SFlt‐1 Splicing Regulation by U2AF65 and JMJD6 in Endothelial Cells

Abstract

The obstetric disorder preeclampsia (PE) is a condition presenting with new onset hypertension and other co‐morbidities such as proteinuria and endothelial dysfunction. Women with preeclamptic pregnancies have significantly increased circulating levels of the soluble vascular endothelial growth factor (VEGF) receptor Flt‐1 (sFlt‐1). Acting as a VEGF antagonist, which is crucial for the growth and proliferation of endothelial cells, sFlt‐1 causes decreased bioavailability of VEGF. The mechanism regulating the alternative splicing of full length Flt‐1 to the soluble variant are not understood, though U2AF65 and JMJD6 are two proteins which have been implicated in splicing event. Previous research has suggested that U2AF65 promotes the splicing of sFlt‐1, while JMJD6 antagonizes splicing by marking U2AF65 for degradation. Using human umbilical vein endothelial cells (HUVECs), we tested the hypothesis that U2AF65 and JMJD6 were both necessary for alternative splicing in endothelial cells. siRNA was utilized to knock down both U2AF65 (1± 0.012 Fold vs 0.136± 0.017 Fold; p<0.001) and JMJD6 (0.87±0.17 Fold vs 0.077 ± 0.019 Fold; p<0.001) when normalized to β‐actin. Surprisingly, when we measured sFlt‐1 variants 2 and 3, the two most common variants in humans, we found no difference in sFlt‐1 expression. Additionally, we measured expression of the full length Flt‐1, and similarly saw no changes. Next, we overexpressed both U2AF65 and JMJD6 and assessed levels by qRT‐PCR. We observed a trend for ~2‐fold increase in U2AF65 200% (0.85 ± 0.20 Fold vs 1.899 ± 0.39 Fold; p=0.22) though this did not reach significance. Interestingly, in addition to significantly overexpressing JMJD6 (1.27 ± 0.27 Fold vs 2.40 ± 0.31 Fold; p<0.01), cells overexpressing JMJD6 also had significantly higher expression of U2AF65 (0.85 ± 0.20 Fold vs 2.63 ± 0.30 Fold; p<0.05). We measured expression of the FLT1 gene, with no changes seen in the expression of Flt‐1 or sFlt‐1 version 2. The sFlt‐1 variant 3, however, was significantly higher in cells overexpressing JMJD6 (1.008 ± 0.09 Fold vs 1.56 ± 0.14 Fold; p<0.05). Despite the message levels remaining unchanged, sFlt‐1 protein was affected by U2AF65 and JMJD6, with strong trends toward decreased sFlt‐1 in with knock down of U2AF65 (5066 ± 644.3 pg/mL vs 3231 ± 193.5 pg/mL; p=0.067) and JMJD6 (5066 ± 644.3 pg/mL vs 3447 ± 605 pg/mL; p=0.09). Cells which had expression of both U2AF65 and JMJD6 knocked down exhibited an additive effect of sFlt‐1 protein decrease (5066 ± 644.3 pg/mL vs 2166 ± 63.6 pg/mL; p<0.01). Similar, though non‐significant, trends of a positive correlation were seen in cells overexpressing U2AF65 and JMJD6 having slightly increased sFlt‐1 in the media. Because the message levels did not differ greatly among the transformed cells, we looked at heparanase (HPSE) expression as a modulator of sFlt‐1 release into the media. Though there was no change in HPSE expression in knockdown cells, significantly higher HPSE expression was seen in cells overexpressing both U2AF65 and JMJD6 (1.0 ± 0.03 Fold vs 1.97 ± 0.15 Fold; p<0.001). These data suggest that U2AF65 and JMJD6 play an important role in the release of sFlt‐1, but may not be part of the alternative splicing machinery. Future experiments will look at other potential modulators regulating both sFlt‐1 and heparanase, such as MMP‐9.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.