PPARγ Gene Expression is Autoregulated in Primary Adipocytes: Ligand, Sumoylation, and Isoform Specificity

Being a key-factor in glucose homeostasis, PPAR gamma transcriptional activity (TA) is of high importance. However, its mediation by ligands and post-translational modifications in insulin target tissues are unclear. We investigated effects of rosiglitazone (Rg) and sumoylation on PPAR gamma-TA by overexpressing expression vectors and promoter- reporters for PPAR gamma 1 and PPAR gamma 2 in primary rat adipocytes. Wild type (WT) PPAR gamma 1 and PPAR gamma 2 dose-dependently repressed transcription from their promoters to a maximum of 40-50 %. PPAR gamma 2 mutants defective in either MAP-kinase phosphorylation (S112A) or the ligand-binding domain (LBD; P495L, L496A/E499A) exhibited decreased repression of PPAR gamma 2 promoter. Rg enhanced repression by S112A, but not by LBD-defective mutants. Sumoylation-defective PPAR gamma 1 mutants K77R and K365R repressed PPAR gamma 2 promoter activity similar to WT, while Rg enhanced repression by K77R but not by K365R. Sumoylation-defective PPAR gamma 2 mutants K107R and K395R exhibited impaired TA and impaired responsiveness to Rg. GLUT4 promoter, previously shown by us to be repressed by WT-PPAR gamma 1 and WT-PPAR gamma 2, was similarly repressed by both sumoylation-defective PPAR gamma 1 mutants, while both sumoylation-defective PPAR gamma 2 mutants exerted reduced repression. Surprisingly, Rg alleviated only WT-PPAR gamma 2-induced repression, while augmenting that induced by WT-PPAR gamma 1 and all sumoylation-defective mutants. Promoter and chromatin immunoprecipitation analyses revealed that PPAR gamma 2 autorepression involves its direct binding to its promoter. In concert with effects at promoter level, Rg decreased endogenous level of PPAR gamma 2 mRNA, while increasing that of GLUT4. We suggest a hypothetical model for PPAR gamma gene regulation in primary adipocytes that is isoformspecific and Rg/sumoylation-dependent. These findings are important due to the role of PPAR gamma and Rg in insulin sensitivity.