Transcriptional regulation of the phosphoenolpyruvate carboxykinase gene in adipocytes

Peroxisome proliferator activated receptor-gamma (PPARy) and three CCAAT/enhancer binding proteins (C/EBPs) are transcription factors required for adipocyte differentiation. PPARy heterodimerizes with 9-cis retinoic acid receptor (RXR). Fatty acid metabolites and the newest class of type 2 diabetic medications are ligands. PPARy is implicated in several diseases including diabetes and cancer. In addition to their effects on adipocytes, C/EBPs regulate carbohydrate metabolism and immunity. C/EBPs and PPARy cooperatively activate adipogenesis; however, their precise mechanism(s) of action are unknown. We study the transcriptional regulation of the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene that controls a-glycerol-phosphate formation used in free fatty acid esterification in adipocytes. Dr. Beale previously identified an adipocytespecific enhancer (ASE) upstream of the PEPCK gene, and found that PPARy regulates PEPCK induction in adipocytes through the ASE. PPARy binds two sites, gAFl/PCKl and PCK2; however, only PCK2 is required for PEPCK gene expression in adipose tissue. Gene ablation studies showed that C/EBPp controls PEPCK transcription in adipocytes by an unknown mechanism. The present studies investigated the mechanism of C/EBP and PPARy transactivation of the PEPCK gene by characterizing the C/EBP and PPARy response elements. We hypothesized that C/EBPp binding to the ASE was required for PEPCK induction. These studies demonstrated that C/EBPp transactivated the PEPCK gene via the ASE. We idendfied three functional C/EBP binding sites flanking PCK2; however, they were not required for C/EBP transactivation. We also found that PPARy/RXR and C/EBP interacted yielding synergic activation or inhibition of the PEPCK gene. The mechanism of this interaction and their roles are still under investigation. We sought to identify the mechanism for the different funcdons of gAFl/PCKl and PCK2. We hypothesized that gAFl/PCKl and PCK2 bound different transcription factors in adipocytes. Chicken ovalbumin upstream promoter-transcription factor II inhibited PPARy/RXR activation through an intact gAFl/PCKl element. Quite surprisingly, PPARy/RXR, is the predominant adipocyte binding species for both gAFl/PCKl and PCK2. We then hypothesized that PPARy/RXR binding had siteselective effects. This was corroborated as ablating PCK2 abolished PPARy/RXRa transactivation, whereas ablating gAFl/PCKl increased maximal PPARy/RXR activation. These studies provide a unique example of antagonism between two PPARy binding elements on a single promoter.