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Originally published In Press as doi:10.1074/jbc.M103594200 on July 26, 2001

J. Biol. Chem., Vol. 276, Issue 41, 38297-38306, October 12, 2001
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Peroxisome Proliferator-activated Receptor gamma  Ligands Differentially Modulate Muscle Cell Differentiation and MyoD Gene Expression via Peroxisome Proliferator-activated Receptor gamma -dependent and -independent Pathways*

John G. HunterDagger , Mark F. van DelftDagger , Richard A. Rachubinski§, and John P. CaponeDagger ||

From the Dagger  Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada and the § Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Received for publication, April 23, 2001, and in revised form, June 20, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The effects of distinct classes of peroxisome proliferator-activated receptor gamma  (PPARgamma ) ligands on myogenesis and MyoD gene expression were examined in mouse skeletal muscle C2C12 myoblasts. Treatment of C2C12 cells with the PPARgamma ligand, 15-deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2), repressed morphologically defined myogenesis and reduced endogenous mRNA levels of the myogenic differentiation markers MyoD, myogenin, and alpha -actin. In contrast, two synthetic PPARgamma ligands, L-805645 and ciglitazone, exhibited no effects. In transient transfection assays, 15d-PGJ2 specifically inhibited the expression of a MyoD promoter-luciferase reporter gene (MyoDLuc) in a cell type- and promoter-specific manner, indicating that 15d-PGJ2 functions in part by repressing MyoD gene transcription. The inhibition of MyoD gene expression by 15d-PGJ2 is mediated by the distal region of the MyoD gene promoter. PPARgamma on its own also inhibited MyoDLuc expression and further augmented the 15d-PGJ2 response. In contrast, L-805645 and ciglitazone did not inhibit MyoDLuc expression on their own but did so in the presence of ectopically expressed PPARgamma . Interestingly, a transdominant inhibitor of PPARgamma (hPPARgamma 2Delta 500) had no effect on the 15d-PGJ2-dependent repression of MyoDLuc expression but overcame L-805645/PPARgamma -dependent repression. Finally, saturating concentrations of L-805645, which did not affect myogenesis, failed to ablate 15d-PGJ2-mediated repression of the myogenic program. Thus, distinct PPARgamma ligands may repress MyoD gene expression through PPARgamma -dependent and -independent pathways, and 15d-PGJ2 can inhibit the myogenic program independent of its cognate receptor, PPARgamma .


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferator-activated receptors (PPARs)1 are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. PPARs regulate a large number of genes that are essential for lipid and metabolic homeostasis, energy balance, and cellular differentiation (1-4). The three PPAR subtypes identified, PPARalpha , PPARdelta (NUC1, FAAR, or PPARbeta ), and PPARgamma (gamma 1, gamma 2, and gamma 3 isoforms) exhibit unique, as well as overlapping, tissue expression and ligand activation profiles and regulate distinct physiological processes (4-11).

PPARgamma is expressed predominantly in adipose tissue, and its central role as a key adipogenic determination factor is well established (12-14). Two classes of synthetic insulin-sensitizing compounds, the thiazolidinediones (TZDs) and phenoxyacetic acids, and the prostaglandin metabolite, 15-deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2) serve as ligands of PPARgamma and can promote the differentiation of cells into adipocytes (11-13, 15, 16). PPARgamma is also expressed in the skeletal muscle of rodents and humans, suggesting that it may play a role in myogenesis and/or muscle cell function (8, 9, 17-19). Adipocytes and myocytes originate from the same mesodermal pluripotent precursor. Like adipogenesis, myogenesis is a multistep process subject to regulation by exogenous and endogenous signals, such as hormones and growth factors (20). This process is orchestrated principally by the myogenic basic helix-loop-helix family of transcription factors that includes MyoD, Myf-5, myogenin, and MRF4 (20-25). Myogenic regulatory factors function in a hierarchical manner and are differentially required for the establishment and maintenance of the myogenic phenotype in proliferating myoblasts and for the activation of muscle-specific gene expression in terminally differentiated muscle cells.

A physiological role for PPARgamma in muscle is suggested by the fact that insulin sensitizers such as the TZDs act in muscle, the main site for insulin-stimulated glucose uptake (26, 27). Also, TZDs have been shown to enhance the expression of adipogenic and lipid metabolic genes in skeletal muscle of type II diabetic patients (18, 19, 28, 29). Moreover, certain PPARgamma ligands, including TZDs and fatty acid derivatives, have been shown to inhibit myogenesis in cultured myogenic cell lines and, under certain conditions, to induce an adipose-like phenotype in these cells (30-32). Interestingly, forced expression of PPARgamma in myoblasts inhibits muscle cell differentiation and, under appropriate conditions, causes their transdifferentiation into mature adipocytes, suggesting that PPARgamma can induce a developmental switch between two specialized cell types (30). The physiological relevance of these findings remains to be established; however, in conditions such as Duchenne muscular dystrophy, mitochondrial myopathy, obesity, and diabetes, fat cell accumulation in muscle tissue is observed. Also, ablation of MyoD and Myf-5 genes in mice results in the loss of muscle and its replacement by fat tissue (33, 34).

PPARgamma ligands such as the TZDs and 15d-PGJ2 have been shown to elicit a diverse range of shared and distinct biological effects, some of which appear to be mediated through pathways that are independent of PPARgamma (35-39). To further explore the role of PPARgamma and its ligands on myogenesis and muscle cell-specific gene expression, we examined the effects of three different classes of PPARgamma ligands, the TZD ciglitazone, the phenoxyacetic acid L-805645, and the prostaglandin derivative 15d-PGJ2, on the differentiation of mouse skeletal muscle C2C12 cells and on the expression of a reporter gene linked to the promoter/enhancer regulatory region of the MyoD gene (MyoDLuc). We show here that 15d-PGJ2 specifically represses the morphological and biochemical differentiation of C2C12 cells. In contrast, L-805645 and ciglitazone exhibited no effects. Moreover, saturating levels of L-805645 did not ablate the 15d-PGJ2-mediated effects on differentiation. In transient transfection assays, 15d-PGJ2 specifically inhibited expression by MyoDLuc and did so independently of exogenously expressed PPARgamma , whereas repression of MyoDLuc by L805645 or ciglitazone was observed only in the presence of exogenously expressed PPARgamma . These findings indicate that distinct PPARgamma ligands can inhibit MyoD gene expression via PPARgamma -dependent and PPARgamma -independent pathways and that the ability of 15d-PGJ2 to inhibit myogenesis is independent of its cognate receptor, PPARgamma .

