A CAMP RESPONSE ELEMENT AND AN ETS MOTIF ARE INVOLVED IN THE TRANSCRIPTIONAL REGULATION OF FLT-1 TYROSINE KINASE (VASCULAR ENDOTHELIAL GROWTH-FACTOR RECEPTOR-1) GENE

The fit-1 gene encodes a transmembrane tyrosine kinase, Fit-1, a recep tor for vascular endothelial growth factor. The expression of fit-1 ge ne is restricted to endothelial cells in vivo, To understand the molec ular mechanism underlying endothelial-specific expression of this gene , we studied the functional significance of transcriptional motifs in the 200-base pair region of the human fit-1 gene promoter, which has b een identified to confer cell type specificity. By point mutation anal ysis using chloramphenicol acetyltransferase plasmids in 293E1 cells, which express significant levels of fit-1 mRNA, me found that an Ets m otif, E4, at -54 to -51 and a cAMP response element (GRE) at -83 to -7 6 are involved in the transcriptional regulation of this gene, Disrupt ion of either this GRE or E4 within the promoter sequence of 90 base p airs resulted in a decrease in chloramphenicol acetyltransferase activ ity of 90%, indicating that co-existence of both of CRE and Ets motif E4 is necessary for transcription of the fit-1 gene. Go-transfection o f an expression vector containing c-ets-l, c-ets-2, or c-erg cDNA with this 90-base pair sequence yielded a 5-8-fold elevation of chloramphe nicol acetyltransferase activity, further supporting the idea that Ets family protein(s) participates in the regulation of the fit-1 gene. G el shift assays using nuclear extracts of 293E1 and endothelial cells demonstrated the existence of protein factor(s) that specifically bind s to CRE and Ets motif E4, respectively. Taken together, our results s trongly suggest cooperation of a GRE and an Ets motif for the function of the fit-1 gene promoter.