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Endocrinology Vol. 140, No. 1 392-397
Copyright © 1999 by The Endocrine Society

ARTICLES

c-Jun N-Terminal Kinase Phosphorylates Peroxisome Proliferator-Activated Receptor-{gamma}1 and Negatively Regulates Its Transcriptional Activity

Heidi S. Camp, Sherrie R. Tafuri and Todd Leff

Department of Cell Biology (H.S.C., T.L.), Department of Molecular Biology (S.R.T.), Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, Michigan 48105; and the Department of Biological Chemistry, University of Michigan Medical School (T.L.), Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Dr. Todd Leff, Parke-Davis, Department of Cell Biology, 2800 Plymouth Road, Ann Arbor, Michigan 48105. E-mail: todd.leff{at}aa.wl.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) transcription factor plays a pivotal role in adipocyte differentiation and metabolic regulation. The transcriptional activity of PPAR{gamma} is positively modulated by ligand binding and negatively regulated by phosphorylation mediated by the MEK/ERK signaling pathway. The phosphorylation of mouse PPAR{gamma}1 at Ser82 by ERK causes a decrease in both basal and ligand-dependent transcriptional activity. In this report we examined the ability of other mitogen-activated protein kinase family members to phosphorylate PPAR{gamma}1. We demonstrate that in vitro, PPAR{gamma}1 is efficiently phosphorylated by JNK/SAPK (c-Jun N-terminal kinase or stress-activated protein kinase) but only weakly phosphorylated by p38. In transfected 293T cells, PPAR{gamma}1 is phosphorylated at Ser82 in response to known JNK activators such as UV irradiation and anisomycin treatment. This phosphorylation is not blocked by either the specific MEK inhibitor PD98059 or the p38 inhibitor SB203580, indicating that it is independent of the MEK/ERK and p38 signaling pathways. Finally, in transient transfection reporter assays, activation of JNK by anisomycin or by overexpression of MKK4 (the upstream JNK kinase) decreased ligand-dependent PPAR{gamma}1 transcriptional activity. These results suggest that the activation of the JNK/SAPK pathway by extracellular signals, perhaps by inflammatory cytokines such as tumor necrosis factor-{alpha}, would result in a reduction of PPAR{gamma} transcriptional activity and reduce the effects of PPAR{gamma} ligands.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEROXISOME proliferator-activated receptors (PPARs) are members of the ligand-activated nuclear receptor superfamily. PPARs bind to specific response elements as heterodimers with the retinoid X receptor (RXR) and activate transcription in response to a variety of endogenous and exogenous ligands, including fatty acids, arachidonic acid metabolites, and synthetic drugs used to treat metabolic disorders (1, 2, 3, 4, 5). Three PPAR isoforms ({alpha}, {gamma}, and {delta}) differ in their tissue distribution and ligand specificity (for a review see Ref. 6). PPAR{alpha} is present in heart, kidney, and liver and appears to primarily regulate genes involved in lipid and lipoprotein metabolism, whereas PPAR{delta} expression is ubiquitous, and its physiological role is unclear. PPAR{gamma} is expressed predominantly in adipose tissue, with lower levels in muscle and liver (7, 8). Although the physiological role of PPAR{gamma} has not been clearly determined, it appears to be an important transcriptional regulator of genes involved in glucose and lipid metabolism.

A central role for PPAR{gamma} in metabolic regulation was demonstrated by the observation that antidiabetic drugs known as the thiazolidinediones (TZDs) were high affinity ligands of PPAR{gamma} (9). TZDs have profound effects on glucose metabolism in diabetic subjects and appear to act primarily through PPAR{gamma}, as indicated by a correlation between the antidiabetic potency of TZDs and receptor affinity (10). In addition, a compound (LG268) that specifically binds to RXR{alpha}, the heterodimeric partner of PPAR{gamma}, has a similar anti-diabetic effect (11). Although many of the details are not clearly understood, a model has emerged in which activated PPAR{gamma} modulates the expression of genes involved in glucose and lipid metabolism. Activation of these PPAR{gamma} target genes would lead directly or indirectly to an improvement in the metabolic defects associated with diabetes (12). This model predicts that endogenous regulators of PPAR{gamma} activity would be key components of the general system that regulates metabolism and energy balance.

