TET3 Mediates Alterations in the Epigenetic Marker 5hmC and Akt pathway in Steroid-Associated Osteonecrosis

Jie Zhao  ^{1   2} , Xin-Long Ma  ^{1   2} , Jian-Xiong Ma  ² , Lei Sun  ² , Bin Lu  ² , Ying Wang  ² , Guo-Sheng Xing  ² , Yan Wang  ² , Ben-Chao Dong  ² , Li-Yan Xu  ^{1   2} , Ming-Jie Kuang  ^{1   2} , Lin Fu  ^{1   2} , Hao-Hao Bai  ² , Yue Ma  ^{3   4} , Wei-Lin Jin  ^{3   4}

Affiliations

• ¹ Tianjin Medical University General Hospital, Tianjin, China.
• ² Institute of Orthopedics, Tianjin Hospital, Tianjin, China.
• ³ Department of Instrument Science and Engineering, Key Lab. for Thin Film and Microfabrication Technology of Ministry of Education, School of Electronic Information and Electronic Engineering, Shanghai Jiao Tong University, Shanghai, China.
• ⁴ National Centers for Translational Medicine, Shanghai Jiao Tong University, Shanghai, China.

Abstract

Steroid-associated osteonecrosis (SAON) is one of the common complications of clinical glucocorticoid (GC) administration, with osteocyte apoptosis appearing as the primary histopathological lesion. However, the precise mechanism underlying SAON remains unknown. Epigenetic modification may be a major cause of SAON. Recently, cumulative research revealed that Ten-Eleven Translocation (TET) proteins can catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and then alter the epigenetic state of DNA. Here, we report that TET3-5hmC was upregulated in the femoral head tissues of SAON patients and MLO-Y4 cells with dexamethasone (Dex) treatment. Knockdown of TET3 in MLO-Y4 cells decreased 5hmC enrichment and rescued Dex-induced apoptosis. Meanwhile, the local intramedullary injection of TET3 siRNA in Sprague-Dawley rats abrogated GC-induced osteocyte apoptosis, histopathological changes, abnormal MRI signals, and bone microstructure declines in the femoral head in vivo. Moreover, a hydroxymethylated DNA immunoprecipitation (hMeDIP)-chip analysis of Dex-treated osteocytes revealed 456 different 5hmC-enriched genes. The Akt pathway was found to mediate the functional effect of Dex-induced dynamic 5hmC change; this was further verified in clinical samples. The loss of TET3 in MLO-Y4 cells abrogated Dex-induced Akt signaling pathway inhibition. Therefore, our data for the first time identify the effect of TET3-5hmC on the Akt pathway and the necessity of this signaling cascade in SAON, identifying a new potential therapeutic target. © 2016 American Society for Bone and Mineral Research.

Keywords: APOPTOSIS; DNA DEMETHYLATION; EPIGENETICS; OSTEONECROSIS; TET3.