Post-transcriptional control of expression of sFlt-1, an endogenous inhibitor of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a major modulator of angiogenesis. Biological effects of VEGF are mediated by endothelial cell-surface receptors, KDR and Flt-1. Alternative Flt-1 RNA processing, involving retention of intron 13 and the use of intronic cleavage-polyadenylation signals, produces a secreted form of Flt-1, sFlt-1, that binds VEGF with high affinity and can inhibit VEGF signaling. To probe mechanisms controlling sFlt-1 expression, we have cloned and sequenced Flt-1 intron 13 from a mouse genomic library and located RNA processing signals potentially involved in sFlt-1 mRNA formation. A minigene construct containing Flt-1 intron 13 directed the expression of both secreted (i.e., cleaved/polyadenylated) and transmembrane (i.e., spliced) forms of Flt-1 mRNA and protein. Using rapid amplification of 3' cDNA ends (T-RACE) and quantitative PCR (QPCR) analysis to test the activity of intronic cleavage-polyadenylation signals, we observed that multiple sites were utilized for sFlt-1 mRNA processing in both native tissues and Flt-1 minigene transfectants. In transfectants, the most distal signal was utilized preferentially. The potential for interaction between pathways leading to sFlt-1 or full-length Flt-1 was evaluated using QPCR to measure relevant mRNAs after transfection with signal mutants. Decreased expression of sFlt-1 mRNA in cleavage-polyadenylation mutants was accompanied by reciprocal increases in full-length FIt-1 mRNA. Multiple sFlt-1 mRNA species are formed that differ by up to 3.9 kb in their T-untranslated regions (UTRs), which contain sites of potential regulatory importance. The reciprocity between sFlt-1 and Flt-1 mRNA expression suggests a novel post-transcriptional mechanism by which sFlt-1 protein production and, thereby, responsiveness to VEGF, may be modulated. (C) 2004 Wiley-Liss, Inc.