Yamashita K, Discher D J, Hu J, Bishopric N H, Webster K A
Department of Molecular and Cellular Pharmacology, University of Miami Medical Center, Miami, Florida 33149, USA.
J Biol Chem. 2001 Apr 20;276(16):12645-53. doi: 10.1074/jbc.M011344200. Epub 2001 Jan 22.
Endothelin-1 (ET-1) is a peptide hormone with potent vasoconstrictor properties which is synthesized and secreted predominantly by vascular endothelial cells. Its production is regulated by numerous stimuli including ischemia and hypoxia, and the enhanced levels that occur during myocardial ischemia may contribute to the progression of heart failure. We reported previously a preliminary characterization of a hypoxia-inducible factor-1 (HIF-1) binding site in the human ET-1 promoter which contributed to the activation of ET-1 expression in endothelial cells. We report here that the HIF-1 binding site alone is not sufficient for the response to hypoxia but requires an additional 50 base pairs of flanking sequence that includes binding sites for the factors activator protein-1 (AP-1), GATA-2, and CAAT-binding factor (NF-1). Mutation of any one of these sites or the HIF-1 site eliminated induction by hypoxia. Mutations of the AP-1 and GATA-2 sites, but not the HIF-1 site, were complemented by overexpressing AP-1, GATA-2, HIF-1alpha, or the activator protein p300/CBP, restoring the response to hypoxia. Binding studies in vitro confirmed physical associations among GATA-2, AP-1, and HIF-1 factors. Overexpression or depletion of p300/CBP modulated the level of ET-1 promoter expression as well as the endogenous ET-1 transcript but did not change the fold induction by hypoxia in either case. Regulation of the ET-1 promoter by hypoxia in non-endothelial cells required overexpression of GATA-2 and HIF-1alpha. The results support essential roles for AP-1, GATA-2, and NF-1 in stabilizing the binding of HIF-1 and promoting recruitment of p300/CBP to the ET-1 hypoxia response complex.
内皮素-1（ET-1）是一种具有强大血管收缩特性的肽类激素，主要由血管内皮细胞合成和分泌。其产生受多种刺激调节，包括缺血和缺氧，心肌缺血期间ET-1水平升高可能促进心力衰竭的进展。我们之前报道了人类ET-1启动子中缺氧诱导因子-1（HIF-1）结合位点的初步特征，该位点有助于内皮细胞中ET-1表达的激活。我们在此报告，仅HIF-1结合位点不足以对缺氧作出反应，还需要额外50个碱基对的侧翼序列，其中包括激活蛋白-1（AP-1）、GATA-2和CAAT结合因子（NF-1）的结合位点。这些位点或HIF-1位点中任何一个发生突变都会消除缺氧诱导作用。AP-1和GATA-2位点发生突变，但HIF-1位点未突变，通过过表达AP-1、GATA-2、HIF-1α或激活蛋白p300/CBP可得到互补，恢复对缺氧的反应。体外结合研究证实了GATA-2、AP-1和HIF-1因子之间存在物理关联。p300/CBP的过表达或缺失调节了ET-1启动子表达水平以及内源性ET-1转录本，但在两种情况下均未改变缺氧诱导倍数。非内皮细胞中缺氧对ET-1启动子的调节需要GATA-2和HIF-1α的过表达。这些结果支持了AP-1、GATA-2和NF-1在稳定HIF-1结合以及促进p300/CBP募集到ET-1缺氧反应复合物中的重要作用。