In addition to being regulated by ligand binding, PPAR{gamma} activity is modulated by phosphorylation. Previously, we and others have shown that PPAR{gamma} is phosphorylated by a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated protein kinase (ERK), and that phosphorylated PPAR{gamma} has significantly reduced transcriptional activity compared with that of the unphosphorylated version (13, 14, 15). Thus, agents that cause an increase in PPAR{gamma} phosphorylation may reduce sensitivity to PPAR{gamma} ligands such as TZDs and may even contribute to the development of insulin resistance.

MAP kinases (MAPKs) are a large family of Ser/Thr kinases that are regulated by extracellular stimuli, including growth factors, mitogens, and cellular stress (16, 17). In addition to ERK1 and ERK2, which are activated by growth factors via the Ras/Raf/MEK pathway, there are three additional MAPK family members that are activated primarily by stress stimuli: JNK (also termed SAPK) (18, 19, 20), p38 (also termed CSBP) (21, 22), and BMK1 (also termed ERK5) (23). All of the MAPK family members display distinct, yet overlapping, substrate recognition specificity. Because some substrates can be phosphorylated by more than one MAPK signaling pathway, we sought to investigate whether other MAPKs could phosphorylate PPAR{gamma}1. In the current study, we demonstrate that JNK phosphorylates PPAR{gamma}1 on Ser82, the same site phosphorylated by ERK, and that in vivo, activation of JNK causes a phosphorylation-dependent decrease in PPAR{gamma}1 transcriptional activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and materials
Cell culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY). The ECL detection system was obtained from Amersham (Arlington Heights, IL). 12-O-Tetraphorbol 12-myristate 13-acetate (TPA) was purchased from Harlan Bioproducts for Science, Inc. (Indianpolis, IN). PD98059 and BRL 49653 were synthesized at Parke-Davis Pharmaceutical Research Division of Warner-Lambert Co., (Ann Arbor, MI) SB203580 was purchased from Alexis Corp. (San Diego, CA).

In vitro phosphorylation assay
Construction of mutant PPAR{gamma}1 (Ser82Ala) was described previously (13). Both the wild-type and the mutant mouse PPAR{gamma}1 were in vitro translated using rabbit reticulocyte lysates in the presence of [35S]Met. One tenth of the in vitro translated proteins were then incubated with either activated ERK2 (New England Biolabs, Inc., Beverley, MA), the activated ß-form of rat JNK (Stratagene, La Jolla, CA), or activated Xenopus MalE-Mpk2/p38/RK (Upstate Biotechnology, Inc., Lake Placid, NY) as recommended by the manufacturers. Incubation reactions contained 10 µM cold ATP and 1 x MAPK buffer (25 mM HEPES, pH 7.5, and 10 mM magnesium acetate). Proteins were resolved in 8 M urea-10% acrylamide gel (100:1, acrylamide-bisacrylamide) (15) that can separate the phosphorylated form of PPAR{gamma} from unphosphorylated PPAR{gamma}. The gel was dried and exposed for autoradiography. Bacterially expressed myelin basic protein (MBP) and c-Jun were used as kinase substrates in control reactions (MBP for ERK2 and p38, and c-Jun for JNK) containing 10 µM [{gamma}-32P]ATP, 25 mM HEPES (pH 7.5), and 10 mM magnesium acetate.

