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J. Biol. Chem., Vol. 276, Issue 48, 44495-44501, November 30, 2001

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p38 Mitogen-activated Protein Kinase Activates Peroxisome Proliferator-activated Receptor

A POTENTIAL ROLE IN THE CARDIAC METABOLIC STRESS RESPONSE^*

Philip M. Barger, Alyssa C. Browning, Ashley N. Garner, and Daniel P. Kelly^§¶

From the Center for Cardiovascular Research, Departments of  Medicine, ^§ Pediatrics, and ^¶ Molecular Biology & Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, June 26, 2001, and in revised form, September 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The expression of enzymes involved in fatty acid -oxidation (FAO), the principal source of energy production in the adult mammalian heart, is controlled at the transcriptional level via the nuclear receptor peroxisome proliferator-activated receptor  (PPAR). Evidence has emerged that PPAR activity is activated as a component of an energy metabolic stress response. The p38 mitogen-activated protein kinase (MAPK) pathway is activated by cellular stressors in the heart, including ischemia, hypoxia, and hypertrophic growth stimuli. We show here that PPAR is phosphorylated in response to stress stimuli in rat neonatal cardiac myocytes; in vitro kinase assays demonstrated that p38 MAPK phosphorylates serine residues located within the NH₂-terminal A/B domain of the protein. Transient transfection studies in cardiac myocytes and in CV-1 cells utilizing homologous and heterologous PPAR target element reporters and mammalian one-hybrid transcription assays revealed that p38 MAPK phosphorylation of PPAR significantly enhanced ligand-dependent transactivation. Cotransfection studies performed with several known coactivators of PPAR demonstrated that p38 MAPK markedly increased coactivation specifically by PGC-1, a transcriptional coactivator implicated in myocyte energy metabolic gene regulation and mitochondrial biogenesis. These results identify PPAR as a downstream effector of p38 kinase-dependent stress-activated signaling in the heart, linking extracellular stressors to alterations in energy metabolic gene expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The expression of enzymes involved in fatty acid -oxidation (FAO),¹ the principal source of energy production in the adult mammalian heart, is tightly controlled at the transcriptional level during cardiac development and in response to physiologic and pathophysiologic stimuli (1-7). The nuclear receptor PPAR has been shown to serve as a key transcriptional regulator of this energy metabolic pathway (Ref. 8; reviewed in Ref. 9). PPAR is a member of the nuclear receptor superfamily of transcription factors and binds cognate response elements as an obligate heterodimer with the retinoid X receptor (RXR). PPAR is ligand-activated by a variety of natural and synthetic agonists, including arachidonic acid derivatives, fibrates, and long-chain fatty acids: metabolic substrates for cardiac FAO enzymes. The important role played by PPAR in cardiac metabolism is underscored by the marked reduction in the basal level of cardiac FAO enzyme gene expression in PPAR / mice (10, 11), leading to reduced long-chain fatty acid uptake and oxidation (12).

Evidence has emerged that PPAR plays a critical role in the energy metabolic stress response in tissues that rely largely on mitochondrial fat oxidation for energy production, such as heart and liver. Under normal physiologic conditions, the expression of cardiac FAO enzyme genes are induced after a short term fast coincident with increased use of fatty acids for myocardial energy production (1, 3). In contrast, PPAR / mice do not exhibit the expected fasting-mediated induction of most FAO enzyme genes, but instead develop hypoglycemia, exhibit inadequate ketogenesis, accumulate neutral lipid in both heart and liver, and have a high death rate relative to wild-type mice (1). In addition, metabolic inhibition experiments and studies of senescent PPAR / mice implicate PPAR in the cardiac and hepatic lipid homeostatic response (10, 13, 14). Finally, PPAR expression and activity are induced by physiologic stimuli known to increase energy demand and mitochondrial oxidative flux such as electrical activation of canine skeletal muscle (15) and in humans subjected to a course of endurance training (16). Taken together, these results suggest that PPAR serves as a metabolic stress response factor to transduce changes in cellular energy demand and fatty acid uptake into oxidative energy-producing capacity via the transcriptional control of FAO enzyme expression.

