Regulation of Peroxisome Proliferator-activated Receptor gamma  Activity by Mitogen-activated Protein Kinase*

(Received for publication, December 31, 1996, and in revised form, February 6, 1997)

Heidi S. Camp and Sherrie R. Tafuri Dagger

From the Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Adipocyte differentiation is regulated both positively and negatively by external growth factors such as insulin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF). A key component of the adipocyte differentiation process is PPARgamma , peroxisomal proliferator-activated receptor gamma . To determine the relationship between PPARgamma activation and growth factor stimulation in adipogenesis, we investigated the effects of PDGF and EGF on PPARgamma 1 activity. PDGF treatment decreased ligand-activated PPARgamma 1 transcriptional activity in a transient reporter assay. In vivo [32P]orthophosphate labeling experiments demonstrated that PPARgamma 1 is a phosphoprotein that undergoes EGF-stimulated MEK/mitogen-activated protein (MAP) kinase-dependent phosphorylation. Purified PPARgamma 1 protein was phosphorylated in vitro by recombinant activated MAP kinase. Examination of the PPARgamma 1 sequence revealed a single MAP kinase consensus recognition site at Ser82. Mutation of Ser82 to Ala inhibited both in vitro and in vivo phosphorylation and growth factor-mediated transcriptional repression. Therefore, phosphorylation of PPARgamma 1 by MAP kinase contributes to the reduction of PPARgamma 1 transcriptional activity by growth factor treatment.


INTRODUCTION

Peroxisome proliferator-activated receptors (PPARs)1 are members of the nuclear hormone receptor superfamily (1). These receptors heterodimerize with retinoic acid-like receptor, RXR, and become transcriptionally active when bound to ligand. The three PPAR isoforms (alpha , delta , and gamma ) differ in their C-terminal ligand binding domains, and each appears to bind and respond to a specific subset of agents including hypolipidemic drugs, long chain fatty acids, aracadonic acid metabolites, and antidiabetic thiazolidinediones (2-4). PPARgamma is expressed predominantly in mouse white and brown fat, with lower levels in liver, whereas PPARalpha is present in heart, kidney, and liver (5, 6). PPARdelta expression is ubiquitous (7, 8).

Ectopic expression of either PPARalpha or PPARgamma in NIH-3T3 cells is sufficient to induce adipocyte differentiation in the presence of PPARgamma activators (9, 10). The rapid induction of PPARgamma during adipocyte differentiation and its enriched expression in adipose tissues suggest that PPARgamma is responsible for the initiation and maintenance of the adipocyte phenotype in vivo (9). Previously two isotypes of PPARgamma (PPARgamma 1 and PPARgamma 2) have been identified in 3T3-L1 adipocytes (11). Zhu et al. (12) have demonstrated that these two isotypes are derived from a single PPARgamma gene by alternative promoter usage and RNA splicing. However, thus far, no functional difference has been found between the two isotypes.

Adipogenesis is a complex process; multiple hormones and factors regulate the conversion of progenitor cells to adipocytes. Insulin and/or insulin-like growth factor enhance the ability of PPAR ligand to induce differentiation of both 3T3-L1- and PPARgamma -overexpressing cell lines (9, 13). In contrast, growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast growth factor inhibit adipocyte conversion (14-18). In this report, we find that activation of EGF and PDGF receptors and subsequent phosphorylation of PPARgamma 1 by the MAP kinase signaling pathway decreases PPARgamma 1 transcriptional activity. This repression is mediated by MAP kinase phosphorylation of Ser82 on PPARgamma 1. These studies identify PPARgamma 1 as a substrate of MAP kinase and provide evidence for regulation of PPARgamma 1 activity by phosphorylation.


EXPERIMENTAL PROCEDURES

Chemicals and Materials

Cell culture reagents were purchased from Life Technologies, Inc. The ECL detection system and carrier-free [32P]orthophosphate were obtained from Amersham Corp. The PDGF was purchased from Intergen, while EGF was from Harlan. PD98059 and BRL49653 were synthesized at Parke-Davis Pharmaceutical Research Division of Warner-Lambert Co.

