Redox Regulation of SERCA2 Is Required for Vascular Endothelial Growth Factor-Induced Signaling and Endothelial Cell Migration

Abstract

Aims: 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 Ca²⁺ ATPase (SERCA) in VEGF-induced signaling and EC migration. Results: 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) ⁴⁵Ca²⁺ uptake, a measure of SERCA activity, and knockdown of SERCA2 prevented VEGF-induced EC migration and ⁴⁵Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ entry through Orai1 were also required for VEGF- and ^•NO-induced EC Ca²⁺ influx. Innovation and Conclusions: These results demonstrate that ^•NO-mediated activation of SERCA2b via S-glutathiolation of cysteine-674 is required for VEGF-induced EC Ca²⁺ influx and migration, and establish redox regulation of SERCA2b as a key component in angiogenic signaling. Antioxid. Redox Signal. 00, 000–000.