Vascular endothelial cell growth factor (VEGF) plays a pivotal role in promoting neovascularization. VEGF gene expression in vascular endothelial cells in normal tissues is maintained at low levels but becomes highly up-regulated in a variety of disease settings including cancers. Tumor necrosis factor superfamily 15 (TNFSF15; VEGI; TL1A) is an anti-angiogenic cytokine prominently produced by endothelial cells in a normal vasculature. We report here that VEGF production in mouse endothelial cell line bEnd.3 can be inhibited by TNFSF15 via microRNA-29b (miR-29b) that targets the 3'-UTR of VEGF transcript. Blocking TNFSF15 activity by using either siRNA against the TNFSF15 receptor known as death domain-containing receptor-3 (DR3; TNFRSF25), or a neutralizing antibody 4-3H against TNFSF15, led to inhibition of miR-29b expression and reinvigoration of VEGF production. In addition, we found that TNFSF15 activated the JNK signaling pathway as well as the transcription factor GATA3, resulting in enhanced miR-29b production. Treatment of the cells either with SP600125, an inhibitor of JNK, or with JNK siRNA, led to eradication of TNFSF15-induced GATA3 expression. Moreover, GATA3 siRNA suppressed TNFSF15-induced miR-29b expression. These findings suggest that VEGF gene expression can be suppressed by TNFSF15-stimulated activation of the JNK-GATA3 signaling pathway which gives rise to up-regulation of miR-29b.

Tumor necrosis factor superfamily‐15 (TNFSF15; VEGI; TL1A) is a negative modulator of angiogenesis for blood vessel homeostasis and is produced by endothelial cells in a mature vasculature. It is known to be downregulated by vascular endothelial growth factor (VEGF), a major regulator of neovascularization but the mechanism of this interaction is unclear. Here we report that VEGF is able to stimulate the production of two microRNAs, miR‐20a and miR‐31, which directly target the 3′‐UTR of TNFSF15. Additionally, we show that two VEGF‐stimulated cell growth signals, Erk and Akt, are responsible for promoting the expression of miR‐20a and miR‐31. Treatment of human umbilical vein endothelial cells (HUVECs) with Akt inhibitor LY294002 results in diminished miR‐20a and miR‐31 production, while Erk inhibitor U0126 prevented VEGF‐stimulated expression of miR‐20a but not that of miR‐31. Furthermore, inactivation of either Erk or Akt signals restores TNFSF15 gene expression. In an angiogenesis assay, elevated miR‐20a or miR‐31 levels in HUVECs leads to enhancement of capillary‐like tubule formation in vitro, whereas lowered miR‐20a and miR‐31 levels results in an inhibition. These findings are consistent with the view that miR‐20a and miR‐31 mediate VEGF‐induced downregulation of TNFSF15. Targeting these microRNA molecules may therefore provide an effective approach to inhibit angiogenesis.

No abstract