(Received for publication, September 17, 1996, and in revised form, January 17, 1997)
From the Department of Molecular Biology & Pharmacology and Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
The inflammatory cytokine interleukin 1
(IL-1
) induces both cyclooxygenase-2 (Cox-2) and the inducible
nitric-oxide synthase (iNOS) with increases in the release of
prostaglandins (PGs) and nitric oxide (NO) from glomerular mesangial
cells. However, the intracellular signaling mechanisms by which IL-1
induces iNOS and Cox-2 expression is obscure. Our current studies
demonstrate that IL-1
produces a rapid increase in p38
mitogen-activated protein kinase (MAPK) phosphorylation and activation.
Serum starvation and SC68376, a drug which selectively inhibits p38
MAPK in mesangial cells, were used to investigate whether p38 MAPK
contributes to the signaling mechanism of IL-1
induction of NO and
PG synthesis. Serum starvation and SC68376 selectively inhibited
IL-1
-induced activation of p38 MAPK. Both SC68376 and serum
starvation enhanced NO biosynthesis by increasing iNOS mRNA
expression, protein expression, and nitrite production. In contrast,
both SC68376 and serum starvation suppressed PG release by inhibiting
Cox-2 mRNA, protein expression, and PGE2
synthesis. These data demonstrate that IL-1
phosphorylates and
activates p38 MAPK in mesangial cells. The activation of p38 MAPK may
provide a crucial signaling mechanism, which mediates the up-regulation
of PG synthesis and the down-regulation of NO biosynthesis induced by
IL-1
.
Interleukin 1 (IL-1)1 is a potent immunoregulatory and proinflammatory cytokine secreted by a variety of cells in response to infection, activated lymphocyte products, microbial toxins, and inflammatory and other stimuli (1). IL-1 induces both Cox-2 and inducible nitric-oxide synthase (iNOS) with increases in the release of PGs and NO from mesangial cells (2).
The iNOS found in several cell types including macrophages, (3-5)
vascular smooth muscle cells (6, 7), and renal mesangial cells (8) is
highly regulated by cytokines, which can facilitate or inhibit the
induction of this enzyme. Stimulatory cytokines such as IL-1 and tumor
necrosis factor increase iNOS mRNA by transcriptional activation.
Once iNOS is induced, it produces tremendous amounts of NO that can
contribute to cell and tissue regulation and damage. However, iNOS gene
expression, mRNA stability, and protein synthesis and degradation
are all amenable to modification by cytokines or other agents such as
growth factors. Transforming growth factor-, for example, reduces
cytokine-induced iNOS activity by inhibiting mRNA translation and
increases iNOS protein degradation, whereas interleukin-4 interferes
with iNOS transcription (9).
The cyclooxygenase is a ubiquitous enzyme that is involved in many inflammatory processes. Cyclooxygenase isoforms, Cox-1 and Cox-2, are the key enzymes that convert arachidonic acid to PGs. Cox-2 is normally undetectable in most tissues but can be rapidly induced in certain cell types by various proinflammatory or mitogenic stimuli (10). This inducible enzyme is thought to be involved in inflammation, cellular differentiation, and mitogenesis by releasing proinflammatory PGs. However, mice lacking Cox-2 have normal inflammatory responses but develop severe nephropathy as the animal ages, suggesting Cox-2 may be critical for normal kidney growth, differentiation, and function (11).
The intracellular signaling mechanisms triggered by IL-1 are not completely defined (12). IL-1 stimulation activates a family of protein kinases known as the mitogen-activated protein kinases (MAPKs) (13). At least four genetically distinct MAPK pathways, which are functionally independent and regulated by distinct protein cascades, have been identified in yeast (14). In mammalian cells, three subgroups of MAPK have been detected and include the extracellular signal-regulated kinase (ERKs), the c-jun amino-terminal kinases (JNKs) and p38 MAPKs (15). These kinases are activated by distinct upstream dual specificity kinases (MAPK kinase/MAPK kinase), which phosphorylate both threonine and tyrosine in a regulatory Thr-X-Tyr motif present in all MAPKs. Once activated, these MAPKs then phosphorylate and activate their specific substrates on serine and/or threonine residues and produce their effects on downstream targets (14, 15).
