Redox regulation of SERCA2 is required for vascular endothelial growth factor-induced signaling and endothelial cell migration

摘要

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 Ca 2+ 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) 45Ca 2+ uptake, a measure of SERCA activity, and knockdown of SERCA2 prevented VEGF-induced EC migration and 45Ca 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. Innovation and Conclusions: 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. Antioxid. Redox Signal. 00, 000-000.