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- Mammalian expression plasmids for mouse PPARgamma 2 (mPPARgamma 2) and human PPARgamma 2 (hPPARgamma 2) and PPARgamma 1, mPPARalpha , human 9-cis-retinoic acid receptor (RXRalpha ), and hPPARgamma 2Delta 500 and the reporter plasmid pAOx(X2)GL, which contains two tandem copies of the peroxisome proliferator response element (PPRE) from the promoter of the rat acyl-CoA oxidase (AOx) gene, have been described (11, 40, 41).

MyoDLuc is a luciferase reporter gene construct that contains a 2.7-kbp fragment of the human MyoD gene, including the proximal promoter and the transcription start site (-2,500 nucleotides to +198 nucleotides), linked to the 4-kbp far upstream enhancer region of the human MyoD gene promoter (42, 43). This plasmid was constructed by subcloning a 6.5-kbp XhoI-SalI fragment from F3/-2.5 MyoDCAT (42, 44) into the HindIII-XhoI sites of the pGL2 luciferase reporter plasmid (Promega, Madison, WI) after first filling the ends with the Klenow fragment of DNA polymerase I. The cloning strategy removes the minimal SV40 promoter present in pGL2. A reporter construct lacking the 4-kbp distal enhancer region of the MyoD gene promoter was generated by digestion of MyoDLuc with EcoRI. A reporter construct containing the distal enhancer region of the MyoD gene promoter fused to the SV40 promoter was generated by insertion of a 4-kbp EcoRI fragment from MyoDLuc, made blunt with the Klenow fragment of DNA polymerase I, into the SmaI site of pGL2. A reporter construct containing the 258-bp MyoD core enhancer (44) was constructed by excising a HindIII-XbaI fragment from the plasmid meiCAT (a gift of Michael Rudnicki, McMaster University, Hamilton, Ontario, Canada), filling in the ends of the fragment with the Klenow fragment of DNA polymerase I and inserting the blunt fragment into the SmaI site of pGL2. MyogLuc contains 200 bp of the proximal promoter, including the transcription start site and enhancer, of the human myogenin gene (21) cloned into the SmaI site of the modified pGL2 vector lacking its SV40 promoter.

Cell Culture and Differentiation-- Mouse skeletal muscle C2C12 myoblasts and C3H10T1/2 mouse embryo fibroblasts were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. For analysis of differentiation, C2C12 cells were plated at a density of 4 × 105 cells/10-cm dish and switched to differentiation medium (Dulbecco's modified Eagle's medium plus 10% horse serum) when the cells reached ~90% confluence (day 0). The medium was changed at days 1 and 3. Cells were supplemented with 10 µM 15d-PGJ2 (Cayman Chemicals, Ann Arbor, MI), 10 µM L-805645 2-(2-(4-phenoxy-2-propylphenoxy)-indole-5-acetic acid) (a gift of Joel Berger; Merck) or 50 µM ciglitazone (BIOMOL, Plymouth Meeting, PA) on days 0, 1, and 2. Control transfections were performed with the equivalent volume of appropriate vehicle: ethanol for 15d-PGJ2 and ciglitazone and dimethyl sulfoxide for L-805645.

For morphological analysis, cells were washed with phosphate-buffered saline (PBS) and fixed with cold 90% methanol for 5 min at day 0 (90% confluence) and at days 2 and 4 postconfluence. Fixed cells were rinsed with PBS and stored in PBS plus 0.01% Thimersol (Sigma). Immunostaining was performed with MF-20, a mouse monoclonal anti-myosin heavy chain antibody (45). Briefly, cells were incubated with MF-20 in 5% nonfat dry milk/PBS for 60 min at room temperature. Plates were washed three times with PBS and incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:1000 dilution) in PBS plus 5% nonfat dry milk with gentle shaking for 60 min at room temperature. Plates were washed three times with PBS and counterstained with 1.7 mM diaminobenzidine, 50 mM Tris-HCl, pH 7.6, 0.06% hydrogen peroxide for 15 min. The reaction was stopped with water, and cells were photographed. Cell nuclei were visualized by staining with hematoxylin (Sigma) for 10 min.

Northern Blot Analysis-- Total RNA was isolated from C2C12 cells using a commercially available kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Ten µg of RNA per sample was separated by electrophoresis on a 1% agarose, 7% formaldehyde gel in borate buffer (0.02 M borate, pH 8.3, 0.2 mM EDTA). RNA was transferred to a nylon membrane and hybridized to radiolabeled probes essentially as described (46). The blot was subjected to autoradiography, and radiolabeled bands were quantitated by PhosphorImager analysis and normalized to the signal from phosphoglycerate kinase (PGK) RNA, which was used as an internal standard for RNA loading. The same blot was used, after extensive washing, for hybridization to each of the probes. Probes were prepared by random primer labeling of plasmid vectors or purified restriction endonuclease fragments from cDNAs for alpha -, beta -, and gamma -actins, PGK, human MyoD, human myogenin (a gift from Michael Rudnicki, McMaster University, Hamilton, Ontario, Canada), and mouse PPARgamma 2, using a commercially available kit (Amersham Pharmacia Biotech) and [alpha -32P]dATP. Probes were purified by gel filtration on Sephadex G-50 prior to use.

Cell Transfection-- C2C12, C3H10T1/2, and BSC40 cells, as indicated in the figure legends, were transfected using the calcium phosphate method as described (40). Briefly, cells (2 × 105 cells/35-mm dish) were incubated in medium lacking phenol red and containing 10% charcoal-stripped fetal bovine serum and transfected with 1.25 µg of luciferase reporter plasmid, 0.5 µg of the indicated receptor expression plasmid, and 0.5 µg of pCH110 (Amersham Pharmacia Biotech), a beta -galactosidase expression vector used as an internal reference. Plasmid and promoter dosage in each case was normalized by the addition of the appropriate amount of corresponding empty vector. The PPARalpha ligand, Wy-14,643, was added to a final concentration of 100 µM, while the PPARgamma ligands were added to a final concentration of 10 µM for 15d-PGJ2 and for L-805645 and of 50 µM for ciglitazone. Luciferase activity was measured 48 h posttransfection as described (5, 40). All transfections were carried out in duplicate at least three independent times or in triplicate at least two independent times.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PPARgamma Ligands Differentially Repress the Myogenesis of C2C12 Cells-- C2C12 cells are a well characterized cell culture model of muscle differentiation. Under conditions permissive for differentiation, such as low serum concentration or confluence, C2C12 myoblasts undergo differentiation to form myocytes, subsequently fusing into multinucleated myotubes. Certain PPARgamma ligands have been shown to inhibit the myogenic program in both C2C12 and G8 myoblast cells (30-32). Different PPARgamma ligands, however, are known to have distinct biological effects and to function, in some cases, via PPAR-independent pathways. To explore the generality of action of PPARgamma ligands on muscle cell differentiation, three distinct classes of PPARgamma ligands were examined for their effects on the myogenesis of C2C12 cells: 15-deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2), a natural cyclopentenone prostanoid (13, 15); L-805645, a phenoxyacetic acid (11); and ciglitazone, a TZD (16). The last two compounds belong to distinct classes of insulin sensitizers.