The response of the postnatal heart to growth and stress stimuli includes activation of a network of signal transduction cascades, including the stress-activated protein kinases, p38 mitogen-activated protein kinase (MAPK) and c-Jun NH₂-terminal kinase (JNK) (reviewed in Refs. 17-19). Evidence is emerging that the p38 MAPK pathway is an important component of the cardiac cellular stress response. p38 MAPK is activated in heart and other tissues by inflammatory and oxidant stressors (reviewed in Ref. 20). In the intact heart, p38 kinase is activated by pressure overload and, in cultured cardiac myocytes, by hypertrophic stimuli, such as ₁-adrenergic agonists and cyclic strain (18, 19, 21). The p38 kinase pathway is also activated by cardiac ischemia or hypoxia and has been linked both to the cardiac myocyte apoptotic program and to the protective effects of ischemic preconditioning (19, 22-24). Given the regulation of PPAR activity during cellular stress, the present study sought to examine whether the p38 stress-activated protein kinase signal transduction cascade influences PPAR activity in heart. Herein, we show that PPAR exists as a phosphoprotein in cardiac myocytes and that p38 activation significantly enhances this state of phosphorylation, leading to an increase in ligand-dependent transactivation of targets and enhanced cooperativity with the transcriptional coactivator PGC-1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Primary Rat Neonatal Cardiac Myocyte Cell Culture-- Ventricular cardiac myocytes were isolated from 1-2 day-old rats as described (4) with the following modifications. Myocytes were maintained on dishes pretreated with 0.1% gelatin (Specialty Media). After 24 h in DMEM (4.5 g/liter glucose) supplemented with 10% horse serum, 5% fetal calf serum, bromodeoxyuridine (100 µM), L-glutamine (2 mM), and Fungizone (250 µg/ml), the medium was changed to serum-free DMEM (1 g/liter glucose) supplemented with bromodeoxyuridine, L-glutamine, Fungizone, transferrin (10 µg/ml), insulin (10 ng/ml), and essentially fatty acid-free BSA (1 mg/ml) (Sigma). Ligands, agonists, and inhibitors were added to the medium after an additional 12 h as described below.

Plasmids and Transient Transfection Studies-- Cardiac myocyte transient transfections were performed using the calcium phosphate method as described (25) with the following modifications: 4 µg of reporter DNA (MCPT.Luc.781 or MCPT.Luc.781.m1; Ref. 13) were used per well in 12-well plates. SB202190 (20 µm) (Calbiochem) or Me₂SO vehicle were added where indicated. CV-1 transient transfections were performed as described previously (26) utilizing 4 µg of reporter plasmid and 500 ng of expression plasmids per well. (ACO)₃TKLuc (4), pCDM-RXR, pCDM() (27), pCDM-PPAR (8), and pcDNA-PGC-1 (28) have been described. The PPAR-GAL4DBD fusion expression vector was created by subcloning a cDNA encoding mouse PPAR tagged with a NH₂-terminal FLAG epitope into pCMXGAL4, which was obtained along with (UAS)₃TKLuc from David D. Moore (Baylor College of Medicine, Houston, TX). Expression vectors for wild-type p38 kinase and constitutively active MKK6 (MKK6b(E)) were obtained from Jiahuai Han (Scripps Research Institute, La Jolla, CA). The expression vector for SRC-1 was a gift of Ming-Jer Tsai and Sophia Y. Tsai (Baylor College of Medicine); the expression vector for PBP was a gift of Janardan K. Reddy (Northwestern University Medical School, Chicago, IL). PPARS6-21A-GAL4DBD, PPARS73-77A-GAL4DBD, and PPARS6-77A-GAL4DBD were created by site-directed mutagenesis of PPARGAL4DBD using the QuikChange kit (Stratagene) according to the manufacturer's protocol. CV-1 cells were maintained in DMEM supplemented with 10% charcoal-stripped fetal calf serum. SB202190 and oleic acid (250 µm)/BSA complex (Sigma) as well as Me₂SO or BSA vehicle were added 12 h after transfection as indicated.

