Molecular analysis of PPARgamma mutations associated with diabetes and lipodystrophy

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor family, acts as a ligand-activated transcription factor to regulate the target gene expression. Numerous studies showed that PPARgamma is a master regulator in adipogenesis. In addition, PPARgamma also regulates glucose and lipid metabolism, which is inspired by the observation that antidiabetic agent known as thiazolidinediones (TZDs) are high-affinity ligands of PPARgamma. In this study, by examining six naturally occurring autosomal dominant PPARgamma mutations which caused type 2 diabetes and lipodystrophy, we are to investigate how specific molecular aspects of these PPARgamma mutations lead to severe physiological phenotypes. Comparative analysis revealed that these mutations were all defective in their abilities to activate transcription and different mutation caused reduced ligand binding affinities to different degrees. Dominant-negative assays revealed that these mutations were classified into two distinct classes, V290M and P467L inhibited the wild-type PPARgamma activity in a dominant-negative manner, while others did not. Moreover, stability assays showed that dominant-negative mutations had higher protein stability than non-dominant-negative mutations. The outcome of these experiments led us to examine the mechanism by which mutant receptor inhibits wild-type receptor in either a dominant-negative or a non-dominant-negative manner. A number of recent molecular studies have proposed that proteasome-mediated degradation of the nuclear receptor from the promoter is essential for ligand-induced transactivation, and that there is a cyclical assembly and disassembly of transcriptional complexes on DNA promoter elements during the transcription. By using chromatin immunoprecipitation (ChIP), we found that dominant-negative mutation P467L had slower promoter turnover leading to prolonged promoter occupancy comparing to wild-type receptor, while non-domintant-negative mutation F388L did not. And blocking receptor degradation by MG132 eliminated the different promoter turnover between wild-type and P467L, indicating that proteasome mediated degradation is required for PPARgamma promoter turnover. Furthermore, saturated amount of rosiglitazone treatment reversed the dominant-negative activity of P467L by increasing its promoter degradation. Therefore, our results demonstrated a molecular mechanism for dominant-negative effect of PPARgamma mutant, reflecting a combination of enhanced promoter occupancy and defective ligand activated transcription.