Vascular Biology Section, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Antioxid Redox Signal. 2012 Oct 15;17(8):1099-108. doi: 10.1089/ars.2011.4022. Epub 2012 May 31.
Vascular endothelial growth factor (VEGF) increases angiogenesis by stimulating endothelial cell (EC) migration. VEGF-induced nitric oxide ((•)NO) release from (•)NO synthase plays a critical role, but the proteins and signaling pathways that may be redox-regulated are poorly understood. The aim of this work was to define the role of (•)NO-mediated redox regulation of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) in VEGF-induced signaling and EC migration.
VEGF-induced EC migration was prevented by the (•)NO synthase inhibitor, N (G)-nitro-L-arginine methyl ester (LNAME). Either VEGF or (•)NO stimulated endoplasmic reticulum (ER) (45)Ca(2+) uptake, a measure of SERCA activity, and knockdown of SERCA2 prevented VEGF-induced EC migration and (45)Ca(2+) uptake. S-glutathione adducts on SERCA2b, identified immunochemically, were increased by VEGF, and were prevented by LNAME or overexpression of glutaredoxin-1 (Glrx-1). Furthermore, VEGF failed to stimulate migration of ECs overexpressing Glrx-1. VEGF or (•)NO increased SERCA S-glutathiolation and stimulated migration of ECs in which wild-type (WT) SERCA2b was overexpressed with an adenovirus, but did neither in those overexpressing a C674S SERCA2b mutant, in which the reactive cysteine-674 was mutated to a serine. Increased EC Ca(2+) influx caused by VEGF or (•)NO was abrogated by overexpression of Glrx-1 or the C674S SERCA2b mutant. ER store-emptying through the ryanodine receptor (RyR) and Ca(2+) entry through Orai1 were also required for VEGF- and (•)NO-induced EC Ca(2+) influx.
These results demonstrate that (•)NO-mediated activation of SERCA2b via S-glutathiolation of cysteine-674 is required for VEGF-induced EC Ca(2+) influx and migration, and establish redox regulation of SERCA2b as a key component in angiogenic signaling.
血管内皮生长因子 (VEGF) 通过刺激内皮细胞 (EC) 迁移来增加血管生成。一氧化氮 ((•)NO) 从一氧化氮合酶释放出来，在 VEGF 诱导的信号转导中发挥关键作用，但对可能受氧化还原调节的蛋白质和信号通路知之甚少。本工作旨在定义 VEGF 诱导的信号转导和 EC 迁移中 (•)NO 介导的还原调节对肌浆/内质网 Ca2+ATP 酶 (SERCA) 的作用。
一氧化氮合酶抑制剂 N(G)-硝基-L-精氨酸甲酯 (LNAME) 可阻止 VEGF 诱导的 EC 迁移。VEGF 或 (•)NO 均可刺激内质网 (ER) ((45)Ca(2+) 摄取，这是 SERCA 活性的一种衡量标准)，而 SERCA2 的敲低则可阻止 VEGF 诱导的 EC 迁移和 (45)Ca(2+) 摄取。免疫化学鉴定的 SERCA2b 的 S-谷胱甘肽加合物增加了 VEGF 的含量，LNAME 或谷胱甘肽过氧化物酶-1 (Glrx-1) 的过表达也可阻止其增加。此外，VEGF 未能刺激过表达 Glrx-1 的 EC 迁移。VEGF 或 (•)NO 可增加 SERCA S-谷胱甘肽化，并刺激过表达野生型 (WT) SERCA2b 的 EC 迁移，但不能刺激过表达 C674S SERCA2b 突变体的 EC 迁移，因为该突变体中的反应性半胱氨酸-674 突变为丝氨酸。VEGF 或 (•)NO 引起的 EC Ca(2+) 内流增加可通过 Glrx-1 或 C674S SERCA2b 突变体的过表达来消除。肌浆网 Ca2+ 释放通过ryanodine 受体 (RyR) 和 Ca2+ 通过 Orai1 的进入也需要 VEGF 和 (•)NO 诱导的 EC Ca(2+) 内流。
这些结果表明，VEGF 诱导的 EC Ca(2+) 内流和迁移需要 (•)NO 介导的通过半胱氨酸-674 的 SERCA2b 的 S-谷胱甘肽化激活，并且确定了 SERCA2b 的氧化还原调节是血管生成信号的关键组成部分。
