, respectively (Fig.
2C).
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Inhibition of ERK1/2 and p38 in IL-1-exposed Islets--
To
dissect the relative contributions of ERK1/2 and p38 signaling pathways
in the IL-1
-induced Elk-1, ATF2, and Hsp25 kinase activities, islets
were incubated with p38i and MEKi 1 h prior to IL-1
exposure
(Fig. 3A). The MEKi inhibited
both basal and IL-1
stimulated Elk-1 kinase activity without
affecting the ATF2 and Hsp25 kinase activities. A maximal inhibition
was seen at 100 µM of MEKi. The p38i inhibited both basal
and IL-1
-induced ATF2 and Hsp25 kinase activities; it was more
effective in Hsp25 kinase inhibition, which was completely blocked at
10 µM, whereas this concentration of p38i inhibited
IL-1
-induced ATF2 kinase activity by 71%. When the inhibitors were
combined, the kinase activities toward the three substrates were
completely blocked.
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Involvement of ERK1/2 and p38 in IL-1-mediated
-Cell
Dysfunction--
MEKi and p38i were then used to evaluate the impact
of ERK1/2 and p38 on IL-1
-mediated
-cell dysfunction. NO
production was not detectable from untreated islets, and neither MEKi
nor p38i added alone or in combination resulted in NO production (Table I). At a low (25 pg/ml) IL-1
concentration, p38i caused a 35% decrease in the IL-1
-induced islet
NO production, whereas MEKi blocked NO production at that concentration
of IL-1
. At a high (150 pg/ml) IL-1
concentration, p38i and MEKi
caused a 25 and 33% reduction, respectively of IL-1
-induced islet
NO production. However, in the presence of the two inhibitors, a
synergistic effect was found, and IL-1
-induced NO synthesis was
completely blocked.
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ERK1/2 and p38 Regulate IL-1-induced Islet NO Synthesis at the
Transcriptional Level--
To investigate whether ERK1/2 and p38
controlled IL-1
-induced islet NO production at the transcriptional
level, reverse transcription-PCR of iNOS mRNA in IL-1
-exposed
islets preincubated with/without the inhibitors was performed. iNOS
mRNA was not detectable in untreated islets (Fig.
4). IL-1
-induced iNOS was expressed to a 3.3-fold greater magnitude at 6 h compared to 24 h of
IL-1
exposure. IL-1
-induced iNOS was inhibited by both of the
inhibitors; the MEKi was slightly more effective at both 6 and 24 h of IL-1
exposure. Combined, the inhibitors reduced IL-1
-induced
iNOS by ~95% at both 6 and 24 h.
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Noncytokine Stimulation of ERK1/2 and p38 Activity and Islet NO
Production--
To investigate whether noncytokine activation of
ERK1/2 and p38 was able to induce islet NO production, islets were
exposed to hyperosmolarity. As shown in Fig.
5A, a weak phosphorylation of
Elk-1, ATF2, and Hsp25 was found in control islets (0', baseline). When
exposed to hyperosmolarity (525 mosM) a marked
time-dependent phosphorylation of the three substrates was
found. Hyperosmolarity induced rapid (within 0.5 min) phosphorylation
with a 2.2- and 2.3-fold activation of ATF2 and Hsp25, respectively. A
3.1-fold activation of Elk-1 was evident within 2.5 min. The time
points for peak values with 3.8-, 4.6-, and 3.6-fold activation of
Elk-1, ATF2, and Hsp25, respectively, were very similar to those
induced by IL-1 (see Fig. 1A). Hsp25 phosphorylation
declined to baseline at 12 h, whereas the phosphorylation of ATF2
and Elk-1 was sustained until at least 12 h. Elk-1 phosphorylation
was more sustained, and Hsp25 phosphorylation showed a more rapid
descent when compared with IL-1
-induced phosphorylation.
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DISCUSSION |
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Our data demonstrate that p38 and ERK1/2 are both activated by
IL-1 in rat islets of Langerhans and in the rat insulinoma cell line
RIN-5AH, implying that the ERK1/2 and p38 activity found in the intact
islet most likely originates from the
-cells, in accordance with the
recently reported p38 activation by IL-1
in the INS-1
-cell line
(39).
MEK- and p38-specific inhibitors individually reduced IL-1
stimulated islet NO synthesis (Table I) and iNOS mRNA (Fig. 4), indicating that both ERK1/2 and p38 are involved in IL-1
-mediated expression of iNOS. iNOS induction by ERK1/2 in
-cells may be explained by Elk-1-induced c-fos expression mediated through
the serum response element (40). c-Fos combines with c-Jun to form the
transcription factor activating protein-1 (41), and two activating
protein-1 binding sites have been identified in the murine iNOS gene
promoter (42). Activated p38 may induce
-cell iNOS via several
transcription factors: 1) through activating protein-1, because
p38-mediated expression of c-jun and c-fos has
been observed (43); 2) through NF-
B, because p38 seems to be
required for NF-
B-mediated transcriptional activation, but it
affects neither NF-
B DNA binding nor phosphorylation of its subunits
(44, 45) and the murine iNOS gene promoter region contains two NF-
B
binding sites (42), and because NF-
B is involved in iNOS expression
in
-cells (36, 46); and 3) through p38-mediated cAMP-responsive
element-binding protein activation (47), because cAMP-responsive
element-binding protein has been reported to be involved in iNOS
induction via the CAAT box in the murine iNOS promoter (48).