C2C12 cells were incubated under conditions permissive for differentiation in the presence of 15d-PGJ2 (10 µM), L-805645 (10 µM), or ciglitazone (50 µM). Myogenesis was monitored at different times by immunostaining with an antibody, MF-20, directed against the myosin heavy chain (45). Extensive myotube formation was observed in untreated cells at days 2 and 4 postconfluence (Fig. 1, panels A), as expected under these conditions. In contrast, 15d-PGJ2 inhibited the ability of these cells to form myocytes and myotubes (Fig. 1, compare panels B with panels A). Conversely, L-805645 and ciglitazone had no observable effect on the differentiation process on either day 2 or day 4 postconfluence (Fig. 1, compare panels C or panels D, respectively, with panels A). To quantify the effect of 15d-PGJ2 on myogenesis, the fusion index was determined. The fusion index is the percentage of nuclei incorporated into myosin heavy chain-positive (MHC+) cells versus the total number of nuclei (21, 47). 15d-PGJ2 treatment decreased the fusion index, and thus the percentage of nuclei associated with myocytes and myotubes, ~4-fold, whereas L-805645 and ciglitazone exhibited no effect (data not shown). Thus, 15d-PGJ2, but not the other two PPARgamma -specific ligands, represses the myogenesis of C2C12 cells.


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  		Fig. 1.   15d-PGJ2, but not L-805645 or ciglitazone, inhibits the morphological differentiation of C2C12 cells. C2C12 cells were grown to 90% confluence and switched to differentiation medium at day 0, with subsequent medium changes at days 1 and 3. 15d-PGJ2 (10 µM final concentration) (panels B), L-805645 (10 µM) (panels C), ciglitazone (50 µM) (panels D), or vehicle (panels A), as indicated, was added at days 0, 1, and 2. Cells were fixed on day 2 or on day 4, as indicated, and immunostained with MF-20, an antibody reactive to myosin heavy chain, to visualize myocytes.

15d-PGJ2 Represses the Expression of mRNAs Encoding Myogenic Factors-- mRNA profiles were examined by Northern analysis to determine if 15d-PGJ2-mediated inhibition of muscle cell differentiation is associated with reduced expression of genes encoding myogenic transcription factors. C2C12 cells were incubated under the different conditions reported above, and mRNA levels for the myogenic markers MyoD, myogenin, and skeletal alpha -actin were determined for cells incubated in the absence or presence of 15d-PGJ2, L-805645, or ciglitazone at day 0 (90% confluence) and at 24-h intervals thereafter. MyoD is present both in myoblasts and myotubes, myogenin is a marker for C2C12 commitment to the differentiation pathway, and alpha -actin is a marker of terminal differentiation (25). Radiolabeled bands were quantified and standardized to the signal for PGK mRNA. Fig. 2, A-C, shows the results of Northern analysis with RNA isolated from 15d-PGJ2-, L-805645-, and ciglitazone-treated cells, respectively. The levels of MyoD mRNA remained constant relative to the signal for PGK mRNA in cells treated with vehicle during the time course of 3 days (compare day 0 with days 1, 2, and 3), while the levels of myogenin and alpha -actin mRNA increased ~30-fold, as has been previously reported (25, 47). An ~50% reduction in the steady state levels of MyoD mRNA was observed at each time interval in the presence of 15d-PGJ2 (Fig. 2A). A 3-4-fold reduction in the levels of myogenin and alpha -actin mRNAs was observed in the presence of 15d-PGJ2 at day 1 (Fig. 2A). The decrease of myogenin and alpha -actin mRNA was slightly less pronounced at days 2 and 3, suggesting that some differentiation of cells had occurred. In contrast to the results observed with 15d-PGJ2, treatment of cells with L-805645 or ciglitazone did not reduce the levels of MyoD, myogenin, or alpha -actin mRNAs relative to the levels found in untreated cells (Fig. 2, B and C). Thus, 15d-PGJ2 selectively repressed the steady state levels of mRNAs encoding different myogenic factors, consistent with the morphological observations reported above.


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  		Fig. 2.   15d-PGJ2 specifically inhibits expression of genes for myogenic differentiation markers in C2C12 cells. C2C12 cells were treated as described in the legend to Fig. 1. Total RNA was isolated from cells treated with vehicle (-) or with 15d-PGJ2 (+) (A), L-806454 (+) (B), or ciglitazone (+) (C) at the days indicated postconfluence and subjected to Northern blot analysis with probes specific for MyoD, myogenin, mPPARgamma 2, and mixed probes for alpha - and beta -actin mRNAs, as indicated. Hybridization to phosphoglycerate kinase (PGK) mRNA was used as an internal loading standard. RNA levels were quantitated by PhosphorImager analysis and normalized to the PGK standard.

We also monitored the expression of PPARgamma mRNA during C2C12 differentiation. Low levels of PPARgamma mRNA were detected in C2C12 myoblast cells, similar to what was previously found for G8 myoblasts (30). There was a slight decrease in PPARgamma mRNA levels during the 3-day time course of differentiation in the untreated cells (Fig. 2, A-C). Treatment with 15d-PGJ2 or ciglitazone led to a modest (2-fold), but reproducible, elevation of PPARgamma mRNA levels at each time interval as compared with the levels of PPARgamma mRNA in C2C12 myoblasts at day 0 (Fig. 2, A and C). Treatment of cells with L-805645 showed no effect on steady-state levels of PPARgamma .

15d-PGJ2 Represses the Activity of the MyoD Gene Promoter-- As reported above, 15d-PGJ2, but not the other PPARgamma ligands tested, inhibited the myogenic program concomitant with a reduction in the steady state levels of mRNAs encoding muscle cell-specific transcription factors. To determine if this regulation occurs at the level of transcription, we carried out transient transfection studies with a luciferase reporter gene linked to the promoter/regulatory region of the MyoD gene (MyoDLuc). MyoD is a central regulator in the determination of somatic cells into the myogenic program. Attenuation of its gene expression prevents myogenesis in C2C12 cells, while overexpression of the MyoD gene leads to the conversion of many cell types to muscle cells (20, 25, 48).

