Interleukin-1β-induced Cyclooxygenase-2 Expression Requires Activation of Both c-Jun NH2-terminal Kinase and p38 MAPK Signal Pathways in Rat Renal Mesangial Cells*

The inflammatory cytokine interleukin-1β (IL-1β) induces cyclooxygenase-2 (Cox-2) expression with a concomitant release of prostaglandins from glomerular mesangial cells. We reported previously that IL-1β rapidly activates the c-Jun NH2-terminal/stress-activated protein kinases (JNK/SAPK) and p38 mitogen-activated protein kinase (MAPK) and also induces Cox-2 expression and prostaglandin E2 (PGE2) production. The current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 JNK2/SAPKβ reduces Cox-2 expression and PGE2 production stimulated by IL-1β. Similarly, overexpression of the kinase-dead form of p38 MAPK also inhibits IL-1β-induced Cox-2 expression and PGE2production. These results suggest that activation of both JNK/SAPK and p38 MAPK is required for Cox-2 expression after IL-1β activation. Furthermore, our experiments confirm that IL-1β activates MAP kinase kinase-4 (MKK4)/SEK1, MKK3, and MKK6 in renal mesangial cells. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1β- induced Cox-2 expression with inhibition of both JNK/SAPK and p38 MAPK phosphorylation. Overexpression of the kinase-dead form of MKK3 or MKK6 demonstrated that either of these two mutant kinases inhibited IL-1β-induced p38 MAPK phosphorylation and Cox-2 expression but not JNK/SAPK phosphorylation and activation. This study suggests that the activation of both JNK/SAPK and p38 MAPK signaling cascades is required for IL-1β-induced Cox-2 expression and PGE2synthesis.

Prostaglandin (PG) 1 H synthase is a homodimer that catalyzes the rate-limiting step in prostaglandin biosynthesis. This bifunctional enzyme forms prostaglandins by catalyzing the conversion of arachidonic acid to prostaglandin G 2 (PGG 2 ) by its inherent cyclooxygenase activity. PGG 2 is subsequently reduced to PGH 2 by the peroxidase activity of this enzyme. PGH 2 serves as a common precursor for prostaglandins, prostacyclins, and thromboxanes (1). Prostaglandins are arachidonic acid metabolites that influence inflammatory responses, bone development, wound healing, hemostasis, reproductive function, glomerular filtration, and renal homeostasis. Furthermore, alterations in prostaglandin production have been linked to cardiovascular disease, chronic and acute inflammation, atherosclerosis, and colon cancer (2,3).
Previous work has demonstrated that both the JNK/SAPK and p38 MAPK cascades are activated by the inflammatory cytokines IL-1 and tumor necrosis factor-␣ as well as by a wide variety of cellular stresses such as ultraviolet light, ionizing radiation, hyperosmolarity, heat shock, and oxidative stress (13). These findings suggest a role for these two kinase pathways as important signaling mechanisms underlying the inflammatory process. We demonstrated previously that p38 MAPK activation is linked to IL-1␤-induced prostaglandin biosynthesis in renal mesangial cells (11). In addition, we have demonstrated that overexpression of a constitutively active truncation mutant of MEKK1, a putative upstream kinase of MKK4/SEK1, can induce Cox-2 expression and prostaglandin biosynthesis (14). Earlier studies demonstrated activation of JNK1 by the v-src oncogene and induction of Cox-2 expression (15). In the current study we have expressed either wild type or dominant negative constructs of both p38 MAPK and JNK, as well as their immediate upstream activators, to evaluate their role in IL-1␤-induced Cox-2 expression.
The data presented in this manuscript suggest a requirement for both p38 MAPK and JNK activity for cytokine-induced Cox-2 expression. Control of Cox-2 expression by IL-1␤ may be linked to elements within the Cox-2 promoter which require activated transcription factor binding (16). In conjunction with previous findings, our observations suggest a potential mechanism for transcriptional activation of the Cox-2 gene which involves the activation and binding of transcription factors induced by both p38 MAPK and JNK to facilitate full expression of Cox-2 in response to IL-1␤ stimulation.