Mammalian p38 MAPK, the homologue of the yeast HOG1, is activated through different receptors by multiple stimuli, such as hyperosmolarity, UV light, heat shock, arsenite, endotoxin, and cytokines (16). However the cellular consequences of this signaling pathway are incompletely understood. Recent studies have suggested that p38 MAPK activates MAPK-activated protein kinase-2, which, in turn, phosphorylates the small heat shock protein 27 (HSP27). The physiological role of this event is controversial but may help the cell to resist thermal stress (17-20). Several other investigators have provided evidence that p38 MAPK may be involved in the regulation of cytokine production (21), neuronal apoptosis (22) and platelet aggregation (23, 24).
We previously demonstrated that IL-1 activates JNK in renal
mesangial cells. In this current study, we examined whether the p38
MAPK signaling pathway also responds to IL-1 activation in this cell
type. Furthermore, we have investigated whether p38 MAPK may be
involved in the regulation of Cox-2 and iNOS.
Human recombinant IL-1 and restriction enzymes
were purchased from Boehringer Mannheim. Myelin basic protein (MBP) and
PGE2 were from Sigma. SC68376, was kindly
provided by Dr. Joe Portnova (G. D. Searle Corporation, St. Louis, MO),
and its structure is shown in Fig. 1. Fetal bovine serum
was purchased from Life Technologies, Inc. Polyclonal rabbit IgG
antibodies against iNOS, Cox-2, p38, and phospho-specific p38 were from
Transduction Laboratories (Lexington, KY), Cayman Chemical Co. Inc.
(Ann Arbor, MI), Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and
New England BioLabs (Beverly, MA), respectively. Murine cDNA probes
ligated in BlueScript SK
for Cox-1 (pBSCOX-1) and Cox-2
(pBSCox-2) were generous gifts of Dr. Karen Seibert (Monsanto
Corporation, St. Louis, MO). Mouse iNOS and rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes were
prepared by reverse transcriptase-PCR using total RNA of rat mesangial
cells as described previously (2). pET28cjdelta, a histidine-tagged
fusion protein expression plasmid that encodes amino acids 1-79 of
NH2-terminal c-jun was generously provided by
Dr. Maryann Gruda (Department of Molecular Biology, Bristol Myers
Squibb Pharmaceutical Research Institute, Princeton, NJ). His-c-jun(1-79) was expressed as a histidine-tagged fusion protein in
Escherichia coli NovaBlue (DE3) and purified by His-bind
resin (Novagen) (25).
Cell Culture
Primary mesangial cell cultures were prepared from male Sprague-Dawley rats as described previously (25). Cells were grown in medium supplemented with 15% heat-inactivated fetal calf serum, 0.3 IU/ml insulin, 100 units/ml penicillin, 100 µg/ml streptomycin, 250 µg/ml amphotericin B, and 15 mM HEPES, pH 7.4. All experiments were performed with confluent cells grown in 25 or 75 cm2 flasks and used at passages 3 through 8. For experiments carried out in serum-free media, cells were starved for 24 h prior to experiments. For all other experiments, serum was reduced from 15 to 5% on the day of the experiment.
Western Blot AnalysisConfluent cells were incubated in
RPMI 1640 media containing 5% fetal calf serum and treated with
IL-1, with or without other pharmacological reagents. Cells were
washed with ice-cold phosphate buffer and lysed in 0.5 ml of Laemmli
sample buffer (10% glycerol, 5% 2-mercaptoethanol, 2.3% SDS, 62.5 mM Tris-HCl, pH 6.8, 10 mM EDTA) or whole cell
extract buffer (25 mM HEPES-NaOH, pH 7.7, 0.3 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM
dithiothreitol (DTT), 20 mM
-glycerophosphate, 100 µM NaVO4, 2 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride) to which 6 × Laemmli
sample buffer was added before heating. After boiling for 5 min, equal
amounts of protein were run on 12% SDS-polyacrylamide gel
electrophoresis. Proteins were transferred to polyvinylidene difluoride
membranes (Immobilon-P; Millipore Corp., Bedford, MA). The membranes
were saturated with 5% fat-free dry milk in Tris-buffered saline (50 mM Tris, pH 8.0, 150 mM NaCl) with 0.05% Tween
20 (TBS-T) for 1 h at room temperature. Blots were then incubated
overnight with anti-iNOS, Cox-2, p38 MAPK, or phospho-specific p38 MAPK antibodies at 1:1000 dilution in 5% bovine serum albumin TBS-T. After
washing with 5% milk TBS-T solution, blots were further incubated for
1 h at room temperature with goat anti-rabbit IgG antibody coupled
to horseradish peroxidase (Amersham) at 1:2000 dilution in TBS-T. Blots
were then washed five times in TBS-T before visualization. Enhanced
chemiluminescence (ECL) kit (Amersham Corp.) was used for
detection.
