The expression of aortic myosin phosphatase during atherosclerosis progression in apoE-knockout mice

• Main Authors: Jung-Chien Cheng, 鄭融鍵
• Other Authors: Meei-Jyh Jiang
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/60686107221879540103

碩士 === 國立成功大學 === 細胞生物及解剖學研究所 === 93 === 　　Abnormal contractility of vascular smooth muscle is an important characteristic of cardiovascular diseases such as hypertension and atherosclerosis. Myosin phosphatase (MP), a major enzyme regulating smooth muscle contractility, is a heterotrimer consisting of a PP1�� catalytic subunit, a 130-kDa myosin binding subunit (MBS) and a 20-kDa subunit of unknown function. Recent studies indicated that MP activity can be regulated by the phosphorylation of MBS and an endogenous phosphatase inhibitor CPI-17. Phosphorylation of MBS at Thr-694 and Thr-852 by Rho-kinase was demonstrated to inhibit MP activity. In atherosclerotic arteries, aberrant activation of Rho-kinase has been shown to mediate hypercontractility of vascular smooth muscle cells. In contrast, relatively little is known on the adaptation of contractile proteins during atherosclerosis progression. This study was aimed to investigate the expression and phosphorylation of MBS during atherosclerosis progression. In addition, the expression of PP1�� was also examined. 　　We first examined the expression levels of MBS and PP1�� during atherosclerosis progression in apoE-knockout (apoE-KO) mice between 8 weeks and 24 weeks by immunoblotting and RT-PCR. The results showed that MBS protein levels in aorta significantly decreased as the age of apoE-KO mice increased especially in aortic arch whereas no difference was observed in B6 mice of similar age. Secondly, the phosphorylation level of MBS during atherosclerosis progression in apoE-KO mice between 8 and 24 weeks of age was examined by immunoblotting using antibodies against phospho-MBS. No difference of MBS phosphorylation at Thr694 and Thr852 was detected. In contrast to decreases in protein expression, MBS mRNA levels did not vary significantly between 8 weeks and 24 weeks, suggesting that decreased MBS protein levels is attributed to changes in protein synthesis or degradation. In the case of PP1��, neither protein nor mRNA expression was changed in apoE-KO mice during atherosclerosis progression. The ubiquitin-proteasome system is the major intracellular protein degradation pathway in eukaryotic cells and has been shown to be up-regulated in atherosclerosis. To examine the ubiquitination of MBS during atherosclerosis progression, immunoprecipitation was performed using anti-ubiquitin antibody and analyzed by immunoblotting using anti-MBS antibody. In contrast to decreased MBS protein levels, no difference in the level of MBS ubiquitination was detected. Reactive oxygen species (ROS) has been reported to regulate genes expression and protein ubiquitination. Treatment of human aortic smooth muscle cells (HASMCs) with H2O2 or LY83583, which generates superoxide production, did not modify the expression of MBS as indicated by immunoblotting analysis. This study demonstrates that the expression of regulatory subunit of myosin phosphatase, MBS, but not catalytic subunit, PP1��, decreased during atherosclerosis progression. Because no difference of MBS mRNA expression level was detected, thus, the decreased MBS protein level is probably attributed to decreased protein synthesis and/or increased protein degradation during atherosclerosis.