胰岛素经PI3K-GSK3β信号通路调节内皮细胞内皮素-1基因表达

发布时间：2018-04-20 08:55

本文选题：糖原合酶激酶-3β + GSK3β　；参考：《浙江大学》2007年博士论文

【摘要】： 内皮素-1(endothelin-1,ET-1)作为强效的血管收缩因子和有丝分裂原,在高血压、冠心病、心肌肥大等疾病的发病中起重要作用。内皮细胞ET-1表达调节主要发生于转录水平。大量文献报道胰岛素能刺激内皮细胞ET-1表达;而胰岛素抵抗时的高胰岛素血症可激活机体内皮素系统,引起的ET-1表达升高,进而导致心血管细胞的增生或肥大以及血管舒缩异常。胰岛素激活PI3激酶(P13K)和丝裂原活化蛋白激酶(MAPK)两条主要信号通路,现已知PI3K/Akt通路介导胰岛素刺激内皮细胞一氧化氮(NO)产生;但不清楚PI3K/Akt通路是否参与胰岛素刺激内皮细胞ET-1基因表达。胰岛素激活PI3K/Akt,进而磷酸化9位丝氨酸残基(Ser~9)导致糖原合酶激酶-3β(GSKβ)灭活。GSK3β是一种多功能的丝氨酸/苏氨酸激酶,可磷酸化多种转录因子并调节其活性,例如:改变转录因子与DNA的结合能力。作为胰岛素信号下游的重要激酶,GSK3β是否参与调节内皮细胞ET-1基因表达,尚未见文献报道。人ET-1基因启动子含TATA框,其活性受多种转录因子调节,如:AP-1、含锌指结构转录因子GATA-2、缺氧诱导因子-1(HIF-1);但这些转录因子的表达并不局限于血管内皮细胞;因而,这些因子单独作用不大可能引起胰岛素诱导的内皮细胞特异性基因ET-1表达。血管内皮锌指-1(Vezf1)与ET-1核心启动子结合,是调节细胞特异性基因ET-1在内皮细胞表达的潜在靶点。目前尚不清楚Vezf1是否参与胰岛素对内皮细胞ET-1表达的调节。本课题研究:(1)胰岛素对GSK3β灭活作用是否引起内皮细胞ET-1基因表达增加;(2)胰岛素上调ET-1启动子活性是否由PI3K介导;(3)Vezf1是否参与胰岛素信号对内皮细胞ET-1基因表达调节。研究目的在于阐明胰岛素调节内皮细胞ET-1表达的分子机制。材料和方法 1.细胞培养人肺动脉内皮细胞(PAEC)生长于含2%胎牛血清(FBS)的EBM-2培养液中。人脐静脉内皮细胞(HUVEC),用含4%FBS的RPMI 1640培养液培养。 2.胰岛素和氯化锂(LiCl)处理单层培养的人肺动脉内皮细胞(PAEC)血清饥饿6h后,加入含0-100nM重组猪胰岛素或20mM LiCl(GSK3β抑制剂)条件培养液,37℃孵育1h。用酶联免疫吸附法(ELISA)检测培养上清液ET-1多肽含量或用Real time定量RT-PCR(qRT-PCR)分析细胞ET-1 mRNA水平。部分细胞经胰岛素或LiCl处理后,用4%甲醛固定,ELISA法检测细胞的Ser~9磷酸化GSK3β和总GSK3β蛋白水平(GSK3p活性)。 3.GSK3βsiRNA和Vezf1 siRNA转染内皮细胞单层PAEC分别转染GSK3βNezf1 siRNA和control siRNA(不针对某种特殊序列)。37℃孵育72h后。用ELISA法测定培养上清液ET-1含量;采用qRT-PCR分析细胞ET-1和GSK3βNezf1基因表达;siRNA对目标蛋白的抑制作用经Western blot分析证实。 4.重组腺病毒Ad5-GSK3β感染内皮细胞人PAEC分别感染染重组共表达GSK3β基因和GFP基因的腺病毒表达载体(Ad5-GSK3β)和仅表达GFP基因的空白腺病毒(Ad5-GFP);细胞感染后在含或不含18nM胰岛素条件培养液中孵育1h。ELISA法检测培养上清液ET-1含量;qRT-PCR分析细胞ET-1基因表达。 5.报告基因质粒构建和荧光素酶检测从人基因组DNA调取ET-1基因启动子(-832/+171bp)片断;将其插入报告基因质粒pGL3-Basic,形成重组质粒pGL3-ET1。以构建好的pGL3-ET1为基础,将其中Vezf1结合位点(-47bp处)“TTACCCCCACTC”突变成“TTACATCCACTC”,形成一个突变质粒pGL3-ET1-m。 HUVEC用Lipofectin Reagent转染pGL3-ET1/pGL3-ET1-m;共转染pRL-SV40(含SV40启动子的海肾荧光素酶报告基因质粒)作为内参照。转染48h后,用100nM胰岛素刺激1、3和6h;部分细胞在胰岛素刺激前30min至刺激结束,培养液中含浓度为100nM的Wortmannin(P13K抑制剂)或10μM的PD-98059(MAPK抑制剂);双荧光素酶报告检测系统(Dual-LuciferseReporter Assay System)检测荧光素酶活性(表示ET-1启动子活性)。结果 1.胰岛素刺激人内皮细胞分泌ET-1 人PAEC用10、100nM胰岛素刺激1h;培养上清液中ET-1含量比对照组明显增加(P＜0.05或P＜0.01),并呈剂量依赖效应。 2.GSK3β抑制剂LiCl模仿胰岛素刺激内皮细胞ET-1基因表达人PAEC用100nM胰岛素或20mM LiCl条件培养液处理1h后,GSK3β的Ser~9磷酸化水平比对照组明显增高(P＜0.01),提示LiCl模仿胰岛素抑制GSK3β活性。