We have recently demonstrated that Related Transcription Enhance Factor-1 (RTEF-1) is a transcription factor in endothelial cells. We have also determined that angiogenic factors such as Vascular Endothelial Growth Factor (VEGF) is a transcriptional target of RTEF-1 in vitro. In addition, we established that RTEF-1 expression is enhanced under hypoxic conditions. The goal in this proposal is to further define the role played by RTEF-1 in endothelial cells. We hypothesize that RTEF-1 is involved in the hypoxia-related angiogenesis process. Presumably RTEF-1 plays an important role in improvement of myocardial ischemic injury via angiogenic activities of its target genes. We propose molecular, genetic, and genomic approaches to determine the cellular and physiologic role of RTEF-1 in regulation of endothelium-dependent events in vitro and in vivo. Specifically, in Aim1, we will identify the target genes of RTEF-1 in endothelial cells and attempt to understand how common GC-rich areas of these genes'promoter interact with RTEF-1. We will focus on FGFR1 and COX-2 promoter activity though the binding of the GC rich area, and aim to define the binding elements interacting with RTEF-1 using electrophoretic mobility shift assays (EMSAs) and mutagenesis methods. We will also study the endothelial cell lines with RTEF-1 overexpression, RTEF-1 dominant-negative construct and RTEF-1 siRNA to determine the role of RTEF-1 in angiogenesis using in vitro angiogenesis assays.
In Aim 2, we will determine the mechanism of regulation of RTEF-1 in hypoxia and attempt to identify signaling pathways in RTEF-1 activation in hypoxic endothelial cells. We will determine the modulation of RTEF-1 expression and phosphorylation in response to hypoxia via MAP kinase pathway using in vivo protein phosphorylation assay, pharmacological inhibitors and immunochemical assays. Further, we will characterize the functional consequences of HIF-1a signaling on RTEF-1 promoter activity. Finally, in Aim 3, we will examine the role of RTEF-1 in myocardial ischemia through the angiogenic effects of its target genes. We will use retrovirus-RTEF-1 and RTEF-1 trangenic mice with VE-cadherin promoter in a myocardial ischemia model to determine angiogenic response. Our approach is a new, multidisciplinary paradigm for the study of transcription factors that will be of an obvious benefit in helping us understand the vascular role of RTEF-1.