In vivo phosphorylation of PPAR{gamma}
293T cells were maintained in DMEM containing 10% FCS (Life Technologies, Inc.). Cells were transfected with mouse full-length wild-type PPAR{gamma}1 or mutant PPAR{gamma}1 (Ser82Ala) using lipofectamine (Life Technologies, Inc.). Cells were harvested in HNTG lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 100 mM NaF, and complete protease inhibitors, used according to the manufacturer’s directions; Sigma Chemical Co., St. Louis, MO]. Proteins were resolved in 8 M urea-10% acrylamide (100:1, acrylamide-bisacrylamide) gel (15). PPAR{gamma} protein was visualized by blotting with a polyclonal anti-PPAR{gamma} antibody raised against recombinant mouse PPAR{gamma}1 (13). Immunological detection of activated kinases by Western blot analysis was carried out using anti-ACTIVE MAPK (Promega Corp., Madison, WI), anti-phospho-SAPK/JNK (New England Biolabs, Inc.), and phospho-specific p38 MAPK (New England Biolabs, Inc.) polyclonal antibodies.

Transient reporter assays
The reporter construct used in the transient transfections contained three copies of the PPRE site from the aP2 enhancer (ARE6) (24) inserted upstream of a minimal thymidine kinase (TK) promoter in the pGL3 (Promega Corp.) luciferase vector. 293T cells were grown in 10% FCS-DMEM and cotransfected with mouse PPAR{gamma}1 (200 ng) and mouse RXR{alpha} (50 ng) expression plasmids, the TK luciferase reporter plasmid (200 ng), and an internal reference plasmid pCMV (CMV, cytomegalovirus) ß-galactosidase (50 ng) using lipofectamine (Life Technologies, Inc.). In some experiments, 200 ng of an expression vector producing MKK4 were transfected into cells along with PPAR{gamma}1. After transfection, cells were treated for 24 h with 25 µM BRL 49653 and further treated with 2 µg/ml anisomycin for the last 6 h or by UV irradiation (0.1 J in a Stratagene UV Stratalinker 1800) that was delivered 6 h before harvesting cells. Luciferase and ß-galactosidase activities were determined using a luciferase assay (Promega Corp.) and Galacto-light system (Tropix, Inc., Bedford, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
JNK phosphorylates PPAR{gamma}1 in vitro
We previously demonstrated that PPAR{gamma}1 is phosphorylated at Ser82 by the MAPK family member ERK2 (13). To determine whether PPAR{gamma}1 can be phosphorylated by other MAPK family members, such as JNK and p38, [35S]Met-labeled wild-type and mutant versions of PPAR{gamma}1 were synthesized by in vitro translation and incubated with the activated forms of various MAPKs. The phosphorylation state of PPAR{gamma}1 was then assessed using a gel shift assay in which phosphorylated PPAR{gamma}1 migrates more slowly than the unphosphorylated protein. Both ERK2 and JNK efficiently phosphorylated PPAR{gamma}1 (22% and 23% phosphorylation, respectively), whereas PPAR{gamma}1 was only weakly phosphorylated by p38 (9% compared with 4% for the control reaction; Fig. 1aGo). The low activity of p38 on PPAR{gamma}1 was not due to inactive kinase as demonstrated by the phosphorylation of MBP by the same preparation of p38 (Fig. 1bGo). Both ERK2 and JNK were also shown to be active in this control assay. The phosphorylation of PPAR{gamma} by ERK2 and JNK was independently confirmed in a direct in vitro phosphorylation assay using [{gamma}-32P]ATP and recombinant PPAR{gamma} protein purified from Escherichia coli (data not shown). Taken together, these results demonstrate that JNK is as active as ERK2 in terms of its ability to phosphorylate PPAR{gamma} in vitro.



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  		Figure 1. In vitro phosphorylation of PPAR{gamma}1 by MAPK family members. A, Approximately 1 µg of either the wild-type or mutant (Ser82Ala) PPAR{gamma}1 DNA was in vitro translated in the presence of [35S]Met, and the products were subjected to an in vitro cold kinase assay in the presence of activated ERK2, JNK, or p38. The final reactions were then resolved in 8 M urea-10% acrylamide gel. B, Control phosphorylation reactions were carried out in the presence of [{gamma}-32P]ATP using MBP as the substrate for ERK2 and p38, and c-Jun as a substrate for JNK.

