A novel mechanism of RhoA activation induced by tumor necrosis factor-α in mouse cerebral microvascular endothelial cells

AIM: To investigate the effects of tumor necrosis factor α (TNF-α) on RhoA activity in mouse cerebral microvascular endothelial cells.METHODS: The bEnd.3 cells, a mouse brain microvascular endothelial cell line, were cultured. RhoA activity was analyzed by pull-down assay 10 min, 30 min and 60 min after TNF-α treatment. Expression of RhoA protein was determined by Western blotting 1 h, 3 h, 6 h, 12 h and 24 h after TNF-α treatment. Small interfering RNA (siRNA) targeting to p115RhoGEF or control nsRNA was transfected into bEnd.3 cells. The expression of p115RhoGEF was determined by Western blotting, and RhoA activity was detected by pull-down assay 30 min after TNF-α treatment.RESULTS: RhoA activity peaked at 30 min after TNF-α treatment(P<0.01) . TNF-α significantly increased the protein expression of RhoA at 12 h and 24 h (P<0.05). Knock-down of p115RhoGEF by siRNA in bEnd.3 cells attenuated TNF-α-induced RhoA activation (P<0.05).CONCLUSION: TNF-α up-regulates RhoA activity and expression. p115RhoGEF may play a role in TNF-α-induced activation of RhoA.