Department of Nephrology, The Fifth People's Hospital of Jinan, Jinan 250022, PR China.
Special Inspection Section, The Fifth People's Hospital of Jinan, Jinan 250022, PR China.
Life Sci. 2021 Jun 1;274:119226. doi: 10.1016/j.lfs.2021.119226. Epub 2021 Feb 17.
O-linked N-acetylglucosaminyltransferase (OGT) is involved in diabetes-related diseases including diabetic nephropathy (DN), and responsible for O-GlcNAcylation. Moreover, O-GlcNAcylation and OGT could be induced by high glucose. Thus, we sought to explore the molecular mechanism of OGT in DN.
Loss- and gain-functions were conducted to determine the roles of OGT, enhancer of zeste homolog 2 (EZH2), hairy and enhancer of split 1 (HES1) and phosphatase and tensin homolog (PTEN) in the viability, cell cycle and fibrosis of mesangial cells (MCs), followed by the assessment using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and Western blot assay (fibrosis-related proteins). The interaction between OGT and EZH2 and the effect on EZH2 glycosylation were verified by chromatin immunoprecipitation (ChIP) and glutathione S-transferase (GST) pull-down assays. EZH2 stability was checked by treatment with cycloheximide.
Expression of OGT was repressed in the DN mice and high glucose-treated MCs. Elevated OGT suppressed viability of high glucose-treated MCs, blocked proliferation characterized by repressed cyclin D1, but enhanced p21 levels, and inhibited fibrosis evidenced by reduced levels of fibronectin (FN) and collagen-4 (col-4). OGT interacted with EZH2 and promoted its glycosylation thus stabilizing the EZH2. EZH2 overexpression enhanced the enrichment of EZH2 and histone H3 Lys27 trimethylation (H3K27me3) in the HES1 promoter. HES1 was upregulated and PTEN was downregulated in DN mice. Transduction of lentivirus vector containing overexpression (oe)-OGT alleviated renal injury in DN mice.
Collectively, OGT stabilizes histone methyltransferases EZH2 to regulate HES1/PTEN thus inhibiting DN.
O-连接的 N-乙酰氨基葡萄糖转移酶（OGT）参与包括糖尿病肾病（DN）在内的糖尿病相关疾病，负责 O-GlcNAcylation。此外，高葡萄糖可诱导 O-GlcNAcylation 和 OGT。因此，我们试图探讨 OGT 在 DN 中的分子机制。
采用失活和激活功能确定 OGT、EZH2、HES1 和 phosphatase and tensin homolog（PTEN）在系膜细胞（MCs）活力、细胞周期和纤维化中的作用，然后通过 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）测定、流式细胞术和 Western blot 测定（纤维化相关蛋白）进行评估。通过染色质免疫沉淀（ChIP）和谷胱甘肽 S-转移酶（GST）下拉测定验证 OGT 与 EZH2 的相互作用及其对 EZH2 糖基化的影响。通过用环己酰亚胺处理检查 EZH2 的稳定性。
在 DN 小鼠和高葡萄糖处理的 MCs 中，OGT 的表达受到抑制。高 OGT 抑制高葡萄糖处理的 MCs 的活力，阻止增殖，表现为细胞周期蛋白 D1 下调，但上调 p21 水平，并抑制纤维化，表现为纤连蛋白（FN）和胶原-4（col-4）水平降低。OGT 与 EZH2 相互作用并促进其糖基化，从而稳定 EZH2。EZH2 的过表达增强了 EZH2 和组蛋白 H3 Lys27 三甲基化（H3K27me3）在 HES1 启动子上的富集。在 DN 小鼠中，HES1 上调，PTEN 下调。转导含过表达（oe）-OGT 的慢病毒载体减轻了 DN 小鼠的肾损伤。
总之，OGT 稳定组蛋白甲基转移酶 EZH2，以调节 HES1/PTEN，从而抑制 DN。
