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 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.

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.

FIG. 1.

VEGF and ^•NO increase endothelial cell migration and SERCA activity. (A) HAEC monolayers were treated with VEGF (50 ng/ml) or DETA NONOate (30 μM), and then migration into a scratch wound was measured over 6 h. The ^•NO synthase inhibitor, LNAME (30 μM), was added just before the scratch (n=3). (B) SERCA activity was assessed by thapsigargin-sensitive ⁴⁵Ca²⁺ uptake in saponin-permeablized HAECs treated with DETA NONOate or VEGF, or VEGF and LNAME (n=4). * p<0.05 versus control. DETA NONOate, diethylenetriamine NONOate; HAEC, human aortic endothelial cell; LNAME, N (G)-nitro-L-arginine methyl ester; ^•NO, nitric oxide; SERCA, sarco/endoplasmic reticulum Ca²⁺ ATPase; VEGF, vascular endothelial growth factor.

FIG. 2.

siRNA-mediated knockdown of SERCA2 prevents VEGF-induced endothelial cell migration. HAECs were treated with specific siRNA to SERCA2. Knockdown was confirmed by qRT-PCR for SERCA2 (A) and immunoblot for SERCA2 [(B), n=4]. HAECs treated with siRNA for SERCA2 were assessed for basal ⁴⁵Ca²⁺ uptake [(C), p<0.001, n=3]. HAEC migration with or without VEGF [(D), 50 ng/ml, n=6]. *p<0.05 versus Control.

FIG. 3.

VEGF-induced S-glutathiolation of SERCA increases migration of HAECs. (A) HAECs were infected with adenoviral vectors to overexpress β-galactosidase (LacZ) or Glrx-1 by ∼10-fold. (B) VEGF (15 min) or DETA-NONOate (1 min) treatment-induced S-glutathiolation of SERCA was assessed by immunoprecipitation with a polyclonal SERCA antibody, and then immunoblotting for protein-bound glutathione adducts using a monoclonal antibody. Bar graph of densitometry results is shown above a representative blot (n=8, *p<0.05). (C) HAECs overexpressing Glrx-1 were treated with VEGF or DETA NONOate, and then scratch wounded for assessment of migration over 6 h (n=4, *p<0.05 vs. control). HAECs overexpressing LacZ or Glrx-1 were seeded on Matrigel in the presence or absence of VEGF (D) and assessed for tube formation over 24 h (n=4, *p<0.05 vs. LacZ control, ***p<0.001). Glrx, glutaredoxin-1.

FIG. 4.

Mutation of SERCA2b-reactive cysteine-674 prevents VEGF- or ^•NO-induced S-glutathiolation and endothelial cell migration. (A) WT SERCA2b or SERCA2b C674S was overexpressed in HAECs by ∼3-fold with adenoviral vectors. (B) S-glutathiolation of either WT SERCA2b or SERCA2b C674S was assessed in HAECs by immunoprecipitation of SERCA2b and immunoblot of protein-bound glutathione (n=4, *p<0.05 vs. control). Migration over 6 h in response to DETA NONOate [(C), n=3, *p<0.05] or VEGF [(D), n=4, *p<0.05] was assessed in HAECs overexpressing WT or C674S SERCA2b. (E) EC tube formation in response to VEGF was assessed at 24 h and quantified by scoring of tube number (*p<0.05 vs. vehicle control, **p<0.01). EC, endothelial cell; WT, wild type.

FIG. 5.

Inhibition of nitric oxide synthase, knockdown of SERCA2, or overexpression of Glrx-1 or SERCA2b C674S mutant decreases VEGF-induced Ca²⁺ entry. (A, B) VEGF or ^•NO was added in the absence of extracellular Ca²⁺, and the maximal increase in intracellular Ca²⁺ associated with Ca²⁺ influx upon Ca²⁺ (2 mM) re-addition was assessed in ECs treated with LNAME (30 μM, n=4, ***p<0.001). Data shown are the representative VEGF response trace with mean±standard error of the mean of Ca²⁺ for measured cells (A) and quantification of change in Fura2 fluorescence ratio between baseline and maximal Ca²⁺ in VEGF- and ^•NO-stimulated cells (B). (C, D) Ca²⁺ influx in HAECs overexpressing LacZ or Glrx-1 representative trace (C) and quantification of maximal Ca²⁺ associated with Ca²⁺ influx [(D), n=at least 4, ***p<0.001]. (E, F) Ca²⁺ influx in EC, in which SERCA2 was knocked down by siRNA [(E), n=4, ***p<0.001] and representative trace of Fura2 in HAECs treated with VEGF before Ca²⁺ addition (F). (G–I) Ca²⁺ influx in ECs overexpressing either WT SERCA2b or SERCA2b C674S [(I), n=at least 4, *p<0.05, ***p<0.001] and representative traces of Fura2 ratio in HAECs treated with VEGF (G) or ^•NO (H) before Ca²⁺ addition.

FIG. 6.

Store-operated Ca²⁺ influx and store-emptying are required for VEGF-induced Ca²⁺ influx. (A–C) Ca²⁺ influx in HAECs in response to VEGF or ^•NO in cells treated with siRNA against Orai1 shown as the representative VEGF (A) or ^•NO gas (B) response trace and quantitation of maximal Ca²⁺ [(C), n=3, ***p<0.001]. (D–F) Ca²⁺ influx in response to VEGF or ^•NO after treatment with ryanodine (100 μM) representative VEGF (D) and ^•NO gas trace (E) and quantification of maximal Ca²⁺ [(F), n=3, ***p<0.001]. Ry, ryanodine.

FIG. 7.

A model for VEGF-induced, SERCA2b C674-dependent Ca²⁺ influx in ECs. VEGF stimulation promotes the production of ^•NO by eNOS, leading to S-glutathiolation of the SERCA2b-reactive cysteine-674 and activation of SERCA2b. Ca²⁺ is pumped by SERCA2b into the ER, where it is then released by the RyR, eliciting continued activation of eNOS. Additionally, activation of the plasma membrane Ca²⁺ channel, Orai1, stimulates Ca²⁺ influx from the extracellular space into the cytosol, further stimulating eNOS and stimulating EC migration. eNOS, endothelial nitric oxide synthase; RyR, ryanodine receptor.