VEGF induces metallothionein 1g promoter by utilizing E2F1

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The E2F family of transcription factors contributes significantly to the regulation of cell cycle progression and plays a pivotal role in several biological functions such as differentiation, transformation and apoptosis. The E2F family members are involved in the transcriptional regulation of many of the cellular genes that are specifically required for the S phase transition of the cell cycle and DNA synthesis. In addition, expression of many pro-apoptotic genes like p73 and caspase 3 are regulated by E2F1. Here we show that the metallothionein 1G (MT1) promoter is upregulated by E2F1 upon VEGF stimulation of human aortic endothelial cells. Metallothioneins are intracellular, low molecular weight, cystein-rich proteins that are induced by heavy metals such as zinc and cadmium. They play a significant role in heavy metal detoxification and homeostasis. They also have anti-apoptotic effects and protect cells from oxidative stress or chemical agent-induced cell death. Analysis of the MT1G promoter showed the presence of many potential E2F binding sites flanked by potential SP1 and MRE (metal response element) sites. MT promoters are induced when cells are exposed to heavy metals such as zinc and cadmium, and the transcriptional induction is mediated by the binding of MTF transcription factor to the MRE. Since we found that MT1 promoter is induced by the angiogenic growth factor, VEGF, we examined the possibility that MT1 is regulated by E2F1. Transient transfection experiments showed that the MT1 promoter could indeed be induced by E2F1. Detailed analysis of the promoter showed that the E2F binding sites are necessary for VEGF-mediated induction of MT1. VEGF stimulation led to increased binding of E2F1 to the MT1 promoter, as seen by Chromatin immunoprecipitation assays. Interestingly, mutation of MRE sites did not affect the VEGF-response of the MT1 promoter, but its metal response was significantly reduced, as expected. Based upon these data we propose that MT1G is induced upon VEGF stimulation and this induction is E2F mediated. We also propose that this VEGF-E2F mediated induction is independent of MTF. We are further exploring the possible role of metallothioneins in angiogenesis and tumor development.