The production of sFlt-1 splice variants is regulated by jumonji domain containing protein 6 (Jmjd6) in the placenta. (#30)
Soluble fms-like tyrosine kinase 1 (sFlt-1) – a splice variant of the vascular endothelial growth factor receptor 1 (Flt-1) – is an anti-angiogenic factor with a key role in the pathogenesis of preeclampsia. Released at elevated levels from hypoxic preeclamptic placenta, it causes maternal endothelial dysfunction and end organ damage. Surprisingly, while there are numerous reports describing the association between sFlt-1 and preeclampsia, the molecular mechanism in placenta that results in elevated production of sFlt-1 has not been discovered. Recently, jumonji domain containing protein 6 (Jmjd6) was shown to be an oxygen-dependent protein involved in angiogenic regulation1. In hypoxic conditions, Jmjd6 is unable to interact and hydroxylate U2AF65, the spliceosome component that appears directly involved in Flt-1 splicing. This alters the splicing pattern to produce sFlt-1. Therefore, we examined whether Jmjd6 may be a key molecule that regulates sFlt-1 production in preeclamptic placenta .
Both RT-PCR and western analysis of severe preeclamptic (n=22) and gestationally matched preterm controls (n=8) confirmed that Jmjd6 is expressed within the placenta. Furthermore, immunofluorescence demonstrated that Jmjd6 co-localises with U2AF65 to the syncytiotrophoblast. Proximity ligation assay (PLA) confirmed direct endogenous protein:protein interaction between U2AF65 and Jmjd6. The effect of hypoxia (1% oxygen) on Jmjd6 and sFlt-1 splice variants i13 and e15a was then determined in HUVEC, JEG-3 and syncytialised BeWo cells. Hypoxia induced a significant decrease in Jmjd6, as expected in HUVECs1, with an increase of both sFlt-1 variants compared to normoxia (20% oxygen) seen for all three cell types (p<0.05). Lastly, Jmjd6 siRNA treatment also resulted in a significant increase in the expression of both sFlt-1 variant mRNAs (p<0.05) confirming Jmjd6 mediates differential sFlt-1 splicing.Our data suggests oxygen-dependent Jmjd6 plays a key role in regulating sFlt-1 production in placental tissues. It may be the key molecular mechanism regulating sFlt-1 production in placenta.