 
JNK activators induce PPAR{gamma} phosphorylation in 293T cells
It has been well documented that exposure of cells to environmental stresses such as UV light, osmotic pressure, or high temperature can cause activation of JNK and/or p38 MAPKs (25, 26). JNK and p38 have also been shown to be activated by protein synthesis inhibitor anisomycin as well as by certain inflammatory cytokines (25, 26, 27, 28). To determine whether PPAR{gamma}1 can be phosphorylated by known JNK/p38 activators, serum-starved 293T cells transfected with PPAR{gamma}1 were treated with anisomycin or with UV light. Whole cell extracts were then prepared, and the phosphorylation state of PPAR{gamma}1 was assessed using a gel shift assay. As shown in Fig. 2aGo, treatment with anisomycin or UV light caused an increase in PPAR{gamma}1 phosphorylation compared to in the untreated control cell extracts (Fig. 2aGo, lanes 5 and 6, and 7 and 8). To determine whether the MEK/ERK signaling pathway is involved or contributes to this phosphorylation, cells were pretreated with PD98059, a specific MEK inhibitor. As shown in Fig. 2aGo, PD98059 did not block anisomycin-induced PPAR{gamma} phosphorylation (lane 6), indicating that this phosphorylation was not caused by activation of the MEK/ERK signaling pathway. However, PD98059 did cause a slight reduction in UV light-induced PPAR{gamma} phosphorylation, suggesting ERK1/2 might contribute to PPAR{gamma} phosphorylation induced by UV light (Fig. 2aGo, lane 8). This interpretation is supported by the control experiments presented in Fig. 2cGo. Although anisomycin treatment specifically activated JNK and p38, UV light treatment caused a slight increase in ERK2 activation in addition to a strong activation of JNK and p38. As a control, cells were also treated with TPA, a known activator of MEK/ERK. As previously reported (15), this treatment caused a significant increase in PPAR{gamma} phosphorylation that was completely blocked by PD98059 (Fig. 2aGo, lanes 3 and 4).



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  		Figure 2. Anisomycin and UV light treatments stimulate PPAR{gamma}1 phosphorylation in vivo. A, 293T cells were transfected with pSG5 (lane 1) or PPAR{gamma}1 (lanes 2–8), serum starved for 8 h, and treated with 0.1 µg/ml TPA (lanes 3 and 4), 2 ng/ml anisomycin (An; lanes 5 and6), or 1000 J UV irradiation (lanes 7 and 8) for 30 min. Some reactions were pretreated with 40 µM PD98059 (PD) for 15 min (lanes 4, 6, and 8). Whole cell lysates were prepared and subjected to 8 M urea-10% PAGE and Western blotted using a polyclonal anti-PPAR{gamma} antibody. The percent phosphorylation was quantified from autoradiograms using BioImage Visage Whole band software (Genomic Solutions, Inc., Ann Arbor, MI). The histogram presents average values derived from three independent experiments. The SEM is indicated. *, Significant difference (P < 0.05) from vehicle control (V), as determined by single factor ANOVA using Dunnett’s test. #, Significant difference (P < 0.05) between PD98059 treatment pairs (with and without PD) as determined by Student’s t test. The slight reduction of phosphorylation caused by PD98059 in anisomycin- and UV-treated cells was not statistically significant. B, 293T cells were transfected with PPAR{gamma}1, serum deprived for 8 h, and treated for 30 min with 0.1 µg/ml TPA or 2 ng/ml anisomycin (An). Some reactions were pretreated with 20 µM SB203580 (SB) for 15 min as indicated. The histogram presents average values derived from multiple independent experiments. The SEM is indicated. C, The blot was reprobed using antibodies either anti-active JNK, anti-active ERK, or anti-active p38, which recognize only the phosphorylated forms of JNK, ERK, or p38, respectively.

 
In the experiment described above, it was not possible to determine whether anisomycin and UV light induced PPAR{gamma}1 phosphorylation via the JNK or the p38 signaling pathways. To examine this question, cells were incubated with either TPA or anisomycin in the absence or presence of a p38 inhibitor SB203580, which was shown previously to specifically block p38 kinase activity (29, 30). Cells were harvested, and the phosphorylation status of PPAR{gamma}1 was determined as described above. As demonstrated in Fig. 2bGo, PPAR{gamma}1 phosphorylation induced by either TPA or anisomycin was not altered in the presence of SB203580. These results suggest that p38 is not responsible for the phosphorylation of PPAR{gamma}1 in response to stress-inducing agents such as anisomycin.