Adenovirus Production and Orthophosphate Labeling-- Adenovirus expressing both green fluorescent protein and murine PPAR tagged with an NH₂-terminal FLAG epitope was created by subcloning the FLAG-PPAR cDNA from PPAR-GAL4DBD into pAdTrackCMV and produced as described (29). Primary cardiac myocytes were infected with FLAG-PPAR-expressing adenovirus 24 h after initial plating at a multiplicity of infection sufficient to infect greater than 95% of the cells based on green fluorescent protein fluorescence. Some plates were treated with SB202190 for 48 h prior to orthophosphate labeling. The cells were then washed and maintained in phosphate-free DMEM supplemented with 1 mCi of H₃³²PO₄ for 3 h. At that time, plates were treated with anisomycin (0.2 µM) (Calbiochem) or Me₂SO vehicle with and without SB202190 for an additional 30 min. Following labeling, the cells were washed and scraped into phosphate-buffered saline supplemented with 1× Complete protease inhibitor mixture (Roche Molecular Biochemicals), Na₃VO₄ (200 µM), Na₄P₂O₇ (100 µM), and phenylmethylsulfonyl fluoride (0.1 mg/ml). Cells were pelleted and lysed in RIPA buffer plus protease and phosphatase inhibitors as above. Lysates were precleared with Protein L-Sepharose (Pierce) and then incubated with anti-FLAG M2 antisera (Sigma) overnight at 4 °C. Immune complexes were collected on Protein L-Sepharose and electrophoresed via SDS-PAGE. The proteins were transferred to nitrocellulose and imaged via phosphorimager. Western blot analysis to demonstrate loading was performed with anti-PPAR antibody (provided by John Woods and Joel Berger, Merck Co.).

In Vitro Kinase Studies-- A murine PPAR cDNA containing an NH₂-terminal FLAG epitope was cloned in-frame with GST in pGEX-4T-1 (Amersham Pharmacia Biotech). Recombinant protein was expressed in BL21 bacteria and partially purified according to the manufacturer's protocol. GST-PPAR protein was left bound to glutathione-Sepharose 4B, and in vitro kinase assays were performed with activated p38 kinase (Upstate Biotechnology, Inc.) in 1× kinase reaction buffer (Stratagene). Reactions were allowed to proceed for 30 min at 30 °C, and products were subjected to SDS-PAGE. Site-directed mutagenesis of pGEX-PPAR was performed as above using the same oligonucleotides to produce recombinant proteins containing the identical mutations present in the PPAR-GAL4DBD plasmid series.

Statistical Analysis-- Values presented in graphs are mean ± standard error of the mean (S.E.). Differences between values were analyzed by a one-factor analysis of variance or unpaired Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of SAPK Pathways Leads to Phosphorylation of PPAR in Cardiac Myocytes-- To determine whether PPAR is a target for SAPK-mediated phosphorylation in cardiac myocytes, ³²P labeling of adenoviral-expressed, epitope-tagged PPAR in primary cultures of neonatal rat cardiac myocytes was performed under serum-free conditions. Immunoprecipitation of ³²P-labeled FLAG-PPAR demonstrated that PPAR exists as a phosphoprotein under basal culture conditions in cardiac myocytes (Fig. 1, lane 1). To determine whether SAPK pathways contribute to the phosphorylation of PPAR in myocytes, phospholabeling experiments were performed in the presence of SB202190, an inhibitor primarily of the p38 kinase pathway. The presence of SB202190 reduced the phosphorylation of FLAG-PPAR (Fig. 1, lane 2). Conversely, a brief exposure to anisomycin, an activator of both p38 and JNK kinases, the major SAPK pathways in cardiac myocytes, dramatically increased levels of phosphorylated PPAR (Fig. 1, lane 3). Finally, SB202190 prevented the anisomycin-induced increase in PPAR phosphorylation (Fig. 1, lane 4). Given that SB202190 is capable of inhibiting p38 MAPK and, under certain conditions, the JNK pathway, these data are consistent with PPAR serving as a downstream target of either the p38 MAPK or JNK pathway in cardiac myocytes.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   PPAR is phosphorylated in cardiac myocytes via SAPK signaling. Orthophosphate-32 labeling and immunoprecipitation of adenovirally expressed FLAG-tagged PPAR was performed in cultured rat neonatal cardiac myocytes. Cells were cultured under serum-free conditions in the presence or absence of the p38 MAPK pathway inhibitor SB202190 (20 µM) for 48 h. Cells were then incubated with H332PO4 for 3 h prior to stimulation with anisomycin (0.2 µM) and/or SB202190. Labeled proteins were immunoprecipitated using anti-FLAG antisera. Labeled PPAR was detected via Western blotting with anti-PPAR antisera.