MyoDLuc was transfected into C2C12 cells in the absence or presence of PPARgamma ligands. As shown in Fig. 3A, MyoDLuc activity was reduced by ~50% in C2C12 cells in the presence of 15d-PGJ2, whereas the addition of L-805645 and ciglitazone showed no effect on MyoDLuc expression. These findings are consistent with the findings of Northern analysis presented in Fig. 2. The addition of Wy-14,643, a peroxisome proliferator and selective activator of PPARalpha , had no effect on expression by MyoDLuc. 15d-PGJ2-mediated repression of MyoDLuc expression was specific, since the expression of an SV40 promoter/enhancer reporter gene construct, pSV2Luc, and of a PPRE-containing reporter gene, pAOx(X2)GL, were unaffected by the presence of 15d-PGJ2 (Fig. 3A). The lack of responsiveness of pAOx(X2)GL to PPAR ligands suggests that endogenous levels of PPARalpha and/or PPARgamma in C2C12 cells are insufficient to activate this reporter gene (see below).


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  		Fig. 3.   The effects of ligands and the presence of PPARgamma on the expression of a reporter gene linked to the MyoD promoter. A, 15d-PGJ2 inhibits expression by MyoDLuc. C2C12 cells were transfected with the indicated luciferase reporter genes, as described under "Experimental Procedures." Where indicated, 15d-PGJ2 (10 µM final concentration), L-805645 (10 µM), or ciglitazone (50 µM) was added to cells 24 h posttransfection. Wy-14,643 (100 µM) was added at the time of transfection and 24 h later. Luciferase activity was measured 48 h posttransfection. B, ectopic expression of PPARgamma inhibits MyoDLuc expression and mediates repression by L-805645 and ciglitazone. C2C12 cells were transfected with the MyoDLuc reporter gene, as above, and with expression vectors for mPPARgamma 2 and/or human RXRalpha in the absence or presence of 15d-PGJ2, L-805645, or ciglitazone, as indicated. C, PPARgamma ligands activate expression from the acyl-CoA oxidase PPRE in C2C12 cells in a PPARgamma -dependent manner. C2C12 cells were transfected as above but with pAOx(X2)GL, a PPAR-responsive reporter gene containing the PPRE of the rat acyl-CoA oxidase gene. Values shown in all panels represent the average luciferase activity ± S.E.M. of at least three independent experiments carried out in duplicate and normalized to the values for the respective untreated cells transfected with reporter gene alone, which was taken as 1 in each case.

To determine the effects of exogenously expressed PPARgamma on MyoDLuc expression, an expression vector containing mPPARgamma 2 was cotransfected with MyoDLuc either in the absence or presence of ligands, and reporter gene activity was assessed. Expression by MyoDLuc was repressed ~2-fold by mPPARgamma 2 on its own, while mPPARgamma 2 and 15d-PGJ2 together mediated an approximately 5-fold repression relative to the value of the control in the absence of any addition (Fig. 3B). However, PPARgamma did not significantly affect the ratio of repression between 15d-PGJ2-treated and -untreated cells. Exogenous expression of the obligate PPAR heterodimerization partner, RXRalpha , had no effect on MyoDLuc expression; nor did it augment repression by PPARgamma and/or 15d-PGJ2. C2C12 cells contain endogenous RXRalpha ; therefore, the RXRalpha expression vector was omitted in subsequent experiments.

MyoDLuc expression was inhibited 5- and 3-fold relative to control values by the addition of L-805645 and ciglitazone, respectively; however, these reductions were entirely dependent on the exogenous expression of mPPARgamma 2 (Fig. 3B). Repression mediated by 15d-PGJ2, L-805645, and ciglitazone was specific, since the expression of the SV40 promoter/enhancer-containing reporter, pSV2Luc, was unaffected by the addition of these compounds in the presence of exogenously expressed mPPARgamma 2 (data not shown).

Interestingly, activation of pAOx(X2)GL by 15d-PGJ2, L-805645, and ciglitazone was also dependent on the exogenous expression of mPPARgamma 2 (Fig. 3C), demonstrating that these compounds effectively transduce a positive PPARgamma -mediated signal under the experimental conditions and concentrations of activator used. Moreover, these findings indicate that the endogenous levels of PPARgamma in C2C12 cells are insufficient to support the activation of a PPRE-dependent promoter. These results suggest that two pathways may act in 15d-PGJ2-mediated repression of the MyoD gene promoter, with one being dependent on, and the other being independent of, PPARgamma (see below).

To further explore the nature and specificity of PPARgamma - and/or PGJ2-mediated repression of MyoDLuc expression, we carried out transient transfection studies in C3H10T1/2 and BSC40 cells. C3H10T1/2 cells are an embryonic mouse fibroblast cell line that does not synthesize endogenous MyoD but retains the capacity to activate endogenous MyoD and enter the myogenic pathway under appropriate conditions. BSC40 cells are a monkey kidney cell line that lacks myogenic potential. Although MyoD is expressed specifically in cells of muscle cell lineage in vivo, in culture model systems MyoD exhibits ubiquitous expression, possibly due to epigenetic regulatory mechanisms (48). In C3H10T1/2 cells, MyoDLuc expression was repressed by the addition of 15d-PGJ2, both in the absence or presence of mPPARgamma 2 and in a fashion similar to that seen in C2C12 cells (Fig. 4A). Interestingly, expression by MyoDLuc was unaffected by the presence of 15d-PGJ2 and/or mPPARgamma 2 in BSC40 cells (Fig. 4A). BSC40 cells are responsive to PPARs and their activators, as determined by activation of pAOx(X2)GL (data not shown) (40). MyoDLuc expression in C2C12 cells was unaffected by exogenous expression of PPARgamma , in either the absence or presence of the PPARgamma -specific ligand, Wy-14,643 (Fig. 4C). As a further test of specificity, we carried out transfection studies of C2C12 cells with MyogLuc, a luciferase reporter gene construct linked to the myogenin gene promoter (21). MyogLuc expression was unaffected by the presence of mPPARgamma 2 and/or 15d-PGJ2 (Fig. 4B).


