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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:783.)
© 2007 American Heart Association, Inc.

Vascular Biology

Cysteine-674 of the Sarco/Endoplasmic Reticulum Calcium ATPase Is Required for the Inhibition of Cell Migration by Nitric Oxide

Jia Ying; XiaoYong Tong; David R. Pimentel; Robert M. Weisbrod; Mario P. Trucillo; Takeshi Adachi; Richard A. Cohen

From the Vascular Biology Unit (J.Y., X.Y.T., R.M.W., M.P.T., R.A.C.) and the Myocardial Biology Unit (D.R.P.), Boston University School of Medicine, Mass, and the Department of Biochemistry & Integrative Medical Biology (T.A.), School of Medicine, Keio University, Japan.

Correspondence to Richard A. Cohen, MD, Vascular Biology Unit, X720, 650 Albany Street, Boston, MA 02118. E-mail racohen{at}bu.edu

Objectives— Nitric oxide inhibits smooth muscle cell migration after arterial injury, but the detailed mechanism is not fully understood. The sarco/endoplasmic reticulum calcium ATPase (SERCA) lowers cell Ca²⁺ by increasing intracellular Ca²⁺ uptake and inhibiting extracellular Ca²⁺ influx. Our previous studies showed that NO causes cyclic GMP-independent arterial relaxation by increasing SERCA activity by inducing reversible S-glutathiolation at cysteine-674. Because Ca²⁺ is an important second messenger for cell migration, we hypothesized that NO also inhibits cell migration through redox regulation of SERCA activity via cysteine-674.

Methods and Results— To test our hypothesis, overexpression of either wild type (WT) or mutant SERCA in which cysteine-674 was mutated to serine was accomplished by stable transfection of HEK 293 or adenoviral expression in rat aortic smooth muscle cells (VSMCs). In the cell models expressing mutant SERCA, biotinylated-iodoacetamide (BIAM) and biotinylated-glutathione labeling of SERCA was decreased, and NO failed to increase SERCA activity or decrease Ca²⁺ influx, thus validating that the expression of mutant SERCA prevents its redox-dependent activation. In the absence of NO, fetal bovine serum stimulated migration of both cell types expressing WT or C674S SERCA at similar rates. The NO donor S-nitrosopenicillamine inhibited migration of cells with WT SERCA, but had no effect on the migration of either HEK cells or VSMCs with C674S SERCA. The same result was obtained in VSMCs in which endogenous NO was produced by iNOS induced by interleukin (IL)-1ß. Blocking cyclic GMP did not prevent the inhibition of migration by NO.

Conclusions— In cells overexpressing SERCA, the cyclic GMP-independent, redox regulation of SERCA cysteine-674 is required for the inhibition of cell migration by both exogenous and endogenously generated NO.

Inhibition of migration of cultured HEK or rat aortic smooth muscle cells by nitric oxide is associated with S-glutathiolation and activation of the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA). The S-glutathiolation and activation of SERCA, as well as the inhibition of migration, was prevented by overexpressing a C674S SERCA mutant.

Key Words: nitric oxide • SERCA • migration • S-gluthiolation

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