Induction of Cyclooxygenase-2 by the Activated MEKK1 right-arrow  SEK1/MKK4 right-arrow  p38 Mitogen-activated Protein Kinase Pathway*

Zhonghong GuanDagger , ShaAvhree Y. BuckmanDagger , Alice P. Pentland§, Dennis J. Templeton, and Aubrey R. MorrisonDagger parallel

From the Dagger  Department of Molecular Biology and Pharmacology and Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, the §  Department of Medicine, Rochester University School of Medicine, Rochester, New York 14642, and the  Institute of Pathology and Program in Cell Biology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The mitogen-activated protein kinase (MAPK) cascade is believed to function as an important regulator of prostaglandin biosynthesis. Previously we reported that interleukin-1beta induces activation of JNK/SAPK and p38 MAPK with concomitant up-regulation of cyclooxygenase (Cox)-2 expression and prostaglandin E2 (PGE2) synthesis. Our experiments demonstrate that overexpression of Delta MEKK1 (a constitutively active truncation mutant of MEKK1 containing the C-terminal 324 amino acids) increases Cox-2 expression and PGE2 production which is completely blocked by SC68376, a pharmacologic inhibitor of p38 MAPK. Delta MEKK1 overexpression results in activation of both c-Jun N-terminal kinases/extracellular signal-regulated kinases (JNK/SAPK) and p38 MAPK. Furthermore, activation of MEKK1 increases SEK1/MKK4 but not MKK3 or MKK6 activity. These findings suggest that MEKK1 right-arrow SEK1/MKK4 may function as an upstream kinase capable of activating both p38 MAPK and JNK/SAPK with subsequent induction of Cox-2 expression and PGE2 production. We also found that overexpression of the constitutively active form of SEK1 (SEK1-ED) increases both p38 MAPK and JNK/SAPK phosphorylation, and increases PGE2 production and Cox-2 expression. By comparison, overexpression of the dominant negative form of SEK1 (SEK1-AL) decreases the phosphorylation of both p38 MAPK and JNK/SAPK and reduces Cox-2 expression. Together, this data suggests a potential role for the MEKK1 right-arrow SEK1/MKK4 right-arrow p38 MAPK right-arrowright-arrow Cox-2 cascade linking members of the MAPK pathway with prostaglandin biosynthesis.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Prostaglandins are ubiquitous compounds involved in various homeostatic and inflammatory processes throughout the body. They are formed by the combined action of a phospholipase A2 (PLA2)1 which liberates arachidonic acid from the sn-2 position of cellular membrane phospholipids and the cyclooxygenase (Cox) which converts arachidonic acid to the endoperoxide intermediate PGH2. PGH2 is subsequently converted to prostaglandins by the action of cell-specific synthases (1). There are two cyclooxygenase enzymes which have been identified (60% homology), Cox-1 and Cox-2. Cox-1 is constitutively expressed in most tissues and mediates physiologic responses such as regulation of renal and vascular homeostasis and cytoprotection of the stomach. By comparison Cox-2 is primarily considered an inducible immediate-early gene product whose synthesis can be up-regulated by mitogenic or inflammatory stimuli including: tumor promoters (2), IL-1beta (3), endotoxins (4), platelet-derived growth factor (5), and serum (6).

The physiological role of the cyclooxygenase has been the topic of much interest. Cyclooxygenases are the main therapeutic target for non-steroidal anti-inflammatory drugs which exhibit their antipyretic, analgesic, and anti-inflammatory effects in humans via inhibition of prostaglandin biosynthesis (7). Non-steroidal anti-inflammatory drugs have been effective in the reduction of inflammatory symptoms in carrageenan-induced rat paw inflammation models (8) and in the reduced incidence of colon cancer (9, 10). Moreover, rat intestinal epithelial cells overexpressing Cox-2 demonstrate resistance to apoptosis (11) and Caco-2 human colon cancer cells demonstrate increased metastatic potential when transfected with Cox-2 (12). Perhaps, the most striking evidence for the pervasive role of Cox-2 is obtained from targeted gene disruption models. Mice which are homozygously deficient for Cox-2 develop severe renal pathology and die prematurely of renal failure (13).

Recently, a novel class of cytokine-suppressive anti-inflammatory drugs have been shown to be inhibitors of endotoxin-stimulated tumor necrosis factor and IL-1 induction (14). These cytokine-suppressive anti-inflammatory drugs have been further shown to inhibit the catalytic activity of p38 MAPK. These and other recent findings serve to potentially link prostaglandin biosynthetic pathways which mediate inflammatory responses, with activation of the mitogen-activated protein kinase (MAPK) signaling cascade.

