Stimulation Of Endothelin-1 Gene Expression By Insulin Via PI3K-GSK3β Signaling In Endothelial Cells

Insulin stimulates secretion of a potent vasoactive and mitogenic peptide endothelin-1(ET-1)in endothelial cells.Regulation of ET-1 secretion is mainly at transcriptional level.Insulin resistance is associated with hyperinsulinemia that has been linked to activation of the endothelin system and increased ET-1 secretion,which contributes to abnormal growth or hypertrophy of cardiovascular cells,as well as the developments of atherosclerosis and hypertension.Insulin action stimulates both MAP Kinase(MAPK)and PI3K/Akt signaling pathways.Despite the mechanism of PI3K-dependent NO production has been well established,little is know about the intracellular signaling events involved in insulin stimulated ET-1 gene expression.Insulin activates PI3K,resulting in Akt activation,which phosphorylates GSK3βat serine 9(ser~9)residue and leads to its inactivation.GSK3βis a multifunctional serine/threonine kinase. One of the many functions of GSK3βis to phosphorylate transcription factors and regulate their activities,for instance,by changing their DNA binding capacities.As an important member of the insulin signaling,whether GSK3βis involved in regulation of ET-1 gene expression is presently unknown.The human ET-1 gene has a TATA-containing promoter,whose activity can be modulated by many transcription factors such as AP-1,the zinc finger transcription factor GATA-2 and hypoxia-induced factor-1.The expression of the identified transcription factors for the ET-1 promoter is not restricted to vascular endothelial cells.Therefore,the action of these factors alone is unlikely to cause insulin-induced stimulation of ET-1 expression that occurs specifically in endothelium.Vezf1 is an essential endothelium-specific transcription factor,binding to the ET-1 promoter core,and is a potential target for regulation of endothelial cell-type specific ET-1 gene expression.In the present study,we focus on:(1)whether insulin inhibition of GSK3βis associated with elevation of ET-1 gene expression in endothelial cells;(2) whether PI3K mediates the insulin-stimulated ET-1 expression.(3)whether Vezf1 is involved in insulin-dependent elevation of ET-1 gene expression.Materials and methods1.Cell culturesHuman pulmonary artery endothelial cells(PAEC)were cultured in EBM-2 medium with 2%fetal bovine serum(FBS)and supplements.Human umbilical venous endothelial cells(HUVEC)were maintained in RPMI 1640 medium supplemented with 4%FBS.2.Insulin and Lithium Chloride(LiCl)treatment of endothelial cellsMonolayers of human PAEC were incubated in serum-free medium for 6 h followed by incubation with 0-100 nM porcine insulin or 20 mM LiCl(GSK3βinhibitor)for 1 h.The ET-1 peptide levels in culture medium or ET-1 mRNA levels in the treated cells were analyzed by ELISA or quantitative real-time RT-PCR(qRT-PCR).Insulin inhibits GSK3βactivity through phosphorylation(ser~9).To determine whether GSK3βactivity is also inhibited by LiCl,human PAEC were treated with insulin or LiCl and subjected to ELISA assay for the relative levels of phosphorylated GSK3β(Ser~9)to total GSK3β.3.Transfection of human PAEC with siRNAMonolayers of human PAEC were transfected with GSK3β/Vezf1 siRNA and control siRNA(non-targeting).The culture medium was used for ELISA assay and the treated cells were subjected to qRT-PCR analysis after 72 h.4.Infection of human PAEC with Ad-GSK3βMonolayers of human PAEC were infected with recombinant adenovirus co-expressing GSK3βand GFP genes(Ad-GSK3β)and sham adenoviruses Ad-GFP(without insert).The infected cells were treated with 18 nM insulin for 1 h.The treated cells and conditioned medium were collected and used for the assessment of ET-1 mRNA expression and ET-1 peptide release.5.Reporter plasmids construction and luciferase assaypGL3-ET1 which contains the firefly luciferase gene under control of human ET-1 promoter(-832/+171bp)was created by inserting fragment of human ET-1 gene 5'-flanking region into pGL3-Basic.Site-directed mutagenesis of the ET-1 promoter was performed by PCR to create the mutated plasmid pGL3-ET1-m,in which,the Vezf1 banding sequence TTACCCCCACTC was mutated to TTACATCCACTC.HUVEC were transfected with pGL3-ET1/pGL3-ET1-m and pRL-SV40 which contains Renilla luciferase gene driven by the SV40 promoter was co-transfected in all experiments.48 h after transfection,the cells were treated with insulin in the absence or presence of 100 nM Wortmannin(PI3K inhibitor) or 10μM PD-98059(MAPK inhibitor)for 1,3 and 6 h.The cells were lysed for luciferase assay using Dual-Luciferase Reporter Assay System.The