PAEC用10、100nM胰岛素或20mM LiCl刺激1h,细胞ET-1mRNA水平明显高于相应对照组(P＜0.05或P＜0.01)。结果表明GSK3β活性抑制与培养内皮细胞ET-1基因表达上调有关。 3.特异性抑制GSK3β表达上调内皮细胞ET-1 mRNA表达和ET-1多肽释放为进一步证实GSK3β对ET-1基因表达的影响,人PAEC转染GSK3βsiRNA/control siRNA后,细胞和培养上清液分别用于qRT-PCR和ELISA分析。GSK3βsiRNA转染的人内皮细胞GSK3βmRNA表达仅为control siRNA转染细胞的20%,证实siRNA成功抑制GSK3β基因表达。GSK3p沉默分别上调ET-1mRNA表达50%(P＜0.01)和ET-1多肽释放100%(P＜0.01)。结果证实GSK3β活性抑制可上调内皮细胞ET-1 mRNA转录和ET-1多肽释放。 4.过表达GSK3β基因减弱胰岛素对ET-1表达的刺激作用为阐明GSK3β在胰岛素调节ET-1产生中的作用,人PAEC感染共表达GSK3β和GFP重组腺病毒后,qRT-PCR和ELISA分别检测细胞ET-1基因转录与ET-1多肽分泌。与空白病毒(无插入片断)感染细胞相比,过表达GSK3β表达明显减弱胰岛素对ET-1 mRNA表达(P＜0.01)和ET-1多肽释放的刺激作用(P＜0.05);结果支持GSK3β负调节内皮细胞ET-1转录与ET-1分泌。 5.Vezf1调节内皮细胞ET-1表达为确定Vezf1是否调节内皮细胞ET-1基因表达,人PAEC转染VeZf1 siRNA后,细胞和培养上清液用于qRT-PCR和ELISA分析。Vezf1 siRNA转染细胞Vezf1 mRNA水平仅为control siRNA转染细胞的9%,而相应ET-1 mRNA表达和ET-1多肽释放仅分别为control siRNA转染细胞的20%和40%(P＜0.01);结果表明Vezf1参与调节内皮细胞ET-1基因转录和ET-1多肽释放。 6.胰岛素上调ET-1基因启动子活性由PI3K介导现有研究已经证明,细胞内胰岛素灭活GSK3β主要由PI3K介导。为确定内皮细胞中胰岛素信号刺激ET-1基因表达是否由PI3K介导,HUVEC共转染报告基因质粒pGL3-ET1和pRL-SV40,37℃孵育48h后,加入含100nM胰岛素培养液37℃,孵育1、3和6h;部分细胞在胰岛素刺激之前30min和刺激期间,培养液中含100nM的Wortmannin或10μM的PD-98059;用双荧光素酶检测试剂盒检测ET-1启动子活性。Wortmannin和PD-98059在无胰岛素刺激条件下对ET-1启动子活性无明显影响(P＞0.05);胰岛素刺激后各时间点ET-1启动子活性比对照组(无胰岛素刺激)明显增强(P＜0.05或P＜0.01);胰岛素+Wortmannin组ET-1启动子活性明显低于胰岛素单独处理组(P＜0.05或P＜0.01),而与对照组相比,无显著性差异(P＞0.05);胰岛素+PD-98059组ET-1启动子活性与胰岛素单独处理组比较,无显著性差异(P＞0.05),且明显高于对照组(P＜0.05或P＜0.01)。结果说明胰岛素上调ET-1基因启动子活性由PI3K介导。 7.Vezf1参与胰岛素对ET-1基因表达的调节为证实胰岛素调节ET-1表达是否通过转录因子Vezf1发挥作用,HUVEC分别转染pGL3-ET1和Vezf1结合序列突变质粒pGL3-ET1-m;转染48h后,加入含100nM胰岛素培养液,37℃孵育6h;双荧光素酶检测试剂盒检测ET-1启动子活性。胰岛素明显上调转染pGL3-ET1内皮细胞的ET-1启动子活性(P＜0.05);而转染pGL3-ET1-m内皮细胞的ET-1启动子活性在胰岛素刺激后与对照组无明显差异。结果说明Vezf1参与胰岛素对ET-1基因表达调节。结论 1.本实验首次发现PI3K-GSK3β信号通路在胰岛素调节内皮细胞ET-1基因表达中起关键作用。PI3K-GSK3β信号通路可能是胰岛素抵抗时高胰岛素血症引起内皮细胞ET-1表达增加的分子基础。 2.内皮细胞特异性转录因子Vezf1参与胰岛素对内皮细胞ET-1基因表达调节。
[Abstract]:Endothelin -1 (endothelin-1, ET-1), as a powerful vasoconstrictor and mitogen, plays an important role in the pathogenesis of hypertension, coronary heart disease, and myocardial hypertrophy. The regulation of ET-1 expression in endothelial cells mainly occurs at the transcriptional level. A large number of literature reports that insulin can stimulate ET-1 expression in endothelial cells, and the high pancreas in insulin resistance. Islet emia can activate the endothelin system and increase the expression of ET-1, which may lead to the proliferation or hypertrophy of cardiovascular cells and abnormal vasomotion.