After experimental maneuvers,
various harvested cells were solubilized in whole cell extract buffer.
Protein kinase assays were performed using modifications of the
procedures of Kameshita and Fujisawa (26) and Cano et al.
(27). Briefly, SDS-polyacrylamide was polymerized in the presence or
absence of 200 µg/ml of His-c-jun (1-79) or 400 µg/ml of MBP.
After electrophoresis, SDS was removed by incubation in 20%
isopropanol in 50 mM Tris-HCl, pH 8.0, for 1 h. The
gel was then washed for 1 h with 1 mM DTT, 50 mM Tris-HCl, pH 8.0. To denature the proteins, gels were
incubated in 6 mM guanidine-HCl, 20 mM DTT, 2 mM EDTA, 50 mM Tris-HCl, pH 8.0, for 1 h.
Proteins were then renatured by incubation overnight in 1 mM DTT, 2 mM EDTA, 0.04% Tween 20, 50 mM Tris-HCl, pH 8.0. For the protein kinase assays, gels
were equilibrated for 1 h in kinase buffer containing 1 mM DTT, 0.1 mM EGTA, 20 mM
MgCl2, 40 mM HEPES-NaOH, pH 8.0, 100 µM NaVO4. The kinase reaction was carried out
for 1 h in kinase buffer with 30 µM ATP and 5 µCi/ml of [-32P]ATP. Finally, the gels were washed
extensively in 5% trichloroacetic acid and 1% sodium pyrophosphate
until washes were free of radioactivity. Autoradiography of dried gel
was performed at
80 °C.
The cell extracts were
immunoprecipitated by incubation overnight with anti-p38 antibody and
then with protein A-Sepharose beads for 3 h at 4 °C. The beads
were washed 3 times with 1 ml of ice-cold whole cell extract buffer.
The immune-complex p38 kinase assay using anti-p38 antibody
immunoprecipitates was performed at 30 °C for 20 min in 30 µl of
kinase reaction buffer (5 µg MBP, 20 µM ATP, 10 µCi
of [-32P]ATP, 25 mM HEPES, and 20 mM MgCl2). The reaction was terminated with
Laemmli sample buffer, and the products were resolved by 12%
SDS-polyacrylamide gel electrophoresis. The phosphorylated MBP was
visualized by autoradiography.
Northern blot analysis was performed
as described previously (25). The cDNA probes of murine iNOS, rat
GAPDH, and murine Cox-1 and Cox-2 were prepared as described previously
(2, 28). Confluent cells grown in 75-cm2 flasks were washed
twice with phosphate-buffered saline. Cells were incubated in RPMI 1640 media containing 5% fetal calf serum with IL-1 and/or
pharmacological agents for 3 h (for Cox-1 and Cox-2) or 12 h
(for iNOS) and harvested. These times represent the peak response of
the respective messages. Total RNA was isolated using the acid
guanidinium thiocyanate-phenol-chloroform method (RNA STAT 60, Tel-Test
"B", Friendswood, TX). 20-30 µg of total RNA was fractionated by
1% agarose-formaldehyde gel electrophoresis, transferred to nylon
membranes (GeneScreen, Du Pont), and immobilized with UV cross-linking.
All probes used in our experiments were radiolabeled with
[32P]dCTP by the random primed labeling method. After
prehybridization for 6 h at 42 °C in 50% deionized formamide,
0.04% polyvinylpyrrolidone, 0.04% bovine serum albumin, 0.04%
Ficoll, 5 × SSC, 1% SDS, and denatured salmon sperm DNA (100 µg/ml), hybridization was performed at 42 °C for 18-24 h in a
solution containing 50% deionized formamide, 0.02%
polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.02% Ficoll,
5 × SSC, 1% SDS, and denatured salmon sperm DNA (100 µg/ml). Membranes were washed twice at room temperature for 5 min in 2 × SSC and twice at 60 °C for 30 min in 2 × SSC and 0.1% SDS and were exposed to film overnight. To control for variability in the
loaded quantity of RNA, all membranes were probed with GAPDH cDNA
to determine the steady-state levels of GAPDH gene-related sequences
and used to normalize the amount of Cox-1, Cox-2, and iNOS mRNA.