ratio of firefly luciferase activity to Renilla luciferase activity in each sample served as a measure of the normalized luciferase activity and represented the ET-1 promoter activity.Results1.Insulin stimulates ET-1 secretion-in endothelial cellsInsulin(10-100 nM)remarkably increased the secretion of ET-1 peptides from human PAEC in a dose-dependent manner(P＜0.05 or P＜0.01).2.LiCl mimics insulin action to stimulate the ET-1 gene expression To investigate the alteration of ET-1 mRNA caused by GSK3βsuppression, the ratios of phosphorylated and total GSK3βin 100 nM insulin or 20 mM LiCl treated cells were measured.LiCl mimics insulin to increase ser~9 phosphorylation of GSK3β(P＜0.01),indicating LiCl suppresses activities of GSK3β.qRT-PCR analysis show both LiCl and insulin treatment remarkably increased the mRNA expression of ET-1(P＜0.05 or P＜0.01).These data suggest that insulin inactivation of GSK3βis linked to elevation of ET-1 gene expression.3.Specific suppression of GSK3βexpression leads to elevation of ET-1 mRNA expression and peptides releaseTo confirm the effect of GSK3βon ET-1 gene expression,human PAEC were transfected with GSK3βsiRNA or control siRNA.The transfected cells and the culture medium were subjected to qRT-PCR and ELISA assays.The level of GSK3βmRNA in the GSK3βsiRNA transfected-cells was only 20%of control siRNA transfected-cells,which confirms siRNA-mediated inhibition of GSK3βexpression.Silencing GSK3βgene led to 50%and 100%increases in the level of ET-1 mRNA and ET-1 peptides,respectively(P＜0.01).These results further support the notion that ET-1 gene expression in endothelial cells can be regulated by GSK3βinactivation.4.GSK3βoverexpression attenuates insulin-dependent elevation of ET-1 expressionTo elucidate the role of GSK3βin modulation of ET-1 epression,human PAEC were infected with the recombinant adenovirus co-expressing GSK3βand GFP genes and assessed for the levels of ET-1 mRNA and ET-1 peptides release by qRT-PCR and ELISA.Overexpression of GSK3βin PAEC downregulated the levels of ET-1 mRNA and ET-1 peptides release in the presence or absence of 18 nM insulin,compared to sham virus-infected cells (P＜0.01 or P＜0.05).These results support that GSK3βnegatively modulates ET-1 transcription in endothelial cells.5.Vezf1 modulates ET-1 expression in endothelial cellsTo determine whether Vezf1 regulates ET-1 expression,human PAEC were transfected with Vezf1 siRNA and subjected to qRT-PCR and ELISA. When the level of Vezf1 mRNA was downregulated to 9%,the relative levels of ET-1 mRNA and ET-1 peptides were decreased to 20%and 40%in the Vezf1 siRNA-transfected cells,compared to control siRNA-transfected cells, respectively(P＜0.01).The data demonstrate that Vezf1 is linked to the transcription of ET-1 gene in endothelial cells.6.Insulin-dependent elevation of ET-1 promoter activity is mediated by PI3KTo determine whether insulin stimulation of ET-1 expression is mediated by PI3K,HUVEC were transfected with pGL3-ET1 and treated with or without 100 nM insulin in the absence or presence of 100 nM Wortmannin or 10μM PD-98059.Either Wortmannin or PD-98059 treatment alone did not significantly influence the basal activity of ET-1 promoter.Insulin stimulation remarkably upregulated ET-1 promoter activities at each time point(1-6 h). ET-1 promoter activities of insulin-stimulated cells in the presence of Wortmannin were significantly lower than treated with insulin alone and not different from control(P＜0.05 or P＜0.01).ET-1 promoter activities of insulin-stimulated cells in the presence of PD-98059 were significantly higher than control(P＜0.05 or P＜0.01)and not different from those treated by insulin alone.These observations indicate that insulin-stimulated ET-1 gene expression is mediated by PI3K.7.Mutation of Vezf1 binding element abolishes insulin-stimulated elevation of ET-1 promoter activityTo determine whether stimulation of ET-1 gene expression by insulin is linked to Vezf1,HUVEC were transfected with pGL3-ET1 or pGL3-ET1-m and stimulated with 100 nM insulin for 6 h.Insulin treatment significantly enhanced ET-1 promoter activities in pGL3-ET1 transfected cells(P＜0.05),but not pGL3-ET1-m transfected cells.The results suggest that Vezf1 is involved in insulin stimulation of ET-1 gene expression in endothelial cells. Conclusion1.These data suggest that PI3K-GSK3βsignaling play a key role in insulin stimulated ET-1 gene expression in endothelial cells.PI3K-GSK3βsignaling may be responsible for insulin stimulation of ET-1 production in insulin resistance associated-hyperinsulinemia.2.Vezf1 may be a target for ET-1 regulation by insulin in endothelial cells.