EXPERIMENTAL PROCEDURES
Materials-Human recombinant IL-1␤ and restriction enzymes were purchased from Boehringer Mannheim. Myelin basic protein (MBP) was purchased from Sigma. Fetal bovine serum was purchased from Life Technologies, Inc. Polyclonal or monoclonal rabbit or mouse IgG antibodies against Cox-2 and Cox-1 were from Cayman Chemical Co. Inc. MKK3, MKK4, MKK6, JNK, phospho-specific JNK, ERK, and p38 MAPK antibodies were from Santa Cruz Biotechnology Inc. Phosphospecific p38 MAPK, MKK4/SEK, and MKK3/MKK6 antibodies were from New England BioLabs. Phospho-specific ERK antibody was from Promega. Anti-FLAG-M2 antibody was from IBI Kodak. pET28cj␦, a histidine-tagged fusion protein expression plasmid that encodes c-jun (1-79) which contains the NH 2 -terminal activation domain of c-jun and a mutant c-jun (1-79), in which Ser 63 and Ser 73 of c-jun (1-79) were mutated to alanine, were generously provided by Dr. Maryann Gruda (Department of Molecular Biology, Bristol Myers Squibb Pharmaceutical Research Institute, Princeton, NJ). Both wild type His-c-jun (1-79) and mutant His-c-jun (1-79, Ala 63 -Ala 73 ) were expressed as histidinetagged fusion proteins in Escherichia coli NovaBlue (DE3) and purified by His-Bind™ resin (Novagen). pGEX ATF-2 (1-96) was obtained from Dr. J. Silvio Gutkind (Molecular Signaling Unit, Laboratory of Cellular Development and Oncology, NIH). MKK4/SEK1 wild type (pCMV SEK1-WT), a constitutively active mutant form of SEK1 (pCMV SEK1-ED), in which Ser 220 and Thr 224 were mutated to glutamic acid and aspartic acid, respectively, and a dominant negative mutant (pCMV SEK1-AL), in which Ser 220 and Thr 224 were mutated to alanine and leucine, respectively, were provided by Dr. Dennis Templeton, Institute of Pathology and Program in Cell Biology, Case Western Reserve University School of Medicine. Wild type or dominant negative mutant p54 SAPK␤ (K55A) in pGEX, MKK3 in pCMV, and MKK6 (S207A/S211L) in pcDNA3 were kindly provided by Dr. Jim Woodgett, Ontario Cancer Institute, Princess Margaret Hospital. Wild type or the dominant negative mutant of JNK1 (T183A/Y185F) in pCMV5 and p38 MAPK (T180A/Y182F) in pGEX were kindly donated by Dr. Roger Davis, Howard Hughes Medical Institute, University of Massachusetts Medical Center. GST-ATF-2 (1-96) and GST-p38 MAPK were expressed as GST fusion proteins in E. coli and purified by GST-binding resin (Amersham Pharmacia Biotech).
Cell Culture-Primary mesangial cell cultures were prepared from male Sprague-Dawley rats as described previously (17). Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 0.3 IU/ml insulin, 100 units/ml penicillin, 100 g/ml streptomycin, 250 g/ml amphotericin B, and 15 mM HEPES, pH 7.4. All experiments were performed with confluent cells grown in 25-cm 2 or 75-cm 2 flasks and used at passages 10 -18. For experiments carried out in serum-free media, cells were starved for 24 h before the experiments. For all other experiments, serum was reduced from 10% to 5% on the day of the experiment.
Infection of Rat Mesangial Cells and NIH 3T3 Cells by the Retroviral Vector-p54 SAPK␤ was subcloned into retroviral vector pLXSN, and 10 g of plasmid DNA was purified and used to transfect PA317 retroviral packaging cells (American Type Culture Collection CRL 9078) with LipofectAMINE (Life Technologies, Inc). Transfected clones were selected in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, 100 g/ml streptomycin, and 500 g/ml G418 (Life Technologies, Inc.) and then isolated with sterile glass cloning rings.
