Abstract 3376: Molecular Mechanism of Alternative VEGFR-1 Gene Expression in Human Endothelial Cell
Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer and its signals are mediated through receptor tyrosine kinases called VEGFR-1 (Flt-1) and VEGFR-2 (KDR/flk-1). From the VEGFR-1 gene, both a membrane type receptor and a soluble form of VEGFR-1 (sVEGFR-1) are expressed. sVEGFR-1 inhibits the function of VEGF by sequestering it. Thus, both angiogenic and anti-angiogenic proteins are expressed from the same gene. However, the precise molecular mechanism of its alternative gene expression and its biological relevance remains to be elucidated. In this study, we investigated the mechanisms of the alternative gene expression in human vascular endothelial cells. Northern blot analysis revealed that sVEGFR-1 was alternatively polyadenylated at several sites of the intron 13 of VEGFR-1 gene, and that there existed ‘a long form of sVEGFR-1’ mRNA which contains almost all of the intron 13. Furthermore, sVEGFR-1 expression was distinctly regulated from full-length VEGFR-1 by various stimuli. Especially, VEGF upregulated only sVEGFR-1 but not full-length VEGFR-1. This induction was mediated by PKC signaling pathways. We then constructed the luciferase reporter gene driven by the VEGFR-1 promoter and followed by the intron 13 of VEGFR-1 gene. The reporter assay revealed that PKC signal did not affect VEGFR-1 promoter activity but upregulated polyadenylation efficiency at the intron 13, suggesting that the induction of sVEGFR-1 was regulated by polyadenylation efficiency. Finally, to evaluate the biological relevance of alternative expression of VEGFR-1 gene, we constructed siRNAs to knockdown only sVEGFR-1, only full-length VEGFR-1 and both of them. The endothelial cells transfected with the siRNA for sVEGFR-1 showed higher chemotactic activity than those tranfected with control siRNA. Furthermore, the tumor transfected with the siRNA for sVEGFR-1 grew larger and was more highly vascularized in mouse subcutaneous tumor model. These results indicate that endogenous sVEGFR-1 may function as a negative feedback system that inhibits excessive angiogenesis in the pathological settings. Thus, the modulation of the switching mechanisms of full-length VEGFR-1 and sVEGFR-1 expression may be a novel target for therapeutic application.