title: Redox regulation of SERCA2 is required for vascular endothelial growth factor-induced signaling and endothelial cell migration.
abstract: OBJECTIVE
  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) (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.
  
  
  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.
authors.affiliation: Vascular Biology Section, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Evangelista
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Thompson
authors.forename: Robert M
authors.initials: RM
authors.lastname: Weisbrod
authors.forename: David R
authors.initials: DR
authors.lastname: Pimental
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Tong
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Bolotina
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cohen
mesh_terms: D000328:Adult; D002118:Calcium / Q000378:metabolism; D002465:Cell Movement; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D005260:Female; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D055815:Young Adult
chemical_list: D042461:Vascular Endothelial Growth Factor A; D009569:Nitric Oxide; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; D002118:Calcium