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

Juyoung Kim; Hyun Jeong Kwak; Ji-Young Cha; Yun-Seung Jeong; Sang Dahl Rhee; Hyae Gyeong Cheon

doi:10.1074/jbc.M113.493650

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

From the ^‡Department of Pharmacology School of Medicine and
^§Department of Molecular Medicine, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon 406-799, Republic of Korea,
the ^¶Bioorganic Science Division, Korea Research Institute of Chemical Technology, Taejeon 305-343, Republic of Korea, and
the ^‖Gachon Medical Research Institute, Gil Medical Center, Incheon 405-760, Republic of Korea

↵2 To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine, Gachon University, Yeonsu-3 Dong, Yeonsu-Gu, Incheon 406-799, Republic of Korea. Tel.: 82-32-820-4761; Fax: 82-32-820-4744, E-mail: hgcheon{at}gachon.ac.kr.

↵1 Both authors contributed equally to this work.

Background: Rosiglitazone, a well known PPARγ 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.

Abstract

Rosiglitazone, a well known insulin sensitizer, stimulates adipocyte differentiation via the activation of peroxisome proliferator-activated receptor γ (PPARγ). 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. PPARγ antagonists (bisphenol A diglycidyl ether or GW9662) blunted the effects of rosiglitazone on PHD regulation. Furthermore, putative PPARγ binding sites were identified in the promoter region of PHDs by ChIP-PCR, implying that rosiglitazone may induce PHD up-regulation directly by PPARγ 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 PPARγ-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.

Footnotes

↵* This work was supported by the Korean Government (Ministry of Education and Science Technology (MEST)) National Research Foundation of Korea (NRF) Grant 2011-0013253.