Components of the MAPK pathway have been implicated as mediators of phosphorylation of intracellular substrates such as protein kinases and transcription factors (15) as well as regulators of cell growth and differentiation (16). In mammalian cells, at least three different subfamilies of MAPK have been identified each having several isoforms. They include the extracellular signal-regulated kinases (ERKs), p44 MAPK (ERK1) and p42 MAPK (ERK2); stress-activated protein kinases (SAPKs), also called c-Jun N-terminal kinases (JNKs): which include JNK2 (p54 SAPKalpha /beta /gamma ) and JNK1 (p45 SAPKalpha /beta ) and the p38 MAPKs (alpha , beta , gamma , and delta ). These kinases are in turn activated by distinct upstream MAPK/ERK kinases (MEKs, MKKs) which recognize and phosphorylate threonine and tyrosine residues within a tripeptide motif (Thr-X-Tyr) required for MAPK activation (17). Once phosphorylated, these MAPKs then activate their specific substrates on serine and/or threonine residues to produce their effects on downstream targets. Recent work has demonstrated that both SAPK/JNK and p38 MAPK cascades are activated preferentially by the inflammatory cytokines IL-1beta and tumor necrosis factor-alpha , as well as by a wide variety of cellular stresses such as ultraviolet light, ionizing radiation, hyperosmolarity, heat shock, oxidative stress, etc. (17). These findings further implicate the role of these two kinase pathways as important signaling mechanisms underlying the inflammatory process.

Recent work has linked activation of the prostaglandin generative pathway with the MAPK pathway. Lin et al. (18) have demonstrated that the activation of cytosolic phospholipase A2 (cPLA2) is mediated by MAPK. Furthermore, Kramer et al. (19) have demonstrated that thrombin activates both ERK and p38 MAPK in human platelets which may utilize cPLA2 as a downstream target. We have shown that IL-1 stimulation of renal mesangial cells mediates prostaglandin E2 production and Cox-2 expression with concomitant activation of p38 MAPK and SAPK-mediated signaling pathways (20, 21). Furthermore, we have demonstrated that inhibition of p38 MAPK with a pyridyl oxazole results in the down-regulation of IL-1 mediated Cox-2 expression and PGE2 production, suggesting a role for p38 in the modulation of prostaglandin synthesis (20). Previous work by Templeton et al. (22) has shown that cells induced to overexpress a constitutively active form of Delta MEKK1 directly activate the SAPK pathway. The availability of this model system encouraged exploration of possible SAPK-mediated effects on prostaglandin production suggested by our previous cytokine stimulation data. In our current study, we describe the ability of Delta MEKK1 expressing cells to induce Cox-2 expression and PGE2 production and analyze the intermediate kinases involved in the regulation of prostaglandin biosynthesis.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
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References

Reagents-- Myelin basic protein (MBP) was purchased from Sigma. The p38 MAPK inhibitor SC68376 was kindly provided by Dr. Joe Portnova (G. D. Searle Corp.) and dissolved in dimethyl sulfoxide. 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.; cPLA2, MEKK1, MKK3, MKK4, MKK6, JNK, phospho-specific JNK, ERK, and p38 MAPK antibodies were from Santa Cruz Biotechnology Inc. Phospho-specific p38 MAPK, SEK/MKK4, and MKK3/MKK6 were from New England BioLabs. Phospho-specific ERK antibody was from Promega. pET28-c-Jun, a histidine-tagged fusion protein expression plasmid that encodes c-Jun-(1-79) which contains the amino-terminal activation domain of c-Jun and a mutant c-Jun-(1-79, S63A/S73A), in which serine 63 and 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, S63A/S73A) were expressed as a histidine-tagged fusion protein 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). pGEX p38 MAPK was 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 a GST fusion protein in E. coli and purified by GST-binding resin (Pharmacia Biotech Inc.).

Cell Culture and Transfection-- All experiments were performed with NIH 3T3 cells as well as NIH 3T3 cells stably transfected with an IPTG inducible EE-epitope-tagged MEKK1 truncation mutant (containing the C-terminal 324 amino acids, Delta MEKK-1) subcloned into the lacSwitch promoter construct (Stratagene) (22, 23). Normal NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 25 mM HEPES. MEKK1 3T3 cells were maintained in the above media with 500 mg/liter of G418 and 200 mg/liter of hygromycin. Delta MEKK1 expression was induced using 1 mM IPTG for various time periods.

SEK1/MKK4 wild type (SEK1-WT), a constitutively active mutation form of SEK1 (SEK1-ED, serine 220 and threonine 224 mutated to glutamic acid and aspartic acid, respectively) or the dominant negative mutation (SEK1-AL, serine 220 and threonine 224 mutated to alanine and leucine, respectively) were subcloned into the popRSV1 mammalian expression vector (Stratagene) and stably transfected in NIH 3T3 cells. Cells were plated and transfected at 50-80% confluence using 20 µg of DNA per 75-cm2 flask using LipofectAMINE (Life Technologies, Inc). Stably transfected isolates were selected in 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 buffer (25 mM HEPES-NaOH (pH 7.7), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM dithiothreitol (DTT), 20 mM beta -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 10% SDS-PAGE. 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 primary 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 or mouse IgG antibody coupled to horseradish peroxidase (Amersham) at 1:3000 dilution in TBS-T. Blots were then washed five times in TBS-T before visualization. Enhanced chemiluminescence (ECL) kit (Amersham) was used for detection.