Vector Constructs and Transient Transfection

For eukaryotic expression of PPARgamma 1 and RXRalpha , the entire PPARgamma 1 or RXRalpha cDNA was inserted 3' to the cytomegalovirus promoter in pSG5 (Stratagene). Constitutively active MAP kinase kinase (CA-MEK), which contains mutations at Ser218 to Glu and Ser222 to Glu was obtained from Dr. S. Decker (Parke-Davis). Site-directed mutagenesis of PPARgamma 1/pSG5 was conducted using the MORPH site-specific plasmid DNA mutagenesis system (5 Prime right-arrow 3 Prime, Inc., Boulder, CO). The oligonucleotide used in mutagenesis was CAAAGTAGAACCTGCAGCTCCACCTTATTATTCTGAAAAGACCC and changed Ser82 to Ala. The reporter construct used in the transfections contained three copies of the PPRE site from the aP2 enhancer (ARE7) inserted upstream of a minimal thymidine kinase (TK) promoter in the pGL3 basic luciferase vector (a gift from Dr. R. Wyborski). All constructs were sequenced prior to use. For the transient transfection, NIH 3T3 cells were grown in 10% fetal calf serum/Dulbecco's modified Eagle's medium and co-transfected with various expression plasmids and pCMV beta -galactosidase plasmid (Clontech) using Lipofectamine (Life Technologies, Inc.). After recovery, cells were placed in 0.5% bovine serum albumin/Dulbecco's modified Eagle's medium for 5 h and then treated with 25 µM BRL49653 and/or 100 ng/ml PDGF for 16 h. Luciferase and beta -galactosidase activities were determined using a Luciferase assay (Promega) and the Galacto-light system (Tropix, Inc.).

Production of PPAR Fusion Proteins and in Vitro Phosphorylation Assay

To express the maltose-binding protein (MBP) fusion proteins in Escherichia coli, the coding regions of PPARgamma 1, PPARdelta , and RXRalpha were inserted downstream of the isopropyl-beta -D-thiogalactopyranoside-inducible MalE-lacZalpha gene fusion in the pMAL-C2 plasmid (New England Biolabs). Protein expression was induced with isopropyl-beta -D-thiogalactopyranoside, and the fusion proteins were partially purified by amylose affinity chromatography (19). In vitro phosphorylation of MBP, MBP-PPARgamma 1, and MBP-PPARdelta by MAP kinase was performed as described previously (20) using a bacterially expressed glutathione S-transferase fusion protein of 44-kDa MAP kinase (GST-MAP kinase) and the 45-kDa MEK (GST-MEK1). Using a PPARgamma -specific polyclonal antibody (produced using the MBP-PPARgamma fusion protein),2 in vitro translated PPARgamma 1 and the mutant PPARgamma 1 (S82A) were immunoprecipitated and phosphorylated by active GST-MAP kinase as described above.

Mobility Shift Assays

Approximately 0.5 µg of the partially pure MBP-PPARgamma 1, phosphorylated or unphosphorylated, and 0.5 µg of MBP-RXRalpha protein were preincubated for 15 min in 1 × mobility shift assay buffer (15 mM Hepes, pH 7.0, 80 mM KCl, 10% glycerol, 1 µg of poly(dI-dC), 0.2 mM EDTA, and 0.4 mM dithiothreitol) to allow heterodimer formation, or MBP-PPARgamma 1 was phosphorylated prior to heterodimerization with MBP-RXRalpha . Approximately 20 fmol of a 32P-labeled double-stranded ARE7 PPRE-containing oligonucleotide probe (5'-AATTCAAGGCAGAAAGTGAACTCTGATCCAGTAAGAAG-3') was added to the protein mix and incubated at room temperature for 20 min. Protein-DNA complexes were analyzed in 5% 1 × TBE polyacrylamide gels.

Cell Transfection and in Vivo Radiolabeling

293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Life Technologies, Inc.) and transfected using a calcium phosphate transfection protocol according to the manufacturer (Stratagene). For in vivo labeling, transfected cells were serum-starved overnight in 0.5% bovine serum albumin/Dulbecco's modified Eagle's medium, pretreated with phosphate-free medium for 1 h, and subsequently incubated in 0.8 mCi of [32P]orthophosphate at 37 °C for 3 h. Cells were preincubated with either BRL49653 (25 µM) or PD98059 (40 µM) for 15 min followed by the addition of EGF (100 ng/ml). EGF stimulation proceeded for 5 or 15 min prior to removal of the media and cell lysis. Cells were harvested in radioimmune precipitation lysis buffer (10% glycerol, 137 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 20 mM Tris, pH 8.0, 2 mM EDTA, complete protease inhibitors, and 20 mM NaVO4). Whole cell extracts were immunoprecipitated with anti-PPARgamma antibody and protein A-Sepharose (Life Technologies, Inc.) for 16 h at 4 °C and resolved in 10% SDS-PAGE. To detect MAP kinase activity in 293T cells, whole cell lysates were prepared and subjected to Western blot analysis using the anti-active MAP kinase antibody (Promega) and ECL system (Amersham).