Previously, we have shown that there is a single MAPK phosphorylation site in the N-terminal region located at Ser82 of mouse PPAR{gamma}1 (13). To determine whether Ser82 is also the residue that is phosphorylated by JNK, 293T cells were transfected with either wild-type PPAR{gamma}1 or a mutant form of PPAR{gamma}1 that contains an alanine residue substituted for serine at position 82 (Ser82Ala). Transfected cells were treated with TPA or anisomycin, and the degree of PPAR{gamma}1 phosphorylation was measured using the gel shift assay described above. As shown in Fig. 3Go, both TPA and anisomycin induced an approximately 5-fold stimulation of wild-type PPAR{gamma}1 phosphorylation, but both agents failed to induce the phosphorylation of the Ser82Ala mutant. These results demonstrate that in vivo, Ser82 of PPAR{gamma}1 is the phosphorylation site for both ERK and JNK.



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  		Figure 3. JNK-mediated PPAR{gamma}1 phosphorylation occurs at Ser82. Wild-type PPAR{gamma}1 or mutant PPAR{gamma}1 (Ser82Ala) complementary DNA was transfected onto 293T cells and treated with TPA or anisomycin (An) in the presence or absence of MEK inhibitor PD98059 (PD) for 30 min. The percent phosphorylated PPAR{gamma}1 protein was quantitated as described in Fig. 2Go, and data are presented as a histogram.

 
Activation of JNK decreases PPAR{gamma}-dependent transcriptional activity
Phosphorylation of PPAR{gamma}1 at Ser82 by ERK has been shown to significantly decrease its transcriptional activity (13, 14, 15). To confirm that JNK activation causes a similar decrease in PPAR{gamma}1 transcriptional activity, a PPAR{gamma}-dependent transient transcription assay was performed. PPAR{gamma} activity was measured using a minimal TK reporter plasmid containing three copies of the PPAR{gamma}-binding site (ARE6) from the aP2 gene promoter (24). 293T cells were transfected with this reporter (ARE6/TKpGL3) together with PPAR{gamma}1 and RXR{alpha} and treated with either anisomycin or UV light. Because anisomycin is a general protein synthesis inhibitor, we performed a dose-response and time-course experiment to identify conditions that did not inhibit protein synthesis, but still activated JNK. Treatment of 293T cells with 2 µg/ml anisomycin for up to 7 h did not change ß-galactosidase expression from a transfected reference plasmid (data not shown). Transfected cells were allowed to recover overnight in the presence or absence of the PPAR{gamma} ligand BRL 49653 and then treated for 6 h with anisomycin or by exposure to UV light 6 h before harvest. As presented in Fig. 4aGo, both anisomycin and UV light treatment decreased ligand-dependent PPAR{gamma}1 transcriptional activity by 50% and 40%, respectively. In this system, treatment with UV light and anisomycin did not affect basal transcriptional activity in the absence of ligand. These results indicate that PPAR{gamma} phosphorylation by the JNK pathway leads to a decrease in ligand-dependent PPAR{gamma} transcriptional activity.



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  		Figure 4. Activation of JNK decreases PPAR{gamma}1 transcriptional activity. A, 293T cells were cotransfected with a PPRE-containing reporter plasmid (ARE6/TKpGL3) and expression plasmids producing RXR{alpha} and PPAR{gamma}1. Transfected cells were then treated with UV light (UV) or anisomycin (An) in the presence or absence of the PPAR{gamma} ligand BRL 49653 (BRL) as described in Materials and Methods. B, 293T cells were cotransfected with ARE6/TKpGL3, RXR{alpha}, and PPAR{gamma}1 in the presence or absence of an MKK4 expression plasmid. In all transfection assays, the expression of pCMV ß-galactosidase plasmid was used as an internal control for the normalization of transfection efficiency. Error bars represent the SEM.

