The nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) regulates transcription in response to prostanoid and thiazolidinedione ligands and promotes adipocyte differentiation. The amino-terminal A/B domain of this receptor contains a consensus mitogen-activated protein kinase site in a region common to PPAR␥1 and -␥2 isoforms. The A/B domain of human PPAR␥1 was phosphorylated in vivo, and this was abolished either by mutation of serine 84 to alanine (S84A) or coexpression of a phosphoprotein phosphatase. In vitro, this domain was phosphorylated by ERK2 and JNK, and this was markedly reduced in the S84A mutant. A wild type Gal4-PPAR␥(A/B) chimera exhibited weak constitutive transcriptional activity. Remarkably, this was significantly enhanced in the S84A mutant fusion. Ligand-dependent activation by full-length mouse PPAR␥2 was also augmented by mutation of the homologous serine in the A/B domain to alanine. The nonphosphorylatable form of PPAR␥ was also more adipogenic. Thus, phosphorylation of a mitogen-activated protein kinase site in the A/B region of PPAR␥ inhibits both ligand-independent and ligand-dependent transactivation functions. This observation provides a potential mechanism whereby transcriptional activation by PPAR␥ may be modulated by growth factor or cytokinestimulated signal transduction pathways involved in adipogenesis.

Activation of peroxisome proliferator-activated receptor (PPAR) gamma, a nuclear receptor highly expressed in adipocytes, induces the differentiation of murine preadipocyte cell lines. Recently, thiazolidinediones (TZDs), a novel class of insulin-sensitizing compounds effective in the treatment of non-insulin-dependent diabetes mellitus (NIDDM) have been shown to bind to PPARgamma with high affinity. We have examined the effects of these compounds on the differentiation of human preadipocytes derived from subcutaneous (SC) and omental (Om) fat. Assessed by lipid accumulation, glycerol 3-phosphate dehydrogenase activity, and mRNA levels, subcultured preadipocytes isolated from either SC or Om depots did not differentiate in defined serum-free medium. Addition of TZDs (BRL49653 or troglitazone) or 15-deoxyDelta12,14prostaglandin J2 (a natural PPARgamma ligand) enhanced markedly the differentiation of preadipocytes from SC sites, assessed by all three criteria. The rank order of potency of these agents in inducing differentiation matched their ability to activate transcription via human PPARgamma. In contrast, preadipocytes from Om sites in the same individuals were refractory to TZDs, although PPARgamma was expressed at similar levels in both depots. The mechanism of this depot-specific TZD response is unknown. However, given the association between Om adiposity and NIDDM, the site-specific responsiveness of human preadipocytes to TZDs may be involved in the beneficial effects of these compounds on in vivo insulin sensitivity.

The nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) promotes adipocyte differentiation, exerts atherogenic and anti-inflammatory effects in monocyte/macrophages, and is believed to mediate the insulin-sensitizing action of antidiabetic thiazolidinedione ligands. As no complete PPAR␥ antagonists have been described hitherto, we have constructed a dominant-negative mutant receptor to inhibit wild-type PPAR␥ action. Highly conserved hydrophobic and charged residues (Leu 468 and Glu 471 ) in helix 12 of the ligand-binding domain were mutated to alanine. This compound PPAR␥ mutant retains ligand and DNA binding, but exhibits markedly reduced transactivation due to impaired coactivator (cAMP-response elementbinding protein-binding protein and steroid receptor coactivator-1) recruitment. Unexpectedly, the mutant receptor silences basal gene transcription, recruits corepressors (the silencing mediator of retinoid and thyroid receptors and the nuclear corepressor) more avidly than wild-type PPAR␥, and exhibits delayed ligand-dependent corepressor release. It is a powerful dominant-negative inhibitor of cotransfected wild-type receptor action. Furthermore, when expressed in primary human preadipocytes using a recombinant adenovirus, this PPAR␥ mutant blocks thiazolidinedione-induced differentiation, providing direct evidence that PPAR␥ mediates adipogenesis. Our observations suggest that, as in other mutant nuclear receptor contexts (acute promyelocytic leukemia, resistance to thyroid hormone), dominant-negative inhibition by PPAR␥ is linked to aberrant corepressor interaction. Adenoviral expression of this mutant receptor is a valuable means to antagonize PPAR␥ signaling.Peroxisome proliferator-activated receptor ␥ (PPAR␥), 1 an orphan member of the nuclear hormone receptor family, was first characterized as a transcription factor that regulates adipocyte-specific gene expression (1) and induces preadipocyte differentiation (2), but is now recognized to have a central role in other biological processes. PPAR␥ mediates inhibition of inflammatory cytokine production (interleukin-6 and tumor necrosis factor ␣) from monocytes (3), and receptor activation by oxidized low density lipoprotein-derived ligands promotes macrophage foam cell formation (4). PPAR␥ activation promotes colonic neoplasia (5), but inhibits the growth of breast cancer cells (6). Thiazolidinediones (TZDs), a novel class of antidiabetic agent that act as insulin sensitizers in vivo, bind PPAR␥ with high affinity (7), and prostaglandin J2 (8) and fatty acids have been proposed to be natural ligands. PPAR␥ regulates target gene transcription as a heterodimer with the retinoid X receptor, and this heterodimeric complex has been shown to be activated synergistically by TZDs and RXR-specific ligands (9). However, no complete synthetic or natural PPAR␥ antagonists have been described hitherto. We have therefore generated a dominant-negative PPAR␥ mutant to inhibit wildtype receptor action.In keeping with other members of the nuclear receptor...