p38 MAPK Directly Phosphorylates PPAR-- The phosphorylation of PPAR shown above could occur as a result either of direct phosphorylation by SAPKs or phosphorylation via other downstream kinases. To examine whether PPAR is a direct substrate of p38 kinase, in vitro kinase assays were performed. Incubation of GST-PPAR fusion proteins with activated p38 kinase resulted in a robust phosphorylation of PPAR (Fig. 2, lane 2). Examination of the primary amino acid sequence of murine PPAR reveals a number of putative MAPK (S/T)P recognition sequences, all of which are located in the NH₂-terminal A/B domain (Fig. 2). To localize the primary phospho-acceptor sites within PPAR, the in vitro kinase assay was repeated with mutant PPAR proteins containing substitutions of nonphosphorylatable alanines for serines at amino acid positions 6/12/21 (S6-21A), 73/76/77 (S73-77A), or all six putative phospho-acceptor serines (S6-77A). Examination of the relative degree of phosphorylation of the mutant PPAR proteins demonstrated that the major serine phosphorylation sites are localized within the grouping of serines at positions 6, 12, and 21. Although S6-21A is still phosphorylated, this occurs at a significantly reduced degree relative to wild-type and the S73-77A mutant. As expected, no phosphorylation of the S6-77A mutant was observed in this assay, effectively localizing all the p38 kinase phospho-acceptor sites to within the A/B domain.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   p38 MAPK directly phosphorylates PPAR on NH2-terminal serine residues. Partially purified bacterially expressed GST-PPAR wild-type and mutant fusion proteins were incubated with [-32P]ATP in the presence (+) or absence () of activated p38 MAPK. Lane 2 demonstrates phosphorylation of wild-type GST-PPAR. Putative MAPK recognition sequences are shown at the bottom, including the specific combinations of serine residues mutated to alanine to create GST-PPAR S6-21A, GST-PPAR S73-77A, and GST-PPAR S6-77A. Labeled GST-PPAR was detected via Western blotting with anti-PPAR antisera.

p38 MAPK Activity Is Necessary for Full PPAR Transactivating Function in Cardiac Myocytes-- To determine whether activated p38 kinase affects PPAR transactivating function in cardiac myocytes, transient transfection studies were performed with a luciferase reporter construct (MCPT.Luc.781) containing the promoter from the human muscle-type carnitine palmitoyltransferase I (M-CPT I or CPT I) gene, a known cardiac PPAR target involved in the mitochondrial FAO pathway (13). MCPT.Luc.781 was transiently transfected into rat neonatal cardiac myocytes in the absence and presence of SB202190. M-CPT I promoter activity was reduced greater than 70% by addition of SB202190 to the medium (Fig. 3). When a M-CPT I promoter-reporter construct containing a mutated PPAR response element (MCPT.Luc.781.m1; Ref. 13) was used in identical experiments, p38 kinase inhibition had no effect, indicating that an intact PPAR binding site is necessary for the p38 MAPK effect (Fig. 3). These results together with the in vitro kinase data suggest that phosphorylation by p38 kinase augments PPAR-mediated activation of M-CPT I gene transcription in cardiac myocytes.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   p38 MAPK activity is necessary for full PPAR transactivation in cardiac myocytes. MCPT.Luc.781 or MCPT.Luc.781.m1 were transfected into cardiac myocytes in the presence or absence of SB202190 (20 µM) for 48 h. The reporter constructs are shown schematically at the top, including the sequence of the wild-type and mutated PPAR response element, FARE-1. The bars represent mean luciferase activity (in relative luciferase units or RLU ± S.E.) normalized (=1.0) to the activity of MCPT.Luc.781 under basal culture conditions. The data represent the mean of at least three independent experiments. The asterisk (*) denotes a significant difference (p < 0.05) between the indicated conditions.