 
To confirm this hypothesis, we examined the effect of activating JNK by a second independent method. Components of the kinase cascade that lead to JNK activation include MKK4 (also termed SEK1 or JNKK) (25, 26, 31) and the multiple isoforms of MAPK kinase kinases (MEKK1, MEKK2, and MEKK3) (32, 33). Because MKK4 is immediately upstream of JNK in the signaling pathway and is known to activate JNK, we transfected 293T cells with PPAR{gamma} and the reporter plasmid described above (ARE6/TKpGL3) together with a plasmid expressing MKK4 and assessed PPAR{gamma}1-dependent transcriptional activity. In the presence of overexpressing MKK4, a slight reduction of basal and a dramatic inhibition of ligand-dependent PPAR{gamma}1 transcriptional activity was observed (Fig. 4bGo). Taken together, these results indicate that JNK activation leads to decreased PPAR{gamma}1 activity as a result of phosphorylation at Ser82 and suggests that physiological stimuli that activate the JNK pathway could have a negative effect on the expression of PPAR{gamma} target genes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PPAR{gamma} is a key component of the system that regulates energy metabolism and glucose homeostasis. The primary evidence for this hypothesis is that activating ligands of PPAR{gamma} can correct many of the defects in glucose metabolism that appear with type II diabetes (12, 34). In the current study we have demonstrated that activation of JNK negatively regulates PPAR{gamma} transcriptional activity by phosphorylation at the same site previously reported to be phosphorylated by ERK. Taken together with previous studies demonstrating that ERK can regulate PPAR{gamma}1, our findings suggest that PPAR{gamma} transcriptional activity may be subject to negative regulation by a variety of signals that activate one or more of the MAPK signaling pathways.

There are several transcription factors that are known to be phosphorylated at the same site by different MAPK family members. The ternary complex transcription factor protein ELK-1 is a target for all three MAPKs: ERK (35), JNK (36), and p38 (27). There are, however, many examples of distinct specificities for substrate site recognition by these kinases. For example, SAP-1 is recognized and phosphorylated by ERK and p38, but not by JNK (37), whereas the transcription factor MEF2C is phosphorylated with much higher efficiency by p38 then by JNK or ERK (38). In the case of PPAR{gamma}, its activity can be clearly regulated by at least two branches of the MAPK signaling cascade, ERK and JNK, which suggests that multiple signals can lead to PPAR{gamma} phosphorylation and subsequent reduction in the sensitivity of PPAR{gamma} to its cognate ligands.

Although the physiological role of PPAR{gamma} regulation by phosphorylation is unclear at this time, it must provide a means for a variety of physiological signals, acting through MAPK signaling pathways, to affect the expression of PPAR{gamma} target genes and thereby alter metabolism. An interesting possibility that is directly related to our finding that JNK kinase can phosphorylate PPAR{gamma}1 is suggested by the observation that at least in some cells the JNK pathway can be activated by the inflammatory cytokine tumor necrosis factor-{alpha} (TNF{alpha}) (20, 33). Several studies have suggested a role for TNF{alpha} in the development of insulin resistance in cells and animal disease models. TNF{alpha} is overexpressed in the adipose tissue of some diabetic animals and patients (39, 40, 41, 42), and in some models TNF{alpha} overexpression appears to cause insulin resistance (43). Although TNF{alpha} has been shown to affect several aspects of insulin action (44, 45), an additional possibility suggested by the findings reported here is that overexpression of TNF{alpha} in a diabetic state could lead to the phosphorylation of PPAR{gamma} via the JNK signaling pathway. Consequently, as we have demonstrated, this phosphorylation would reduce the effectiveness of activating ligands and could contribute to the development of insulin resistance.


   Acknowledgments
 
We thank Dr. Kevin Pumiglia for helpful discussions, Dr. Stuart Decker for the MKK4 complementary DNA clone, Dr. Alan Saltiel for discussion and for reading the manuscript, and Dr. David G. Taylor for help with the statistical analysis.

Received April 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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