Zhang Hong-hai, Lechuga Thomas J, Tith Tevy, Wang Wen, Wing Deborah A, Chen Dong-bao
Departments of Obstetrics and Gynecology (H-h.Z., T.J.L., T.T., W.W., D.A.W., D-b.C.) and Pathology (T.J.L., D-b.C.), University of California, Irvine, Irvine, California 92697.
Mol Endocrinol. 2015 Mar;29(3):434-44. doi: 10.1210/me.2014-1297. Epub 2015 Jan 30.
Rapid nitric oxide (NO) production via endothelial NO synthase (eNOS) activation represents a major signaling pathway for the cardiovascular protective effects of estrogens; however, the pathways after NO biosynthesis that estrogens use to function remain largely unknown. Covalent adduction of a NO moiety to cysteines, termed S-nitrosylation (SNO), has emerged as a key route for NO to directly regulate protein function. Cofilin-1 (CFL1) is a small actin-binding protein essential for actin dynamics and cytoskeleton remodeling. Despite being identified as a major SNO protein in endothelial cells, whether SNO regulates CFL-1 function is unknown. We hypothesized that estradiol-17β (E2β) stimulates SNO of CFL1 via eNOS-derived NO and that E2β-induced SNO-CFL1 mediates cytoskeleton remodeling in endothelial cells. Point mutation studies determined Cys80 as the primary SNO site among the 4 cysteines (Cys39/80/139/147) in CFL1. Substitutions of Cys80 with Ala or Ser were used to prepare the SNO-mimetic/deficient (C80A/S) CFL1 mutants. Recombinant wild-type (wt) and mutant CFL1 proteins were prepared; their actin-severing activity was determined by real-time fluorescence imaging analysis. The activity of C80A CFL1 was enhanced to that of the constitutively active S3/A CFL1, whereas the other mutants had no effects. C80A/S mutations lowered Ser3 phosphorylation. Treatment with E2β increased filamentous (F)-actin and filopodium formation in endothelial cells, which were significantly reduced in cells overexpressing wt-CFL. Overexpression of C80A, but not C80S, CFL1 decreased basal F-actin and further suppressed E2β-induced F-actin and filopodium formation compared with wt-CFL1 overexpression. Thus, SNO(Cys80) of cofilin-1 via eNOS-derived NO provides a novel pathway for mediating estrogen-induced endothelial cell cytoskeleton remodeling.
通过内皮型一氧化氮合酶（eNOS）激活快速产生一氧化氮（NO）是雌激素发挥心血管保护作用的主要信号通路；然而，雌激素发挥作用的NO生物合成后的途径仍 largely 未知。NO 部分与半胱氨酸的共价加合，称为 S-亚硝基化（SNO），已成为 NO 直接调节蛋白质功能的关键途径。丝切蛋白-1（CFL1）是一种小的肌动蛋白结合蛋白，对肌动蛋白动力学和细胞骨架重塑至关重要。尽管被确定为内皮细胞中的主要 SNO 蛋白，但 SNO 是否调节 CFL-1 功能尚不清楚。我们假设雌二醇-17β（E2β）通过 eNOS 衍生的 NO 刺激 CFL1 的 SNO，并且 E2β 诱导的 SNO-CFL1 介导内皮细胞中的细胞骨架重塑。点突变研究确定 Cys80 是 CFL1 中 4 个半胱氨酸（Cys39/80/139/147）中的主要 SNO 位点。用 Ala 或 Ser 取代 Cys80 来制备 SNO 模拟/缺陷（C80A/S）CFL1 突变体。制备重组野生型（wt）和突变型 CFL1 蛋白；通过实时荧光成像分析确定它们的肌动蛋白切断活性。C80A CFL1 的活性增强到组成型活性 S3/A CFL1 的活性，而其他突变体没有影响。C80A/S 突变降低了 Ser3 磷酸化。用 E2β 处理增加了内皮细胞中丝状（F）-肌动蛋白和丝状伪足的形成，在过表达 wt-CFL 的细胞中显著减少。与 wt-CFL1 过表达相比，C80A 而非 C80S CFL1 的过表达降低了基础 F-肌动蛋白并进一步抑制了 E2β 诱导的 F-肌动蛋白和丝状伪足形成。因此，通过 eNOS 衍生的 NO 对丝切蛋白-1 进行 SNO（Cys80）为介导雌激素诱导的内皮细胞骨架重塑提供了一条新途径。