The involvement of ERK1/2 and p38 in IL-1 regulation of iNOS seems to
be cell-specific because 1) IL-1 activation of ERK1/2 is necessary for
iNOS expression in rat cardiac microvascular endothelial cells (23), 2)
p38, but not ERK1/2, is involved in IL-1-mediated iNOS expression in
mouse astrocytes (49), and 3) IL-1 stimulation of p38 down-regulates
iNOS in rat messangial cells (50). Our data support this concept by
showing that both ERK1/2 and p38 are required for IL-1-mediated iNOS
expression in rat pancreatic -cells.
ERK1/2 and p38 activation was found to be necessary for
IL-1-mediated islet NO synthesis (Table I) and iNOS induction (Fig. 4). However, the observation that hyperosmolarity caused a maximal Elk-1, ATF2, and Hsp25 phosphorylation and ERK1/2 and p38 activation (Fig. 5) similar to that caused by IL-1
without inducing islet NO
synthesis suggests that ERK1/2 and p38 activation is not sufficient to
cause IL-1
-mediated islet NO synthesis. Thus, other signaling events
activated by IL-1
, but not provided by hyperosmolarity, are
necessary. These signals could be involved in I
B degradation and
NF-
B translocation to the nucleus because a recently identified IL-1
receptor associated kinase shares similarity with a protein kinase essential for activation of a NF-
B homologue in
Drosophila (51). Another signal could be provided by
interferon response factor-1 expression because interferon response
factor-1 has been implicated in IL-1
-induced iNOS expression in rat
-cells (52).
p38 but not ERK1/2 blockade attenuated inhibition of glucose-stimulated
insulin release by 150 pg/ml of IL-1 (Table II), but because the
MEKi and p38i were equally potent in decreasing
-cell NO synthesis
induced by 150 pg/ml of IL-1
, and because MEKi was even more
efficient in decreasing iNOS mRNA than p38i, p38 must be involved
in signaling of NO-independent effects of IL-1
in
-cells. The
inhibitory effect of p38 activation on insulin release could be due to
a direct action on the
-cell stimulus-secretion coupling, because
p38 is slightly activated (1.5-fold) by 15 mM glucose (our
media contained 11 mM glucose) in the INS-1
-cell line
(39). However, the observation that p38 inhibition did not affect
glucose-stimulated insulin release from islets not exposed to IL-1
(Table II), suggests that p38 is not involved in signaling pathways of
glucose-stimulated insulin release. Rather, the findings that diverse
classes of DNA-damaging agents activate p38 (53), that a sustained
activation of p38 has been associated with apoptosis (54, 55), and that
IL-1
induces apoptosis in
-cells (37, 56) and the present finding
of a sustained (>12 h) IL-1
-mediated p38 activation in islets make
it more likely that p38 induces an apoptotic signal in
-cells. We
are currently investigating the possible involvement of p38 on
IL-1
-mediated
-cell cytotoxicity.
Even though combination of MEKi and p38i inhibited IL-1-induced
islet iNOS expression by 95% and completely blocked islet NO
synthesis, the IL-1
-mediated inhibition of insulin release was only
attenuated by 33%, indicating that islet NO production is not
sufficient in causing
-cell death and that other mediators of
NO-independent signaling pathways, in addition to p38, are involved. A
possible mediator could be JNK, which is activated by IL-1
in both
islets (Fig. 3) and RIN-cells (12) and is involved in apoptosis (54,
57).
In conclusion, this study suggests that ERK1/2 and p38 are involved in
IL-1 signaling in
-cells, that they are necessary but not
sufficient in causing IL-1
-induced
-cell NO synthesis by
controlling iNOS gene transcription, and, furthermore, that p38 is
involved in signaling of NO-independent effects of IL-1
in
-cells. These observations make p38 inhibition a powerful approach
to further elucidate the involvement of p38 in the pathogenesis of
insulin-dependent diabetes mellitus.
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ACKNOWLEDGEMENTS |
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We acknowledge Roger J. Davis for the gift of GST-ATF2, Peter Angel for GST-c-Jun, and Peter Shaw for GST-Elk-1. We thank Susanne Munch, Birgitte Born, and Hanne Foght for excellent technical assistance and Anne Brock Rasmussen for secretarial help.
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FOOTNOTES |
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* This work was supported by the Danish Diabetes Association and Novo Nordisk.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.
¶ Recipient of a Postdoctoral Fellowship from Juvenile Diabetes Foundation International.
Supported by NIH Grant AI 15614.
§§ To whom correspondence should be addressed. Tel.: 45-44439101; Fax 45-44438232; E-mail: tmpo{at}novo.dk.
1
The abbreviations used are: IL, interleukin;
ATF2, activating transcription factor 2; ERK, extracellular
signal-regulated kinase; GST, glutathione S-transferase;
Hsp25, 25-kDa heat shock protein; NO, nitric oxide; iNOS, inducible NO
synthase; JNK, c-Jun NH2-terminal kinase; MAPK,
mitogen-activated protein kinase; MAPKAP-K2, MAPK-activated protein
kinase-2; MEKi, MAPK/ERK kinase inhibitor; NF-B, nuclear
factor-
B; p38i, p38 inhibitor; RIN, rat insulinoma; PAGE,
polyacrylamide gel electrophoresis; CM, complete medium; PCR,
polymerase chain reaction.
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REFERENCES |
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