ZNF580, A Human C2H2-zinc Finger Gene, Is Unregulated By S1P And Promotes Migration And Proliferation Of Endothelial Cells

Objective:To investigate whether a novel human C2H2-zinc finger gene ZNF580(GenelD:51157) is involved in the migration and proliferation of endothelial cells induced by sphingosine-1-phosphate (SIP).Methods:The methods used in this study included cell culture, RT-PCR, Western blot, cell migration assay, MTT colorimetric assay, Gelatin zymography, hVEGF ELISA and RNA interference. The expression patterns of SIP receptors (S1P1, S1P2, S1P3, S1P4, S1P5) mRNA in EA.hy926cells were examined by using RT-PCR analysis. We investigated the effects of S1P on ZNF580mRNA and protein levels in EA.hy926cells to determine whether SIP regulates ZNF580expression. Cells were incubated with different concentrations of S1P or with S1P (0.5μM) for different time intervals. Total RNA or protein from treated EA.hy926cells was harvested and subjected to RT-PCR or Western blot analysis. To determine whether the p38MAPK pathway has been implicated in the upregulation of ZNF580expression by SIP, EA.hy926cells were pre-treated with SB203580(the chemical inhibitor of p38MAPK) and then treated with0.5μM SIP for4h. ZNF580cDNA cloned into C1vectors were transfected into EA.hy926cells by liposomal transfection with LipofectamineTM2000to obtain the EA.hy926cells of overexpression of ZNF580. SiRNA-ZNF580s were transfected into EA.hy926cells by liposomal transfection with LipofectamineTM2000to obtain the EA.hy926cells of downexpression of ZNF580. Cell migration assay and MTT colorimetric assay were designed to investigate the effects of ZNF580on endothelial cell motility and growth. We investigated whether ZNF580regulates matrix metalloproteinase-2(MMP-2) expression in endothelial cells by RT-PCR and gelatin zymography on10%sodium dodecyl sulphate (SDS) gels incorporating1mg/ml gelatin. We investigated whether ZNF580regulates vascular endothelial growth factor (VEGF) expression in endothelial cells by RT-PCR and hVEGF ELISA analysis.Results:Our study shows that EA.hy926endothelial cells express S1P1, S1P3and S1P5; however, no significant expression of either S1P2or S1P4was detected. Furthermore, SIP upregulates both ZNF580mRNA and protein levels in a concentration-and time-dependent manner. SB203580, the specific inhibitor of the p38MAPK pathway blocks the SIP-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580in EA.hy926cells leads to the enhancement/decrease of MMP-2and VEGF expression, the migration and proliferation of EA.hy926endothelial cells.Conelusion:These results elucidate that ZNF580plays an important role in the process of the migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.