Li Jiaqi, Sun Lu, Li Yinghui
Department of Medical Genetics, School of Life Science, China Medical University, Shenyang, Liaoning 110122, P.R. China.
Department of Clinical Genetics, Shengjing Hospital, China Medical University, Shenyang, Liaoning 110004, P.R. China.
Exp Ther Med. 2021 Feb;21(2):114. doi: 10.3892/etm.2020.9546. Epub 2020 Dec 3.
Nitric oxide (NO) serves a crucial role in the kidney and is synthesized by NO synthase (NOS). Asymmetrical dimethylarginine is an endogenous inhibitor of NOS that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). To investigate the role of acetylation in DDAH2 expression, 293 cells were treated with trichostatin A (TSA), a deacetylase inhibitor and the mRNA and protein levels were assessed using quantitative PCR and western blotting respectively. Its promoter activity was detected using a luciferase assay. The effect of TSA on NF-κB acetylation was tested after immunoprecipitation. The binding of NF-κB to the DDAH2 promoter was analyzed using an electrophoretic mobility shift assay and chromatin immunoprecipitation. TSA upregulated DDAH2 expression and transcriptional activity of the DDAH2 promoter through a NF-κB responsive element, which is located at the -1582 to -1573 position of the DDAH2 promoter. Furthermore, TSA treatment promoted NF-κB acetylation, resulting in enhanced NF-κB binding affinity to its binding site both and . Taken together, the present study demonstrated that NF-κB acetylation upregulated DDAH2 expression by enhancing the binding ability of NF-κB to the DDAH2 promoter, resulting in increased promoter activity. The results provided a possible mechanism underlying the regulation of NO production in renal cells and a potential target for treating certain NO-associated renal disorders.
一氧化氮（NO）在肾脏中发挥着关键作用，由一氧化氮合酶（NOS）合成。不对称二甲基精氨酸是NOS的内源性抑制剂，可被二甲基精氨酸二甲胺水解酶（DDAH）代谢。为了研究乙酰化在DDAH2表达中的作用，用脱乙酰酶抑制剂曲古抑菌素A（TSA）处理293细胞，分别使用定量PCR和蛋白质印迹法评估mRNA和蛋白质水平。使用荧光素酶测定法检测其启动子活性。免疫沉淀后检测TSA对NF-κB乙酰化的影响。使用电泳迁移率变动分析和染色质免疫沉淀分析NF-κB与DDAH2启动子的结合。TSA通过位于DDAH2启动子-1582至-1573位置的NF-κB反应元件上调DDAH2表达和DDAH2启动子的转录活性。此外，TSA处理促进了NF-κB乙酰化，导致NF-κB对其结合位点的结合亲和力增强。综上所述，本研究表明，NF-κB乙酰化通过增强NF-κB与DDAH2启动子的结合能力上调DDAH2表达，从而导致启动子活性增加。这些结果为肾细胞中NO产生的调节提供了一种可能的机制，并为治疗某些与NO相关的肾脏疾病提供了一个潜在的靶点。