RESULTS

Growth Factors Decrease the Transcriptional Activity of PPARgamma 1

Transcription reporter assays were used to determine the effect of growth factors on the transcriptional activity of PPARgamma 1. The luciferase reporter constructs used in NIH3T3 cells contained the TK promoter (TKpGL3) or three copies of ARE7 PPRE elements upstream of the TK promoter (ARE7-TKpGL3). In the absence of co-transfected PPARgamma 1 and RXRalpha expression plasmids, no PPARgamma ligand (BRL49653)-dependent transcription was observed from either the TkpGL3 or ARE7-TKpGL3 (Fig. 1A). In the presence of PPARgamma 1 and RXRalpha , a 2-fold increase in transcription was observed from ARE7-TK reporter after 16 h of treatment with BRL49653. The addition of 100 ng/ml PDGF to these cells decreased both the basal and BRL49653-activated transcription from the ARE7. This suggests that at least a fraction of the activity from the ARE7-TKpGL3 plasmid in the absence of exogenously added ligand is due to the activation of the PPARgamma 1·RXRalpha heterodimer by endogenous ligands. This activity was also reduced by PDGF treatment.


Fig. 1. PDGF treatment decreases the basal and ligand-mediated transactivation of PPARgamma 1. A, NIH 3T3 cells were co-transfected with either control (TKpGL3) or PPRE-containing (ARE7/TKpGL3) reporter plasmid and pSG5 or PPARgamma 1 (PPARgamma 1/pSG5) and RXRalpha expression plasmids (RXRalpha /pSG5). pCMV beta -galactosidase plasmid was used as an internal control for the transfection efficiency. 24-h serum-starved, transfected cells were treated with Me2SO (open bars), 25 µM BRL49653 (dark hatched bars), BRL plus 100 ng/ml PDGF (light hatched bars), or PDGF alone (solid bars) for 16 h. Transactivation is reported as the ratio of luciferase activity to beta -galactosidase activity to normalize for transfection efficiency. The results are expressed as mean and S.E. of triplicate measurements. The data shown represent three independent experiments. B, co-transfection with ARE7/TKpGL3 and PPARgamma 1/pSG5 and RXRalpha /pSG5 in the presence of increasing concentrations of CA-MEK plasmid.
[View Larger Version of this Image (22K GIF file)]


Close examination of the PPARgamma amino acid sequence revealed that PPARgamma 1 contains one serine residue, Ser82, whose surrounding amino acids correspond to the consensus phosphorylation site for MAP kinase (Fig. 2A) (22). This site is absolutely conserved between human and mouse PPARgamma 1. A variation of the MAP kinase consensus site is also found in mouse PPARalpha at a similar position in the amino acid sequence. PPARdelta lacks this site altogether (Fig. 2A). Since both EGF and PDGF are known to activate MAP kinase in vivo, a CA-MEK that constitutively activates MAP kinase was co-transfected with ARE7-TKpGL3, PPARgamma 1, and RXRalpha expression plasmids. As shown in Fig. 1B, CA-MEK decreased both the basal and the ligand-dependent PPARgamma 1 transcriptional activity in a dose-dependent manner. No significant effect was seen with the TKpGL3 parental reporter construct. This suggests that the intracellular signaling pathways activated by PDGF or EGF can modulate PPARgamma 1-dependent transcriptional activity.


