||The expression of aortic myosin phosphatase during atherosclerosis progression in apoE-knockout mice
||Institute of Cell Biology and Anatomy
血管平滑肌的不正常收縮是心血管疾病，包括高血壓、血管痙攣和動脈粥狀硬化的重要指標。肌凝蛋白去磷酸酶乃調控平滑肌收縮的一個主要酵素，是由三個次單元所組成，包括具有催化作用的PP1、分子量為130 kDa，可與肌凝蛋白結合的調節次單元MBS、和分子量為20 kDa，功能尚不明的次單元。近年來的研究指出，肌凝蛋白去磷酸酶的活性可以受到MBS，與內生性的去磷酸酶抑制蛋白CPI-17的磷酸化所調控。目前已知，Rho蛋白激酶可以磷酸化MBS的Thr694和Thr852，進而抑制肌凝蛋白去磷酸酶的活性。在動脈粥狀硬化的血管中，血管平滑肌的過度收縮，已知係Rho蛋白激酶不正常的活化所致；但是血管內負責調控收縮的蛋白在動脈粥狀硬化過程中表現量的變化目前所知仍有限。因此，本實驗主要的目的在探討肌凝蛋白去磷酸酶中MBS與PP1，在動脈粥狀硬化過程中其表現量的變化。
本實驗首先利用免疫轉漬法和RT-PCR，來偵測8週到24週大的apoE基因剔除小鼠中MBS和PP1蛋白和mRNA的表現量。結果顯示，隨著apoE基因剔除小鼠週齡的增加，MBS蛋白在主動脈中的表現量有顯著下降的情形，尤其是在主動脈弓的位置；而在B6小鼠中MBS的表現量隨著週齡的增加並沒有變化。在MBS的磷酸化方面，利用可以偵測特定磷酸化位置的抗體，來偵測MBS在Thr694與Thr852磷酸化的情形。結果顯示，在這兩個位置的磷酸化程度在8到24週的apoE基因剔除小鼠並無差異。相對於MBS蛋白顯著的減少，利用RT-PCR的方法我們發現MBS的mRNA表現量並沒有伴隨著減少，這也意味著MBS蛋白的減少可能與蛋白的合成或降解有關。另外，PP1不論是蛋白質或是mRNA的表現量，在apoE基因剔除小鼠的動脈粥狀硬化過程中都沒有顯著的變化。Ubiquitin-proteasome系統是真核細胞負責蛋白降解主要的機制之一，而且在動脈粥狀硬化中這個機制的作用有增加的現象。因此，我們利用ubiquitin的抗體進行免疫沉澱，再以MBS的免疫轉漬法來偵測MBS 的ubiquitination。雖然MBS蛋白的表現量在apoE基因剔除小鼠的動脈粥狀硬化過程中有明顯下降，但是MBS ubiquitination的程度在12至24週之間並無顯著差異。另外，氧活化中間產物已被證實可調控基因的表現與蛋白的ubiquitination。在本實驗，將人類主動脈的平滑肌細胞以過氧化氫或可以刺激細胞產生超氧化物的LY83583處理後，並未減少MBS蛋白的表現量。本實驗主要發現主動脈之肌凝蛋白去磷酸酶中的MBS次單元其蛋白的表現量在apoE基因剔除小鼠的動脈粥狀硬化過程中顯著下降，但是另一個次單元PP1蛋白的表現並無變化。另外，由於MBS mRNA的表現量並無顯著差異，因此推測在動脈粥狀硬化的過程中血管平滑肌細胞內MBS蛋白的減少可能與蛋白合成減少或是蛋白降解增加有關。
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.
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