In-gel Protein Kinase Assay-- Harvested cells were solubilized in whole cell extract buffer. Protein kinase assays were performed using our previously described methods (20). Briefly, SDS-polyacrylamide was polymerized in the presence or absence of 200 µg/ml His-c-Jun-(1-79), His-c-Jun-(1-79, S63A/S73A), or 400 µg/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 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 [gamma -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.

Immunecomplex p38 MAPK Activity Assay-- The cell extracts were immunoprecipitated by incubation overnight with anti-p38 MAPK 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 immunecomplex p38 MAPK activity assay using MBP or GST-ATF-2-(1-96) as the substrate was performed at 30 °C for 20 min in 30 µl of kinase reaction buffer (5 µg of MBP or GST-ATF-2-(1-96), 20 µM ATP, 10 µCi of [gamma -32P]ATP, 25 mM HEPES, and 20 mM MgCl2). The reaction was terminated with Laemmli sample buffer and the products were resolved by 10% SDS-PAGE. The phosphorylated MBP or GST-ATF-2 was visualized by autoradiography.

Immunecomplex MKK3, MKK4, and MKK6 Activity Assay-- The cell extracts were immunoprecipitated by incubation overnight with anti-MKK3, MKK4, or MKK6 antibodies and then incubated 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 immunecomplex MKK3, MKK4, or MKK6 activity assay using GST-p38 MAPK (10 µg/reaction) as the substrate was performed at 30 °C for 20 min in 30 µl of kinase reaction buffer (10 µg of GST-p38 MAPK, 100 µM ATP, 25 mM HEPES, and 20 mM MgCl2). The reaction was terminated with Laemmli sample buffer and the products were resolved by 10% SDS-PAGE. The phosphorylated p38 MAPK was detected by anti-phospho-specific p38 MAPK antibody by Western blot analysis and detected by enhanced chemiluminescence.

PGE2 Determination-- PGE2 in the overlying culture media was measured by a PGE2 ELISA Kit (Cayman Chemical).

Statistical Analysis-- All the experiments were performed at least three times. Data 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.

    RESULTS
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Introduction
Procedures
Results
Discussion
References

Delta MEKK1 Activation Induces PGE2 Production and Cox-2 Expression-- A MEKK1 inducible model system was utilized in which NIH 3T3 cells were stably transfected with a constitutively active truncated fragment of MEKK1 (the C-terminal 324 amino acids, Delta MEKK-1) under the control of an IPTG inducible promoter. Confluent normal NIH 3T3 cells as well as Delta MEKK1 3T3 cells were incubated for varying periods of time with 1 mM IPTG to confirm overexpression of Delta MEKK1 protein by Western blot assay using an anti-MEKK1 antibody. Normal NIH 3T3 cells were virtually unaffected by IPTG stimulation. By comparison, in MEKK1 3T3 cells, Delta MEKK1 expression was minimally detected under basal conditions and was maximally induced at 12-36 h following IPTG stimulation (Fig. 1). Notably, the MEKK1 antibody detected duplicate bands of equal intensity following IPTG stimulation. Previous data suggests that the lower band represents untagged Delta MEKK1 (resulting from an internal initiation product), whereas the upper band represents the EE epitope-tagged Delta MEKK1 (24). In this study, we examined whether activation of Delta MEKK1 expression induces prostaglandin biosynthesis. Comparison of untreated Delta MEKK1 3T3 cells with cells induced by IPTG demonstrates an approximately 4-fold enhancement in PGE2 production (Fig. 2B). Since cyclooxygenase is a key rate-limiting enzyme which mediates prostaglandin biosynthesis, we further examined whether the enhancement of PGE2 production was correlated with changes in the expression of these enzymes. As shown in Fig. 2A, induction of Delta MEKK1 by IPTG resulted in a significant increase in Cox-2 expression. However, induction of Delta MEKK1 did not increase either Cox-1 or cPLA2 protein expression (data not shown). To demonstrate whether MEKK1-induced PGE2 synthesis is mediated by the activation of Cox-2, a Cox-2 specific inhibitor, NS-398, was utilized. As shown in Table I, NS-398, at a concentration of 0.1 µM, completely blocked PGE2 production induced by the activation of MEKK1. These findings suggest that Cox-2 plays an important role in MEKK1-induced PGE2 biosynthesis.


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Fig. 1.   Delta MEKK1 overexpression in MEKK1 inducible NIH 3T3 cells. Western blot assay for validation of Delta MEKK1 overexpression in NIH 3T3 cells. NIH 3T3 cells were stimulated with 1 mM IPTG for the time periods indicated and Delta MEKK1 protein expression was detected by Western blot assay using an anti-MEKK1 antibody. Lane 1, normal NIH 3T3 cells without IPTG stimulation; lanes 2-5, MEKK1 inducible 3T3 cells treated with 1 mM IPTG for 0 (lane 2), 12 (lane 3), 24 (lane 4), and 36 (lane 5) h.