To further examine the functional interaction between p38 kinase and PPAR, MCPT.Luc.781 transfections were performed in CV-1 cells, which are functionally null for PPAR, RXR, and activated p38 kinase. Activation of p38 MAPK was achieved by cotransfection with a constitutively active upstream kinase of p38 kinase (MKK6b(E)) and wild-type p38. Fig. 4A shows that neither p38 kinase activation, via cotransfection of MMK6b(E) with p38, nor treatment with SB202190 affected the basal activity of MCPT.Luc.781 in CV-1 cells. Ligand-mediated activation of cotransfected PPAR and RXR was demonstrated with the addition of oleic acid, a known PPAR ligand, to the culture medium (Fig. 4A). In the presence of activated p38 MAPK, the ligand-mediated PPAR induction of M-CPT I promoter activity was significantly greater relative to treatment with ligand alone (20-fold versus 6-fold; Fig. 4A). This p38 kinase mediated-enhancement of PPAR activity was inhibited by SB202190, confirming that the effect was specific for the p38 kinase pathway. Thus, activated p38 kinase significantly enhances the transactivation properties of the PPAR/RXR heterodimer.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Activation of p38 MAPK enhances transactivation by PPAR/RXR heterodimers in a promoter- and cell context-independent manner. A, MCPT.Luc.781 was co-transfected into CV-1 cells with or without expression vectors for PPAR and RXR and/or MKK6b(E) and p38 as indicated. Cells were maintained in media supplemented with 10% charcoal-stripped fetal calf serum, the PPAR ligand oleic acid (250 µM), SB202190 (20 µM), or vehicle control (BSA and/or Me2SO). The bars represent mean RLU normalized (=1.0) to the activity of MCPT.Luc.781 under basal culture conditions. The data represent the mean of at least three independent experiments. The asterisk (*) denotes a significant difference (p < 0.05) between the indicated conditions. B, (ACO)3TKLuc was co-transfected into CV-1 cells with or without expression vectors for PPAR and RXR and/or MKK6b(E) and p38. The bars represent mean RLU normalized (=1.0) to the activity of (ACO)3TKLuc under basal culture conditions. The data represent the mean of at least three independent experiments. The asterisk (*) denotes a significant difference (p < 0.05) between the indicated conditions.

To exclude the possibility that the p38 kinase effects are mediated by PPAR-independent pathways via elements other than the PPAR response element (FARE-1) within the M-CPT I promoter, the cotransfection experiments were repeated with a reporter containing an independent PPAR response element derived from the peroxisomal acyl-CoA oxidase (ACO) gene, upstream of a heterologous promoter ((ACO)₃TKLuc) (Fig. 4B). As was observed with MCPT.Luc.781, PPAR/RXR-mediated transactivation of (ACO)₃TKLuc was significantly increased by cotransfection of p38 kinase and MKK6b(E) (Fig. 4B). In this series of experiments, MKK6b(E)/p38 cotransfection activated PPAR/RXR heterodimers both in the absence and presence of exogenous ligand.

To confirm that PPAR rather than its heterodimeric partner RXR was the direct functional target of activated p38 kinase in the transfection experiments described above, a modified mammalian one-hybrid system was employed. For these experiments, a full-length PPAR-GAL4 DNA-binding domain fusion protein (PPAR-GAL4DBD) was cotransfected with a GAL4-responsive reporter ((UAS)₃TKLuc) and MKK6b(E)/p38. The PPAR-GAL4DBD fusion protein retains the ability to be activated by PPAR ligand to a similar degree as that observed earlier in the PPAR/RXR heterodimer transfections (Fig. 5). Cotransfection of MKK6b(E)/p38 with PPAR-GAL4DBD revealed that, in the absence of PPAR ligand, p38 kinase does not activate PPAR-GAL4DBD (Fig. 5). However, a significant increase in PPAR-GAL4DBD activity is seen with p38 activation in the presence of PPAR ligand, demonstrating a ligand-mediated induction of ~20-fold in the presence of p38 MAPK activation versus 6-fold ligand-mediated induction in the absence of p38 MAPK activation. To exclude the possibility that these results were caused by spurious activation of the JNK pathway, the (UAS)₃TKLuc cotransfections were repeated with an expression vector for a c-Jun-GAL4DBD hybrid protein, a known JNK-specific target. c-Jun-GAL4DBD was not activated by MKK6b(E)/p38 but was increased (5-fold) by cotransfection of JNK and its activator MEKK (data not shown), indicating that the observed activation of PPAR-GAL4DBD in CV-1 cells was the result of the specific effects of the p38 kinase pathway. To confirm that the PPAR-GAL4DBD fusion protein does not heterodimerize with endogenous RXR in CV-1 cells, parallel control experiments were performed with addition of the RXR ligand, 9-cis-retinoic acid, in the presence or absence of cotransfected RXR. Addition of 9-cis-retinoic acid with or without cotransfection of RXR had no effect on the target reporter activity in the presence of PPAR-GAL4DBD (data not shown), confirming that RXR is not interacting with PPAR-GAL4DBD in this system and, therefore, is not the mediator of the effect of p38 kinase on PPAR/RXR heterodimer transactivation. These results indicate that p38 MAPK activates PPAR in a RXR-independent manner. Moreover, these data demonstrate that the activation of PPAR by p38 kinase is independent of effects on DNA binding.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   p38 MAPK increases PPAR ligand-dependent transactivation in the absence of DNA binding and heterodimerization with RXR. (UAS)3 TKLuc and PPAR-GAL4DBD were cotransfected into CV-1 cells in the presence or absence of expression vectors for MKK6b(E) and p38, oleic acid (250 µM), SB202190 (20 µM), or vehicle control (BSA and/or Me2SO) as indicated. The bars represent mean RLU normalized (=1.0) to the activity of (UAS)3TKLuc cotransfected with an expression vector encoding only GAL4DBD under basal culture conditions. The data represent the mean of at least three independent experiments. The asterisk (*) denotes a significant difference (p < 0.05) between the indicated conditions.

