- Paper Report
- Open Access
Controlling flk-1/KDRgene expression and angiogenesis
- Peter Terry1
© Biomed Central Ltd 2001
- Received: 26 March 2001
- Accepted: 18 September 2001
- Published: 18 September 2001
- Angiogenesis, flk-1/KDR, TGF-?, transcription regulation, VEGF
Vasculogenesis and angiogenesis depend on binding between vascular endothelial growth factor (VEGF) and VEGF receptor-2, which is expressed by the flk-1/KDR gene, only on the endothelium in these contexts. Transforming growth factor (TGF)-? is a central modulator of inflammation and its resolution. At low concentrations, TGF-? enhances VEGF-induced angiogenesis. At high concentrations, TGF-? downregulates flk-1/KDR mRNA synthesis, inhibiting angiogenesis. This paper examined whether TGF-? influences flk-1/KDR expression by changing the binding of transcription factors to DNA in the flk-1/KDR promoter.
TGF-? suppressed activation of a flk-1/KDR promoter fragment spanning base pairs -115 to +296, which region contains a palindromic GATA site in the 5' untranslated region (UTR). Mutation of this GATA site abolished TGF-?-induced suppression of flk-1/KDR. Electrophoretic mobility shift assay (EMSA) confirmed at least one GATA protein, GATA-2, binds to this site, and TGF-? attenuated this binding. GATA-2 transactivated the flk-1/KDR promoter construct more than GATA-1, and TGF-? attenuated this activation. Therefore, TGF-? negatively regulates flk-1/KDR promoter activity by attenuating GATA-2 binding in the 5' UTR.
Cell culture, transient transfection with luciferase reporter gene, PCR cloning and mutagenesis, RNA isolation and RNase protection assay, EMSA