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  		Fig. 4.   Repression of expression by MyoDLuc is ligand-, cell type-, promoter-, and PPAR subtype-specific. A, PPARgamma - and 15d-PGJ2-mediated repression of expression by MyoDLuc is cell type-specific. C2C12, C3H10T1/2, and BSC40 cells were transfected with MyoDLuc and with a mPPARgamma 2 expression vector in the absence or presence of 15d-PGJ2, as indicated. B, PPARgamma and/or 15d-PGJ2 do not affect the activity of the myogenin gene promoter. A luciferase reporter gene linked to the myogenin gene promoter (MyogLuc) was transfected into C2C12 cells in the absence or presence of an expression vector for mPPARgamma 2 and 15d-PGJ2, as indicated. C, PPARalpha does not affect the expression by MyoDLuc. C2C12 cells were transfected with MyoDLuc as above, but in the absence or the presence of an expression vector for mouse PPARalpha and the PPARalpha activator, Wy-14,643, as indicated. All values reported above are the averages of three transfections carried out in duplicate ± S.E.M. and were normalized to untreated cells transfected with the respective reporter gene construct alone.

The transfection experiments reported above were carried out using mPPARgamma 2; however, the predominant PPARgamma isoform in skeletal muscle cells is PPARgamma 1 (17-19, 26, 27). We therefore compared the effects on expression by MyoDLuc in the presence of both human and mouse PPARgamma 1 and gamma 2 isoforms in transient transfections of C2C12 cells. We found neither qualitative nor quantitative differences in the repression profile of MyoDLuc expression as mediated by PPARgamma 1 and gamma 2, either in the absence or presence of 15d-PGJ2, suggesting an equivalence of these receptors in the assays used (data not shown).

The foregoing results show that repression of expression by MyoDLuc by 15d-PGJ2 and/or PPARgamma is specific to this promoter, rather than the result of a generalized repressive or cytotoxic effect, and that repression is dependent on PPAR subtype, ligand, and cell type.

Repression of MyoDLuc Expression Occurs via PPARgamma -independent and PPARgamma -dependent Pathways-- The above results indicate that PPARgamma and 15d-PGJ2 can independently repress expression by MyoDLuc. To investigate this further, we carried out dose-response experiments with both MyoDLuc and pAOx(X2)GL in C2C12 cells. In the absence of exogenously expressed mPPARgamma 2, titration of 15d-PGJ2 led to a dose-dependent repression of MyoDLuc activity with an IC50 of ~5 µM (Fig. 5A). The addition of a constant amount of mPPARgamma 2 shifted the IC50 value for 15d-PGJ2 to ~1.1 µM (Fig. 5A). This correlates with the EC50 of activation of 1 µM of 15d-PGJ2 on pAOx(X2)GL in the presence of exogenously expressed mPPARgamma 2 (Fig. 5B), consistent with previous observations (13). L-805645 exhibited a similar activation and inhibition profile in the presence of mPPARgamma 2. The IC50 of repression of MyoDLuc expression for L-805645 was ~0.45 µM (Fig. 5A), while the EC50 of activation of pAOx(X2)GL was ~0.5 µM (Fig. 5B). Ciglitazone was not as efficacious an activator of PPARgamma , as has been observed by others (16, 49), and it did not reach maximal inhibition of MyoDLuc expression, as evidenced by only a 2-fold repression of expression achieved by 100 µM ciglitazone compared with a 3-fold repression mediated by L-805645 and 15d-PGJ2 (Fig. 5A). However, ciglitazone-mediated activation and repression of pAOx(X2)GL and MyoDLuc expression, respectively, occurred within the same range of concentration.


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  		Fig. 5.   15d-PGJ2 inhibition of MyoDLuc expression is dose-dependent and is enhanced in the presence of PPARgamma . Transfections of C2C12 cells with MyoDLuc or pAOx(X2)GL were carried out as above with varying concentrations of 15d-PGJ2, L-805465, or ciglitazone, in the absence or presence of a constant amount of cotransfected mPPARgamma 2 expression vector, as indicated. Values represent the averages from two separate experiments ± S.E.M. carried out in triplicate and normalized to untreated cells or cells transfected with PPARgamma 2 expression vector in the absence of drug. In the absence of cotransfected mPPARgamma 2 expression vector, L-805645 or ciglitazone had no effect on reporter gene activity at any of the concentrations of drug tested.

The close correspondence between half-maximal activation and inhibition concentrations for both 15d-PGJ2 and L-805645 in the presence of exogenously expressed PPARgamma suggests that these compounds exert their effects through a PPARgamma -dependent pathway. In comparison with 15d-PGJ2, L-805645 is slightly more potent at either activating pAOx(X2)GL or inhibiting MyoDLuc, (Fig. 5, compare A with B). The fact that L-805645 is unable to repress MyoDLuc expression in the absence of exogenously expressed PPARgamma suggests that a PPARgamma -independent pathway exists for the observed 15d-PGJ2-mediated repression.

To address the above question more directly, we made use of a transdominant human PPARgamma 2 receptor, hPPARgamma 2Delta 500, that lacks its five carboxyl-terminal amino acids (41). This mutant receptor is able to interact with RXR and bind to a PPRE but cannot interact with ligand or transactivate a PPAR-responsive element (41). Consistent with these findings, transient transfection of C2C12 cells with hPPARgamma 2Delta 500 repressed the basal level activity of pAOx(X2)GL 2-3-fold (Fig. 6B). Similarly, hPPARgamma 2-receptor- and ligand-mediated transactivation of pAOx(X2)GL was inhibited in a dose-dependent manner by increasing amounts of hPPARgamma 2Delta 500. It should be noted that in order to accommodate a 20-fold molar excess of hPPARgamma 2Delta 500 expression vector, 10% of the usually transfected amount of hPPARgamma 2 expression vector was used, and, accordingly, the absolute -fold induction of activity is correspondingly reduced. Thus, hPPARgamma 2Delta 500 can effectively function as a transdominant inhibitor and repress PPARgamma -mediated transactivation in C2C12 cells.


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  		Fig. 6.   A transactivation-defective mutant of PPARgamma does not inhibit MyoDLuc expression and is unable to overcome the repressive effects of 15d-PGJ2. C2C12 cells were transfected with MyoDLuc (A) or pAOx(X2)GL (B), together with vectors expressing hPPARgamma 2 or a transdominant derivative, hPPARgamma 2 Delta 500, as shown. The hPPARgamma 2 expression vector was added at 10% the usual concentration (0.05 µg) in order to accommodate a 20-fold molar excess of hPPARgamma 2 Delta 500 expression vector. Values shown are the average ± S.E.M. of four independent transfections carried out in triplicate and normalized to untreated cells transfected with the respective reporter gene.

