Published online before print March 20, 2003, doi: 10.1161/01.RES.0000067929.01404.03
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© 2003 American Heart Association, Inc.
Molecular Medicine |
MAP Kinase Kinase 6–p38 MAP Kinase Signaling Cascade Regulates Cyclooxygenase-2 Expression in Cardiac Myocytes In Vitro and In Vivo
From the Division of Vascular Surgery (N.D., E.S., M.C., T.F.L., B.B.R.), Toronto General Hospital, Toronto, Ontario; San Diego State University Heart Institute and the Department of Biology (J.M., D.J.T., C.C.G.), San Diego State University, San Diego, Calif; and Renal Division and Department of Medicine (J.E.F., P.A.M.), St Michael’s Hospital and University of Toronto, Toronto, Ontario.
Correspondence to Barry B. Rubin, Division of Vascular Surgery, 200 Elizabeth St, EC5-302a, Toronto General Hospital, Toronto, Ontario, Canada M5G-2C4; e-mail barry.rubin{at}uhn.on.ca or Christopher C. Glembotski, Department of Biology, San Diego State University, 5500 Campanile Dr, San Diego, CA 92182; e-mail cglembotski@sunstroke.sdsu.edu
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Abstract |
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Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in delayed prostaglandin biosynthesis. The purpose of this study was to evaluate the role of the MAP kinase kinase 6 (MKK6)–p38 MAPK signaling cascade in the regulation of myocardial COX-2 gene expression, in vitro and in vivo. RT-PCR analysis identified p38
and p38ß2 MAPK mRNA in rat cardiac myocytes. Interleukin-1ß induced the phosphorylation of p38 and p38ß2 MAPK in cardiomyocytes and stimulated RNA polymerase II binding to the COX-2 promoter, COX-2 transcription, COX-2 protein synthesis, and prostaglandin E2 (PGE2) release. Infecting cardiomyocytes with adenoviruses that encode mutant, phosphorylation-resistant MKK6 or p38ß2 MAPK inhibited interleukin-1ß–induced p38 MAPK activation, COX-2 gene expression, and PGE2 release. Treatment with the p38 and p38ß2 MAPK inhibitor, SB202190, attenuated interleukin-1ß–induced COX-2 transcription and accelerated the degradation of COX-2 mRNA. Cells infected with adenoviruses encoding wild-type or constitutively activated MKK6 or p38ß2 MAPK, in the absence of interleukin-1ß, exhibited increased cellular p38 MAPK activity, COX-2 mRNA expression, and COX-2 protein synthesis, which was blocked by SB202190. In addition, elevated levels of COX-2 protein were identified in the hearts of transgenic mice with cardiac-restricted expression of wild-type or constitutively activated MKK6, in comparison with nontransgenic littermates. These results provide direct evidence that MKK6 mediated p38 MAPK activation is necessary for interleukin-1ß–induced cardiac myocyte COX-2 gene expression and PGE2 biosynthesis in vitro and is sufficient for COX-2 gene expression by cardiac myocytes in vitro and in vivo.
Key Words: MAP kinase kinase 6 • prostaglandins • recombinant proteins • transgenic mice
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Introduction |
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Cyclooxygenase (COX) catalyzes the rate-limiting step in prostaglandin (PG) biosynthesis.1 Two isoforms of COX have been identified, COX-1 and COX-2.2 In myocardium, COX-1 is constitutively expressed, whereas COX-2 expression is induced by proinflammatory cytokines, such as interleukin-1ß (IL-1ß).3 Serum IL-1ß and myocardial COX-2 levels are elevated in patients with congestive heart failureR4-127475 4,5 and ischemia,6 and COX-2 colocalizes with areas of myocardial inflammation and scarring.5 Therefore, IL-1ß–induced COX-2 gene expression and prostaglandin biosynthesis may play a role in the pathogenesis of some cardiac diseases. In contrast, COX-2 metabolites protect against endotoxin-induced cardiac contractile dysfunction7 and oxidant-mediated cardiomyocyte injury,8 and mediate the protective effects of ischemic preconditioning.9 Therefore, COX-2 may exert both beneficial and deleterious effects in the heart.6
Mitogen-activated protein kinase (MAPK) enzymes, including p38 MAPK, p42/44 MAPK, and c-Jun N-terminal kinase (JNK), have been implicated in the regulation of COX-2 gene expression in a variety of tissues.R8-127475 R10-127475 8,10,11 However, the signal transduction pathways and transcription factors that regulate the induction of COX-2 gene expression are extremely diversified and are both cell- and species-specific.11 For example, the promoter region of the rat COX-2 gene contains a binding site for NF-
B,10 whereas the mouse COX-2 promoter does not,11 and a cAMP response element (CRE) is necessary for the induction of COX-2 transcription in murine fibroblasts,12 whereas the rat COX-2 promoter lacks a CRE.11 Similarly, the transcription factor C/EBPß is essential for the inducible expression of the COX-2 gene in murine macrophages, but not in murine fibroblasts.13 These findings emphasize the differences that exist in the regulation of COX-2 gene expression in different cells and illustrate the need to specifically evaluate the molecular mechanisms that regulate COX-2 gene expression in cardiac myocytes.