p38 MAPK-mediated Activation of PPAR Maps to Phosphorylation Sites within the A/B Domain-- To determine whether the phosphorylation sites identified within the PPAR A/B domain by in vitro kinase studies confer the functional effects shown above, the mammalian one-hybrid experiments were repeated using full-length PPAR-GAL4DBD expression vectors containing the same mutations used for the in vitro kinase assays. Fig. 6A shows that, as predicted by the results of the in vitro kinase assays, the S6-21A and S6-77A mutants are not responsive to p38 kinase in the absence or presence of PPAR ligand (Fig. 6A and data not shown). However, MKK6b(E)/p38-mediated activation of the S73-77A fusion protein is similar to the wild-type protein (Fig. 6A), a result that is also consistent with the results of the in vitro phosphorylation studies. Fig. 6B shows that the mutant PPAR-GAL4DBD fusion proteins retain the ability to be activated by PPAR ligand, indicating that A/B domain phosphorylation is not necessary for ligand-dependent AF-2 function in CV-1 cells. These data indicate that phosphorylation of PPAR by p38 kinase on one or more of the serines at position 6, 12, or 21 is responsible for p38-mediated enhancement of PPAR transactivation function.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Mutation of serine residues within the NH2-terminal domain of PPAR prevents activation by p38 MAPK. A, phosphorylation of serines 6, 12, and/or 21 is necessary for the p38 MAPK-dependent increase in PPAR liganddependent transactivation. (UAS)3TKLuc and PPAR-GAL4DBD expression vectors encoding wild-type (WT) or mutant PPAR harboring serine-to-alanine mutations in amino acid positions 6/12/21 (S6-21A), positions 73/76/77 (S73-77A), or all six positions (S6-77A) were transfected in CV-1 cells in the absence and presence of expression vectors for MKK6b(E) and p38. The bars represent the -fold activation mediated by cotransfection of MKK6b(E) and p38 relative to cotransfection of empty expression vectors in cells maintained in medium supplemented with oleic acid (250 µM) as determined by luciferase activities. The data represent the mean of at least three independent experiments. The asterisk (*) denotes a significant difference (p < 0.05) between the indicated mutant PPAR-GAL4DBD construct and wild type. B, ligand activation is preserved in PPAR phosphorylation mutants. (UAS)3TKLuc and PPAR-GAL4DBD expression vectors encoding mutant PPAR (S6-21A, S73-77A, or S6-77A) were transfected into CV-1 cells. The solid bars represent mean RLU normalized (=1.0) to the activity of (UAS)3TKLuc cotransfected with an expression vector encoding the mutant PPAR-GAL4DBD under basal culture conditions with aqueous BSA vehicle. The hatched bars represent the same transfections in the presence of oleic acid (250 µM). The data represent the mean of at least three independent experiments. The asterisk (*) denotes a significant difference (p < 0.05) between the indicated conditions.

