Abstract
The flt-1 gene encodes a transmembrane tyrosine kinase, Flt-1, a receptor for vascular endothelial growth factor. The expression of flt-1 gene is restricted to endothelial cells in vivo. To understand the molecular 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 flt-1 gene promoter, which has been identified to confer cell type specificity. By point mutation analysis using chloramphenicol acetyltransferase plasmids in 293E1 cells, which express significant levels of flt-1 mRNA, we found that an Ets motif, E4, at -54 to -51 and a cAMP response element (CRE) at -83 to -76 are involved in the transcriptional regulation of this gene. Disruption of either this CRE or E4 within the promoter sequence of 90 base pairs resulted in a decrease in chloramphenicol acetyltransferase activity of 90%, indicating that co-existence of both of CRE and Ets motif E4 is necessary for transcription of the flt-1 gene. Co-transfection of an expression vector containing c-ets-1, c-ets-2, or c-erg cDNA with this 90-base pair sequence yielded a 5-8-fold elevation of chloramphenicol acetyltransferase activity, further supporting the idea that Ets family protein(s) participates in the regulation of the flt-1 gene. Gel shift assays using nuclear extracts of 293E1 and endothelial cells demonstrated the existence of protein factor(s) that specifically binds to CRE and Ets motif E4, respectively. Taken together, our results strongly suggest cooperation of a CRE and an Ets motif for the function of the flt-1 gene promoter.
Highlights
Introduction of point mutation into eithercAMP response element (CRE) or Ets motif E4 in p90WTCAT resulted in a strong suppression of chloramphenicol acetyltransferase (CAT) activity by ϳ90% (Fig. 4), indicating that both CRE and Ets motif are indispensable for the promoter activity of the flt-1 gene
Expression of the flt-1 mRNA in 293E1 Cells Is Mainly Regulated at Transcriptional Level—One of the difficulties in studying the Vascular endothelial growth factor (VEGF)-Flt system is that, far, there exist no endothelial-derived cell lines that express high levels of flt-1 and exhibit biological responses in a VEGF-dependent manner
As described previously [10, 39], we found that placental tissues, sinusoidal endothelial rat liver cells, and 293E1 cells derived from human embryonic kidney express flt-1 mRNA at significant levels
Summary
CRE or Ets motif E4 in p90WTCAT resulted in a strong suppression of CAT activity by ϳ90% (Fig. 4), indicating that both CRE and Ets motif are indispensable for the promoter activity of the flt-1 gene. Induced Ets Family Proteins Trans-activate the flt-1 Promoter—To examine more directly the role of Ets family proteins on the flt-1 promoter, we carried out co-transfection experiments using HeLa cells as the target for transient DNA transfection. This cell line expresses almost undetectable levels of the flt-1 gene; it appeared to be suitable for induction of this gene. This trans-activation appeared to be specific to Ets family proteins, since co-transfection of c-fos and c-jun expression vectors had little or no effect
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