Studies with pharmacological inhibitors have suggested a role for p38 and p42/44 MAPK in the regulation of IL-1ß–induced cardiomyocyte COX-2 gene expression in vitro,3 but may be complicated by the potential lack of specificity of the pharmacological agents that were employed.R14-127475 14,15 p38 MAPK activity is increased by phosphorylation on Thr181 and Tyr183 by two MKK enzymes, MKK3 and MKK6. MKK6 activity is increased by phosphorylation on Ser207 and Thr211. By overexpressing a phosphorylation-resistant MKK6 mutant, we showed that MKK6-mediated p38 MAPK phosphorylation is necessary for IL-1ß induced group IIA phospholipase A2 (PLA2) expression in rat cardiomyocytes.16 The role of MKK6 activation in COX-2 gene expression in cardiomyocytes in vitro, and the molecular events that regulate COX-2 expression in the heart in vivo have not been explored.
In this study, we show that activation of the MKK6–p38 MAPK signaling cascade: (1) stimulates COX-2 mRNA transcription and promotes COX-2 mRNA stability, (2) is sufficient to induce COX-2 gene expression by cardiomyocytes in vitro and transgenic mice in vivo, and (3) is necessary for IL-1ß–induced COX-2 gene expression and prostaglandin biosynthesis by cardiomyocytes in vitro.
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Materials and Methods |
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Cell Culture and Experimental Protocol
Rat neonatal cardiac myocytes were isolated from the hearts of 1- to 2-day-old Sprague-Dawley rats.16 Cells were incubated with inhibitors or adenoviral vectors, as indicated in the Figure legends, and then treated with vehicle or IL-1ß (10 ng/mL) for up to 48 hours. In all studies, isolated cells had characteristic features of cardiac myocytes and beat spontaneously. The methodology for RNA isolation, Northern, and Western blot analysis has been described.16 All studies were approved by the Animal Care Committee of the Toronto General Hospital.
Plasmids
pcDNA3 FLAG-MKK6(Glu) codes for activated human MKK6 and has Glu substitutions at Ser207 and Thr211. pcDNA3 HA-MKK6b(A) contains Ala substitutions at Ser207 and Thr211. Sr3 HA-p38-2 codes for wild-type human p38-2 MAPK. p38-2 MAPK is distinct from p38, p38
, and p38
but is identical to human p38ß2 MAPK.17 pcDNA3 HA-p38-ß2(AGF) was prepared by converting Thr181 and Tyr183 to Ala and Phe, respectively, using site-directed mutagenesis.18
Adenoviruses
The preparation of recombinant adenoviruses encoding FLAG-tagged human MKK6(wt) [ad-MKK6(wt)], ad-MKK6(Glu), ad-MKK6(A), HA-tagged p38ß2 MAPK(wt) [ad-p38ß2 MAPK(wt)], and ad-p38ß2 MAPK(AGF) was performed as described.16 Viral titers were determined by observing GFP fluorescence of primary cultures of neonatal cardiomyocytes, and the minimum quantity of viral stock that afforded 100% infection efficiency was used.
