Rationale:Disturbed flow occurring in arterial branches and curvatures induces vascular endothelial cell (EC) dysfunction and atherosclerosis. We postulated that disturbed flow plays important role in modulating phosphoprotein expression profiles to regulate endothelial functions and atherogenesis.Objective:The goal of this study is to discover novel site-specific phosphorylation alterations induced by disturbed flow in ECs to contribute to atherosclerosis.Methods and Results:Quantitative phosphoproteomics analysis of ECs exposed to disturbed flow with low and oscillatory shear stress (0.5±4 dynes/cm²) versus pulsatile shear stress (12±4 dynes/cm²) revealed that oscillatory shear stress induces phospho-YY1^S118 (serine [S]118 phosphorylation of Yin Yang 1) in ECs. Elevated phospho-YY1^S118 level in ECs was further confirmed to be present in the disturbed flow regions in experimental animals and human atherosclerotic arteries. This disturbed flow-induced EC phospho-YY1^S118 is mediated by CK2α (casein kinase 2α) through its direct interaction with YY1. Yeast 2-hybrid library screening and in situ proximity ligation assays demonstrate that phospho-YY1^S118 directly binds ZKSCAN4 (zinc finger with KRAB [krüppel-associated box] and SCAN [SRE-ZBP, CTfin51, AW-1 and Number 18 cDNA] domains 4) to induce promoter activity and gene expression of HDM2 (human double minute 2), which consequently induces EC proliferation through downregulation of p53 and p21^CIP1. Administration of apoE-deficient (ApoE^−/−) mice with CK2-specific inhibitor tetrabromocinnamic acid or atorvastatin inhibits atherosclerosis formation through downregulations of EC phospho-YY1^S118 and HDM2. Generation of novel transgenic mice bearing EC-specific overexpression of S118-nonphosphorylatable mutant of YY1 in ApoE^−/− mice confirms the critical role of phospho-YY1^S118 in promoting atherosclerosis through EC HDM2.Conclusions:Our findings provide new insights into the mechanisms by which disturbed flow induces endothelial phospho-YY1^S118 to promote atherosclerosis, thus indicating phospho-YY1^S118 as a potential molecular target for atherosclerosis treatment.

中文翻译：

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S118 处的内皮阴阳 1 磷酸化诱导流动下的动脉粥样硬化

基本原理：动脉分支和弯曲处发生的血流紊乱会导致血管内皮细胞 (EC) 功能障碍和动脉粥样硬化。我们假设干扰流在调节磷蛋白表达谱以调节内皮功能和动脉粥样硬化形成中起重要作用。目的：本研究的目的是发现由 ECs 中的干扰流引起的新的位点特异性磷酸化改变，从而导致动脉粥样硬化。方法和结果：对暴露于低振荡剪切应力 (0.5±4 达因/cm ² ) 与脉动剪切应力 (12±4 达因/cm ² ) 的扰动流的 EC 进行定量磷酸蛋白质组学分析，结果表明振荡剪切应力诱导磷酸化 YY1 ^S118（丝氨酸 [S]118 的阴阳 1 磷酸化）在 ECs 中。进一步证实 ECs 中磷酸化 YY1 ^S118水平升高存在于实验动物和人类动脉粥样硬化动脉的受干扰流动区域中。这种扰动的流动诱导的 EC 磷酸-YY1 ^S118由 CK2α（酪蛋白激酶 2α）通过其与 YY1 的直接相互作用介导。酵母 2-杂交文库筛选和原位邻近连接测定表明 phospho-YY1 ^S118直接结合 ZKSCAN4（锌指与 KRAB [krüppel 相关框] 和 SCAN [SRE-ZBP、CTfin51、AW-1 和 18 号 cDNA] 域4) 诱导 HDM2 (human double minute 2) 的启动子活性和基因表达，从而通过下调 p53 和 p21 ^CIP1诱导 EC 增殖. 用 CK2 特异性抑制剂四溴肉桂酸或阿托伐他汀给予 apoE 缺陷型 ( ApoE ^-/- ) 小鼠可通过下调 EC phospho-YY1 ^S118和 HDM2 来抑制动脉粥样硬化的形成。在ApoE ^-/-小鼠中产生具有 EC 特异性 S118-nonphosphorylatable 突变体的 YY1 过表达的新型转基因小鼠证实了磷酸化 YY1 ^S118在通过 EC HDM2 促进动脉粥样硬化中的关键作用。结论：我们的研究结果提供了对机制的新见解扰动的流动诱导内皮磷酸化-YY1 ^S118促进动脉粥样硬化，从而表明磷酸化-YY1 ^S118 作为动脉粥样硬化治疗的潜在分子靶点。