Myosin light chain phosphatase is a downstream target of Rho-kinase in endothelin-1-induced transactivation of the TGF-β receptor.

BACKGROUND: Rho-kinase (ROCK) regulates actomyosin contraction, coronary vasospasm, and cytoskeleton dynamics. ROCK and of NADPH oxidase (NOX) play an essential role in cardiovascular disease and proteoglycan synthesis, which promotes atherosclerosis by trapping low density lipoprotein. ROCK is activated by endothelin-1 (ET1) and transactivates the transforming growth factor beta receptor (TGFβR1), intensifying Smad signaling and proteoglycan production. This study aimed to identify the role of myosin light chain phosphatase (MLCP) as a downstream target of ROCK in TβR1 transactivation.

METHODS: Vascular smooth muscle cells were treated with ET1 and inhibitors of ROCK and MLCP were added. The phosphorylation levels of Smad2C, myosin light chain (MLC), and MLCP were monitored by western blot, and the mRNA expression of chondroitin 4-O-sulfotransferase 1 (C4ST1) was assessed by quantitative real-time PCR.

RESULTS: We examined ROCK's role in ET1-induced TGFβR1 activation. ROCK phosphorylated MLCP at the MYPT1 T853 residue, blocked by the ROCK inhibitor Y27632. ROCK also increased MLC phosphorylation and actomyosin contraction in response to ET1, enhanced by the phosphatase inhibitor Calyculin A. Calyculin A also increased C4ST1 expression, GAG-chain synthesizing enzymes.

CONCLUSIONS: This work suggests that ROCK is involved in ET1-mediated TβR1 activation through increased MLCP phosphorylation, which leads to Smad2C phosphorylation and stimulates C4ST1 expression.