Activated p38 MAPK Enhances Coactivation of PPAR by PGC-1-- Ligand activation of nuclear receptors leads to recruitment of transcriptional coactivators. We sought to test whether the activation of PPAR by p38 MAPK phosphorylation involved the action of specific coactivators. Cotransfection experiments were performed with PPAR-GAL4DBD expression vectors for wild-type or mutant PPARs and known PPAR coactivators, including PGC-1 (28), SRC-1 (30), and PBP (31). As we have shown previously (28), the wild-type PPAR-GAL4DBD fusion protein was activated by PGC-1 in the presence of ligand (Fig. 7). When cotransfection of PGC-1 was combined with MKK6b(E)/p38 in the presence of ligand, a dramatic increase in PPAR coactivation was seen, nearly 6-fold relative to PGC-1 cotransfection in the absence of p38 MAPK activation (Fig. 7). In striking contrast, although the PPAR-S6-21A-GAL4DBD mutant was PGC-1-responsive to the same degree as wild-type PPAR-GAL4DBD, it was not activated further by MKK6b(E)/p38 in the presence or absence of ligand (Fig. 7). Unlike the results with PGC-1, neither SRC-1 nor PBP coactivation of PPAR-GAL4DBD was influenced by activation of p38 MAPK in these experiments (data not shown), suggesting that phosphorylation of PPAR serves to enhance coactivation by a specific subset of coactivators.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   p38 MAPK enhances coactivation of PPAR by PGC-1. (UAS)3TKLuc and PPAR-GAL4DBD wild-type (solid bars) or S6-21A mutant (hatched bars) were co-transfected into CV-1 cells with or without expression vectors for PGC-1, MKK6b(E), and p38. Cells were maintained in media supplemented with oleic acid (250 µM) or vehicle control (BSA). The bars represent mean RLU normalized (=1.0) to the activity of (UAS)3TKLuc in the presence of PPAR-GAL4DBD wild-type (solid bars) or S6-21A mutant (hatched bars) under basal culture conditions. The data represent the mean of at least three independent experiments. The asterisk (*) denotes a significant difference (p < 0.05) between the indicated conditions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PPAR, a lipid-activated transcription factor, plays a critical role in the control of cellular energy metabolism in a variety of physiologic and pathologic states. Evidence has emerged that PPAR activity is modulated in heart and liver during diverse stress responses, including fasting (1), cardiac hypertrophy (4), and cellular hypoxia (32). This implies that upstream signaling events activated by cellular stressors are linked to changes in PPAR activity, which in turn regulates mitochondrial energy metabolism. Members of the p38 kinase family, so-called "stress-activated protein kinases," represent likely candidates to serve as upstream regulators of PPAR. In this report, we show that p38 kinase-mediated phosphorylation activates PPAR in a ligand-influenced manner and results in enhanced functional cooperation with the transcriptional coactivator PGC-1. These results suggest that p38 kinase signaling promotes cardiac mitochondrial fatty acid -oxidation during periods of stress.

Certain pathologic conditions lead to a decrease in myocardial oxidative energy production through reduced FAO enzyme gene expression linked to antagonism of PPAR activity. For example, the PPAR gene regulatory pathway is deactivated during ₁-adrenergic agonist stimulation of cardiac myocyte hypertrophy (4). We have shown that PPAR activity is diminished by a post-transcriptional mechanism mediated by ERK-MAPK, confirming that signal transduction cascades linked to G-protein-coupled receptors can affect the activity of PPAR. Similarly, the related nuclear receptor, PPAR, is deactivated by ERK-mediated phosphorylation through a mechanism that reduces affinity for ligand (33-36). Given these previous findings, the results shown here indicating that phosphorylation of PPAR by p38 MAPK leads to activation of PPAR function was surprising. Taken together with the results of the ERK-MAPK studies (4), we conclude that distinct limbs of the MAPK network, namely ERK and p38, have opposing effects with respect to PPAR activity in the heart. The molecular mechanism(s) underlying this differential response of PPAR to MAPK signaling remains unknown. Our results do not exclude the possibility that, in certain cellular contexts, including cardiac myocytes, the JNK pathway may also alter PPAR activity.