The RT-PCR analysis of p38 and p38ß2 MAPK mRNA, assessment of phosphorylated and total p38 and p38ß2 MAPK levels, FLAG immunoprecipitation and MKK6 kinase assay, HA immunoprecipitation and p38 MAPK kinase assay, measurement of RNA Pol II recruitment to the COX-2 promoter, nuclear run off assays, assessment of COX-2 mRNA stability, generation of MKK6(wt) and MKK6(Glu) transgenic mice, preparation of lysates of cultured rat cardiomyocytes and ventricular tissue from transgenic mice, and statistical analysis are described in the expanded Materials and Methods available in the online data supplement at http://www.circresaha.org.
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IL-1ß Induces COX-2 mRNA Expression, COX-2 Protein Synthesis, and PGE2 Release by Rat Neonatal Cardiomyocytes
COX-2 mRNA was not detected in unstimulated cardiomyocytes after incubation for up to 48 hours. Treatment with IL-1ß induced an increase in COX-2 mRNA that peaked by 24 hours, and had no effect on GAPDH mRNA levels (online Figures 1A and 1B, available in the online data supplement at http://www.circresaha.org). IL-1ß had no effect on cellular COX-1 protein levels, but resulted in a progressive increase in COX-2 protein and PGE2 release, which peaked after 24 hours (online Figures 1C through 1E, respectively). These results are consistent with the notion that IL-1ß induces COX-2 mRNA transcription, COX-2 protein synthesis, and PGE2 release by cardiomyocytes.
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3 independent experiments are shown.
p38 and p38ß2 MAPK Are Both Phosphorylated in Rat Neonatal Cardiomyocytes After Exposure to IL-1ß
To determine if p38 and/or p38ß2 MAPK exist in rat myocardium, RT-PCR analysis of rat neonatal cardiac myocyte RNA was carried out. RT-PCR products of approximately 700 and 600 bp were identified when primer sets based on the sequences of rat p38 MAPK and mouse p38ß2 MAPK were used (Figure 1A). Sequence analysis demonstrated 100% homology of the 706-bp RT-PCR product with nucleotides 33 to 738 of rat p38 MAPK mRNA, and 94% homology of the 602-bp RT-PCR product with nucleotides 128 to 729 of mouse p38ß2 MAPK. These results are consistent with the notion that rat cardiomyocytes express p38 and p38ß2 MAPK mRNA.
Treatment with IL-1ß induced the phosphorylation of p38 MAPK, but had no effect on total cellular p38 MAPK protein levels (Figures 1B and 1C). The phospho-p38 MAPK-specific antiserum that was used to identify phosphorylation of p38 MAPK did not reliably detect p38ß2 MAPK phosphorylation, and we are not aware of any phospho-p38ß2 MAPK-specific antiserum. To identify p38ß2 MAPK phosphorylation, cell lysates were immunoprecipitated with anti-p38ß2 MAPK antiserum, followed by Western blotting with anti-phosphothreonine antiserum. IL-1ß induced p38ß2 MAPK threonine phosphorylation (Figure 1D), but had no effect on total cellular p38ß2 MAPK protein levels (Figure 1E). These results demonstrate that IL-1ß induces the phosphorylation of both p38 and p38ß2 MAPK in rat neonatal cardiomyocytes.
Infection With ad-MKK6(A) Inhibits IL-1ß–Induced COX-2 mRNA Expression, COX-2 Protein Synthesis, and PGE2 Release by Cardiomyocytes
Infection with the adenovirus encoding the constitutively activated MKK6 mutant, ad-MKK6(Glu), leads to a 6-fold increase in p38 MAPK phosphorylation and an 8-fold increase in MAPKAP-K2 activity in rat neonatal cardiac myocytes.R16-127475 16,18 In contrast, infection with the adenovirus encoding the phosphorylation-resistant MKK6 mutant, ad-MKK6(A), which functions as a dominant-negative mutant for IL-1ß–induced group IIA PLA2 expression in neonatal cardiac myocytes,16 abrogates IL-1ß–induced increases in cellular MKK6 activity and p38 MAPK phosphorylation, as described later.
