Jumonji domain-containing protein6 (Jmjd6) regulates the mRNA splicing of sFlt1 and thereby 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 JmjC 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 in vitro (tube formation: 46±18% reduction; spheroid formation: 74±9% reduction) and impaired migration (43±1% reduction). Consistently, Jmjd6+/- mice exhibit 68±9% reduced FITC-lectin perfused vessels in matrigel assay in vivo (p<0.05). In addition, cultured lung endothelial cells 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-array. Jmjd6 siRNA treated cells showed a dysregulation of several spliced gene variants. In particular, a significant increase in soluble VEGF receptor1 (sFlt1) expression, a splice variant of Flt1, was observed (p=0.006). These findings were validated by using specific taqman probes (2-fold increase) as well as by northern blot. Moreover, inhibition of the catalytic function of Jmjd6 by reducing oxygen augmented splicing of sFlt1 in vitro and in vivo (both 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. Since the enzymatic activity of Jmjd6 required oxygen, we further evaluated the influence of hypoxia on sFlt1 splicing. Indeed, hypoxia (0.1 %) or mimicking hypoxia by DFO also increased sFlt1 splicing. However, maintained hypoxia (>48h) lead to a compensatory up-regulation of Jmjd6 expression and normalization of sFlt1 splicing. In conclusion, these results show that the oxygen-dependent enzyme Jmjd6 has an essential role in the regulation of angiogenesis by controlling splicing of Flt1 mRNA. The biphasic time dependent regulation of Jmjd6 enzymatic activation and expression may contribute to the control of the VEGF/sFlt1 balance during angiogenesis.