The peak response of Cox-2 mRNA occurs at 3 h, while for iNOS
mRNA, this time point is 12 h. Therefore, these times were
chosen for Northern analysis.
PGE2 in the culture media was measured by stable isotope gas chromatography-mass spectrometry as described previously (28). At the end of predetermined times, medium was removed and spiked with 25 ng of tetradeuterated PGE2 (d4-PGE2). The media was then acidified to pH 3.5, and PGE2 was extracted with 1- ml octadecyl columns (Baker Co., Sanford, ME). Extracts were derivatized for gas chromatography-mass spectrometry analysis. The samples were analyzed as the pentafluorobenzyl ester methoxime trimethylsilyl ether by negative ion chemical ionization using methane as the reagent gas. Ions monitored were m/z 524 (d0-PGE2) and m/z 528 (d4-PGE2). Mass spectrometry was performed on a Hewlett-Packard 5985B spectrometer using a 25 M Ultra 1 (Hewlett-Packard Co., Palo Alto, CA) capillary column, and data collection and analysis were performed using Vector 2 (Teknivent, St. Louis, MO) software. PGE2 production was normalized for protein as determined by the micro-bicinchoninic acid assay (29).
Nitrite DeterminationThe conditioned incubation medium was collected, and nitrite content in the supernatant was measured by the addition of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric acid) (30). The absorbance at 550 nm was measured, and the amount of nitrite was obtained by extrapolation from a standard curve using sodium nitrite as a standard. The nitrite production was corrected for protein determined as described above.
Statistical AnalysisData were expressed as the mean ± S.E. Statistical analysis was performed by using a paired or unpaired Student's t test. A difference with a p value of 0.05 was considered statistically significant.
Previous
studies in our laboratory have demonstrated that IL-1 activates
several protein kinases, such as tyrosine kinases (31), JNK (25), and
protein kinase C (32) in renal mesangial cells. To investigate whether
the p38 MAPK pathway is involved in IL-1
signal transduction in
glomerular mesangial cells, we examined whether the activation of p38
MAPK was induced by IL-1
. Cultured mesangial cells were treated with
100 units/ml IL-1
, and an immune-complex assay was performed to
directly measure p38 activity. As shown in Fig. 2,
IL-1
rapidly and transiently activated p38 MAPK. The increase of p38
activity induced by IL-1
was detected within 1 min of exposure to
IL-1
, reached a peak in 5-10 min, and declined in 15 min toward
base line. Western blot analysis using the anti-phospho-specific p38
MAPK also showed that IL-1
quickly increased phosphorylation of p38
MAPK in mesangial cells (Fig. 2). The time course of IL-1-induced
phosphorylation of p38 MAPK was highly consistent with that of p38 MAPK
activation. Together, these results establish that the p38 MAPK pathway
is rapidly activated by the proinflammatory cytokine IL-1
in rat mesangial cells and that phosphorylation of Tyr-182 is associated with
the activation of p38 MAPK in mesangial cells stimulated by
IL-1
.
Effect of Serum on IL-1
To demonstrate whether serum was necessary to
express the full response of IL-1 with respect to p38 MAPK activity,
we serum starved the cells for at least 24 h and then measured p38
MAPK activity in response to IL-1
stimulation by immune-complex p38 kinase assay. IL-1
failed to significantly activate p38 MAPK in rat
mesangial cells when cells were serum starved for 24 h (data not
shown). Western blot analysis using phospho-specific p38 MAPK antibody
(which detected p38 MAPK only when phosphorylated on Try-182) showed
that, in serum-replete mesangial cells, IL-1
increased the
phosphorylation of p38 MAPK (Fig. 3). In serum-starved cells, however, IL-1
did not stimulate phosphorylation of p38 MAPK.