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Fig. 2.   Delta MEKK1 induces Cox-2 expression and PGE2 production. A, Western blot assay for Cox-2 protein expression. MEKK1 inducible cells were stimulated by 1 mM IPTG for the indicated time periods and Cox-2 expression was detected by Western blot analysis using an anti-Cox-2 antibody. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-4, MEKK1 inducible 3T3 cells treated by 1 mM IPTG for 0 (lane 2), 12 (lane 3), and 24 (lane 4) h. B, MEKK1 NIH 3T3 cells were stimulated with or without 1 mM IPTG for 0-36 h and PGE2 in the culture media was measured by enzyme-linked immunosorbent assay. White bars, MEKK1 inducible NIH 3T3 cells without IPTG stimulation. Black bars, MEKK1 inducible NIH 3T3 cells with IPTG stimulation.

                              
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Table I
Effects of NS-398, a Cox-2 specific inhibitor, on MEKK1-induced PGE2 production
MEKK1 cells were stimulated with or without 1 mM IPTG in the present of 100 nM NS-398. PGE2 in the media was determined at 24 h of exposure to drugs. Results are expressed at mean ± S.E. (n = 3).

Delta MEKK1 induces p46 SAPK and p54 SAPK Activity-- To investigate the signal transduction pathway responsible for mediating induction of COX-2 expression in MEKK1 cells, in-gel kinase assays using His-c-Jun-(1-79) as a substrate were performed to measure SAPK activity following Delta MEKK1 induction by IPTG. Fig. 3A demonstrates that two bands corresponding to the molecular weights of p46 SAPK and p54/55 SAPK were maximally induced in MEKK1 3T3 cells by 24 h after IPTG induction. Similar experiments utilizing mutated His-c-Jun-(1-79, S63A/S73A) as a substrate demonstrated no phosphorylation of this substrate (Fig. 3B). Similarly, Western blot analysis using an anti-phospho-specific JNK which recognizes both phosphorylated (and presumably active) p46 SAPK and p54/55 SAPK further illustrated that Delta MEKK-1 induction could increase phosphorylation of both p46 SAPK and p54/55 SAPK, respectively (Fig. 3C). Western blot analysis employing a pan-JNK antibody (which also recognizes murine SAPK) indicated that Delta MEKK1 induction did not influence total protein expression of p46 SAPK or p54/55 SAPK (Fig. 3D).


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Fig. 3.   Activation and phosphorylation of SAPK by Delta MEKK1 induction. A, in-gel SAPK kinase assay. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods and SAPK activity was measured by in-gel kinase assay using His-c-Jun-(1-79) as the substrate. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-4, MEKK1 inducible 3T3 cells treated with 1 mM IPTG for 0 (lane 2), 12 (lane 3), and 24 (lane 4) h. B, negative control for the in-gel SAPK kinase assay. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG and the kinase assay was performed with mutant His-c-Jun-(1-79, S63A/S73A) as the substrate. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-4, MEKK1 inducible 3T3 cells treated by 1 mM IPTG for 0 (lane 2), 12 (lane 3), and 24 (lane 4) h. C, Western blot assay for SAPK phosphorylation. NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods and SAPK phosphorylation was determined by Western blot analysis using an anti-phospho-specific JNK antibody (which recognizes murine SAPK). Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-4, MEKK1 inducible NIH 3T3 cells treated by 1 mM IPTG for 0 (lane 2), 12 (lane 3), and 24 (lane 4) h. D, Western blot assay for SAPK protein expression. MEKK1 inducible cells were stimulated with 1 mM IPTG for the indicated time periods and SAPK expression was determined by Western blot analysis using an anti-pan-JNK antibody (which recognizes murine SAPK). Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-4, MEKK1 inducible 3T3 cells treated by 1 mM IPTG for 0 (lane 2), 12 (lane 3), and 24 (lane 4) h.

Delta MEKK1 Induces p38 MAPK Activity-- Recent data generated in our laboratory has suggested that the p38 MAPK activated pathway may contribute to the signaling mechanism for IL-1beta -induced Cox-2 expression and prostaglandin synthesis in renal mesangial cells (20). With this data in mind and the above observations linking Cox-2 expression with MEKK-1 activation, we assessed IPTG-induced Delta MEKK1 3T3 cells for p38 MAPK activity. As shown in Fig. 4, p38 MAPK activity increased within 24-36 h after Delta MEKK1 induction which was examined by immunocomplex kinase assay using MBP (Fig. 4A) or GST-ATF-2-(1-96) (Fig. 4B) as the substrate. This increase in activity was further correlated with Western blot analysis illustrating increased phosphorylation of p38 MAPK at 24 h following IPTG stimulation (Fig. 4D). Western blot analysis using anti-p38 MAPK indicated that total protein expression was unaffected by Delta MEKK1 induction (Fig. 4C). Together, these results demonstrate a role for MEKK1, a kinase heretofore considered upstream of the SAPK pathway, as an activator of p38 MAPK in an intact cell system.