Fig. 2. MAP kinase phosphorylates PPARgamma 1 but not PPARdelta or mutant PPARgamma 1 (S82A) in an in vitro kinase assay. A, schematic diagram of PPARgamma structure (A/B region-putative ligand-independent transactivation domain, DBD-DNA binding domain, and LBD-ligand binding domain). A comparison of mouse and human PPAR isoforms containing the putative MAP kinase phosphorylation site (PASP) is shown. B, approximately 0.5 µg of MBP-PPARgamma 1 or 0.5 µg of MBP-PPARdelta fusion proteins were phosphorylated with active GST-MAP kinase. C, Coomassie-stained gel of the autoradiogram shown in B. D, both the wild type and the mutant PPARgamma 1 were in vitro translated using rabbit reticulocyte lysates and immunoprecipitated with a PPARgamma -specific antibody followed by in vitro kinase assay using active GST-MAP kinase. E, Western blot analysis of the in vitro translated wild type and mutant Ser82 right-arrow Ala PPARgamma 1 using the PPARgamma antibody.
[View Larger Version of this Image (28K GIF file)]


MAP Kinase Phosphorylates PPARgamma 1 but Not PPARdelta in Vitro

To determine if PPARgamma 1 can be phosphorylated by MAP kinase in vitro, partially purified MBP, MBP-PPARgamma 1, or MBP-PPARdelta fusion proteins were incubated with preactivated GST-MAP kinase and [gamma -32P]ATP under conditions that phosphorylate myelin basic protein, a known MAP kinase substrate. As shown in Fig. 2B, MAP kinase efficiently phosphorylated PPARgamma 1 but not MBP-PPARdelta or maltose-binding protein (data not shown). Coomassie staining verified that nearly equal amounts of intact proteins were loaded (Fig. 2C). To determine if Ser82 is the residue phosphorylated in vitro, a mutation was introduced into PPARgamma 1 that changed Ser82 to Ala. Both the wild type PPARgamma 1 and the mutant PPARgamma 1 (S82A) were in vitro translated and immunoprecipitated with a polyclonal anti-PPARgamma antibody. The immunoprecipitated products were used as substrates in the in vitro MAP kinase assay. Mutation at Ser82 to Ala completely abolished the MAP kinase-dependent phosphorylation of PPARgamma 1 (Fig. 2D), indicating that the Ser82 is the only amino acid in PPARgamma 1 that is phosphorylated by MAP kinase. Fig. 2E represents the PPARgamma antibody Western blot of the in vitro translated proteins and shows that both proteins were expressed in the reticulocyte extracts.

PPARgamma 1 Is Phosphorylated by EGF Treatment

To determine if PPARgamma 1 is phosphorylated by growth factor treatment, 293T cells were transfected with PPARgamma 1, serum-starved for 24 h, and incubated with [32P]orthophosphate. To maintain more physiologic conditions, no phosphatase inhibitors were added to the cells prior to lysis. Whole cell lysates were prepared after EGF treatment and immunoprecipitated with a PPARgamma -specific antibody. PPARgamma 1 was weakly phosphorylated in the absence of sera and growth factors; however, treatment with 100 ng/ml of EGF for 5 or 15 min increased PPARgamma 1 phosphorylation 1.5- and 1.8-fold, respectively (Fig. 3A, lanes 3 and 4). In 293T cells, EGF treatment stimulated MAP kinase activity as determined by Western blot analysis with the anti-active MAP kinase antibody (Fig. 3B, lanes 1 and 2). To determine if the MAP kinase signaling pathway is involved in the phosphorylation of PPARgamma 1, the transfected cells were pretreated with 40 µM PD98059, a specific MEK inhibitor (20) for 15 min prior to EGF treatment. PD98059 prevented EGF-stimulated phosphorylation of PPARgamma 1 (Fig. 3A, lane 6), suggesting that MAP kinase activation is involved in the phosphorylation of PPARgamma 1. At this concentration, PD98059 inhibited MAP kinase activation by EGF (Fig. 3B, lane 3). Interestingly, pretreatment of the cells with 25 µM BRL49653 for 15 min also reduced the EGF-dependent phosphorylation of PPARgamma 1 (Fig. 3A, lane 5) without affecting the ability of EGF to stimulate MAP kinase activity (Fig. 3B, lane 4). This implies that occupation of the ligand binding domain may inhibit the ability of MAP kinase to recognize and/or phosphorylate PPARgamma 1.