Infection with ad-MKK6(wt) or ad-MKK6(Glu) resulted in the expression of COX-2 mRNA in the absence of IL-1ß (Figure 2A). The IL-1ß induced increase in COX-2 mRNA was augmented by infection with ad-MKK6(Glu), but was significantly attenuated by infection with ad-MKK6(A) (Figure 2A). ad-GFP, ad-MKK6(wt), ad-MKK6(A), or ad-MKK6(Glu) infection did not have a significant effect on cellular GAPDH mRNA levels (Figure 2B). Infection with ad-MKK6(Glu), and to a lesser extent ad-MKK6(wt), led to COX-2 protein synthesis in the absence of IL-1ß. In the presence of IL-1ß, ad-GFP–, ad-MKK6(wt)–, and ad-MKK6(Glu)–infected cells expressed markedly elevated levels of COX-2 protein. In contrast, infection with ad-MKK6(A) significantly attenuated the IL-1ß–induced increase in COX-2 protein synthesis (Figure 2C). IL-1ß– and ad-MKK6(Glu)–induced COX-2 protein synthesis were both attenuated by preincubation with the p38 and p38ß2 inhibitor, SB20219019 (online Figure 2).
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To document the functionality of ad-MKK6(wt), ad-MKK6(A), and ad-MKK6(Glu), lysates of infected cardiomyocytes were immunoprecipitated with anti-FLAG antiserum, and the ability of the immunoprecipitate to phosphorylate the kinase-dead MKK6 substrate, HA-p38ß2 MAPK(K53R), was assessed. Infection with ad-MKK6(Glu) resulted in more HA-p38ß2 MAPK(K53R) phosphorylation than when cells were infected with ad-MKK6(wt), whereas no HA-p38ß2 MAPK(K53R) phosphorylation was noted in ad-MKK6(A)–infected cells (Figure 3D). HA-p38ß2 MAPK(K53R) phosphorylation was not detected in lysates of ad-GFP infected cells, as these cells do not contain FLAG-tagged MKK6. IL-1ß increased HA-p38ß2 MAPK(K53R) phosphorylation in ad-MKK6(wt)–infected cells, and had no effect on HA-p38ß2 MAPK(K53R) phosphorylation in ad-MKK6(Glu)–infected cells. In contrast, no HA-p38ß2 MAPK(K53R) phosphorylation was identified in ad-MKK6(A)–infected, IL-1ß–treated cells. To assess total cellular MKK6 activity (ie, FLAG-tagged and endogenous MKK6), lysates were incubated with HA-p38ß2 MAPK(K53R) and then evaluated by Western blotting with anti-phospho p38 MAPK antiserum. We found that total cellular MKK6 activity was identical to the MKK6 activity measured with the FLAG-IP assay (data not shown). Differences in cellular COX-2 mRNA expression, COX-2 protein synthesis, and MKK6 activity in cells infected with ad-MKK6(wt), ad-MKK6(Glu), or ad-MKK6(A) were not due to differences in the expression of the wild-type or mutant MKK6 enzymes (anti-FLAG immunoblot, Figure 2E). Infection with ad-GFP, ad-MKK6(wt), ad-MKK6(Glu), or ad-MKK6(A) had no effect on cellular levels of tubulin, a constitutively expressed protein (Figure 2F).
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To assess the role of MKK6 in cardiac myocyte PGE2 release, cells were infected with ad-GFP, ad-MKK6(wt), ad-MKK6(Glu), or ad-MKK6(A) and treated with vehicle or IL-1ß. Infection with ad-MKK6(Glu), without IL-1ß, resulted in PGE2 release (Figure 2G). IL-1ß–induced PGE2 release was significantly increased by infection with ad-MKK6(wt) or ad-MKK6(Glu) in comparison with ad-GFP–infected cells. In contrast, infection with ad-MKK6(A) decreased IL-1ß–induced PGE2 release by approximately 50% (Figure 2G). These results provide direct evidence that (1) MKK6 activation is sufficient for COX-2 mRNA expression, COX-2 protein synthesis, and PGE2 release by cardiomyocytes, and (2) that MKK6 activation is necessary for IL-1ß–induced COX-2 mRNA expression, COX-2 protein synthesis, and PGE2 release by cardiomyocytes in vitro.