Simultaneous immunoblots with an anti-p38 antibody demonstrated equivalent amounts of p38 MAPK in each lane. These results demonstrate that, in primary mesangial cells, IL-1
-induced p38 MAPK activation and phosphorylation are dependent on serum.
Effect of Serum on IL-1
Our laboratory previously reported that IL-1
increases in Cox-2 mRNA expression and PGE2
biosynthesis are dependent on serum (31). To investigate the effects of
serum on IL-1
-induced nitric oxide synthesis, we measured the nitric
oxide released in response to IL-1
as a function of time in
serum-starved and serum-replete cells. As shown in Fig.
4, IL-1
increased nitric oxide secreted into the media both in the
presence and absence of serum. Of note, however, was that in
serum-starved cells, IL-1
produced significantly more NO than in
serum-replete cells by 12 h of incubation. In previous
experiments, we have demonstrated that IL-1
induced iNOS mRNA
expression in serum-replete cells, which peaked at 12 h after
IL-1
stimulation and declined to basal levels by 24 h (2). In
contrast, when cells were serum starved for 24 h, iNOS mRNA
expression was markedly enhanced. IL-1
significantly induced iNOS
expression within 6 h of IL-1
stimulation, and remained at high
levels for 24 h after exposure of cells to IL-1
(data not
shown). Similar experiments carried out in serum-replete and serum-starved cells followed by immunoblot analysis of iNOS protein is
demonstrated in Fig. 5. It demonstrates that in
serum-replete media, iNOS is transiently expressed in response to
IL-1
; whereas in serum-starved cells, iNOS protein expression is
enhanced and sustained. Together, all of the above data suggest that,
in rat mesangial cells, serum starvation inhibits IL-1
-induced
PGE2 synthesis and, in contrast, increases IL-1
-induced
nitric oxide biosynthesis, indicating that some additional "unknown
factors" existing in the serum regulate IL-1
-induced nitric oxide
and PGE2 production in reciprocal ways.
SC68376 Selectively Inhibits p38 MAPK Activity
Recently, a
group of pyridinyl imidazole compounds have been identified that bind
to p38 MAPK and inhibit its activity (21). SC68376 was kindly provided
to us by G. D. Searle and has similar properties. Therefore, in our
experiments, we determined the specificity of this compound by
assessing its ability to inhibit the MAP kinase family of enzymes. We
first incubated differing concentrations of SC68376 directly with p38
MAPK, which was immunoprecipitated with anti-p38 MAPK antibody and
tested the ability of the immune complex to phosphorylate myelin basic
protein. Fig. 6A clearly shows that SC68376
dose dependently inhibited p38 MAPK activity, with an
IC50 of about 2-5 µM. Fig. 6B
shows similar data except that the antibody used to immunoprecipitate
was anti-phospho-p38 antibody. Mesangial cells were preincubated with
various concentrations of SC68376 for 30 min, and then IL-1 was or
was not added to stimulate the cells for another 30 min with the final
concentration of 100 IU/ml of IL-1
. Western blot assays demonstrated
that SC68376 did not significantly decrease IL-1
stimulated p38 MAPK
phosphorylation (data not shown). In-gel kinase assays using myelin
basic protein or His-c-jun(1-79) as substrate showed that SC68376
failed to significantly inhibit either basal ERK or IL-1 activated JNK
activity, respectively. These results demonstrate that SC68376 directly inhibits p38 MAPK catalytic activity. Thus, this compound is a potentially useful pharmacological tool to explore the physiological and pathophysiological function of p38 MAPK.