15d-PGJ2-dependent repression of expression by MyoDLuc in C2C12 cells was unaffected by the expression of hPPARgamma 2Delta 500 (Fig. 6A). The fact that 15d-PGJ2 still represses MyoDLuc expression in the presence of hPPARgamma 2Delta 500 but is abrogated in its ability to transactivate pAOx(X2)GL (Fig. 6B) is consistent with the existence of a PPARgamma -independent pathway for 15d-PGJ2 inhibition of expression by MyoDLuc.

Human PPARgamma 2 can also function to repress MyoDLuc expression in the absence of ligand when supplied exogenously to C2C12 cells, as was also observed with mPPARgamma 2 (Fig. 6A). Repression was not observed with hPPARgamma 2Delta 500, suggesting that repression requires the integrity of the ligand-dependent transactivation domain (Fig. 6A). To further demonstrate a role for PPARgamma in repressing MyoDLuc expression, we examined the effect of hPPARgamma 2Delta 500 on PPARgamma -dependent, L-805645-mediated repression. A 60% repression in MyoDLuc activity was observed when 10% of the usual amount of hPPARgamma 2 expression vector was transfected into C2C12 cells in the presence of L-805645. Increasing concentrations of hPPARgamma 2Delta 500 expression vector abrogated the hPPARgamma 2/L-805645-mediated repression in a dose-dependent manner. Thus, repression mediated by L-805645 is dependent upon PPARgamma . These results support the existence of both PPARgamma -dependent and PPARgamma -independent pathways in PPARgamma activator-mediated repression of MyoDLuc expression.

15d-PGJ2 Does Not Require Activation of PPARgamma for Repression of Myogenesis-- Hu et al. (30) have previously shown that inhibition of myogenesis by the PPARgamma activator, eicosatetraynoic acid, requires ectopic expression of PPARgamma . Our data indicating that 15d-PGJ2 can inhibit MyoDLuc expression through pathways that are both dependent and independent of exogenously expressed PPARgamma leaves open the question of the role of PPARgamma in mediating the 15d-PGJ2-dependent repression of myogenesis in C2C12 cells. To address this question, we investigated the repression of myogenesis by 15d-PGJ2 in the manner reported in Fig. 1, but now in the absence or presence of a saturating concentration of L-805645, a compound that is unable to repress myogenesis (see above). C2C12 cells were fixed and immunostained with MF-20, and the fusion index was quantified 4 days postconfluence. In the absence of 15d-PGJ2, ~45% of nuclei were incorporated into myocytes (Fig. 7). The addition of 15d-PGJ2 led to a dose-dependent inhibition of myogenesis, with maximum inhibition observed at 10 µM. Under these conditions, only ~5% of the nuclei were associated with myocytes. The addition of 50 µM L-805645 did not alter the dose-response curve of 15d-PGJ2; nor did it alter the maximum repression observed (Fig. 7). Taken together, these results indicate that 15d-PGJ2 can repress the myogenic program in C2C12 cells independently of endogenous PPARgamma .


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  		Fig. 7.   Inhibition of muscle cell differentiation by 15d-PGJ2 is not ablated by addition of L-805645. C2C12 cells were cultured in various concentrations of 15d-PGJ2 as described in the legend to Fig. 1 and in the absence or presence of a saturating concentration of L-805645 (50 µM). The extent of differentiation was determined by the fusion index ((number of nuclei within MHC+ cells/total number of nuclei) × 100). Quantitation of nuclei was performed at day 2 postconfluence. The results represent the average of 10 random fields observed at × 200 magnification from two independent experiments and normalized to the value obtained with untreated cells, which was taken as 1. Myocytes were stained with MF-20 antibodies, and nuclei were stained with hematoxylin.

15d-PGJ2 Mediates Inhibition of MyoD Gene Expression through the Distal Region of the MyoD Gene Promoter-- We generated a series of reporter constructs containing fragments of the MyoD gene promoter and tested their transcriptional activity by transient transfection assay to begin to unravel the mechanisms by which 15d-PGJ2 inhibits MyoD gene expression and to determine whether inhibition of MyoD expression by 15d-PGJ2 and PPARgamma can be uncoupled at the level of the MyoD promoter. The various MyoD reporter constructs consisting of the luciferase gene linked to the 2.7-kbp proximal region, the 4-kbp distal region, and the 258-bp core enhancer element of the MyoD gene promoter are shown in Fig. 8A. The 258-bp core enhancer element is located ~20 kbp upstream of the MyoD gene transcription start site and directs cell type and spatio-temporal expression of the MyoD gene (49).


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  		Fig. 8.   15d-PGJ2 inhibition of MyoD gene expression is mediated via the distal region of the MyoD promoter and is independent of coexpressed PPARgamma 2. A, schematic diagram of reporter gene constructs. The MyoDLuc reporter construct, which is shown for comparison, contains the 2.7-kbp proximal promoter element of the human MyoD gene promoter linked to a 4-kbp distal enhancer element of the human MyoD gene promoter (see "Experimental Procedures"). The proximal reporter construct lacks the 4-kbp distal enhancer element. The distal reporter construct contains the distal enhancer element fused to the SV40 promoter element of the plasmid pGL2. The core reporter construct contains a 258-bp control element, which is found within the distal enhancer element and is fused to the SV40 promoter element of the plasmid pGL2. B, C2C12 cells were transfected with the indicated luciferase reporter constructs in the absence or presence of mPPARgamma 2 and/or 15d-PGJ2 (10 µM final concentration), as indicated. Values shown are the average ± S.E.M. of at least three transfections carried out in duplicate and normalized to untreated cells transfected with the respective reporter gene alone, which was taken as 1 in each case.

The addition of 15d-PGJ2 reduced by ~60% the activity of the reporter construct containing the distal region of the MyoD gene promoter (Fig. 8B), similar to what was observed with the MyoDLuc reporter construct (see Fig. 3). However, the addition of 15d-PGJ2 had little effect on the expression of the reporter constructs containing either the proximal region or the core enhancer of the MyoD gene promoter (Fig. 8B). These data show that the inhibitory effect of 15d-PGJ2 on MyoD gene expression is mediated by specific element(s) that are located within the distal region of the MyoD gene promoter and that are distinct from the core enhancer element.

In contrast to the selective effects observed with 15d-PGJ2 alone, expression of exogenous mPPARgamma 2 led to a generalized reduction of 40-60% in the activities of the different reporter gene constructs. The addition of 15d-PGJ2 further reduced the activity of the reporter construct containing the distal region of the MyoD gene promoter; however, the relative extent of inhibition was not significantly different in either the absence or presence of exogenously expressed mPPARgamma 2. Our findings establish that 15d-PGJ2 inhibits MyoD gene expression through distinct promoter elements and further confirms that this inhibition is independent of PPARgamma .

