The Regulatory Mechanism Of Transcription Factor GATA2 On VWF Expression In Vascular Endothelial Cells

Background:von Willebrand factor(vWF)as a large multifunctional glycoprotein,plays an important role in hemostasis,thrombosis,atherosclerosis and angiogenesis.vWF in plasma is mainly derived from vascular endothelial cells secreted to the injured sites from Weibel-Palade bodies during the process of cell injury to participating in the adhesion and aggregation of platelets.GATA is a family of transcription factors with two zinc finger structures.Studies have shown that the GATA family plays an important role in regulating endothelial cell-specific gene expression.GATA3 and GATA6 positively mediate vWF expression accross binding to the vWF promoter in+220 GATA site in endothelial cells.In mammals,GATA2 is widely expressed in a variety of endothelial cells and is involved in vascular development and angiogenesis by regulating the expression of endothelial specific genes.Current studies have shown that GATA2 can bind to the+220 GATA site of vWF promoter in endothelial cells,but the regulatory mechanism of GATA2 on vWF expression remains unclear.Objective:Explore the regulation and mechanism of GATA2 in the expression of vWF in vascular endothelial cells.Methods:1.The mRNA levels of vWF and GATA2 in human umbilical vein endothelial cells(HUVECs),human pulmonary microvascular endothelial cells(HPMECs),human hepatocytes(MIHAs)and human embryonic kidney cells(HEK293Ts)were detected.2.GATA2 siRNA or Control siRNA was transfected into HUVECs,and the mRNA and protein levels of GATA2 and vWF were measured.3.HUVECs were transfected with GATA2 overexpression plasmid or Control plasmid to detect mRNA and protein levels of GATA2 and vWF.4.The supernatant of cell culture medium was collected after transfection GATA2 siRNA or GATA2 overexpression plasmid to HUVECs 48 hours.vWF secretion in the culture medium was detected by Western Blot,and Coomassie brilliant blue staining was used as an internal control.The content of vWF in the culture medium was detected by Enzyme linked immunosorbent assay(ELISA).5.Chromatin immunoprecipitation(ChIP)assay was used to detect the situation of GATA2 binding to the vWF promoter.6.The luciferase reporter plasmid containing the vWF promoter fragment(-487 to+246)was constructed.And GATA2 siRNA or GATA2 overexpression plasmid and the luciferase reporter plasmid were co-transfected into HUVECs to detect luciferin activity.Results:1.The mRNA levels of GATA2 and vWF in HUVECs and HPMECs were higher than those in MIHAs and HEK293Ts,and the mRNA levels of GATA2 and vWF in HPMECs were higher than those in HUVECs.The differences were statistically significant.2.After HUVECs were transfected with GATA2 siRNA,the mRNA and protein levels of GATA2 and vWF were lower than those of the control group.The differences were statistically significant.3.After HUVECs were transfected with GATA2 overexpression plasmid,the mRNA and protein levels of GATA2 and vWF were significantly higher than those of the control group,and the differences were statistically significant.4.Western Blot and ELISA experiments showed that the content of vWF in the culture medium after GATA2 siRNA transfection was lower than that in the control group,and the difference was statistically significant.The content of vWF in the culture medium after GATA2 overexpression was higher than that in the control group,and the difference was statistically significant.5.ChIP assay showed that GATA2 could bind to the fragment containing+220 GATA site of vWF promoter in HUVECs.6.The luciferase activity was decreased after interfering GATA2 in HUVECs.The luciferase activity was increased after GATA2 overexpression.The differences were statistically significant.Conclusions:1.vWF and GATA2 are high expression in endothelial cells.2.The expression of vWF is positively regulated by GATA2 in HUVECs.3.vWF secretion in the culture medium of HUVECs is positively regulated by GATA2.4.GATA2 could bind to the frgment of vWF promoter containing+220 GATA binding site in HUVECs.5.GATA2 positively regulates the promoter activity of vWF in HUVECs.