Insulin activates two main signaling pathways of PI3 kinase (P13K) and mitogen activated protein kinase (MAPK). It is known that the PI3K/Akt pathway mediates insulin stimulation of nitric oxide (NO) production in endothelial cells, but it is not clear whether the PI3K/Akt pathway participates in the insulin stimulation of the ET-1 gene expression in endothelial cells. Insulin activates PI3K/Akt and then phosphorylates 9 bits of silk. Amino acid residue (Ser~9) causes glycogen synthase kinase -3 beta (GSK beta) inactivation.GSK3 beta as a multifunctional serine / threonine kinase, which can phosphorate a variety of transcription factors and regulate its activity, for example, to change the binding capacity of transcription factors and DNA. As an important kinase downstream of insulin signals, GSK3 beta is involved in regulating the ET-1 gene of endothelial cells. The expression has not been reported in the literature.
The human ET-1 gene promoter contains the TATA frame and its activity is regulated by a variety of transcription factors, such as AP-1, zinc finger structure transcription factor GATA-2, and hypoxia inducible factor -1 (HIF-1), but these transcription factors are not limited to vascular endothelial cells; therefore, these factors are not likely to cause insulin induced endothelial specific genes alone. ET-1 expression. The binding of -1 (Vezf1) to ET-1 core promoter is a potential target for regulating the expression of cell specific gene ET-1 in endothelial cells. It is not clear whether Vezf1 is involved in the regulation of insulin on the expression of ET-1 in endothelial cells.
The study: (1) whether the effect of insulin on GSK3 beta inactivation induces the increase of ET-1 gene expression in endothelial cells; (2) whether insulin up ET-1 promoter activity is mediated by PI3K; (3) whether Vezf1 participates in the regulation of ET-1 gene expression in endothelial cells by insulin signal. The aim of the study is to clarify the molecular mechanism of insulin regulating the ET-1 expression of endothelial cells. System.
