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.

Fat cell differentiation is a critical aspect of obesity and diabetes. Dietary fatty acids are converted to arachidonic acid, which serves as precursor of prostaglandins (PGs). PGJ2 derivatives function as activating ligands for peroxisome proliferator-activated receptor ␥ (PPAR␥), a nuclear hormone receptor that is central to adipogenic determination. We report here that PGF2␣ blocks adipogenesis through activation of mitogen-activated protein kinase, resulting in inhibitory phosphorylation of PPAR␥. Both mitogen-activated protein kinase activation and PPAR␥ phosphorylation are required for the anti-adipogenic effects of PGF2␣. Thus, PG signals generated at a cell surface receptor regulate the program of gene expression required for adipogenesis by modulating the activity of a nuclear hormone receptor that is directly activated by other PG signals. The balance between PGF2␣ and PGJ2 signaling may thus be central to the development of obesity and diabetes.Altered levels of free fatty acids or their metabolites commonly occur in obesity and diabetes (1, 2), and fatty acid uptake is increased in these disorders (3). Levels of arachidonic acid (AA), 1 which is derived from dietary essential fatty acids, are high relative to other fatty acids in obesity and diabetic states (4), and high levels of AA may exacerbate diabetes by negatively regulating glucose uptake (5). AA serves as precursor for eicosanoid signaling molecules including leukotrienes, hydroxyeicosatetraenoic acids, and prostaglandins (PGs) (6). Many eicosanoids signal via cell surface G-protein-coupled receptors (GPCRs) (7). Others including 8 S hydroxyeicosatetraenoic acids, leukotriene B4, and a number of PGs including PGJ2 and derivatives such as 15-deoxy-⌬12,14-PGJ2 (15d-PGJ2) bind and activate members of the nuclear hormone receptor superfamily (8) called peroxisome proliferator-activated receptors (PPARs) ␣ and ␥ (9 -12).Obesity is due to increased size and number of adipocytes. PPAR␥, the nuclear receptor for PGJ2 derivatives, plays a central role in adipogenesis (12)(13)(14). PPAR␥ is the target of thiazolidinediones, an exciting new class of antidiabetic drugs that function as direct ligands for PPAR␥ and have also been shown to be adipogenic (10,(15)(16)(17). An endogenous PPAR␥ ligand is therefore likely to be an important metabolic regulator.The rate-limiting step in PGJ2 biosynthesis is catalyzed by cyclooxygenase (COX) (18). The actions of different enzymes upon the COX product PGH2 lead to numerous PGs that have different effects on growth, differentiation, and function of many tissues, including PGF2␣ (7). PGF2␣ is known to be synthesized by preadipocytes but does not activate PPAR␥ (9) and by contrast has a potent inhibitory effect upon adipocyte differentiation (19,20). Thus, products of AA metabolism downstream of COX have opposing effects upon adipogenesis. Although the adipogenic effects of PGJ2 derivatives involve direct activation of nuclear PPAR␥, PGF2␣ utilizes a specific GPCR on the cell surface to initiate intracellular signal...

Continuous use of nonsteroidal anti-inflammatory drugs (NSAIDs) lowers the relative risk of colorectal cancer in humans and decreases tumor yield in rodents treated with carcinogens. One well documented target for NSAIDs is prostaglandin endoperoxide synthase (cyclooxygenase) and two isoforms of this enzyme have been identified, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). COX enzymes produce eicosanoid products, some of which have recently been shown to activate transcription mediated by the nuclear hormone receptor peroxisome proliferator activated receptor gamma (PPARgamma), whose expression is largely restricted to adipose tissue. The present study was undertaken to determine if PPARgamma was expressed in colonic tumors. PPARgamma messenger RNA (mRNA) and protein levels were assayed in colonic tumors and normal adjacent mucosa, as well as in a variety of human colon cancer cell lines. There was a marked increase in PPARgamma RNA levels in four out of four of the colonic tumors compared to paired normal mucosa, where little expression of PPARgamma was detected. Western blotting analysis showed that PPARgamma protein was expressed in four out of five colonic tumor samples. PPARgamma was also expressed in a subset of polyps, and in certain human colon cancer cell lines as well. Additionally, we were able to demonstrate that an eicosanoid, 15 deoxy-delta12,14 PGJ2, transactivated transcription of a PPRE-driven promoter in CaCo-2 cells. Thus, we have shown that PPARgamma gene and protein expression is elevated in rodent colon tumors, in selected human colon cancer cell lines and that the PPARgamma receptor is functional in CaCo-2 cells. Since PPARgamma is a ligand-modulated transcription factor, it may provide a novel target for chemopreventive strategies for colorectal cancer.

Thiazolidinediones (TZDs) constitute an exciting new class of antidiabetic compounds, which function as activating ligands for peroxisome proliferator-activated receptor ␥ (PPAR␥). Until now, there has been an excellent correlation between in vivo hypoglycemic potency and in vitro binding and activation of PPAR␥ by TZDs. We have characterized MCC-555, a novel thiazolidinedione ligand for PPAR␥ with unique functional properties. The antidiabetic potency of this compound is greater than that of other TZDs, including BRL49653, yet its binding affinity for PPAR␥ is less than 1 ⁄10 that of BRL49653. The effect of MCC-555 binding on PPAR␥ transcriptional activity is highly context-specific such that it can function as a full agonist, partial agonist, or antagonist depending on the cell type or DNA binding site. These transcriptional properties are partly explained by unique partial agonism of coactivator recruitment to PPAR␥. The properties of MCC-555 are mechanistically distinct from those of the estrogen receptor partial agonist and antagonist tamoxifen because the N terminus of PPAR␥ is not required for activation by MCC-555, and MCC-555 does not stimulate corepressor recruitment to PPAR␥. The context selectivity of MCC-555 may contribute to its enhanced hypoglycemic potency in vivo despite reduced affinity for PPAR␥ relative to other TZDs. Nuclear hormone receptors (NHRs)1 constitute a class of transcription factors with activity that is regulated by natural or synthetic lipophilic ligands (1). A number of NHRs are involved in developmental and/or metabolic processes, and modulation of NHR activity is an effective strategy in the treatment of a variety of cancers, such as breast cancer (2), prostate cancer (3), and acute promyleocytic leukemia (4), as well as metabolic diseases including thyroid disease (5) and diabetes. Non-insulin-dependent diabetes mellitus is a major cause of morbidity and mortality in industrialized nations and is characterized by a post-insulin receptor defect that has been difficult to target therapeutically until the recent discovery that thiazolidinediones (TZDs) enhance the actions of insulin at a level distal to the insulin receptor (6).The mechanism of TZD action is not completely understood, but a number of lines of evidence point to their function as ligands for a member of the NHR superfamily called peroxisome proliferator-activated receptor ␥ (PPAR␥), the natural ligand of which may be derived from or related to prostaglandin J2 (7-9). One of the most potent TZDs, BRL49653, binds to PPAR␥ with an affinity in the nanomolar range (10), and the rank order of TZD potency for in vivo plasma glucose lowering correlates well with their rank order potency for PPAR␥ activation (11, 12). Nevertheless, a number of questions remain with regard to the mechanism of TZD potentiation of insulin action. The main problem is that PPAR␥ is primarily expressed in adipose tissue (13, 14), whereas muscle is ordinarily the main site of insulin-dependent glucose disposal in mammals. This apparent paradox h...