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Fig. 4.   Activation and phosphorylation of p38 MAPK by Delta MEKK1 induction. A, immunocomplex p38 MAPK assay utilizing MBP as the substrate. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods. p38 MAPK was immunopurified by an anti-p38 MAPK antibody and p38 MAPK activity was detected by immunocomplex kinase assay using MBP as the substrate. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-5, MEKK1 inducible NIH 3T3 cells treated by 1 mM IPTG for 0 (lane 2), 12 (lane 3), 24 (lane 4), and 36 (lane 5) h. B, immunocomplex p38 MAPK assay utilizing GST-ATF-2 as the substrate. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods. p38 MAPK was immunopurified by an anti-p38 MAPK antibody and p38 MAPK activity was detected by immunocomplex kinase assay with GST-ATF-1-(1-96) as the substrate. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-5, MEKK1 inducible NIH 3T3 cells treated with 1 mM IPTG for 0 (lane 2), 12 (lane 3), 24 (lane 4), and 36 (lane 5) h. C, Western blot assay for p38 MAPK protein expression. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods and p38 MAPK expression was determined by Western blot analysis using an anti-p38 MAPK antibody. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-4, MEKK1 inducible 3T3 cells treated by 1 mM IPTG for 0 (lane 2), 12 (lane 3), and 24 (lane 4) h. D, Western blot assay for p38 MAPK phosphorylation. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods and p38 MAPK phosphorylation was determined by Western blot analysis using an anti-phospho-specific p38 antibody. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-4, MEKK1 inducible NIH 3T3 cells treated by 1 mM IPTG for 0 (lane 2), 12 (lane 3), and 24 (lane 4) h.

Previous in vitro and overexpression studies suggest that MEKK1 is able to activate and phosphorylate MEK which in turn, can phosphorylate and activate ERK (23, 25). However, similar to observations by Yan et al. (22), we found that overexpression of Delta MEKK1 at the functional time points failed to activate ERK (data not shown) although SAPK and p38 MAPK were phosphorylated and activated.

p38 MAPK Mediates MEKK1-induced COX-2 Expression-- To ascertain whether p38 MAPK signaling pathways mediate PGE2 production and cyclooxygenase expression induced by Delta MEKK1 induction, we studied the effects of SC68376, an inhibitor of p38 MAPK. SC68376 directly inhibits p38 MAPK catalytic activity (IC50 of 2-5 µM) without influencing ERK and JNK activity. When Delta MEKK1 3T3 cells were treated with 1 mM IPTG and 10 µM SC68376 for 24 h, the inhibitor completely eliminated Cox-2 expression and concomitant PGE2 production normally induced by Delta MEKK1 induction. As shown in Fig. 5, SC68376 exhibits selectivity for inhibition of Cox-2 protein expression. There was negligible influence on MEKK-1, SAPK, and cPLA2 expression (data not shown). Similarly, PGE2 production induced by Delta MEKK1 was completely inhibited by the presence of 10 µM SC68376 (Fig. 5). The above observations confirm that the activation of p38 MAPK provides a crucial signaling mechanism which promotes Cox-2 expression and prostaglandin biosynthesis.


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Fig. 5.   SC68376, a selective p38 MAPK inhibitor, inhibits Delta MEKK1 induced Cox-2 expression and PGE2 production. MEKK1 NIH 3T3 cells were treated with or without 1 mM IPTG in the presence or absence of 10 µM of SC68376 for 24 h. PGE2 in the culture media was measured by ELISA. Cox-2 protein expression were detected by immunoblot assay and densitometric analysis performed and results are indicated above. White bars, PGE2 production. Black bars, Cox-2 protein expression.

SEK1/MKK4 Mediates MEKK1-induced COX-2 Expression-- Previous data has suggested that SEK1/MKK4 is the immediate upstream kinase of SAPK/JNK (26, 27) whereas MKK3 and MKK6 are immediately upstream of p38 MAPK (26, 28-30). Therefore, we decided to investigate whether this proposed signaling cascade was responsible for expression of Cox-2 and activation of p38 MAPK in Delta MEKK1 inducible cells. In accordance with previous findings, IPTG induction of Delta MEKK1 produced phosphorylation of SEK1/MKK4 (Fig. 6A). Furthermore, our experiments also demonstrated that the active Delta MEKK1 resulted in an increase of SEK1/MKK4 activity which was demonstrated by its ability to enhance p38 MAPK phosphorylation (Fig. 6B). However, phosphorylation of both MKK3 and MKK6 (as evidenced using an anti-phospho-MKK3/MKK6 antibody) was not induced by IPTG stimulation (Fig. 7A). Furthermore, these findings were reinforced by immunocomplex kinase assays using p38 MAPK as the substrate for either MKK3 (Fig. 7B) or MKK6 activities (Fig. 7C). As a positive control, anisomycin (31) activated MKK3/MKK6 phosphorylation and activation in NIH 3T3 cells (Fig. 7A, right panel). Thus in both circumstances, either via kinase activity assay or direct phosphorylation analysis, MKK3 and MKK6 were not activated with IPTG induction of p38 MAPK activity. These findings suggest a signaling mechanism for activation of p38 MAPK involving activation of SEK1/MKK4 induced by MEKK1.