A diverse array of molecular consequences have been attributed to nuclear receptor phosphorylation, including increased or decreased ligand-dependent and ligand-independent activation (reviewed in Ref. 37), enhanced recruitment of cofactors (38-40), reduced affinity for ligand (33), increased or decreased capacity for DNA binding (reviewed in Ref. 37), enhanced or inhibited heterodimerization (41, 42), and susceptibility to proteosomal degradation (43). Our results indicate that p38 MAPK-mediated phosphorylation of PPAR leads to an increase in ligand-dependent transactivating function. We also found that p38-mediated phosphorylation of PPAR results in a strong functional cooperation with PGC-1, a known ligand-influenced PPAR coactivator (28). Accordingly, we conclude that enhanced interaction with coactivator rather than increased DNA binding or heterodimerization with RXR is the primary mechanism responsible for increased activity. Interestingly, the mechanism described here does not extend to several other ligand-recruited coactivators, including SRC-1 and PBP. We speculate that phosphorylation of PPAR by p38 MAPK not only increases its trans-activating properties but also dictates coactivator selectivity. PGC-1, as an activator of cardiac mitochondrial function and biogenesis (44, 45), is a likely component of the energy metabolic stress responses.

Previous studies have demonstrated that PPAR exists as a phosphoprotein in primary rat adipocytes (46) and that insulin signaling leads to phosphorylation of the A/B domain and enhanced AF-1 activity (47). Our results suggest an alternative mechanism. The earlier studies reported activation of AF-1 activity using a PPAR A/B domain-GAL4DBD fusion construct as the target (47). However, our results demonstrate that the full-length PPAR-GAL4DBD fusion protein has no constitutive (ligand-independent) AF-1 activity in CV-1 cells (Fig. 5). Moreover, p38 MAPK-mediated phosphorylation does not activate PPAR-GAL4DBD in the absence of ligand, indicating that AF-1 function per se within the context of the full-length molecule, is not enhanced by A/B domain phosphorylation. These results suggest a functional, if not physical interaction between the AF-1 and AF-2 regions of PPAR following phosphorylation in the context of engaged ligand. It is possible that AF-1 activity is increased by A/B domain phosphorylation only when PPAR is ligand-bound, leading to enhanced interaction with specific coactivators, such as PGC-1. This is similar to the mechanism by which phosphorylation of SF-1 in the AF-1 domain enhances cofactor recruitment only when the ligand-binding domain is present, although SF-1 is not known to be ligand-activated (39). Alternatively, phosphorylation of the AF-1 region may lead to direct recruitment of coactivators to AF-1 as is seen with ER (40). However, there is no evidence that PGC-1 interacts with the A/B domain of PPAR, although a separate PPAR/PGC-1 interacting protein could serve as an adaptor.

In summary, we have shown that p38 MAPK phosphorylates and activates the transcription factor PPAR, leading to enhanced ligand-mediated coactivation by the transcriptional coactivator PGC-1. These results identify PPAR as a target of stress-activated signaling. In cardiac myocytes, p38 MAPK activation would be predicted to increase the capacity for energy production by the mitochondrial fatty acid -oxidation pathway, as a component of the metabolic response to diverse physiologic stressors.

	ACKNOWLEDGEMENTS

We thank Jiahuai Han, Janardan K. Reddy, David D. Moore, Ming-Jer Tsai, Sophia Y. Tsai, John Woods, and Joel Berger for providing plasmids and reagents. We especially thank Mary Wingate for assistance with manuscript preparation.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants K08 HL03808 (to P. M. B.), RO1 HL58493, P50 HL61006, P30 DK56341, and P30 DK52574.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.

To whom correspondence should be addressed: Center for Cardiovascular Research, Box 8086, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8908; Fax: 314-362-0186; E-mail: dkelly@imgate.wustl.edu.

Published, JBC Papers in Press, September 27, 2001, DOI 10.1074/jbc.M105945200

	ABBREVIATIONS

The abbreviations used are: FAO, fatty acid -oxidation; PPAR, peroxisome proliferator-activated receptor ; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; PGC-1, peroxisome proliferator-activated receptor  coactivator-1; RXR, retinoid X receptor; M-CPT I, muscle-type carnitine palmitoyltransferase I; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PBP, peroxisome proliferator-activated receptor-binding protein; SRC, steroid receptor coactivator; ACO, acyl-CoA oxidase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; JNK, c-Jun NH₂-terminal kinase; AF, activating function; ERK, extracellular signal-regulated kinase; FARE-1, fatty acid response element 1; RLU, relative light unit(s); DBD, DNA binding domain.

	REFERENCES

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