Infection With ad-p38ß2 MAPK(AGF) Inhibits IL-1ß–Induced COX-2 mRNA Expression, COX-2 Protein Synthesis, and PGE2 Release by Cardiomyocytes
Infection with the adenovirus encoding wild type p38ß2 MAPK, ad-p38ß2 MAPK(wt), leads to a dramatic increase in cellular p38 MAPK activity. Conversely, infection with the adenovirus encoding the phosphorylation-resistant p38ß2 MAPK mutant, p38ß2 MAPK(AGF), which functions as a dominant-negative mutant for B-crystallin gene expression in neonatal cardiac myocytes,18 abrogates IL-1ß–induced increases in cellular p38 MAPK activity, as described later.
Infection with ad-p38ß2 MAPK(wt) induced COX-2 mRNA expression in the absence of IL-1ß (Figure 3A). COX-2 mRNA levels were higher in ad-p38ß2 MAPK(wt)–infected, IL-1ß–treated cells than ad-GFP–infected, IL-1ß–treated cells, but were significantly attenuated in ad-p38ß2 MAPK(AGF)–infected, IL-1ß–treated cells (Figure 3A). Infection with ad-GFP, ad-p38ß2 MAPK(wt), or ad-p38ß2 MAPK(AGF) had no effect on cellular GAPDH mRNA levels (Figure 3B). Exposure to IL-1ß or infection with ad-p38ß2 MAPK(wt) both induced COX-2 protein synthesis. The IL-1ß–induced increase in COX-2 protein synthesis was significantly attenuated by infection with ad-p38ß2 MAPK(AGF) (Figure 3C).
To measure the kinase activity of HA-tagged p38ß2 MAPK(wt) and HA-tagged p38ß2 MAPK(AGF), cell lysates were immunoprecipitated with an anti-HA antibody, and phosphorylation of the p38ß2 MAPK substrate ATF2 was assessed. Infection with ad-p38ß2 MAPK(wt) resulted in significant cellular p38ß2 MAPK activity, which was abrogated when cells were infected with ad-p38ß2 MAPK(AGF) and then treated with vehicle or IL-1ß (Figure 3D). ATF2 phosphorylation was not detected in lysates of ad-GFP–infected cells, which do not contain HA-tagged p38ß2 MAPK (Figure 3D). Differences in cellular p38ß2 MAPK activity, COX-2 mRNA expression, and COX-2 protein synthesis in cells infected with ad-p38ß2 MAPK(wt) or ad-p38ß2 MAPK(AGF) were not due to differences in cellular levels of the expressed wild-type or mutant HA-tagged p38ß2 MAPK enzymes (anti-HA immunoblot, Figure 3E). Infection with ad-GFP, ad-p38ß2 MAPK(wt), or ad-p38ß2 MAPK(AGF) had no effect on cellular levels of tubulin (Figure 3F).
To assess the role of p38ß2 MAPK in cardiac myocyte PGE2 release, cells were infected with ad-GFP, ad-p38ß2 MAPK(wt), or ad-p38ß2 MAPK(AGF) and then treated with vehicle or IL-1ß. Infection with ad-p38ß2 MAPK(wt), in the absence of IL-1ß, resulted in PGE2 release (Figure 3G). Infection with ad-p38ß2 MAPK(wt) significantly increased IL-1ß–induced PGE2 release, whereas infection with ad-p38ß2 MAPK(AGF) decreased IL-1ß–induced PGE2 release 65% (Figure 3G). These results provide direct evidence that p38ß2 MAPK activation is sufficient for COX-2 mRNA expression, COX-2 protein synthesis, and PGE2 release by unstimulated cardiomyocytes, and that p38ß2 MAPK activation is necessary for IL-1ß–induced COX-2 mRNA expression, COX-2 protein synthesis, and PGE2 release by rat cardiomyocytes in vitro.
Infection with ad-MKK6(A) or ad-p38ß2 MAPK(AGF) obliterated IL-1ß–induced p38 MAPK(K53R) (Figure 2) and ATF2 phosphorylation, respectively, but only partially inhibited IL-1ß–induced COX-2 protein synthesis and PGE2 release. Therefore, signaling cascades other than the MKK6–p38 MAPK pathway are likely to participate in the regulation of IL-1ß–induced COX-2 protein synthesis and PGE2 biosynthesis in cardiac myocytes.