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Fig. 6.   Delta MEKK1 induces SEK/MKK4 phosphorylation and activation. A, Western blot assay for SEK/MKK4 phosphorylation. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods and SEK/MKK4 phosphorylation was determined by Western blot analysis using an anti-phospho-specific SEK/MKK4 antibody. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-5, MEKK1 inducible NIH 3T3 cells treated with 1 mM IPTG for 0 (lane 2), 1 2 (lane 3), 24 (lane 4), and 36 (lane 5) h. B, immunocomplex SEK1/MKK4 activity assay utilizing GST-p38 MAPK as the substrate. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods. SEK1/MKK4 was immunopurified by an anti-MKK4 antibody and SEK1/MKK4 activity was detected by immunocomplex kinase assay with GST-p38 MAPK as the substrate. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-5, MEKK1 inducible NIH 3T3 cells treated with 1 mM IPTG for 0 (lane 2), 12 (lane 3), 24 (lane 4), and 36 (lane 5) h.


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Fig. 7.   MEKK1 does not induce MKK3 and MKK6 phosphorylation and activation. A, Western blot assay for MKK3/MKK6 phosphorylation. MEKK1 inducible NIH 3T3 cells were stimulated with either 1 mM IPTG or 100 µg/ml anisomycin (positive control) for the indicated time periods and MKK3/MKK6 phosphorylation was determined by Western blot analysis using an anti-phospho-specific MKK3/MKK6 antibody. Lanes 1-5, MEKK1 inducible NIH 3T3 cells treated with 1 mM IPTG for 0 (lane 1), 6 (lane 2), 12 (lane 3), 24 (lane 4), and 36 (lane 5) h; lanes 6-10, NIH 3T3 cells treated by 100 µg/ml anisomycin for 0 (lane 6), 5 (lane 7), 15 (lane 8), 30 (lane 9), and 60 (lane 10) minutes. B, immunocomplex MKK3 activity assay utilizing GST-p38 MAPK as the substrate. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods. MKK3 was immunopurified by an anti-MKK3 antibody and MKK3 activity was detected by immunocomplex kinase assay with GST-p38 MAPK as the substrate. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-5, MEKK1 inducible NIH 3T3 cells treated with 1 mM IPTG for 0 (lane 2), 12 (lane 3), 24 (lane 4), and 36 (lane 5) h. C, immunocomplex MKK6 activity assay utilizing GST-p38 MAPK as the substrate. MEKK1 inducible NIH 3T3 cells were stimulated with 1 mM IPTG for the indicated time periods. MKK6 was immunopurified by an anti-MKK6 antibody and MKK6 activity was detected by immunocomplex kinase assay with GST-p38 MAPK as the substrate. Lane 1, NIH 3T3 cells without IPTG stimulation; lanes 2-5, MEKK1 inducible NIH 3T3 cells treated with 1 mM IPTG for 0 (lane 2), 12 (lane 3), 24 (lane 4), and 36 (lane 5) h.

To further demonstrate whether SEK1/MKK4 mediates Delta MEKK1-induced p38 MAPK activation and Cox-2 expression, SEK1-WT (wild type), SEK1-ED (constitutively active form), and SEK1-AL (kinase dead form) were stably transfected in NIH 3T3 cells, and protein expression was verified by Western blot assay using an anti-MKK4 antibody (Fig. 8A). In comparison to empty vector and SEK1-WT, expression of the constitutively active SEK1-ED increased both p38 MAPK (Fig. 8B) and JNK/SAPK (data not shown) phosphorylation, whereas SEK1-AL decreased both p38 MAPK (Fig. 8B) and JNK/SAPK phosphorylation. Notably, SEK1-ED expression enhanced Cox-2 expression (Fig. 8C) and PGE2 production (Fig. 8D) which were completely blocked by p38 inhibitor, SC68376 at the dose of 10 µM (data not shown). In contrast, expression of SEK-1AL inhibited Cox-2 expression (Fig. 8C) and PGE2 production (Fig. 8D). This data clearly suggests that SEK1/MKK4 is an important intermediate kinase which modulates MEKK1-induced p38 MAPK activation and Cox-2 expression. Together, we believe that MEKK1 right-arrow SEK1/MKK4 right-arrow p38 MAPK cascade is an important upstream signaling mechanism promoting Cox-2 expression and PGE2 production.


