Shie Jue-Lon, Wu Guifu, Wu Jiaping, Liu Fen-Fen, Laham Roger J, Oettgen Peter, Li Jian
Division of Cardiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 2004 Jun 11;279(24):25010-6. doi: 10.1074/jbc.M403103200. Epub 2004 Apr 8.
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor known to be up-regulated in ischemic heart and hypoxic endothelial cells. However, the transcriptional regulation of VEGF in hypoxia-induced angiogenesis is not fully understood. Transcriptional enhancer factor-1 (TEF-1) is a transcriptional factor family that can regulate many genes expressed in cardiac and skeletal muscle cells by binding to myocyte-specific chloramphenicol acetyltransferase heptamer elements in the promoters of these genes. In this study, we demonstrated that related TEF-1 (RTEF-1), a member of the TEF-1 family, is up-regulated in hypoxic endothelial cells. Overexpression of RTEF-1 increases VEGF promoter activity and VEGF expression. Sequential deletion and site-directed mutation analyses of the VEGF promoter demonstrated that a GC-rich region containing four Sp1 response elements, located between -114 and -50, was essential for RTEF-1 function. This region is beyond the hypoxia-inducible factor-1alpha binding site and does not consist of M-CAT-related elements. By electrophoretic mobility shift assay, RTEF-1 was found to interact with the first Sp1 residue (-97 to -87) of the four consecutive Sp1 elements. Binding activity of RTEF-1 to VEGF promoter is also confirmed by chromatin immunoprecipitation. In addition, induction of VEGF promoter activity by RTEF-1 results in an increase of angiogenic processes including endothelial cells proliferation and vascular structure formation. These results indicate that RTEF-1 acts as a transcriptional stimulator of VEGF by regulating VEGF promoter activity through binding to Sp1 site. In addition, RTEF-1-induced VEGF promoter activity was enhanced in a hypoxic condition, indicating that RTEF-1 may play an important role in the regulation of VEGF under hypoxia.
血管内皮生长因子（VEGF）是一种血管生成生长因子，已知在缺血性心脏和缺氧内皮细胞中上调。然而，VEGF在缺氧诱导的血管生成中的转录调控尚未完全了解。转录增强因子-1（TEF-1）是一个转录因子家族，它可以通过与这些基因启动子中的肌细胞特异性氯霉素乙酰转移酶七聚体元件结合来调节许多在心肌和骨骼肌细胞中表达的基因。在本研究中，我们证明了TEF-1家族成员相关TEF-1（RTEF-1）在缺氧内皮细胞中上调。RTEF-1的过表达增加了VEGF启动子活性和VEGF表达。对VEGF启动子的连续缺失和定点突变分析表明，位于-114至-50之间的包含四个Sp1反应元件的富含GC的区域对于RTEF-1功能至关重要。该区域超出缺氧诱导因子-1α结合位点，且不包含与M-CAT相关的元件。通过电泳迁移率变动分析，发现RTEF-1与四个连续Sp1元件的第一个Sp1位点（-97至-87）相互作用。染色质免疫沉淀也证实了RTEF-1与VEGF启动子的结合活性。此外，RTEF-1对VEGF启动子活性的诱导导致包括内皮细胞增殖和血管结构形成在内的血管生成过程增加。这些结果表明，RTEF-1通过与Sp1位点结合来调节VEGF启动子活性，从而作为VEGF的转录刺激因子。此外，在缺氧条件下，RTEF-1诱导的VEGF启动子活性增强，表明RTEF-1可能在缺氧条件下VEGF的调节中起重要作用。