IL-1ß Induces RNA Pol II Recruitment to the COX-2 Promoter
In order to elucidate the effect of IL-1ß treatment on RNA Pol II recruitment to the COX-2 proximal promoter, chromatin immunoprecipitation was performed using an RNA Pol II-specific antibody. No significant loading of RNA Pol II was detected at the COX-2 promoter in unstimulated cells. In contrast, treatment with IL-1ß for 4 hours led to an increase in RNA Pol II association with the rat COX-2 promoter (Figure 4A).
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p38 MAPK Partially Regulates COX-2 mRNA Transcription and COX-2 mRNA Stability
To define the potential role of p38 MAPK in COX-2 mRNA transcription, cells were preincubated with vehicle or SB202190 for 30 minutes and then incubated with DMSO or IL-1ß for 4 hours. COX-2 and GAPDH mRNA transcriptions were then assessed by nuclear run-off assay.20 IL-1ß increased COX-2 transcription 16-fold (Figures 4B and 4C), a result consistent with the robust increase in RNA Pol II recruitment to the COX-2 promoter after exposure to IL-1ß. Preincubation with SB202190 had no effect on COX-2 transcription in vehicle-treated cells, but decreased COX-2 transcription 58% in cells treated with IL-1ß (Figures 4B and 4C).
To assess the potential role of p38 MAPK in the stabilization of COX-2 mRNA, cells were incubated with IL-1ß for 16 hours, treated with vehicle or SB202190, and then incubated with actinomycin D, which arrests transcription. Exposure to actinomycin D for 1 hour decreased the ratio of COX-2 to GAPDH mRNA by 40%. In contrast, treatment with SB202190 and subsequent exposure to actinomycin D for 1 hour decreased the ratio of COX-2 to GAPDH mRNA by 90% (Figures 5A and 5B).
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To selectively study the role of p38 MAPK in COX-2 mRNA stability, cells were infected with ad-p38ß2 MAPK(wt) for 16 hours, treated with vehicle or SB202190, and then incubated with actinomycin D. Exposure to actinomycin D for 1 hour decreased the ratio of COX-2 to GAPDH mRNA by 48%. In contrast, infection with ad-p38ß2 MAPK(wt), followed by incubation with SB202190 and subsequent exposure to actinomycin D for 1 hour, decreased the ratio of COX-2 to GAPDH mRNA by 87% (Figures 5C and 5D). Taken together, these results provide direct evidence that p38 MAPK partially regulates COX-2 mRNA transcription and COX-2 mRNA stability in rat neonatal cardiomyocytes.
Cardiac-Restricted Expression of MKK6(wt) or MKK6(Glu) Results in Ventricular COX-2 Protein Synthesis
To begin to assess the role of the MKK6–p38 MAPK signaling cascade in myocardial COX-2 expression in vivo, ventricular tissue from tg-MKK6(wt) or tg-MKK6(Glu) mice, or from nontransgenic control littermates, was evaluated for COX-2, MKK6, and tubulin protein levels. Trace amounts of COX-2 protein were identified in ventricular tissues from nontransgenic mice (Figure 6A). In contrast, there was a marked increase in COX-2 protein in ventricular tissue from both tg-MKK6(wt) and tg-MKK6(Glu) mice. MKK6 protein was not identified in ventricular tissue from control mice, was significantly elevated in ventricular tissue from tg-MKK6(wt) mice, and was about 5-fold lower in ventricular tissue from tg-MKK6(Glu) mice, in comparison with tg-MKK6(wt) mice (Figure 6B). This is consistent with the observation that ventricular tissue from tg-MKK6(wt) mice has approximately 3- to 5-fold higher MKK6 activity, as measured by the ability of ventricular lysates to phosphorylate p38ß2 MAPK(K53R), than ventricular tissue from tg-MKK6(Glu) mice (J. Martindale, C. Glembotski, unpublished data, 2003). All mice expressed similar levels of tubulin in ventricular tissue (Figures 6C). Taken together, these results constitute the first direct evidence that overexpression of MKK6(wt) or MKK6(Glu) is sufficient to induce COX-2 protein synthesis in ventricular tissue in vivo.