Fig. 3. EGF stimulates PPARgamma 1 phosphorylation in vivo. A, 293T cells were transfected with pSG5 (lane 1) or PPARgamma 1/pSG5 (lanes 2-6), serum-starved for 24 h, and incubated in phosphate-free media with 0.8 mCi of [32P]orthophosphate for 3 h without phosphatase inhibitors. Subsequently, the cells were pretreated with 25 µM BRL49653 (lane 5) or 40 µM PD98059 (lane 6) for 15 min followed by 100 ng/ml EGF treatment (lanes 3-6) for the indicated time. Lysates were immunoprecipitated with PPARgamma antibody and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The histogram shown below is the quantitation of PPARgamma 1 phosphorylation measured with the PhosphorImager (Molecular Dynamics). The EGF-mediated PPARgamma phosphorylation is presented as the mean ± S.E. of three independent experiments. B, EGF stimulates MAP kinase activity in 293T cells. Cells were serum-starved overnight and stimulated with EGF or pretreated with either BRL49653 or PD98059 for 15 min prior to EGF stimulation. Whole cell lysates were prepared and blotted with anti-active MAP kinase antibody, which recognizes only the phosphorylated form of MAP kinases.
[View Larger Version of this Image (24K GIF file)]


Mutation of Ser82 to Ala in PPARgamma 1 Prevents in Vivo Phosphorylation of PPARgamma 1 and Transcriptional Repression by PDGF

To determine if Ser82 is the residue-phosphorylated in vivo in response to EGF treatment, the Ser82 right-arrow Ala PPARgamma 1 mutant was introduced into 293T cells, and in vivo labeling was performed in the presence and absence of 100 ng/ml EGF (Fig. 4A). Although phosphorylation of the wild type PPARgamma 1 was enhanced by EGF treatment as before, phosphorylation of the mutant was unaffected. Similar amounts of both mutant and wild type protein were expressed in the transfected cells, as shown by Western blot analysis (Fig. 4B). Since all PPARgamma 1 phosphorylation was abolished by this mutation, this result demonstrates that the MAP kinase site at Ser82 is the only phosphorylation site on PPARgamma 1.


Fig. 4. Mutant Ser82 right-arrow Ala PPARgamma 1 is resistant to in vivo phosphorylation. A, in vivo [32P]orthophosphate labeling of cells transfected with wild type PPARgamma 1 (WT) or mutant Ser82 right-arrow Ala PPARgamma 1 in the presence and absence or 100 ng/ml EGF. B, Western blot with PPARgamma antibody showing the levels of wild type and mutant PPARgamma 1 in 293T transfected cells.
[View Larger Version of this Image (21K GIF file)]


To verify that the negative regulation of PPARgamma 1 by growth factors was dependent upon PPARgamma 1 phosphorylation, NIH 3T3 cells were co-transfected with either the wild type PPARgamma 1 or Ser82 right-arrow Ala PPARgamma 1 mutant and ARE7-TKpGL3. Transfected cells were then treated with BRL49653 in the presence or absence of PDGF. Neither basal nor BRL49653-stimulated activity was affected by the Ser82 right-arrow Ala mutant. In contrast, the activity of the Ser82 right-arrow Ala mutant PPARgamma 1 was resistant to PDGF-mediated repression (Fig. 5).


Fig. 5. Mutant Ser82 right-arrow Ala PPARgamma 1 is resistant to growth factor-mediated transcription repression. NIH 3T3 cells were co-transfected with reporter (ARE7-TKpGL3) and either PPARgamma 1 or Ser82 right-arrow Ala PPARgamma 1 expression plasmids as described in Fig. 1. Serum-starved cells were stimulated with appropriate treatments as indicated. Open bar, Me2SO; hatched bar, BRL49653; solid bar, BRL49653 plus PDGF. WT, wild type.
[View Larger Version of this Image (32K GIF file)]


Phosphorylation of PPARgamma 1 Does Not Alter Its DNA Binding Activity

To determine if phosphorylation affects PPARgamma 1 DNA binding, a mobility shift assay was performed on a labeled double-stranded oligonucleotide containing the ARE7 PPRE with both in vitro phosphorylated and unphosphorylated MBP-PPARgamma 1. As previously reported, PPARgamma 1 (Fig. 6, lane 2) alone did not bind to the ARE7 element (6). However, in the presence of RXRalpha , both the phosphorylated and unphosphorylated forms of PPARgamma 1 bound equally well to the ARE7 probe (Fig. 6, lanes 3 and 5). In addition, phosphorylation of preformed PPARgamma 1·RXRalpha heterodimer prior to mobility shift assay did not alter PPARgamma 1 DNA binding.


