Heme oxygenase-1/carbon monoxide induces vascular endothelial growth factor expression via p38 kinase-dependent activation of Sp1.
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.
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan.
Heng-Huei
HH
Lin
Shao-Chuan
SC
Lai
Lee-Young
LY
Chau
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism*; D002460:Cell Line; D051547:Heme Oxygenase-1 / Q000378:metabolism*; D006801:Humans; D008297:Male; D032342:Muscle Cells / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D016329:Sp1 Transcription Factor; D042461:Vascular Endothelial Growth Factor A; D002248:Carbon Monoxide; D051547:Heme Oxygenase-1; D048051:p38 Mitogen-Activated Protein Kinases