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

15d-PGJ2 is a bioactive cyclopentenone prostanoid involved in a variety of diverse biological processes such as adipogenesis, osteogenesis, apoptosis, inflammation, atherosclerosis, carcinogenesis, cellular adhesion, and cellular migration (35, 49-56). As a ligand and activator of PPARgamma (13, 15), 15d-PGJ2 mediates many of its activities through this receptor. However, recent studies have indicated that some 15d-PGJ2-mediated effects occur independently of PPARgamma (38, 39, 54, 57). We now add the repression of myogenesis in vitro to the list of 15d-PGJ2-mediated biological functions and provide evidence that this repression occurs independently of PPARgamma , the cognate receptor for 15d-PGJ2,. Moreover, we show that distinct classes of PPARgamma ligands mediate repression of MyoD promoter activity through pathways that are both dependent on and independent of PPARgamma .

The presence of 15d-PGJ2 repressed C2C12 differentiation as determined both morphologically and at the level of myogenic gene expression. The nearly complete inhibition of C2C12 differentiation was accompanied by a 50% decrease in the steady state levels of mRNA for myogenic factors such as MyoD. It is not known whether the inhibition of C2C12 differentiation was a consequence solely of reduced levels of MyoD. Experiments in mice harboring knockouts in myogenic factors suggest that threshold levels of myogenic factors are required for the progression of the myogenic program, since compensatory roles are seen for the factors in single knockouts but are lost in double knockouts (22, 24, 34). Therefore, a 50% repression in MyoD gene expression, coupled with a reduction in compensatory factors such as Myf-5 and E box myogenic factors, may be sufficient to efficiently repress the myogenic program. Interestingly, two different classes of synthetic ligands of PPARgamma , the phenoxyacetic acid L-805645 and the TZD ciglitazone, did not repress the myogenic program or reduce expression of the endogenous MyoD gene in C2C12 cells, indicating that distinct PPARgamma ligands can differentially affect the myogenic program. Moreover, saturating concentrations of L-805645 did not ablate the inhibition of myogenesis by 15d-PGJ2, suggesting that the inhibition by 15d-PGJ2 is independent of endogenous PPARgamma .

Certain PPARgamma ligands, including TZDs, have been shown not only to inhibit muscle cell differentiation but also to transdifferentiate myoblast cells into adipose-like cells under conditions permissive for adipogenesis (30, 31). While 15d-PGJ2 has been shown to be adipogenic in fat cell precursors (12), we did not observe lipid droplet formation in C2C12 cells as monitored by oil red O staining, even under conditions permissive for adipogenesis (i.e. treatment of cells with dexamethasone, insulin, and 3-isobutyl-1-methylxanthine) (data not shown). This suggests that the effects of 15d-PGJ2 on myogenesis may be separable from those that promote adipogenesis.

The decrease in MyoD mRNA levels in cells in the presence of 15d-PGJ2 occurred in part as a result of changes at the level of gene transcription, as determined by transient transfection assays with the MyoD promoter-linked reporter gene, MyoDLuc. These studies uncovered both PPARgamma -dependent and PPARgamma -independent pathways of repression. Inhibition mediated by either of these pathways was specific to the MyoD promoter and PPARgamma subtype used and was restricted to cells capable of committing to the myogenic lineage. Importantly, the myogenic promoter, MyogLuc was unaffected by PPARgamma under transient transfection experiments. It should be noted that this finding seems at odds with the Northern analysis, showing the repression of myogenin by 15d-PGJ2. However, explanations for this may stem from different culture conditions used (transient transfections were carried out under growth conditions where myogenin is expressed at basal levels versus the Northern analysis carried out under differentiation conditions where myogenin is expressed at enhanced levels), or perhaps the natural promoter contains elements that are responsive to 15d-PGJ2 are lacking in the 200-bp element of MyogLuc. Alternatively, inhibition of myogenin gene expression may be a consequence of the reduced levels of MyoD found in the presence of 15d-PGJ2. Taken together, these findings intimate the existence of cell type-specific factors that mediate the repressive effects observed.

Several lines of evidence indicate that 15d-PGJ2 can selectively repress expression from the MyoD promoter in a PPARgamma -independent manner. For example, 15d-PGJ2, but not L-805645 or ciglitazone, was able to repress MyoDLuc expression independently of exogenously expressed PPARgamma , whereas activation of a PPAR-responsive promoter, pAOx(X2)GL, required exogenously expressed PPARgamma . Furthermore, a dominant-negative PPARgamma derivative, hPPARgamma 2Delta 500, did not abrogate the 15d-PGJ2-mediated repression, as would be expected if 15d-PGJ2 were dependent on endogenous PPARgamma to mediate its repressive effects. Finally, preliminary promoter analysis has indicated that the inhibitory effects of 15d-PGJ2 on MyoD gene expression are mediated by specific elements in the distal region of the MyoD gene promoter and that this inhibition can be distinguished from generalized repressive effects on gene expression mediated by exogenously expressed PPARgamma . 15d-PGJ2 has been shown to mediate repressive effects in other systems in a PPARgamma -independent manner, including the inhibition of inducible nitric-oxide synthase (54, 58), of the beta 2 integrin-dependent oxidative burst by adherent human neutrophils (57), and of proinflammatory cytokines in activated monocytes and macrophages (39). 15d-PGJ2 has also been shown to antagonize NF-kappa B activity (38, 39), thus repressing the transactivation of NF-kappa B target genes. All the effects of 15d-PGJ2 noted above occur in the low micromolar range of concentration, similar to our findings of an IC50 of ~5 µM for the repression of MyoD promoter activity. Whether this finding reflects a similar pathway(s) of repression remains to be determined.