Virus was harvested by placing 5 ml of mesangial medium RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 0.3 unit/ml insulin, 15 mM HEPES, 100 units/ml penicillin, and 100 g/ml streptomycin on confluent, 10-cm plates of transfected PA317 cultures. 1-24 h later the culture supernatant was removed and filtered through a 0.45-m membrane (Gelman Sciences) and diluted 1:3 with mesangial medium. Hexadimethrine bromide (Polybrene) was then added to a final concentration of 8 g/ml (18). Primary rat mesangial cells were cultured as described previously (17). 1 ml of the viruscontaining solution was added to primary rat mesangial cells at 50 -60% confluence. This procedure was repeated at 12 h. At 24 h the virus-containing medium was removed and replaced by normal mesangial medium. At 48 -72 h, G418 was added to the medium at a concentration of 500 g/ml. The medium was subsequently changed every 72 h. After two passages G418 was reduced to 250 g/ml.
Transfection of Rat Mesangial Cells and NIH 3T3 Cells with Lipo-fectAMINE-MKK4/SEK1 wild type (SEK1-WT), a constitutively active mutant form of SEK1 (SEK1-ED), the dominant negative mutant of SEK1 (SEK1-AL), MKK3 wild type, kinase-dead form of MKK3, MKK6 wild type, or dominant negative mutant MKK6 was subcloned into the popRSV1 mammalian expression vector (Stratagene). The wild type or dominant negative mutant form of p38 MAPK or JNK1 was subcloned into the pcDNA3 mammalian expression vector. Primary cultures of rat mesangial cells were plated and transfected at 50 -80% confluence with 20 g of DNA/75-cm 2 flask using LipofectAMINE. Stably transfected isolates were selected in 500 g/ml of G418 for several weeks.
Western Blot Analysis-At the time of harvest, cells were washed with ice-cold phosphate buffer and lysed in whole cell extract (WCE) buffer (25 mM HEPES-NaOH, pH 7.7, 0.3 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM dithiothreitol (DTT), 20 mM ␤-glycerophosphate, 100 M NaVO 4 , 2 g/ml leupeptin, and 100 g/ml phenylmethylsulfonyl fluoride) to which 6 ϫ Laemmli sample buffer was added before heating. After boiling for 5 min, equal amounts of protein were run on 10% 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-HCl, 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 primary antibodies at 1:1000 dilution in 5% bovine serum albumin TBS-T. After washing with 5% milk TBS-T solution, blots were incubated further for 1 h at room temperature with goat anti-rabbit or mouse IgG antibody coupled to horseradish peroxidase (Amersham Pharmacia Biotech) at a 1:3,000 dilution in TBS-T. Blots were then washed five times in TBS-T before visualization. An Enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech) was used for detection.
In-gel Protein Kinase Assay-Harvested cells were solubilized in WCE buffer. Protein kinase assays were performed using our previously described methods (11). Briefly, SDS-polyacrylamide was polymerized in the presence or absence of 200 g/ml His-c-jun (1-79), His-cjun (1-79, Ala 63/73 ), or 400 g/ml MBP. After electrophoresis, SDS was removed by incubation in 20% isopropyl alcohol 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 M guanidine-HCl, 20 mM DTT, 2 mM EDTA, 50 mM Tris-HCl, pH 8.0, for 1 h. Proteins were then renatured by overnight incubation 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 MgCl 2 , 40 mM HEPES-NaOH, pH 8.0, 100 M NaVO 4 . The kinase reaction was carried out for 1 h in kinase buffer with 30 M ATP and 5 Ci/ml of [␥-32 P]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 Ϫ80°C.
Immunocomplex p38 MAPK or JNK Activity Assay-The cell extracts were immunoprecipitated by incubation overnight with anti-p38 MAPK or anti-JNK antibody and then with protein A-Sepharose beads for 3 h at 4°C. The beads were washed three times with 1 ml of ice-cold WCE buffer. p38 MAPK activity was assayed using MBP or GST-ATF-2 (1-96) as the substrate at 30°C for 20 min in 30 ml of kinase reaction buffer (5 g of MBP or GST-ATF-2 (1-96) for p38 activity assay or His-c-jun (1-79) for JNK activity assay, 20 M ATP, 10 Ci of [␥-32 P]ATP, 25 mM HEPES, and 20 mM MgCl 2 ). The reaction was terminated with Laemmli sample buffer, and the products were resolved by 10% SDS-polyacrylamide gel electrophoresis. The phos-phorylated His-c-jun, MBP, or GST-ATF-2 was visualized by autoradiography.
