Wang Jiaojiao, Liu Zhiping, Lu Jing, Zou Jiami, Ye Weile, Li Hong, Gao Si, Liu Peiqing
College of Pharmacy, Jinan University, Guangzhou 510632, China; Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China; National-Local Joint Engineering Lab of Druggability and New Drugs Evaluation, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China; Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
College of Pharmacy, Jinan University, Guangzhou 510632, China; Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, China; National-Local Joint Engineering Lab of Druggability and New Drugs Evaluation, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China; Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
Biochem Pharmacol. 2023 Mar;209:115439. doi: 10.1016/j.bcp.2023.115439. Epub 2023 Jan 30.
SIRT6, an NAD-dependent protein deacetylase, is a key modulator of various biological functions. However, the precise role of SIRT6 in the regulation of endothelial function is still not fully understood. The current study sought to determine whether SIRT6 modulates NOS3 activity to regulate endothelium-dependent relaxations in the arterial wall and, if so, to investigate the potential underlying mechanism (s).
ApoE mice and Sprague-Dawley rats had their aortic rings isolated for a vascular reactivity assay. Endothelial cells were cultured before qRT-PCR, western blot, immunoprecipitation, NO bioavailability, and acetylation/deacetylation assays were performed.
SIRT6 expression was significantly reduced in the aorta of ApoE mice fed a high-cholesterol diet, as was endothelium-dependent relaxation. Endothelial dysfunction could be corrected by delivering a SIRT6 overexpression construct via an adenovirus. In cultured endothelial cells, siRNA knockdown of SIRT6 decreased NOS3 catalytic activity, whereas adenoviral overexpression of SIRT6 increased NOS3-derived nitric oxide (NO) generation. SIRT6 interacted with and deacetylated human NOS3 at lysines 494, 497, and 504 of the calmodulin-binding domain, allowing calmodulin to bind to NOS3 and stimulate NOS3 activity. SIRT6 knockdown also reduced NOS3 expression by inhibiting Kruppel-Like Factor 2 (KLF2).
We identified SIRT6 as a new regulator of the activity of NOS3, with functional implications for endothelial-dependent relaxation.
SIRT6是一种依赖烟酰胺腺嘌呤二核苷酸（NAD）的蛋白质脱乙酰酶，是多种生物学功能的关键调节因子。然而，SIRT6在调节内皮功能中的具体作用仍未完全明确。本研究旨在确定SIRT6是否通过调节一氧化氮合酶3（NOS3）的活性来调控动脉壁中内皮依赖性舒张功能，若如此，则进一步探究其潜在的作用机制。
分离载脂蛋白E（ApoE）小鼠和Sprague-Dawley大鼠的主动脉环进行血管反应性测定。培养内皮细胞，随后进行实时定量逆转录聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法、免疫沉淀、一氧化氮（NO）生物利用度以及乙酰化/去乙酰化检测。
喂食高胆固醇饮食的ApoE小鼠主动脉中SIRT6表达显著降低，内皮依赖性舒张功能亦如此。通过腺病毒递送SIRT6过表达构建体可纠正内皮功能障碍。在培养的内皮细胞中，小干扰RNA（siRNA）敲低SIRT6可降低NOS3催化活性，而腺病毒介导的SIRT6过表达则增加了NOS3衍生的NO生成。SIRT6与钙调蛋白结合域赖氨酸494、497和504位点的人NOS3相互作用并使其去乙酰化，从而使钙调蛋白能够结合到NOS3并刺激NOS3活性。SIRT6敲低还通过抑制Kruppel样因子2（KLF2）降低了NOS3的表达。
我们确定SIRT6是NOS3活性的新调节因子，对内皮依赖性舒张具有功能意义。