Materials and methods
1. cell culture
Human pulmonary artery endothelial cells (PAEC) were grown in EBM-2 culture medium containing 2% fetal bovine serum (FBS). Human umbilical vein endothelial cells (HUVEC) were cultured in RPMI 1640 medium containing 4%FBS.
2. insulin and lithium chloride (LiCl) treatment
After 6h was starved in human pulmonary artery endothelial cells (PAEC) in single cultured human pulmonary artery, 0-100nM recombinant pig insulin or 20mM LiCl (GSK3 beta inhibitor) conditioned medium was added. The content of ET-1 polypeptide in cultured supernatant was detected by enzyme linked immunosorbent assay (ELISA) at 37 degrees or ELISA, or Real time quantitative RT-PCR (Real time). After treatment with insulin or LiCl, the cells were fixed with 4% formaldehyde, and the levels of Ser~9 phosphorylated GSK3 beta and total GSK3 beta protein (GSK3p activity) were detected by ELISA.
Transfection of 3.GSK3 beta siRNA and Vezf1 siRNA to endothelial cells
The monolayer PAEC was incubated with GSK3 beta Nezf1 siRNA and control siRNA respectively (no specific sequence) was incubated at.37 C at.37 C. The ET-1 content of the culture supernatant was determined by ELISA method, and the cell ET-1 and the expression of the gene were analyzed by qRT-PCR, and the inhibitory effect on the target protein was confirmed by the analysis.
4. recombinant adenovirus Ad5-GSK3 beta infection of endothelial cells
Human PAEC infected respectively the recombinant adenovirus expression vector (Ad5-GSK3 beta) and the GFP gene expression vector (Ad5-GSK3 beta) and the blank adenovirus (Ad5-GFP) that only expressed the GFP gene, respectively, and incubated the ET-1 content of the supernatant in the culture medium containing or without 18nM insulin condition culture, and qRT-PCR analyzed the expression of the ET-1 gene of the cells after the infection.
5. report gene plasmid construction and luciferase detection
The ET-1 gene promoter (-832/+171bp) fragment was extracted from the human genome DNA, and was inserted into the reporter gene plasmid pGL3-Basic to form a recombinant plasmid pGL3-ET1., based on the constructed pGL3-ET1, and the Vezf1 binding site (-47bp) "TTACCCCCACTC" was mutated into a "TTACATCCACTC", forming a mutant plasmid pGL3-ET1-m..
HUVEC transfected pGL3-ET1/pGL3-ET1-m with Lipofectin Reagent, CO transfected pRL-SV40 (the sea kidney luciferase reporter gene plasmid containing SV40 promoter) as internal reference. After transfection of 48h, 100nM insulin was used to stimulate 1,3 and 6h. Or 10 M PD-98059 (MAPK inhibitor); double Luciferase Report detection system (Dual-LuciferseReporter Assay System) detected luciferase activity (indicating the activity of ET-1 promoter).
Result
1. insulin stimulates human endothelial cells to secrete ET-1
Human PAEC was stimulated by 10100nM insulin, and ET-1 content in culture supernatant increased significantly (P < 0.05 or P < 0.01), and showed a dose-dependent effect in the culture supernatant (P < 0 or P < 0.01).
2.GSK3 beta inhibitor LiCl mimics insulin stimulated ET-1 gene expression in endothelial cells
After 1h was treated with 100nM insulin or 20mM LiCl conditioned medium, the level of Ser~9 phosphorylation of GSK3 beta was significantly higher than that of the control group (P < 0.01), suggesting that the LiCl mimic insulin inhibition GSK3 beta activity was stimulated by 10100nM insulin or 20mM. The level of the cells was significantly higher than that in the corresponding control group (< 0.05 or 0.01). The inhibition of K3 beta activity was related to the up regulation of ET-1 gene expression in cultured endothelial cells.
3. specific inhibition of GSK3 beta expression upregulated ET-1 mRNA expression and ET-1 peptide release in endothelial cells.