Immunocomplex MKK3, MKK4, and MKK6 Activity Assay-Cell extracts were immunoprecipitated by incubation overnight with anti-MKK3, MKK4, or MKK6 antibody and then incubated with protein A-Sepharose beads for 3 h at 4°C. The beads were washed three times with 1 ml of ice-cold WCE buffer. The immunocomplex MKK3, MKK4, or MKK6 activity assay using GST-p38 MAPK (10 g/reaction) as the substrate was performed at 30°C for 20 min in 30 l of kinase reaction buffer (10 g of GST-p38 MAPK, 100 M ATP, 25 mM HEPES, and 20 mM MgCl 2 ). The reaction was terminated with Laemmli sample buffer, and the products were resolved by 10% SDS-polyacrylamide gel electrophoresis. Phosphorylated p38 MAPK was analyzed by Western blot using anti-phospho-specific p38 MAPK antibody and detected by enhanced chemiluminescence. The phosphorylation level of p38 MAPK was used to reflect MKK3, MKK6, or MKK4/SEK1 activity. PGE 2 Determination-PGE 2 in the overlying culture media was measured with a PGE 2 enzyme-linked immunosorbent assay kit (Cayman Chemical).
Statistical Analysis-Data were expressed as the mean Ϯ S.E. Statistical analysis was performed using paired or unpaired Student's t test. A difference with a p value of 0.05 was considered statistically significant.

JNK/SAPK Mediates IL-1␤-induced Cox-2 Expression-To
determine whether the activation of JNK/SAPK in response to IL-1␤ is required for induction of Cox-2 protein expression and PGE 2 biosynthesis, stably transfected cells overexpressing JNK/SAPK in rat glomerular mesangial cells as well as NIH 3T3 cells were used. We first investigated whether a catalytically inactive form of JNK1 would function as a dominant inhibitor of IL-1␤ induction of Cox-2 expression. Overexpression of both wild type and dominant negative mutant JNK1 in pcDNA3 was verified by a Western blot assay using an anti-JNK antibody. An immunocomplex JNK activity assay demonstrated that overexpression of the kinase-dead form of JNK1 resulted in decreased IL-1␤-induced JNK activity (data not shown). As shown in Fig. 1, the kinase-dead mutant JNK1 inhibited Cox-2 protein expression and PGE 2 production in response to IL-1␤ stimulation. In our experiments, we also evaluated whether the kinase-negative mutant of JNK2/p54 SAPK␤ could inhibit Cox-2 expression and PGE 2 production after IL-1␤ stimulation. Rat mesangial cells and NIH 3T3 cells transfected with either wild type JNK2/p54 SAPK␤ or the JNK2/p54 SAPK␤ kinase-inactive mutant were stimulated with IL-1␤. Overexpression of JNK2/p54 SAPK␤ was verified by the Western blot analysis followed by immunocomplex JNK activity assays that revealed that the kinase-negative form of p54 SAPK␤ inhibited total JNK activity induced by IL-1␤ (data not shown). Similar to JNK1, the dominant negative JNK2/p54 SAPK␤ expressed in pLXSN blocked IL-1␤-induced Cox-2 expression and PGE 2 production in renal mesangial cells (Fig. 2). This finding was also demonstrated in NIH 3T3 cells in which Cox-2 expression and PGE 2 production were inhibited by the kinase-dead form of JNK2/p54 SAPK␤ (data not shown). In experiments in which mesangial cells were infected with empty pLXSN we observed enhanced basal levels of Cox-2 expression and PGE 2 production and a blunting of the response to IL-1␤. Nevertheless kinase-dead JNK2 inhibited both basal and IL-1␤-induced Cox-2 expression and PGE 2 production. These results demonstrate that JNK/SAPK is important for IL-1␤ activation of Cox-2 protein expression and that the activation of JNK/SAPK is necessary for IL-1␤-induced Cox-2 expression and PGE 2 production.
p38 MAPK Is Involved in the Regulation of Cox-2 Expression Induced by IL-1␤-Previously we demonstrated that IL-1␤ increases p38 MAPK phosphorylation and activation. Pharmacological inhibition of p38 MAPK can effectively block Cox-2 expression and PGE 2 release stimulated by IL-1␤ in renal mesangial cells. To confirm further the physiological function of p38 MAPK in the regulation of Cox-2 protein expression, we analyzed the effects of overexpression of the kinase-inactive p38␣ MAPK mutant on IL-1␤-induced Cox-2 expression and PGE 2 production. As shown in Fig. 3, the dominant negative mutant form of p38␣ MAPK blocked Cox-2 expression and PGE 2 production after IL-1␤ stimulation. These results demonstrate further the physiologic function of p38 MAPK in the regulation of IL-1␤-stimulated Cox-2 induction and PGE 2 synthesis.