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Discussion |
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In this study, we have presented 3 independent lines of evidence that demonstrate that activation of the MKK6–p38 MAPK signaling cascade is sufficient to induce COX-2 expression in cardiac myocytes. First, overexpression of MKK6(wt), MKK6(Glu), or p38ß2 MAPK(wt) increased COX-2 mRNA expression and COX-2 protein synthesis by cardiomyocytes in the absence of IL-1ß. Second, infection with ad-MKK6(Glu) induced an increase in COX-2 protein synthesis that was attenuated by the selective p38
and p38ß2 MAPK inhibitor, SB202190.19 Third, cardiac-restricted expression of MKK6(wt) or MKK6(Glu) in transgenic mice, which results in increased ventricular MKK6 protein levels (Figure 6) and increased MKK6 activity (J. Martindale, C. Glembotski, unpublished data, 2003), induced myocardial COX-2 protein synthesis in vivo. To our knowledge, this is the only study that has documented the role of MKK6 in COX-2 gene expression in cardiomyocytes, in vitro or in vivo. Exposure to IL-1ß led to recruitment of RNA Pol II to the COX-2 promoter and to transcription of the COX-2 gene. We have presented 3 independent lines of evidence that demonstrate that activation of the MKK6–p38 MAPK signaling cascade is necessary for IL-1ß–induced COX-2 gene expression in cardiomyocytes. Thus, overexpression of either MKK6(A) or p38ß2 MAPK(AGF), mutated enzymes that cannot be phosphorylated and activated by their respective upstream kinases, inhibited IL-1ß–induced increases in COX-2 mRNA expression, COX-2 protein synthesis, and PGE2 release by cardiomyocytes. Therefore, MKK6(A) and p38ß2 MAPK(AGF) functioned as dominant-negative mutants for IL-1ß–induced COX-2 gene expression and prostaglandin biosynthesis by cardiomyocytes. In addition, pretreatment with the p38 MAPK inhibitor SB202190 attenuated IL-1ß–induced cardiac myocyte COX-2 mRNA transcription in vitro and COX-2 protein synthesis in intact cardiac myocytes. These results provide direct evidence that IL-1ß stimulates MKK6–p38 MAPK–dependent myocardial COX-2 gene expression and PGE2 biosynthesis. The findings that p38 MAPK regulates COX-2 gene expression in cardiac myocytes by increasing COX-2 mRNA transcription and by stabilizing COX-2 mRNA are consistent with previous reports in other cell types.R21-127475 21,22
Whereas our results provide clear evidence that the MKK6–p38 MAPK signaling cascade participates in the regulation of COX-2 gene expression, 4 independent lines of evidence support the notion that other signaling cascades also participate in the regulation of the COX-2 gene in cardiac myocytes. First, infection with ad-MKK6(A) or ad-p38ß2 MAPK(AGF), which obliterated IL-1ß–induced p38ß2 MAPK(K53R) and ATF2 phosphorylation, respectively, only partially inhibited IL-1ß–induced COX-2 protein synthesis and PGE2 release. Second, COX-2 mRNA and COX-2 protein levels, and PGE2 release, were significantly higher in ad-GFP–infected, IL-1ß–treated cells than in ad-MKK6(Glu)–infected, vehicle-treated cells (Figure 2). Third, ad-MKK6(Glu)-infected, IL-1ß–treated cells had significantly higher levels of COX-2 mRNA and COX-2 protein, and released more PGE2 than ad-MKK6(Glu)–infected, vehicle-treated cells, but had similar degrees of MKK6 activity. Fourth, treatment with PD098059, which inhibits MEK1,2-mediated extracellular signal-regulated kinase (ERK) 1,2 and MEK5-mediated ERK5 phosphorylation,14 has been shown to inhibit IL-1ß–induced COX-2 protein synthesis in cardiomyocytes.3 Taken together, these finding indicate that IL-1ß–induced COX-2 gene expression and PGE2 release by cardiomyocytes involves the activation of p38 MAPK and additional signaling molecules, such as ERK or JNK, which participate in the regulation of COX-2 gene expression in some cells.23
p38 MAPK may promote cardiomyocyte apoptosis, whereas p38ß2 MAPK may induce myocardial hypertrophy and cell survival.24 Therefore, it is possible that p38 and p38ß2 MAPK, which are both phosphorylated after cardiac myocytes are treated with IL-1ß, may play distinct roles in the regulation of myocardial genes, such as COX-2. Overexpression of p38ß2 MAPK(AGF) inhibited IL-1ß–induced COX-2 gene expression in cultured cardiomyocytes (Figure 3), thereby implicating the p38ß2 MAPK isoform in the regulation of myocardial COX-2 gene expression. p38ß2 MAPK(AGF) overexpression did not inhibit IL-1ß–induced p38 MAPK phosphorylation in rat neonatal cardiomyocytes (N. Degousee, B. Rubin, unpublished observation, 2003). However, the results of these experiments do not permit us to definitively conclude that p38ß2 MAPK selectively regulates cardiac myocyte COX-2 gene expression, as the comparatively high levels of p38ß2 MAPK(AGF) that are expressed in myocytes that were infected with ad-p38ß2 MAPK(AGF) could competitively inhibit the activity of phosphorylated p38 MAPK in these cells. Pharmacological inhibitors that selectively attenuate p38 or p38ß2 MAPK activity, or viable animals with functional deletions of the p38 or p38ß2 MAPK genes, are required to delineate the precise roles of p38 and p38ß2 MAPK in the regulation of myocardial COX-2 gene expression.
