The Role of Prolyl Hydroxylase Domain Protein (PHD) during Rosiglitazone-induced Adipocyte Differentiation

Abstract

Background: Rosiglitazone, a well known PPAR agonist, stimulates adipocyte differentiation. Results: In C3H10T1/2 cells, rosiglitazone induces prolyl hydroxylase domain proteins 1, 2, and 3, resulting in the degradation of anti-adipogenic proteins such as GATA-3, KLF-2, and TAZ. Conclusion: Three isoforms of the prolyl hydroxylase domain protein play a key role in rosiglitazone-induced adipocyte differentiation. Significance: Novel mechanisms involved in rosiglitazone-induced adipogenesis would provide a better understanding of adipocyte biology. Rosiglitazone, a well known insulin sensitizer, stimulates adipocyte differentiation via the activation of peroxisome proliferator-activated receptor (PPAR). Previous two-dimensional proteomics studies using C3H10T1/2 murine mesenchymal pluripotent stem cells revealed that prolyl hydroxylase domain protein (PHD) levels significantly increased during rosiglitazone-induced adipocyte differentiation (RIAD). In this study, we investigated the functional role played by PHD during RIAD. Three PHD isoforms (PHD1, 2, and 3) were found to be up-regulated in C3H10T1/2 cells during RIAD, whereas PHD knockdown and treatment with PHD inhibitors (dimethyloxalyl glycine or ethyl-3,4-dihydroxybenzoate) blocked RIAD. PHD inhibition was found to be associated with increases in the levels of anti-adipogenic proteins such as GATA-3, KLF-2, and transcriptional coactivator with PDZ binding motif (TAZ), with their reduced ubiquitination, suggesting that PHDs evoke the ubiquitination/proteasomal degradation of anti-adipogenic proteins. On the other hand, MG-132 (a proteasomal inhibitor) prevented the degradation of anti-adipogenic proteins and retarded RIAD. PPAR antagonists (bisphenol A diglycidyl ether or GW9662) blunted the effects of rosiglitazone on PHD regulation. Furthermore, putative PPAR binding sites were identified in the promoter region of PHDs by ChIP-PCR, implying that rosiglitazone may induce PHD up-regulation directly by PPAR activation. Consistent with in vitro results, oral administration of rosiglitazone to ob/ob mice for 2 weeks increased adipose PHD levels and decreased anti-adipogenic protein levels by increasing their ubiquitination. These results suggest that rosiglitazone increases PHD expression in a PPAR-dependent manner and that this leads to the commitment of anti-adipogenic proteins to the ubiquitination-proteasomal pathway and to the subsequent induction of adipocyte differentiation.