In order to further confirm the effect of GSK3 beta on the expression of ET-1 gene, after transfection of human PAEC to GSK3 beta siRNA/control siRNA, the cells and culture supernatant were used for qRT-PCR and ELISA to analyze.GSK3 beta siRNA transfected respectively. The GSK3 beta mRNA expression was only 20% of the transfected cells. Do not increase ET-1mRNA expression 50% (P < 0.01) and ET-1 polypeptide release 100% (P < 0.01). The results showed that the inhibition of GSK3 beta activity could up regulate the ET-1 mRNA transcription of endothelial cells and the release of ET-1 polypeptide.
4. overexpression of GSK3 beta gene weakens insulin's stimulatory effect on ET-1 expression.
In order to elucidate the role of GSK3 beta in the insulin regulation of ET-1 production, after human PAEC infection co expressed GSK3 beta and GFP recombinant adenovirus, qRT-PCR and ELISA detected the transcription of ET-1 gene and the secretion of ET-1 polypeptide respectively. Compared with the blank virus (no inserted fragment) infected cells, overexpression of GSK3 beta reduced the expression of insulin to ET-1 mRNA (< 0.01). The stimulatory effect of ET-1 peptide release (P < 0.05) results in GSK3 beta negative regulation of ET-1 transcription and ET-1 secretion in endothelial cells.
5.Vezf1 regulates the expression of ET-1 in endothelial cells
In order to determine whether Vezf1 regulates the expression of ET-1 gene in endothelial cells, after transfection of human PAEC to VeZf1 siRNA, cells and culture supernatant are used for qRT-PCR and ELISA to analyze.Vezf1 siRNA transfected cells with Vezf1 mRNA level only 9%, while the corresponding expression and polypeptide release are only 20% of the transfected cells respectively. And 40% (P < 0.01); the results showed that Vezf1 was involved in regulating ET-1 gene transcription and ET-1 peptide release in endothelial cells.
6. insulin upregulates ET-1 gene promoter activity mediated by PI3K.
The current research has shown that intracellular insulin inactivation of GSK3 beta is mainly mediated by PI3K. To determine whether the ET-1 gene expression of insulin signal in endothelial cells is mediated by PI3K, HUVEC co transfection reporter gene plasmid pGL3-ET1 and pRL-SV40,37 C incubate 48h, adding 100nM insulin culture medium at 37 C to incubate 1,3 and 6h; some of the cells are in the pancreas. Before the stimulation of 30min and stimulation, the culture medium contained 100nM Wortmannin or 10 M PD-98059, and the double luciferase detection kit was used to detect the activity of ET-1 promoter.Wortmannin and PD-98059 on the ET-1 promoter activity without insulin stimulation (P > 0.05), and the activity of ET-1 promoter at each time point after insulin stimulation. Compared with the control group (P < 0.05 or P < 0.01), the activity of ET-1 promoter in insulin +Wortmannin group was significantly lower than that in the insulin treatment group (P < 0.05 or P < 0.01), but there was no significant difference compared with the control group (P > 0.05), and the activity of ET-1 promoter in the insulin + PD-98059 group was not significantly higher than that of the individual insulin treatment group. Sexual difference (P > 0.05) was significantly higher than that in the control group (P < 0.05 or P < 0.01). The results showed that the up regulation of ET-1 gene promoter activity was mediated by PI3K.
7.Vezf1 participates in the regulation of ET-1 gene expression by insulin
To verify whether insulin regulated ET-1 expression through the transcription factor Vezf1, HUVEC transfected pGL3-ET1 and Vezf1 binding sequence mutation plasmid pGL3-ET1-m respectively. After transfecting 48h, adding 100nM insulin culture solution and incubating 6h at 37 C, double luciferase detection kit was used to detect ET-1 promoter activity. Insulin obviously up-regulated transfection pGL3-ET1 inside The ET-1 promoter activity of the skin cells (P < 0.05), and the activity of ET-1 promoter transfected with pGL3-ET1-m endothelial cells was not significantly different from that of the control group after insulin stimulation. The results showed that Vezf1 was involved in the regulation of the expression of the ET-1 gene in the insulin.
conclusion
1. for the first time, we found that PI3K-GSK3 beta signaling pathway plays a key role in the expression of ET-1 gene in insulin regulated endothelial cells. The.PI3K-GSK3 beta signaling pathway may be the molecular basis for the increase of ET-1 expression in endothelial cells induced by hyperinsulinemia in insulin resistance.
2. endothelial cell specific transcription factor Vezf1 is involved in the regulation of insulin on ET-1 gene expression in endothelial cells.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341

【共引文献】