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.
Adipocyte differentiation is thought to involve sequential induction of the transcription factors C/EBP, peroxisome proliferator-activated receptor ␥ (PPAR␥), and C/EBP␣. C/EBP␣ expression is both necessary and sufficient for adipocyte differentiation. Here we report that ectopic expression of either C/EBP␣ or C/EBP induces PPAR␥ expression and adipogenesis and that retinoic acid (RA) completely inhibits adipogenesis by either form of C/EBP. In studies of normal preadipocytes, RA does not prevent C/EBP induction but blocks induction of PPAR␥, C/EBP␣, and adipogenesis. In transient transfection studies, liganded RA receptor (RAR) specifically blocks transcriptional activation by either C/EBP␣ or C/EBP. These results strongly suggest that C/EBP␣ substitutes for C/EBP to induce adipocyte differentiation and that liganded RAR inhibits adipogenesis by blocking C/EBP-mediated induction of downstream genes.
Binding to receptors in the cell nucleus is crucial for the action of lipophilic hormones and ligands. PPAR-gamma (for peroxisome proliferator-activated receptor) is a nuclear hormone receptor that mediates adipocyte differentiation and modulates insulin sensitivity, cell proliferation and inflammatory processes. PPAR-gamma ligands have been implicated in the development of atherogenic foam cells and as potential cancer treatments. Transcriptional activity of PPAR-gamma is induced by binding diverse ligands, including natural fatty acid derivatives, antidiabetic thiazolidinediones, and non-steroidal anti-inflammatory drugs. Ligand binding by PPAR-gamma, as well as by the entire nuclear-receptor superfamily, is an independent property of the carboxy-terminal ligand-binding domain (LBD) of the receptor. Here we show that ligand binding by PPAR-gamma is regulated by intramolecular communication between its amino-terminal A/B domain and its carboxy-terminal LBD. Modification of the A/B domain, for example by physiological phosphorylation by MAP kinase, reduces ligand-binding affinity, thus negatively regulating the transcriptional and biological functions of PPAR-gamma. The ability of the A/B domain to regulate ligand binding has important implications for the evaluation and mechanism of action of potentially therapeutic ligands that bind PPAR-gamma and that are likely to extend to other members of the nuclear-receptor superfamily.
Terminal differentiation of stem cells is characterized by cessation of cell proliferation as well as changes in cell morphology associated with the differentiated state. For adipocyte differentiation, independent lines of evidence show that the transcription factors peroxisome proliferator activated receptor ␥ (PPAR␥) and CCAAT/ enhancer-binding protein ␣ (C/EBP␣) as well as the tumor suppressor retinoblastoma (Rb) protein are essential. How these proteins promote adipocyte conversion and how they function cooperatively during the differentiation process remain unclear. We have used retinoic acid (RA) inhibition of adipogenesis to investigate these issues. RA blocked adipogenesis of 3T3-L1 cells induced to differentiate by ectopic expression of PPAR␥ and C/EBP␣ independently or together. However, under these circumstances RA was only effective at preventing adipogenesis when added prior to confluence, suggesting that factors involved in regulation of the cell cycle might play a role in establishing the commitment state of adipogenesis that is insensitive to RA. During differentiation of wild type 3T3 L1 preadipocytes, we found that Rb protein is hyperphosphorylated early in adipogenesis, corresponding to previously quiescent cells reentering the cell cycle, and later becomes hypophosphorylated. The data suggest that, together with the coexpression of PPAR␥ and C/EBP␣, permanent exit from the cell cycle establishes the irreversible commitment to adipocyte differentiation.
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