Abstract 15489: Jmjd6 Regulates the Splicing of sFlt1 and Modulates Angiogenesis
Proteins containing a Jumonji C (JmjC) domain have a central role in the epigenetic control of gene expression. Several molecular functions have been described for this enzyme family. They can either act as protein hydroxylases or histone demethylases as well as by regulating splicing. The hydroxylation reaction catalyzed by Jmj proteins depends on oxygen. Therefore, we examined the function of Jmjd6 in angiogenesis. RNAi-mediated inhibition of Jmjd6 lead to a significant reduction of angiogenesis in endothelial cells (EC) in vitro (tube formation: 46±18% reduction; spheroid formation: 74±9% reduction) and impaired migration (43±1% reduction). Consistently, Jmjd6+/− mice exhibited a significant reduction (68±9%)in FITC-lectin perfused vessels in matrigel assays in vivo. In addition, cultured lung EC derived from Jmjd6+/− mice exhibited impaired network forming activity ex vivo (p<0.05 vs wt). To elucidate the mechanisms underlying the requirement of Jmjd6 for angiogenesis, we performed an exon-splicing-array. Jmjd6 siRNA treatment of EC induced a dysregulation of several spliced gene variants. In particular, a significant increase in soluble VEGF receptor 1 (sFlt1) expression, a splice variant of Flt1, was observed. These findings were validated by using specific taqman probes (2-fold increase) and by measuring sFlt1 protein in the supernatant (increase: + 553±58 pg/ml). Moreover, inhibition of the catalytic function of Jmjd6 by reducing oxygen augmented splicing of sFlt1 in vivo (p<0.05 vs normoxia). Since sFlt1 acts as anti-angiogenic trap for VEGF and PLGF, we tested the functional contribution of sFlt1 and demonstrated that saturating concentrations of VEGF or PLGF or a specific antibody against sFlt1 significantly reduced the inhibition of sprouting caused by Jmjd6 knockdown in vitro (p<0.05). Jmjd6 was recently reported to hydroxylate the splicing factor U2AF65. Indeed, immunoprecipitated U2AF65 binds to sFlt1 mRNA. Moreover, Jmjd6 co-immunoprecipitated with U2AF65 further supporting a link between Jmjd6 and U2AF65-mediated splicing of Flt1. In conclusion, these results demonstrate an essential role of the oxygen-dependent enzyme Jmjd6 in the regulation of angiogenesis by controlling splicing of Flt1 mRNA.
- © 2010 by American Heart Association, Inc.