Heme Oxygenase-1/Carbon Monoxide Induces Vascular Endothelial Growth Factor Expression via p38 Kinase-dependent Activation of Sp1
Heme Oxygenase-1/Carbon Monoxide Induces Vascular Endothelial Growth Factor Expression via p38 Kinase-dependent Activation of Sp1
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DOI：
10.1074/jbc.m110.168831
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发表时间：
2011-02-04
影响因子：
4.8
通讯作者：
Chau, Lee-Young
中科院分区：
文献类型：
--
作者：
Lin, Heng-Huei;Lai, Shao-Chuan;Chau, Lee-Young
血红素加氧酶-1 （HO-1）是一种应激诱导酶，可催化血红素氧化降解为游离铁、一氧化碳和胆绿素。既往研究表明，HO-1过表达可促进缺血心脏中VEGF的表达和血管生成。然而，潜在的机制仍然难以捉摸。本研究表明腺病毒介导的大鼠原代心肌细胞和H9C2心肌细胞的HO-1转导可显著诱导VEGF表达，在直接暴露于CO气体或CO释放化合物三羰基二氯钌（II）二聚体的细胞中也可观察到类似的效果。HO-1/ co通过p38激酶的药理抑制显著抑制VEGF的表达，但对AKT的活化没有作用。VEGF启动子-荧光素酶报告基因检测、电泳迁移量转移检测、超转移检测和染色质免疫沉淀显示，co诱导的VEGF启动子激活需要Sp1转录因子与位于VEGF启动子上的顺式调控序列结合。Western blot分析和免疫染色实验表明，HO-1/CO诱导p38依赖性Sp1在体外和体内Thr-453和Thr-739位点磷酸化。在Thr-453或Thr-739位点发生丙氨酸突变的Sp1蛋白过表达可抑制co诱导的Sp1与VEGF启动子的结合及其转录激活。总的来说，这些数据表明p38依赖性Sp1在Thr-453/Thr-739位点的磷酸化对于HO-1/ co诱导的肌细胞中VEGF表达至关重要。
Heme oxygenase-1 (HO-1) is a stress-inducible enzyme catalyzing the oxidative degradation of heme to free iron, CO, and biliverdin. Previous studies demonstrated that HO-1 overexpression promoted VEGF expression and angiogenesis in the ischemic heart. However, the underlying mechanism remained elusive. Here we show that adenovirus-mediated HO-1 transduction of rat primary cardiomyocytes and H9C2 myocytes resulted in significant induction of VEGF expression, and a similar effect was seen in cells directly exposed to CO gas or a CO-releasing compound, tricarbonyldichlororuthenium (II) dimer. HO-1/CO-induced VEGF expression was significantly suppressed by pharmacological inhibition of p38 kinase, but not of AKT, activation. VEGF promoter-luciferase reporter assays, electrophoretic mobility shift assays, supershift assay, and chromatin immunoprecipitation showed that CO-induced VEGF promoter activation requires the binding of the Sp1 transcriptional factor to a cis-regulatory sequence located at the VEGF promoter. Western blot analysis and immunostaining experiments demonstrated that HO-1/CO induced p38-dependent phosphorylation of Sp1 at Thr-453 and Thr-739 both in vitro and in vivo. Overexpression of Sp1 protein with an alanine mutation at Thr-453 or Thr-739 suppressed CO-induced Sp1 binding to the VEGF promoter and its transcriptional activation. Collectively, these data suggest that p38-dependent phosphorylation of Sp1 at Thr-453/Thr-739 is crucial for HO-1/CO-induced VEGF expression in myocytes.