PGE2 synthesis is catalyzed by the sequential action of PLA2, COX, and PGE2 synthase. The coordinate, MKK6–p38 MAPK-dependent expression of group IIA PLA216 and COX-2 induced by IL-1ß may synergistically increase myocardial PGE2 biosynthesis, as cotransfection of group IIA PLA2 and COX-2 in HEK293 cells dramatically increases IL-1ß–induced prostanoid biosynthesis.25 Group IV PLA2 (cPLA2) may also supply arachidonic acid to COX-2 in cardiac myocytes, as exposure to IL-1ß increases cPLA2 expression in these cells,16 and cotransfection of cPLA2 and COX-2 increases IL-1ß–induced PGE2 biosynthesis.25
Delayed PGE2 synthesis is mediated by a functional association between COX-2 and membrane PGE2 synthase (mPGES).26 As mPGES colocalizes with COX-2 in the perinuclear envelope, and mPGES expression is induced by IL-1ß,26 it is likely that mPGES catalyzes the conversion of PGH2 to PGE2 in IL-1ß–treated cardiomyocytes. Treating cardiomyocytes with IL-1ß likely results in more PGE2 biosynthesis than infection with ad-MKK6(Glu) or ad-p38ß2 MAPK(wt) (Figures 2 and 3) because IL-1ß induces mPGES expression in rat cardiac myocytes, whereas adMKK6(Glu) and ad-p38ß2 MAPK(wt) do not (N. Degousee, B. Rubin, unpublished observation, 2003). Recently, p38 MAPK (and ERK1,2) were shown to regulate IL-1ß–induced mPGES expression in orbital fibroblasts.27 The role of the MKK6–p38 MAPK signaling cascade in IL-1ß–induced mPGES expression in cardiac myocytes is currently being explored.
In summary, our results provide direct evidence that activation of the MKK6–p38 MAPK signaling cascade is sufficient to induce COX-2 gene expression by cardiac myocytes, in vitro and in vivo. In addition, we have shown that activation of the MKK6–p38 MAPK signaling cascade is necessary for IL-1ß–induced cardiac myocyte COX-2 gene expression and PGE2 biosynthesis in vitro. These observations may lead to the development of novel pharmacogenomic therapies that could be used to modulate the expression of COX-2, the rate-limiting enzyme in prostaglandin biosynthesis in the heart.
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Acknowledgments |
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This work was supported by grants from the Heart and Stroke Foundation of Canada (NA-4387, B.B.R.), the Canadian Institutes of Health Research (53297 and 37778, B.B.R. and P.M., respectively), the physicians of Ontario through The P.S.I. Foundation (98-049 and 01-12, T.F.L. and B.B.R., respectively), and the National Institutes of Health (HL-63975 and NS/HL-25037, C.C.G.). B.B.R. is a Wylie Scholar in Academic Vascular Surgery, Pacific Vascular Research Foundation, San Francisco.
Received September 23, 2002; revision received February 4, 2003; accepted March 11, 2003.
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References |
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