MKK3 and MKK6 Function Regulate Cox-2 Expression Stimulated by IL-1␤-MKK3 and MKK6 are upstream kinases that activate and phosphorylate p38 MAPK in vitro and in vivo. To verify whether MKK3 and MKK6 are involved in IL-1␤ signaling, we first measured MKK3 and MKK6 activity by an immunocomplex kinase assay using GST-p38 MAPK as the substrate and phosphorylation of p38 MAPK with an anti-phospho-specific p38 MAPK antibody. Fig. 4, A and B  plasmids encoding either epitope-tagged MKK3 or MKK6 wild type or the kinase-negative mutant. Stable overexpression of MKK3 (Fig. 5A) and MKK6 (data not shown) in popRSV1were detected by Western blot analysis using anti-FLAG tag antibody. Transfection of cells with dominant negative MKK3 (Fig.  5B) or MKK6 (data not shown) inhibited p38 MAPK after IL-1␤ stimulation. Importantly however, JNK phosphorylation was unaffected (data not shown). These data verify that MKK3 and MKK6 can activate p38 MAPK after IL-1␤ stimulation in renal mesangial cells. Furthermore, we examined the effects of the kinase-inactive mutant forms of MKK3 or MKK6 on Cox-2 expression and PGE 2 production stimulated by IL-1␤. Interestingly, we observed that overexpression of either kinase-negative mutant (MKK3 or MKK6) resulted in the inhibition of IL-1␤-induced Cox-2 expression and PGE 2 synthesis in renal mesangial cells (Figs. 5 and 6). One explanation for these results is that the expressed dominant negative protein binds the p38 MAPK substrates and prevents phosphorylation by either of the activated native MKK3 or MKK6 proteins. These results demonstrate that both MKK3 and MKK6 may mediate IL-1␤-induced p38 MAPK activation as well as Cox-2 protein expression and PGE 2 production.

MKK4/SEK1 Mediates IL-1␤-induced Cox-2 Expression through Both JNK/SAPK and p38 MAPK Mechanisms-Our
previous studies have demonstrated that MKK4/SEK1 activates and phosphorylates both JNK/SAPK and p38 MAPK. Overexpression of the kinase-negative mutant form of MKK4/ SEK1 inhibits basal Cox-2 expression and PGE 2 production in NIH 3T3 cells cultured in serum-containing media (14). To determine whether MKK4/SEK1 was involved in the IL-1 signal transduction mechanism, we measured MKK4/SEK1 activity after IL-1␤ stimulation. We analyzed the MKK4 activity by an immunocomplex kinase assay using GST-p38 MAPK as the substrate. We found that IL-1␤ can enhance MKK4/SEK1 activity in mesangial cells (Fig. 7A). This finding suggests that MKK4/SEK1 may be involved in IL-1␤ signaling. We therefore tried to determine whether activation of MKK4/SEK1 is required for activation of JNK/SAPK and p38 MAPK and subsequent prostaglandin biosynthesis in mesangial cells. We established permanently transfected mesangial cells containing the  (Fig. 7, B and C). These results suggest that MKK4/ SEK1 can mediate IL-1␤-induced JNK/SAPK and p38 MAPK activation in the intact mesangial cell. More importantly, our experiments show that the kinase-negative mutant form of MKK4/SEK1 (SEK-AL) inhibits IL-1␤-induced Cox-2 expression and PGE 2 production. By comparison, the constitutively active mutant form of MKK4/SEK1 (SEK-ED) enhanced IL-1␤induced Cox-2 expression and PGE 2 production (Fig. 7, D and  E). Together, these results suggest a role for MKK4/SEK1 in IL-1␤-induced JNK/SAPK and p38 MAPK activation and modulation of prostaglandin biosynthesis in renal mesangial cells.