Fig. 6. Phosphorylation of PPARgamma 1 does not alter its DNA binding activity. A gel shift assay was performed using approximately 20 fmol of the 32P-labeled oligonucleotide probe containing the ARE7 alone (lane 1), with purified PPARgamma 1 alone (lane 2), PPARgamma 1 and RXRalpha heterodimer complex (lane 3), PPARgamma 1 and RXRalpha heterodimer complex in MAP kinase buffer without active GST-MAP kinase (lane 4), phosphorylated PPARgamma 1 and RXRalpha heterodimer complex with active GST-MAP kinase (lane 5), or phosphorylated PPARgamma 1 prior to heterodimerization with RXRalpha (lane 6).
[View Larger Version of this Image (49K GIF file)]



DISCUSSION

The complexity of gene expression requires the utilization of multiple regulatory mechanisms to control both the quantity and activity of all components of the transcription machinery including upstream enhancer proteins. In this study, we have shown that activation of the MAP kinase signaling pathway by EGF and PDGF induces the phosphorylation of PPARgamma 1 on Ser82 and that this event decreases the ability of PPARgamma 1 to activate transcription. Mutation of the phosphorylated residue (Ser82) prevents PPARgamma 1 phosphorylation as well as the growth factor-mediated repression of PPARgamma 1-dependent transcription. This phosphorylation-mediated transcriptional repression is not due to a reduced capacity of the PPARgamma 1·RXRalpha complex to heterodimerize or recognize its DNA binding site but is due to its ability to become transcriptionally activated by ligand.

The activity of several nuclear hormone receptors is regulated by phosphorylation. Okadaic acid-induced phosphorylation of the human beta 1 thyroid receptor enhances the DNA binding capacity of the protein and increases the ligand-mediated transcription (23). Phosphorylation of retinoic acid receptor alpha  and RXRalpha modulates heterodimerization of the receptors and consequently increases DNA binding activity (24). In addition, the MAP kinase-dependent phosphorylation of Ser118 on the estrogen receptor causes a 1.8-2.3-fold increase in transcriptional activation by the AF1 domain (25). Taken together, these data suggest that in general phosphorylation of nuclear receptors enhances their transcriptional activity. In contrast, our data suggest that MAP kinase phosphorylation of PPARgamma 1 negatively regulates its function.

EGF, PDGF, and fibroblast growth factor inhibit the conversion of 3T3-L1 preadipocytes to adipocytes (15, 17, 18). Moreover, primary rat adipogenic precursor cells are also inhibited from becoming adipocytes in the presence of EGF (14), and EGF-treated animals show retardation of the development of adipose tissue (16). Although the precise mechanism of this inhibition is unknown, growth arrest is required for adipogenesis. It is presumed that activation of the intracellular signaling cascades by growth factors must interfere with the activity of the factors involved in differentiation. We suggest that this interference occurs with the activation of MAP kinase. The activation of MAP kinase by EGF or PDGF induces the phosphorylation of PPARgamma 1, which negatively regulates its activity, thereby preventing the progression of adipocyte differentiation.

The one piece still missing in this puzzle is how insulin promotes adipocyte differentiation. Insulin, like other growth factors, induces MAP kinase activity in 3T3-L1 adipocytes. In fact, two recent publications suggest that insulin stimulation does induce the PPARgamma 1, PPARgamma 2, and PPARalpha phosphorylation (26, 27). However, in contrast to our data, both groups present data suggesting that the insulin induced phosphorylation enhances the transcriptional activity of the PPARs. The use of different growth factors and different cell lines may explain this discrepancy. Yet, Zhang et al. (27) reported that mutation of the phosphorylated serine does not prevent the activation of PPARgamma by insulin. In addition, expression of dominant negative MEK blocks the activity of PPARdelta that is not phosphorylated by MAP kinase. This suggests that the activation of transcription by insulin in their system occurs through a mechanism independent of the MAP kinase-induced phosphorylation of PPARgamma . Tontonoz et al. (9) have shown that deletion of the N-terminal portion of PPARgamma 2, which lacks Ser82, enhances the ability of PPARgamma to induce adipocyte differentiation. Moreover, recently Hu et al. (28) demonstrated that the ectopic expression of a mutant PPARgamma 2 (a serine to alanine mutation at position 112 in PPARgamma 2, which is equivalent to Ser82 of PPARgamma 1) enhanced sensitivity to ligand-induced adipogenesis. These results strongly support the conclusion of the present paper.

