ROS-mediated downregulation of MYPT1 in smooth muscle cells: a potential mechanism for the aberrant contractility in atherosclerosis

Abstract

Reactive oxygen species (ROS) mediates the aberrant contractility in hypertension. Abnormal contractility occurs in atherosclerotic vessels but changes in proteins that regulate contractility remain poorly understood. Myosin phosphatase (MP) activity, which regulates smooth muscle relaxation, is regulated by the phosphorylation of its regulatory subunit, MP targeting subunit 1 (MYPT1). In the present study, we examined the roles of ROS in MP subunit expression both in cultured human aortic smooth muscle cells (HASMCs) and during atherosclerosis progression in apolipoprotein E-knockout (apoE-KO) mice. Furthermore, the effect of decreased MYPT1 on actin cytoskeleton and cell migration activity was assessed in HASMCs. Short hairpin RNA-mediated knockdown of MYPT1 increased stress fibers and attenuated platelet-derived growth factor-induced cell migration in HASMCs. Superoxide anion-inducing agent LY83583 downregulated MYPT1 mRNA and protein levels, but did not affect the phosphorylation of MYPT1 and catalytic subunit of MP, PP1d. The LY83583-induced decrease in MYPT1 was abolished by co-treating with superoxide dismutase or by inhibiting NADPH oxidase with diphenyleneiodonium. Treatment of peroxynitrite, but not hydrogen peroxide (H 2 O 2 ), downregulated MYPT1 protein expression and induced MYPT1 phosphorylation without affecting mRNA levels. Co-treatment with a proteasome inhibitor, MG-132, eliminated peroxynitrite-induced MYPT1 downregulation. In apoE-KO mice, MYPT1 protein, but not mRNA, levels were markedly decreased in 16-week-and 24-week-old mice. Oral estrogen treatment, which was previously shown to decrease aortic ROS levels, upregulated aortic MYPT1 expression. Moreover, reduction in MYPT1 expression correlated with increased aortic sensitivity toward vasoconstrictors. These results suggested that during atherosclerosis progression oxidative stress mediates the downregulation of MYPT1, which may inhibit smooth muscle cell migration and contribute to the aberrant contractility.Laboratory Investigation (2013) 93, 422-433; doi:10.1038/labinvest.2013 published online 18 February 2013 KEYWORDS: atherosclerosis; contractility; myosin phosphatase; reactive oxygen species; smooth muscle At the molecular level, phosphorylation of the 20-kDa myosin light chains (MLC 20 ) is a key determinant for smooth muscle contraction. MLC 20 phosphorylation levels are determined by the activity ratio between myosin light chain kinase (MLCK) and myosin phosphatase (MP). 1 Although MLCK activation depends on cytoplasmic calcium concentration, MP activity is subject to modulation by various signaling molecules. 2 MP is a heterotrimer comprised of a 37-to 38-kDa catalytic subunit (PP1d), a 110-to 130-kDa regulatory subunit (MP targeting subunit 1; MYPT1), and a 20-kDa subunit of unknown function. The phosphorylation of MYPT1 by protein kinases is a major regulatory mechanism for MP that results in either the inhibition or enhancement of MP activity. 3 Small GTPase RhoA and its effecter, Rho-kinase, inhibit MP activity through ...

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