DISCUSSION
The inflammatory cytokine IL-1 is a potent immunoregulatory and proinflammatory agent involved in a variety of pathological processes such as the response to infection, activated lymphocyte products, microbial toxins, and other stimuli (19). In glomerular inflammation, infiltrating macrophages produce IL-1, which activates renal mesangial cells and promotes glomerular injury. Our laboratory has reported previously that IL-1␤ induces Cox-2 protein expression with concomitant synthesis of prostaglandins such as PGE 2 in renal mesangial cells (11,20,21). The induction of this key mediator may provide a critical mechanism involved in IL-1-induced renal inflammation. Recent studies suggest that activation of Cox-2 is not only involved in the pathogenesis of renal inflammatory diseases but may also play a critical role in normal kidney development, differentiation, and function (22).
Although much effort has been made to identify the intracellular signaling pathways triggered by IL-1, the signal trans- duction mechanisms by which IL-1 induces Cox-2 protein expression and prostaglandin production are still unclear. Several recent reports indicate that an important group of protein kinases, the MAPKs, may be involved in these signaling processes. The MAPK pathways have been implicated as a mechanism by which signals are transduced from the cell surface to the nucleus in response to a variety of different stimuli and participate in intracellular processes by further inducing the phosphorylation of intracellular substrates such as other protein kinases and transcription factors. This signaling mechanism is believed to control a wide spectrum of cellular physiological and pathophysiological processes including cell growth, differentiation, and stress responses (12). Recent work has demonstrated that both JNK/SAPK and p38 MAPK cascades are activated by the inflammatory cytokines IL-1␤ and tumor necrosis factor-␣ as well as by a wide variety of cellular stresses such as ultraviolet light, ionizing radiation, hyperosmolarity, heat shock, and oxidative stress (13). These findings suggest an important role for these two kinase pathways in the signaling mechanisms recruited as part of the inflammatory process.
Recent work suggests that the MAPK pathway is also involved in regulating prostaglandin biosynthesis. For example, activation of cytosolic phospholipase A 2 by thrombin involves activation of both ERK and p38 MAPK (23,24). Furthermore, we have shown previously that IL-1 stimulation of renal mesangial cells increases PGE 2 production and Cox-2 expression concomitant with activation of the p38 MAPK and JNK signaling pathways. We have demonstrated that pharmacological inhibition of p38 MAPK dose-dependently inhibits IL-1␤-mediated Cox-2 expression and PGE 2 production (11). In the current study, overexpression of the kinase-inactive mutant p38␣ MAPK inhibits IL-1␤-induced Cox-2 expression and PGE 2 production, thus confirming further that activation of p38␣ MAPK is required for Cox-2 expression and PGE 2 production.
Herschman and his colleagues (15,16) reported previously that activation of the JNK/SAPK pathway plays an important role in v-src-induced PGH synthase-2 (Cox-2) gene expression. To elucidate the physiological function of JNK/SAPK, we overexpressed both wild type and kinase-dead forms of JNK1 and JNK2/p54 SAPK␤ in renal mesangial cells. The kinase-dead form of both JNK constructs markedly inhibits IL-1␤-induced Cox-2 expression and PGE 2 release, thus confirming the requirement of JNK/SAPK activity for cytokine-induced prostaglandin biosynthesis.
Previous data have suggested that MKK4/SEK1 is an immediate upstream kinase activating the JNK pathway (25,26). We recently reported that overexpression of a constitutively active mutant form of MKK4/SEK1 increases both JNK and p38 MAPK activity and phosphorylation (14). Because IL-1␤ can activate MKK4/SEK1, we tested the effects of transfection of either constitutively active or dominant negative MKK4/ SEK1. We observed that the dominant negative mutant MKK4/ SEK1 inhibited IL-1␤-induced JNK/SAPK and p38 MAPK activation, whereas the constitutively active form activated both JNK and p38 MAPK. Furthermore, overexpression of dominant negative MKK4/SEK1 resulted in inhibition of IL-1␤-induced cyclooxygenase expression and prostaglandin biosynthesis.