Additional studies on adipocyte function show that although insulin activates MAP kinase in 3T3-L1 adipocytes, insulin-dependent metabolic responses such as glucose uptake, glycogen synthesis, and lipogenesis are unaffected by the inhibition of MAP kinase with the MEK inhibitor PD98059 (29, 30). In addition, the MEK inhibitor does not prevent or delay 3T3-L1 adipocyte differentiation (data not shown). Since many of the effects of insulin in adipocytes do not utilize the MAP kinase signaling cascade, we suggest that other signaling events induced by insulin during adipogenesis more strongly regulate PPARgamma activity than direct phosphorylation by MAP kinase.

The molecular mechanism of inhibition of PPARgamma via phosphorylation is yet to be determined. Data presented here show that under equilibrium conditions DNA binding of recombinant PPARgamma 1·RXRalpha complexes is unaffected by phosphorylation, implying that heterodimerization of the complex is also unaffected. This suggests that transcriptional activation by PPARgamma 1 is regulated by phosphorylation. Transcriptional activation by nuclear receptors is modulated upon the association of the receptors with co-activators (31, 32) and co-repressors (21, 33-34). Because of allosteric changes in the receptor, ligand-bound receptor has a greater affinity for the co-activator than the co-repressor and thus enhances transcription (21, 33). Since pretreatment with BRL49653 decreased receptor phosphorylation in cell culture, we speculate that phosphorylation, possibly by hindering ligand binding or preventing changes in receptor conformation, plays a role in the selectivity and/or affinity of PPARgamma for the cofactors.


FOOTNOTES

*   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.
Dagger    To whom all correspondence and reprint requests should be addressed: Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI 48105. Tel.: 313-998-5840; Fax: 313-996-5668; E-mail: Tafuris{at}aa.wl.com.
1   The abbreviations used are: PPAR, peroxisomal proliferator-activated receptor; MAP, mitogen-activated protein; RXR, retinoic acid-like receptor; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; MEK, MAP kinase kinase; CA-MEK, constitutively active MEK; MBP, maltose-binding protein; TK, thymidine kinase; PPRE, peroxisome proliferator response element; ARE7, adipocyte regulatory factor response element.
2   H. Camp, A. L. Whitton, and S. T. Tafuri, submitted for publication.

ACKNOWLEDGEMENT

We thank Drs. S. Decker, R. Herrera, T. Leff, K. Pumiglia, and A. Saltiel for review of the manuscript and for helpful discussions. We thank Dr. D. Dudley for providing GST-MEK and GST-MAP kinase fusion proteins and Dr. S. Decker for CA-MEK and helpful suggestions.