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Fig. 8.   Effects of SEK1/MKK4 on p38 MAPK phosphorylation and Cox-2 expression. A, Western blot assay for validation of SEK1/MKK4 overexpression in NIH 3T3 cells. popRSV1 (lane 1), popRSV1 SEK-WT (lane 2), popRSV1 SEK-AL (lane 3), or popRSV1 SEK ED (lane 4) were stably transfected in NIH 3T3 cells and SEK1 protein expression was detected by Western blot assay using an anti-MKK4 antibody. B, Western blot assay for p38 MAPK phosphorylation. popRSV1 (lane 1), popRSV1 SEK-WT (lane 2), popRSV1 SEK-AL (lane 3), or popRSV1 SEK-ED (lane 4) were stably transfected in NIH 3T3 cells and p38 MAPK phosphorylation was detected by Western blot assay using an anti-phospho-specific p38 MAPK antibody. C, Western blot assay for Cox-2 expression: popRSV1 (lane 1), popRSV1 SEK-WT (lane 2), popRSV1 SEK-AL (lane 3), or popRSV1 SEK-ED (lane 4) were stably transfected in NIH 3T3 cells and Cox-2 protein expression was detected by Western blot assay using an anti-Cox-2 antibody. D, PGE2 production induced by SEK1 overexpression. NIH 3T3 cells were stably transfected by popRSV1, popRSV1 SEK-WT, popRSV1 SEK-AL, or popRSV1 SEK-ED and PGE2 in the culture media was measured by enzyme-linked immunosorbent assay.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The MAPK family consists of at least three different subgroups which include: ERKs, JNKs (SAPKs), and p38 MAPK kinases. Among these groups, the JNK/SAPK and p38 MAPK pathways share activation by inflammatory cytokines, bacterial endotoxins, and environmental stress (17, 32-34), whereas the ERKs are preferentially activated by mitogens of the receptor tyrosine kinase family. Once activated, these MAPKs can phosphorylate and activate transcription factors which regulate gene expression. However, the physiological consequences for the cell following activation of these kinases remain unclear. Investigators have implicated the role of the JNK/SAPK and p38 MAPK pathway in the regulation of inflammatory mediators, cytokine production (14), platelet aggregation (35), and neuronal apoptosis (36). These kinases are in turn activated by upstream mitogen-activated protein/Erk kinases (MEKs) such as SAPK/ERK kinase-1 (SEK1, also known as MKK4) which is upstream of JNK/SAPK and MKK3/MKK6 which are upstream of p38 MAPK. These MEKs are in turn activated by MEK kinases of which MEKK 1, 2, 3, and 5 have been characterized to date (25, 37). Previous data has implicated MEKK1 and MEKK2 as preferential activators of the JNK/SAPK pathway. MEKK 3 exhibits preference for activation of the ERK pathway in vivo (37). Currently, the identity of the MEKK (or MEKKs) upstream of the p38 MAPK pathway is unknown. Although SEK1/MKK4 can activate p38 in vitro (26), previous studies failed to demonstrate that inducible expression of MEKK1 can activate p38 MAPK in vivo.

The data presented in this paper provides evidence for the role of MEKK1 as an upstream activator of both p46 and p54 SAPK as well as p38 MAPK. More importantly, we provide evidence that cells overexpressing Delta MEKK1 can result in phosphorylation and activation of p38 MAPK. The activation and phosphorylation of both p46 and p54 SAPK as well as p38 MAPK peak within 24 h of IPTG exposure. Delta MEKK1 induction does not affect the phosphorylation and activation of ERK. These results provide an important indication that intact mammalian cells induced to overexpress Delta MEKK1 can phosphorylate and activate p38 MAPK, suggesting that MEKK1 is one of the upstream kinases regulating the p38 MAPK pathway.

Interestingly, our experiments also demonstrate that Delta MEKK1 induction by IPTG results in increased activity of SEK1/MKK4, but not MKK3 or MKK6. Co-transfection of 293 cells with MKK4 and p38 MAPK demonstrated activation of the p38 MAPK (38). Similarly, our results indicate that overexpression of the constitutively active form of SEK1 not only increases JNK/SAPK activity but also enhances p38 MAPK activity. However, overexpression of the kinase dead form of SEK1 decreases both JNK/SAPK and p38 MAPK activity. The above results clearly suggest that the MEKK1 right-arrow SEK1/MKK4 kinase cascade not only activates JNK/SAPK, but may also function as an upstream activator p38 MAPK pathway in vivo. Since several reports suggest that MKK3 and MKK6 are the immediate upstream kinases which activate p38 MAPK, it remains a possibility that MKK3/MKK6 utilizes an alternative pathway for activation of p38 MAPK.

Prostaglandins are formed by the dual action of a phospholipase A2 which releases arachidonic acid from membrane phospholipids and cyclooxygenase which converts its substrate to prostaglandin H2 which is subsequently converted to prostaglandins by cell-specific synthases. Cyclooxygenases (Cox-1 and Cox-2) function as rate-limiting enzymes in the biosynthesis of prostaglandins. Cox-2 has been identified as an inducible enzyme stimulated by cytokines, tumor promoters, and hormones (39). Previous work from our laboratory (21) has suggested that Cox-2 can be activated by IL-1 stimulation of renal mesangial cells. Cytokine treatment induces concomitant activation of JNK/SAPK and p38 MAPK (20, 21). Further evidence has demonstrated that pharmacologic inhibition of p38 MAPK by SC68376 inhibited cytokine-induced stimulation of Cox-2 and PGE2 synthesis, suggesting that p38 MAPK pathway is one of the important signaling mechanisms modulating cytokine-induced Cox-2 gene expression and prostaglandin biosynthesis (20).