FIG. 7. Effects of MKK4 overexpression on IL-1␤-induced
Cox-2 expression and PGE 2 production. Panel A, primary mesangial cell cultures were treated with 100 units/ml IL-1␤, and MKK4 activity in response to IL-1␤ stimulation was measured by immunocomplex kinase assay using p38 MAPK as substrate. Panels B-E, popRSV vector, popRSV MKK4 (WT), popRSV MKK4 constitutively active mutant (ED), or popRSV MKK4 dominant negative mutant (AL) was stably transfected into primary mesangial cell cultures. Panel B, transfected cells were then stimulated with or without 100 units/ml IL-1␤. JNK phosphorylation was detected by Western blot assay using an antiphospho-specific JNK antibody. Panel C, p38 MAPK phosphorylation detected by Western blot assay using an anti-phospho-specific p38 MAPK antibody. Panel D, Cox-2 protein expression in transfected mesangial cells in response to IL-1␤ stimulation analyzed by Western blot assay using an anti-Cox-2 antibody. Panel E, PGE 2 production in culture media released by MKK4 overexpressing mesangial cells treated with or without IL-1␤. Therefore, we believe that MKK4/SEK1 is an important upstream kinase that influences both p38 MAPK and JNK/SAPK activity, resulting in regulation of Cox-2 expression.
Previous data have indicated that both MKK3 and MKK6 can activate and phosphorylate p38 MAPK (27,28). Our experiments demonstrate that IL-1␤ increases the activity of both MKK3 and MKK6 in renal mesangial cells. To ascertain whether MKK3 and MKK6 function in the regulation of p38 MAPK activation and Cox-2 expression induced by IL-1␤, wild type and kinase-dead MKK3 or MKK6 constructs were utilized. The data presented demonstrate that activation of either MKK3 or MKK6 can activate p38 MAPK and increase Cox-2 expression and PGE 2 synthesis with IL-1 stimulation. Overexpression of the dominant negative mutant forms of either MKK3 or MKK6 results in marked inhibition of p38 MAPK activation and Cox-2 expression induced by IL-1␤. Importantly the kinase-dead mutants do not affect activation of JNK by IL-1␤.
Overall, our data suggest that MKK3, MKK4, and MKK6 are involved in cytokine-induced activation of p38 MAPK and resultant Cox-2 expression. The observation that p38␣ MAPK can be equally activated by MKK3, MKK4, or MKK6 (29) suggests that p38␣ MAPK may function as a common substrate for these three MAPK kinases. Overexpression of the dominant negative mutant form of any of these MAPK kinases may result in inhibition of p38 MAPK activity by either 1) competing with endogenous MAPK kinases for binding and activation of p38 MAPK or 2) by competing for activation by a putative upstream MAPK kinase kinase. The latter seems unlikely because dominant negative p38␣ does not affect MKK4 and JNK activation. The consequence of inhibition of MKK3, MKK4, or MKK6 activity is inhibition of IL-1␤-induced Cox-2 expression.
The aforementioned results suggest that the activation of JNK/SAPK and p38 MAPK is necessary both for induction of Cox-2 protein expression and for PGE 2 production in the renal mesangial cells when induced by IL-1␤. This conclusion is based on the observation that the inhibition of either p38␣ MAPK or the JNK/SAPK pathway results in significant inhibition of IL-1␤-induced Cox-2 expression and PGE 2 production. Based on previous observations and our current findings, Fig. 8 depicts a hypothetical model for the combined role of p38 MAPK and JNK activation in the modulation of Cox-2 expression when mesangial cells are exposed to IL-1␤.
In summary, we demonstrate that activation of both SAPK/ JNK and p38 MAPK is required for Cox-2 expression and PGE 2 production after IL-1␤ stimulation. Furthermore, we demonstrate that MKK4/SEK1, MKK3, and MKK6 are all involved in IL-1␤-induced prostaglandin biosynthesis. MKK3 and MKK6 function as upstream regulators of p38 MAPK, whereas MKK4/ SEK1 can function as the upstream kinase of both p38 MAPK and SAPK/JNK. We believe that the activation of both SAPK/ JNK and p38 MAPK signaling cascades together are crucial intracellular mechanisms that mediate Cox-2 expression and PGE 2 synthesis induced by cytokine stress.