Effects of SC68376 on IL-1
To determine whether the p38 MAPK
signaling pathway is involved in the induction of PGE2 and
nitric oxide synthesis by IL-1, we studied the effects of SC68376,
an inhibitor of p38 MAPK, on IL-1
-induced nitric oxide and
PGE2 biosynthesis in renal mesangial cells. SC68376, in the
range 0.1-100 µM, dose dependently enhanced the effect
of IL-1
on nitrite production (Fig. 7A),
iNOS protein synthesis (Fig. 7B), and iNOS mRNA
expression (Fig. 8). The experiments suggested an
apparent discrepancy between the amount of protein detected by Western
analysis and the amount of NO produced. We have no clear explanation
for this; however, one may speculate that some of the NO produced is
converted to nitrite, which we measured, and some further metabolized
to nitrate, which is not measured in the assay. Furthermore, at high
concentrations of NO the potential for covalent nitrosylation of
mesangial proteins exists. In contrast, SC68376 markedly inhibited
IL-1
induced PGE2 production (Fig. 7C), Cox-2
protein synthesis (Fig. 7D), and Cox-2 mRNA expression
(Fig. 8). Quantitative densitometry of iNOS and Cox-2 protein
expression seen in Fig. 8 is reported in Table I. In
previous studies in our laboratory, we found that PGE2
negatively modulates the induction of nitric oxide synthesis by IL-1
(1). To rule out the possibility that the effects of SC68376 on IL-1
induced nitric oxide synthesis was secondary to the decrease of
PGE2 production, we treated cells with various concentrations of SC68376 with and without the addition of 25, 250, or
2500 ng/ml PGE2. We found that exogenous PGE2
did partially reverse the effect of SC68376 on IL-1
-stimulated
nitrite production (Fig. 9), which was consistent with
earlier observations (2). However, this reversal was only about 20% at
the highest concentration of PGE2, thus suggesting that
SC68376 facilitates the induction of nitric oxide synthesis by IL-1
and that the up-regulation is not mediated by the decrease in
PGE2 production. These observations indicate that p38 MAPK
inhibitor SC68376 negatively modulates the cyclooxygenase pathway and
positively modulates the nitric-oxide synthase pathway.
|
IL-1 is involved in several pathological processes of the renal
glomerulus. The "activated" phenotype of mesangial cells stimulated by IL-1 could play vital roles in the further progression of glomerular inflammatory injury. In primary cultures of mesangial cells, we demonstrated previously that IL-1 induced iNOS and Cox-2 expression, which in turn, increased NO and PGE2 production. However,
the signaling pathways by which IL-1 induces NO and PG biosyntheses are
not well understood (2, 28, 32). Recently, much effort has been
directed at defining the signal transduction pathways utilized by IL-1.
Cellular responses to IL-1 stimulation trigger a cascade of protein
kinases that transmit signals from the cell surface to the nucleus and
that ultimately regulate gene expression. The MAPKs are a family of
serine/threonine kinases activated by dual phosphorylation of Thr and
Tyr within a Thr-X-Tyr motif. There are at least three
distinct types of MAPKs in mammalian cells, each of which has a
distinct cascade of activation and distinct functions (33, 34). p38
MAPK is a member of the MAPK group of signal transduction kinases. It
is activated by environmental stress and proinflammatory cytokines
including IL-1
. In the present study, we have demonstrated the
activation of p38 MAPK in IL-1
-treated rat mesangial cells by an
immune-complex kinase assay. Our results indicate that IL-1
activates p38 MAPK in mesangial cells. An increase in p38 MAPK activity
was detected 1 min after exposure of the cells to 100 units/ml IL-1
.
The maximal activity occurred in 5-10 min, followed by a rapid
decline in activity toward basal levels within 15 min. To assess
whether the phosphorylation of Tyr-182 of p38 MAPK is also associated
with its activation in mesangial cells, we examined the phosphorylation
of p38 MAPK in response to IL-1
stimulation by using
phospho-specific p38 MAPK (Tyr-182) antibody. Similar to the
IL-1
-induced p38 MAPK activity, IL-1
significantly enhances p38
MAPK tyrosine phosphorylation. The increase of phosphorylated p38 MAPK
was detectable 1 min after IL-1
stimulation, reached the peak at
5-10 min, and recovered to the basal level in 60 min. These data
indicated that the activation of p38 MAPK signal cascade is an early
event in mesangial cells in response to IL-1
stimulation. We
therefore hypothesized that the activation of p38 MAPK signaling
pathway may mediate IL-1
signal amplification and modulation that
results in later events such as NO and PG production.
