Heme Oxygenase-1/Carbon Monoxide Induces Vascular Endothelial Growth Factor Expression via p38 Kinase-dependent Activation of Sp1

Heng-Huei Lin,Shao-Chuan Lai,Lee-Young Chau

doi:10.1074/jbc.m110.168831

Heng-Huei Lin, Shao-Chuan Lai + Show 1 more

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https://doi.org/10.1074/jbc.m110.168831

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Abstract

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.

Highlights

To confirm the effect of CO on VEGF expression, H9C2 myocytes were exposed to CO gas or treated with a CO-releasing compound, CORM-2, for 24 h, which resulted in induction of VEGF gene expression in a dose-dependent manner (Fig. 1, C and D), whereas CORM-2 inactivated by overnight incubation at 37 °C in DMEM had no effect (Fig. 1D)
Two GC-rich sequences located between nucleotides Ϫ73 and Ϫ62 in the rat VEGF promoter are required for the Sp1 binding and promoter activation induced by p38 and JNK overexpression in rat cardiomyocytes [26]
In an attempt to obtain mechanistic insights into CO-induced VEGF gene transcription, we performed a reporter assay using a series of 5Ј-deletion constructs of the human VEGF promoter in H9C2 cells, and the results showed that a regulatory sequence located at Ϫ238/Ϫ233 (GGGCGG) was required for VEGF gene induction by CO

Summary

Introduction

Consistent with these mRNA expression results, levels of VEGF protein were substantially increased in the culture medium of CORM-2-treated H9C2 cells (supplemental Fig. S2). P38 Mediates HO-1/CO-induced VEGF Expression—To identify the signaling pathway involved in induction of VEGF expression by HO-1/CO, the phosphorylation states of the kinases p38 and AKT, which have been shown to be activated by HO-1/CO in cardiomyocytes [13], were examined in H9C2 myocytes transduced with Adv-HO-1 or treated with CORM-2.

Results

Conclusion