In this study we show that 3T3 cells activated to overexpress Delta MEKK1 exhibit up-regulated expression of Cox-2 and increased PGE2 synthesis. The Cox-2 specific inhibitor, NS-398 (0.1 µM), completely inhibited PGE2 production induced by the activation of MEKK1. Thus, we believe that the activation of Cox-2 plays a critical role in MEKK1-induced PGE2 biosynthesis. However, overexpression of Delta MEKK1 does not result in up-regulation of Cox-1 or cPLA2 protein expression. In order to further determine whether Cox-2 expression was mediated by either the SAPK/JNK or the p38 MAPK pathway, we utilized SC68376, an oxazole compound which inhibits p38 MAPK (IC50 of 2-5 µM). This compound does not affect p38 MAPK phosphorylation, however, it inhibits the ability of p38 MAPK to activate downstream targets. Furthermore, the selectivity of this inhibitor has been shown by its inability to affect either ERK or SAPK/JNK activity or phosphorylation. Using SC68376, we demonstrated that inhibition of p38 MAPK completely blocked the effects of Delta MEKK1 overexpression on Cox-2 expression and prostaglandin biosynthesis. These observations clearly implicate the role of p38 MAPK as an important mediator of MEKK1-induced Cox-2 expression and PGE2 production. Previous data from Herschman's (40) laboratory demonstrated that the JNK pathway is involved in platelet-derived growth factor-induced transcriptional regulation of Cox-2. Therefore, their results in addition to our own, suggest that the activation of both p38 MAPK and JNK pathways are involved in MEKK1-induced Cox-2 expression and PGE2 synthesis.

How does MEKK1 activate p38 MAPK which, in turn, induces Cox-2 expression? Since our results indicate that MEKK1 activation increases SEK1/MKK4 activity without affecting MKK3 or MKK6 activity, our hypothesis is that SEK1/MKK4 may mediate this signaling mechanism. Overexpression of the constitutively active form of SEK1 results in increased phosphorylation of JNK/SAPK and p38 MAPK. The kinase negative mutation, SEK1-AL, inhibits phosphorylation of both aforementioned kinases. This evidence suggests that SEK1/MKK4 is the upstream kinase of both JNK/SAPK and p38 MAPK pathway. Furthermore, our experiments also demonstrate that activated SEK1 induces Cox-2 expression and PGE2 production whereas dominant negative SEK1 inhibits this response. SEK1-induced Cox-2 expression and PGE2 production are completely inhibited by p38 MAPK inhibitor. Together, these results illustrate the ability of SEK1/MKK4 to mediate MEKK1-induced Cox-2 expression and PGE2 production and serve as an upstream kinase modulating p38 MAPK activation.

The implications of the data presented link the MAPK pathway and the prostaglandin generative pathway. Previous work has identified ERK as an activator of cPLA2 in Chinese hamster ovary cells (18). Furthermore, recent data generated in thrombin-stimulated platelets indicate the possible role of p38 MAPK as an activator of cPLA2 (19, 35). In contrast, our data demonstrates that NIH 3T3 cells stimulated to overexpress Delta MEKK1 and activate p38 MAPK do not exhibit any perceptible changes for cPLA2 total protein. We cannot exclude the possibility that increased cPLA2 activity may be involved in the regulation of MEKK1-induced prostaglandin biosynthesis. However, our data demonstrate that the Cox-2 inhibitor can completely block MEKK1-induced PGE2 production. Therefore, we believe that even if cPLA2 is involved in this regulation, it would have a minimal role in PGE2 production when Cox-2 is inhibited. These results suggest that cPLA2 is not the only target for activation by the MAPK pathway, but Cox-2 is a key mediator.

Our experiments demonstrate that overexpression of Delta MEKK1 increases Cox-2 expression and PGE2 production which is completely blocked by SC68376, a pharmacologic inhibitor of p38 MAPK. Delta MEKK1 overexpression results in activation of both JNK/SAPK and p38 MAPK. Induction of Delta MEKK1 increases SEK1/MKK4 but not MKK3 or MKK6 activity. Overexpression of the constitutively active form of SEK1 increases both p38 MAPK and JNK/SAPK phosphorylation, and enhances PGE2 production and Cox-2 expression which can be completely inhibited by a p38 MAPK inhibitor. Overexpression of the dominant negative SEK1-AL decreases the phosphorylation of both p38 MAPK and JNK/SAPK and reduces Cox-2 expression. Collectively, this data suggests that MEKK1 right-arrow SEK1/MKK4 may function as an upstream kinase pathway which activates both p38 MAPK and JNK/SAPK and, in turn, induces Cox-2 expression and PGE2 production. Together, our current findings suggest a novel signaling pathway by which MEKK1 right-arrow SEK/MKK4 right-arrow p38 MAPK right-arrowright-arrowright-arrow Cox-2. This pathway provides further evidence for the role of the MAPK pathway in the mediation of prostaglandin biosynthesis and as a potential target for modulation of the inflammatory response.

    FOOTNOTES

* This work was supported by United States Public Health Awards DK 38111 and DK 50606.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.

parallel To whom correspondence should be addressed: Professor of Medicine and Molecular Biology and Pharmacology, Barnes Jewish Hospital, 216 South Kingshighway, Renal Div., Box 8305, St. Louis, MO 63110. Tel.: 314-454-8495; Fax: 314-454-8430; E-mail: morrison{at}pharmdec.wustl.edu.

1 The abbreviations used are: PLA2, phospholipase A2; Cox, cyclooxygenase; PGE2, prostaglandin E2; IL, interleukin; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MBP, myelin basic protein; GST, glutathione S-transferase; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MEK, mitogen-activated protein/Erk kinases.

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Abstract
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Discussion
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