Although p38 MAPK has been found to be activated by several forms of
environmental stress and cytokines, the physiological and
pathophysiological function of this kinase in mammalian cells is still
unclear. Recently, a role for p38 MAPK in the regulation of HSP27,
cytokine biosynthesis, platelet aggregation, and neural apoptosis has
been reported (17-24). To demonstrate whether p38 MAPK pathway
mediates and regulates IL-1-induced NO and PG synthesis, SC68376 was
used in our studies to inhibit p38 MAPK. We found that SC68376
selectively inhibited both basal and IL-1
-activated p38 MAPK
activity in vitro but did not affect its phosphorylation in
intact cells. In contrast, this agent failed to inhibit ERK and JNK
activity. We therefore used this p38 MAPK inhibitor to explore the role
of p38 MAPK in IL-1
-induced NO and PG production. Previous studies
in our laboratory have found that, in mesangial cells, serum is crucial
for the full induction of PGE2 biosynthesis by IL-1
(31). We hypothesized that some "unknown serum factors" are
necessary to trigger the IL-1
-induced signal transmitting mechanisms
leading to the phosphorylation/dephosphorylation cascades. However, our
early data suggested that IL-1
-activated JNK signaling pathways are
independent of serum (25). Surprisingly, the present results show that
in mesangial cells, serum starvation significantly reduces
IL-1
-activated p38 MAPK phosphorylation and activation and clearly
demonstrate that IL-1
activates p38 MAPK pathway in a
serum-dependent manner. The unknown serum factors may
converge on this cascade upstream of p38 MAPK. Thus, serum starvation
is another experimental probe to determine the function of p38 MAPK in
that serum starvation selectively inhibits this kinase response to
IL-1
in the renal mesangial cell.
To evaluate whether the p38 MAPK pathway is important for the
regulation of IL-1-induced PGE2 synthesis, we examined
the effects of SC68376 in addition to serum starvation on
IL-1
-induced Cox-2 gene and protein expression and PGE2
production. In agreement with our previous results (2, 28, 31), in
mesangial cells starved for 24 h of serum, the effects of IL-1
on Cox-2 mRNA expression, protein expression, and PGE2
production were significantly reduced. Similar to the effects of serum
starvation, SC68376 dose dependently inhibited Cox-2 mRNA and
protein expression and PGE2 formation. These results
indicate that selective inhibition of p38 MAPK suppresses
IL-1
-induced Cox-2 expression and PGE2 production, suggesting that IL-1
-induced PGE2 synthesis is
up-regulated by the activation of p38 MAPK pathway.
IL-1 stimulates NO generation by enhancing iNOS expression with
subsequent elevation of intracellular cyclic GMP. The increased formation of NO and cyclic GMP in mesangial cells may not only alter
the contractile responses of cells but may also cause tissue injury and
thus contribute to the pathogenesis of certain forms of
glomerulonephritis (2, 28, 35). To test our hypothesis that p38 MAPK
mediates and regulates IL-1
-induced NO synthesis, we evaluated the
effects of SC68376 and serum starvation on IL-1
-induced NO formation
in our current studies. In the presence of serum, IL-1
-induces Cox-2
and PGE2 synthesis and also increases iNOS mRNA and
protein expression and NO generation. The peak response of the mRNA
for iNOS occurred at 12 h and returned toward basal levels by
24 h. However, in mesangial cells serum starved for 24 h,
IL-1
markedly induced iNOS mRNA and protein expression as well
as NO production. The mRNA and protein remained elevated at 24 h. As with serum starvation, SC68376 produced
dose-dependent increases in IL-1
-induced iNOS expression
and NO release in mesangial cells. The current studies demonstrate that
the effects of SC68376 on IL-1
-induced NO production were not
reversed by PGE2. Thus, the effects of p38 MAPK inhibition
on the amplification of IL-1
-induced NO generation are not secondary
to its effects on the decrease of PGE2 formation. Together,
the above data demonstrate that inhibition of p38 MAPK promotes the
IL-1
-induced iNOS expression and NO production in mesangial cells,
suggesting that the signaling mechanism of IL-1
-induced NO synthesis
is down-regulated by the activation of the p38 MAPK signal transmitting
pathway.
In summary, our present studies demonstrate that IL-1 phosphorylates
and activates p38 MAPK in mesangial cells. The activation of p38 MAPK
provides a crucial signaling transduction mechanism, which may
positively regulate IL-1
-induced PG synthesis but negatively regulate IL-1
-induced NO biosynthesis. These opposing actions of the
p38 MAPK pathway may suggest the possibility that a p38 MAPK mechanism
may play an important role in the regulation and balance of function of
this important proinflammatory cytokine. However, because other
mechanisms are involved in IL-1
signaling, the final response to
IL-1
stimulation may be dependent on the integration of multiple
signaling pathways.