Zhou Weilin, Negash Sewite, Liu Jie, Raj J Usha
Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, CA 90502, USA.
Am J Physiol Lung Cell Mol Physiol. 2009 May;296(5):L780-9. doi: 10.1152/ajplung.90295.2008. Epub 2009 Feb 27.
We have previously reported that in ovine fetal pulmonary venous smooth muscle cells (FPVSMC), decreased expression of cGMP-dependent protein kinase (PKG) by hypoxia could explain hypoxia-induced SMC phenotype modulation. In this study, we investigated the role of myocardin, a possible downstream effector of PKG, in SMC phenotype modulation induced by 1 and 24 h of hypoxia. Hypoxia for 1 h induced the phosphorylation of E-26-like protein 1 (Elk-1), indicating a quick activation of Elk-1 after hypoxia. Either hypoxia (1 h) or treatment with DT-3, a PKG inhibitor, increased associations of Elk-1 with myosin heavy chain (MHC) gene and serum response factor (SRF), which was paralleled by a decrease in association of myocardin with MHC gene and SRF. Exposure to hypoxia of FPVSMC for 24 h significantly decreased the promoter activity of multiple SMC marker genes, downregulated protein and mRNA expression of myocardin, and upregulated mRNA expression of Elk-1, but had no significant effects on the phosphorylation of Elk-1. Inhibition of myocardin by siRNA transfection downregulated the expression of SMC marker proteins, while overexpression of myocardin prevented the hypoxia-induced decrease in expression of SMC marker proteins. Inhibition of PKG by siRNA transfection downregulated the expression of myocardin, but upregulated that of Elk-1. Overexpression of PKG prevented hypoxia-induced effects on protein expression of myocardin and Elk-1. These data suggest that PKG induces displacement of myocardin from SRF and upregulates myocardin expression, thus activating the SMC genes transcription. The inhibitory effects of hypoxia on PKG may explain hypoxia-induced SMC phenotype modulation by decreasing the effects of PKG on myocardin.
我们之前报道过，在绵羊胎儿肺静脉平滑肌细胞（FPVSMC）中，缺氧导致的环磷酸鸟苷依赖性蛋白激酶（PKG）表达降低可解释缺氧诱导的平滑肌细胞表型调节。在本研究中，我们调查了心肌素（PKG的一种可能的下游效应物）在1小时和24小时缺氧诱导的平滑肌细胞表型调节中的作用。缺氧1小时诱导了E-26样蛋白1（Elk-1）的磷酸化，表明缺氧后Elk-1迅速激活。缺氧（1小时）或用PKG抑制剂DT-3处理均增加了Elk-1与肌球蛋白重链（MHC）基因和血清反应因子（SRF）的结合，同时心肌素与MHC基因和SRF的结合减少。FPVSMC暴露于缺氧环境24小时显著降低了多个平滑肌细胞标记基因的启动子活性，下调了心肌素的蛋白和mRNA表达，并上调了Elk-1的mRNA表达，但对Elk-1的磷酸化无显著影响。通过小干扰RNA转染抑制心肌素下调了平滑肌细胞标记蛋白的表达，而心肌素的过表达则阻止了缺氧诱导的平滑肌细胞标记蛋白表达的降低。通过小干扰RNA转染抑制PKG下调了心肌素的表达，但上调了Elk-1的表达。PKG的过表达阻止了缺氧对心肌素和Elk-1蛋白表达的影响。这些数据表明，PKG诱导心肌素从SRF上移位并上调心肌素表达，从而激活平滑肌细胞基因转录。缺氧对PKG的抑制作用可能通过降低PKG对心肌素的作用来解释缺氧诱导的平滑肌细胞表型调节。