In addition to the PPARgamma -independent pathway described above, our findings indicate the existence of a PPARgamma -dependent pathway for repression of the MyoD promoter by 15d-PGJ2 and other receptor ligands. For example, ectopic expression of PPARgamma decreased the IC50 required to repress MyoDLuc expression by 15d-PGJ2 by almost 5-fold (from 5 µM to 1.1 µM). This IC50 was similar to the EC50 of 15d-PGJ2-mediated activation of pAOx(X2)GL, suggesting that this activity was PPARgamma -dependent. The presence of L-805645 or ciglitazone did not repress MyoDLuc expression in transfections of C2C12 cells; however, both compounds were capable of inhibiting MyoDLuc expression or activating pAOx(X2)GL expression in the presence of exogenously expressed PPARgamma . Activation and repression occurred in a similar range of drug concentration and was abrogated by a transactivation-defective mutant of PPARgamma , hPPARgamma 2Delta 500. Importantly, PPARgamma on its own repressed MyoDLuc activity in the absence of added ligand, whereas the transactivation-defective mutant of PPARgamma was inactive. These findings are consistent with a scenario in which a PPARgamma -dependent pathway exists for the regulation of the MyoD promoter and are in agreement with the findings of Hu et al. (30), who demonstrated that the repression of myogenesis and of expression of muscle-specific mRNAs, including that encoding MyoD, in G8 myoblasts by the PPARgamma activator eicosatetraynoic acid was dependent on the exogenous expression of PPARgamma . Our findings also add PPARgamma to the growing list of nuclear hormone receptors, including retinoic acid receptor, thyroid hormone receptor 36, RXR, chicken ovalbumin upstream promoter transcription factor I, and retinoid orphan receptor alpha , that have been shown to attenuate the transcription of the MyoD gene (59-62).

The mechanism by which PPARgamma represses MyoD gene expression remains to be determined. The expression of the MyoD gene is subject to complex transcriptional regulation, including autoregulation (44, 48). One possibility is that PPARgamma acts directly through a functional PPRE in the MyoD gene promoter; however, such an element has not been identified. Preliminary analysis of distal, proximal, and core enhancer elements of the MyoD gene promoter present in the reporter plasmid MyoDLuc showed that PPARgamma had a generalized repressive effect on MyoD gene expression regardless of promoter context, consistent with an absence of a specific DNA target in the MyoD gene promoter. Alternatively, PPARgamma may attenuate autoregulation of the MyoD gene by MyoD through direct interaction with MyoD itself or through auxiliary cofactors (63), as has been observed for other nuclear receptors such as RXR, TRbeta , retinoid orphan receptor alpha , and chicken ovalbumin upstream promoter transcription factor (59, 60, 62). This possibility is probably unlikely, since C3H10T1/2 cells, which do not express MyoD, are still permissive for PPARgamma -dependent repression of expression by MyoDLuc. There is evidence from other systems to suggest that PPARgamma can transrepress through competition with coactivators, including SRC-1 and the CREB-binding protein (64). Cofactors such as SRC-1 bind PPARgamma in a ligand-dependent manner, consistent with our observation that repression of MyoDLuc expression was enhanced in the presence of PPARgamma ligands. Finally, PPARgamma may mediate its repressive effects on MyoD gene expression indirectly through interference with the expression or function of cellular factors that positively regulate MyoD gene expression. In this context, PPARgamma has been shown to antagonize the activities of AP-1, Sp1, STAT, and NF-kappa B (65), many of which are known to be important for muscle cell differentiation and the regulation of the MyoD gene promoter (48).

Our work shows that the exogenous expression of PPARgamma in a muscle tissue culture system attenuates the expression of MyoD, a key myogenic factor. The physiological role of PPARgamma in muscle cells is unknown; however, there is mounting evidence that the low levels of PPARgamma expressed in muscle tissue may play some role in muscle development or muscle pathology. Thus, mRNA for PPARgamma is up-regulated in the skeletal muscle of obese or type II diabetic individuals (26, 28), and in human skeletal muscle, expression levels of PPARgamma correlate with the expression levels of genes involved in lipid metabolism, which are putative targets of PPARgamma (29). Moreover, genetic polymorphisms of PPARgamma have been linked to insulin resistance, obesity, and diabetes (66-69), and troglitazone has been shown to increase the level of expression of PPARgamma mRNA in the skeletal muscle of diabetic patients (19). In agreement with the latter observation, we found that both 15d-PGJ2 and ciglitazone enhanced the expression of PPARgamma mRNA in C2C12 cells consistent with the suggestion that PPARgamma ligands may participate in a positive feedback mechanism that elevates PPARgamma mRNA expression. However, L-805645 did not induce the expression of PPARgamma mRNA in C2C12 cells. This finding is consistent with a number of findings showing that different PPARgamma ligands not only demonstrate differential effects on the activation and function of PPARgamma , but also on the expression of its encoding gene (6, 7, 19, 37). Taken together, it is possible that under certain conditions, MyoD may serve as a molecular target underpinning some of the physiological effects of PPARgamma and PPARgamma ligands that have been observed in muscle tissue.

In summary, the findings reported herein add to the growing body of evidence supporting a physiological role for PPARgamma and PPARgamma ligands in myogenesis and in muscle cell-specific gene expression and demonstrate that distinct PPARgamma ligands exert differential effects, some of which are independent of the cognate nuclear receptor. Future studies directed toward unraveling the molecular basis for these ligand- and receptor-dependent effects should prove highly relevant to our understanding of the development of normal muscle function and of the adult-onset pathologies related to the dysregulation of this process.

    ACKNOWLEDGEMENTS

We thank Michael Rudnicki, Robert Perry, and Hansa Patel for helpful discussions and for providing reagents and plasmids. We also thank Joel Berger for generously providing L-805645.

    FOOTNOTES

* This work was supported by a grant from the Heart and Stroke Foundation of Canada (to J. P. C. and R. A. R.).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.

Senior Scientist of the Canadian Institutes for Health Research and an International Research Scholar of the Howard Hughes Medical Institute. To whom correspondence may be addressed: Dept. of Cell Biology, University of Alberta, MSB 5-14, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-9868; Fax: 780-492-9278; E-mail: rick.rachubinski@ualberta.ca.

|| Senior Scientist of the National Cancer Institute of Canada. To whom correspondence may be addressed: Dept. of Biochemistry, McMaster University, 1200 Main St. W., Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 22184); Fax: 905-546-0800; E-mail: caponej@fhs.csu.mcmaster.ca.

Published, JBC Papers in Press, July 26, 2001, DOI 10.1074/jbc.M103594200

    ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor gamma ; TZD, thiazolidinedione; 15d-PGJ2, 15-deoxy-Delta 12,14-prostaglandin J2; mPPAR, mouse PPAR; hPPAR, human PPAR; RXRalpha , 9-cis-retinoic acid receptor; PPRE, peroxisome proliferator response element; AOx, acyl-CoA oxidase; kbp, kilobase pair(s); bp, base pair(s); PBS, phosphate-buffered saline; PGK, phosphoglycerate kinase; STAT, signal transducers and activators of transcription.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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