REFERENCES

  1. Evans, R. M. (1988) Science 240, 889-895 [Medline] [Order article via Infotrieve]
  2. Issemann, I., and Green, S. (1990) Nature 347, 645-650 [CrossRef][Medline] [Order article via Infotrieve]
  3. Forman, B., Tontonoz, P., Chen, J., Brun, R., Spiegelman, B., and Evans, R. (1995) Cell 83, 803-812 [Medline] [Order article via Infotrieve]
  4. Kliewer, S., Lenhard, J., Willson, T., Patel, I., Morris, D., and Lehmann, J. (1995) Cell 83, 813-819 [Medline] [Order article via Infotrieve]
  5. Chawla, A., and Lazar, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1786-1790 [Abstract]
  6. Tontonoz, P., Hu, E., Graves, R., Budavari, A., and Spiegelman, B. (1994) Genes & Dev. 1224-1234
  7. Kliewer, S. A., Forman, B. M., Blumberg, B., Ong, E. S., Borgmeyer, U., Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7355-7359 [Abstract]
  8. Amri, E. Z., Bonino, F., Ailhaud, G., Abumrad, N. A., and Grimaldi, P. A. (1995) J. Biol. Chem. 270, 2367-2371 [Abstract/Free Full Text]
  9. Tontonoz, P., Hu, E., and Spiegelman, B. (1994) Cell 79, 1147-1156 [Medline] [Order article via Infotrieve]
  10. Brun, R., Tontonoz, P., Forman, B., Ellis, R., Chen, J., Evans, R., and Spiegelman, B. (1996) Genes & Dev. 10, 974-984 [Abstract]
  11. Tontonoz, P., Graves, R., Budavari, A., Erdjument-Bromage, M., Hu, E., Tempst, P., and Spiegelman, B. (1994) Nucleic Acids Res. 22, 5623-5634
  12. Zhu, Y., Qi, C., Korenberg, J., Chen, X., Noya, D., Rao, S., and Reddy, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7921-7925 [Abstract]
  13. Tafuri, S. R. (1996) Endocrinology 137, 4706-4712 [Abstract]
  14. Serrero, G. (1987) Biochem. Biophys. Res. Commun. 146, 194-202 [Medline] [Order article via Infotrieve]
  15. Navre, M., and Ringold, G. M. (1989) J. Cell Biol. 109, 1857-1863 [Abstract]
  16. Serrero, G., and Mills, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3912-3916 [Abstract]
  17. Adachi, H., Kurachi, H., Homma, H., Adachi, K., Imai, T., Morishige, K., Matxuzawa, Y., and Miyake, A. (1994) Endocrinology 135, 1824-1830 [Abstract]
  18. Brauer-Krieger, H. I., and Kather, H. (1995) Biochem. J. 307, 549-556 [Medline] [Order article via Infotrieve]
  19. Maina, C. V., Riggs, P. D., Grandea, A. G., Slatko, B. E., Moran, L. S., Tagliamonte, J. A., McReynolds, L. A., and Guan, C. D. (1988) Gene (Amst.) 74, 365-373 [CrossRef][Medline] [Order article via Infotrieve]
  20. Dudley, D., Pang, L., Decker, S., Bridges, A., and Saltiel, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689 [Abstract]
  21. Chen, J. D., and Evans, R. M. (1995) Nature 377, 454-457 [CrossRef][Medline] [Order article via Infotrieve]
  22. Gonzalez, F. A., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 22159-22163 [Abstract/Free Full Text]
  23. Lin, K., Ashizawa, K., and Cheng, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7737-7741 [Abstract]
  24. Lefebvre, M.-P., Gaub, M., Tahayato, A., Rochette-Egly, C., and Formstecher, P. (1995) J. Biol. Chem. 270, 10806-10816 [Abstract/Free Full Text]
  25. Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., Metzger, D., and Chambon, P. (1995) Science 270, 1491-1494 [Abstract]
  26. Shalev, A., Siegrist-Kaiser, C., Yen, P., Wahli, W., Burger, A., Chin, W., and Weier, C. (1996) Mol. Endocrinol. 137, 4499-4502
  27. Zhang, B., Berger, J., Zhou, G., Elbrecht, A., Biswas, S., White-Carrington, S., Szalkowski, D., and Moller, D. (1996) J. Biol. Chem. 271, 31771-31774 [Abstract/Free Full Text]
  28. Hu, E., Kim, J. B., Sarraf, P., and Spiegelman, B. M. (1996) Science 274, 2100-2103 [Abstract/Free Full Text]
  29. Lazar, D. F., Wiese, R. J., Brady, M. J., Mastick, C. C., Waters, S. B., Yamauchi, K., Pessin, J. E., Cuatrecasas, P., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 20801-20807 [Abstract/Free Full Text]
  30. Wiese, R. J., Mastic, C. C., Lazar, D. F., and Saltiel, A. R. (1995) J. Biol. Chem 270, 3442-3446 [Abstract/Free Full Text]
  31. Halachmi, S., Marden, E., Martin, G., Mackay, H., Abbondanza, C., and Brown, M. (1994) Science 264, 1455-1458 [Medline] [Order article via Infotrieve]
  32. Cavailles, V., Dauvois, S., Danielian, P. S., and Parker, M. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10009-10013 [Abstract/Free Full Text]
  33. Horlein, A. J., Naar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., Soderstrom, M., Glass, C. K., and Rosenfeld, M. G. (1995) Nature 377, 397-403 [CrossRef][Medline] [Order article via Infotrieve]
  34. Kurokawa, R., Soderstrom, M., Horlein, A., Halachmi, S., Brown, M., Rosenfeld, M. G., and Glass, C. K. (1995) Nature 377, 451-454 [CrossRef][Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.