DIFFERENTIAL TRANSCRIPTIONAL REGULATION OF THE 2 VASCULAR ENDOTHELIALGROWTH-FACTOR RECEPTOR GENES - FLT-1, BUT NOT FLK-1 KDR, IS UP-REGULATED BY HYPOXIA/

Vascular endothelial growth factor (VEGF) and its two endothelial cell -specific receptor tyrosine kinases, Flk-1/KDR and Flt-1, play a key r ole in physiological and pathological angiogenesis. Hypoxia has been s hown tee be a major mechanism for up-regulation of VEGF and its recept ors in vivo. When we exposed human umbilical vein endothelial cells to hypoxic conditions in vitro, we observed increased levels of Flt-1 ex pression, In contrast, Flk-1/KDR mRNA levels were unchanged or slightl y repressed, These findings suggest a differential transcriptional reg ulation of the two receptors by hypoxia, To identify regulatory elemen ts involved in the hypoxic response, promoter regions of the mouse Flt -1 and Flk-1/KDR genes were isolated and tested in conjunction with lu ciferase reporter gene. In transient transfection assays, hypoxia led to strong transcriptional activation of the Flt-1 promoter, whereas Fl k-1/KDR transcription was essentially unchanged, Promoter deletion ana lysis demonstrated a 430-bp region of the Flt-1 promoter to be require d for transcriptional activation in response to hypoxia, This region i ncludes a heptamer sequence matching the hypoxia-inducible factor-1 (H IF) consensus binding site previously found in other hypoxia-inducible genes such as the VEGF gene and erythropoietin gene, We further narro wed down the element mediating the hypoxia response to a 40-base pair sequence including the putative HIF binding site, We show that this el ement acts like an enhancer, since it activated transcription irrespec tive of its location or orientation in the construct, Furthermore, mut ations within the putative HIF consensus binding site lead to impaired transcriptional activation by hypoxia, These findings indicate that, unlike the KDR/Flk-1 gene, the Flt-1 receptor gene is directly up-regu lated by hypoxia via a hypoxia-inducible enhancer element located at p ositions -976 to -937 of the Flt-1 promoter.