NO-sGC-cGMP信号通路通过cGK调控PAI-2基因表达
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摘要
目的:体外分析环磷酸鸟苷是否(cGMP)通过cGMP依赖性蛋白激酶(cGK)调控人纤溶酶原激活物抑制剂2(PAI-2)基因启动子活性,阐明PAI-2基因表达的分子调控机制。方法:构建cGK的真核过表达载体及其小干扰RNA(siRNA)敲减体系,以及PAI-2基因启动子缺失突变序列双萤光素酶报告载体,瞬时转染人肺动脉平滑肌细胞(HPASMCs),在可溶性鸟苷酸环化酶(sGC)特异性刺激剂BAY 41-2272处理前后,real-time PCR检测PKG1(cGK编码基因)和PAI-2基因的mRNA水平。结果:cGK过表达能显著上调细胞内PKG1的mRNA水平和蛋白表达水平,cGK siRNA则能显著降低其表达(P<0.01);BAY 41-2272处理HPASMCs后,PKG1基因和PAI-2基因的mRNA水平均明显升高,而cGK siRNA则能抑制该上调作用(P<0.01);双萤光素酶报告基因实验显示,cGK过表达显著上调PAI-2基因启动子活性,而cGK敲减组其活性与对照组比较差异无统计学显著性;BAY 41-2272处理后PAI-2基因启动子缺失突变体PAI-2-Prom-M1组和PAI-2-Prom-M2组的萤光素酶相对活性显著增强,其它启动子缺失突变体组其活性与对照组比较差异无统计学显著性。结论:NO-sGC-cGMP信号通路通过cGK调控PAI-2表达,PAI-2基因转录起始位点5’侧翼区的-1 000 bp～-800 bp是其表达调控的关键区域之一。
AIM: To analyze the expression level of human plasminogen activator inhibitor 2(PAI-2) gene and the activity of PAI-2 gene promoter regulated by cyclic guanosine monophosphate(cGMP)-dependent protein kinase(cGK) in vitro. METHODS: The eukaryotic expression vector of cGK, cGK siRNA knockdown systems and dual-luciferase reporter vector of PAI-2 gene promoter were transiently transfected into human pulmonary arterial smooth muscle cells(HPASMCs). The mRNA levels of both PAI-2 and cGK encoding gene PKG1 were detected by real-time PCR before and after soluble guanylate cyclase(sGC) specific stimulator BAY 41-2272 treatment. RESULTS: The PKG1 expression at mRNA and protein levels was significantly up-regulated following cGK over-expression and was significantly reduced after cGK siRNA treatment(P<0.01). The mRNA levels of PKG1 and PAI-2 were remarkably increased after BAY 41-2272 treatment, while the up-regulation was suppressed by cGK siRNA(P<0.01). Dual-luciferase reporter assay results demonstrated that the activity of PAI-2 gene promoter was dramatically up-regulated following the over-expression of cGK in HPASMCs, while its activity in cGK knockdown group showed no significant difference compared with control group. The activity of PAI-2-Prom-M1 group and PAI-2-Prom-M2 group was significantly up-regulated after BAY 41-2272 treatment, while the activity of other promoter variants revealed no significant difference compared with control group. CONCLUSION: cGMP up-regulates the expression of PAI-2 dependent on cGK via modulating PAI-2 gene promoter. The key regulatory promoter region is-1 000 bp to-800 bp of the 5' flanking region upstream of the transcription initiation site.
AIM: To analyze the expression level of human plasminogen activator inhibitor 2(PAI-2) gene and the activity of PAI-2 gene promoter regulated by cyclic guanosine monophosphate(cGMP)-dependent protein kinase(cGK) in vitro. METHODS: The eukaryotic expression vector of cGK, cGK siRNA knockdown systems and dual-luciferase reporter vector of PAI-2 gene promoter were transiently transfected into human pulmonary arterial smooth muscle cells(HPASMCs). The mRNA levels of both PAI-2 and cGK encoding gene PKG1 were detected by real-time PCR before and after soluble guanylate cyclase(sGC) specific stimulator BAY 41-2272 treatment. RESULTS: The PKG1 expression at mRNA and protein levels was significantly up-regulated following cGK over-expression and was significantly reduced after cGK siRNA treatment(P<0.01). The mRNA levels of PKG1 and PAI-2 were remarkably increased after BAY 41-2272 treatment, while the up-regulation was suppressed by cGK siRNA(P<0.01). Dual-luciferase reporter assay results demonstrated that the activity of PAI-2 gene promoter was dramatically up-regulated following the over-expression of cGK in HPASMCs, while its activity in cGK knockdown group showed no significant difference compared with control group. The activity of PAI-2-Prom-M1 group and PAI-2-Prom-M2 group was significantly up-regulated after BAY 41-2272 treatment, while the activity of other promoter variants revealed no significant difference compared with control group. CONCLUSION: cGMP up-regulates the expression of PAI-2 dependent on cGK via modulating PAI-2 gene promoter. The key regulatory promoter region is-1 000 bp to-800 bp of the 5' flanking region upstream of the transcription initiation site.
引文
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[2] Medcalf RL,Stasinopoulos SJ.The undecided serpin.The ins and outs of plasminogen activator inhibitor type 2[J].FEBS J,2005,272(19):4858-4867.
[3] Stringer B,Udofa EA,Antalis TM.Regulation of the human plasminogen activator inhibitor type 2 gene:cooperation of an upstream silencer and transactivator[J].J Biol Chem,2012,287(13):10579-10589.
[4] Zhang S,Zou L,Yang T,et al.The sGC activator inhibits the proliferation and migration,promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2[J].Exp Cell Res,2015,332(2):278-287.
[5] Zou L,Xu X,Zhai Z,et al.Identification of downstream target genes regulated by the nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate signal pathway in pulmonary hypertension[J].J Int Med Res,2016,44(3):508-519.
[6] Schmidt HH,Hofmann F,Stasch JP.cGMP:generators,effectors and therapeutic implications[M].Berlin:Sprin-ger,2009:v-vi.
[7] Hoffmann LS,Chen Horng H.cGMP:transition from bench to bedside:a report of the 6th International Conference on cGMP Generators,Effectors and Therapeutic Implications[J].Naunyn Schmiedebergs Arch Pharmacol,2014,387(8):707-718.
[8] 邹丽辉,张帅,许小毛,等.肺动脉高压患者外周血细胞中PAI-2 mRNA水平及sGC激活剂对其表达的影响[J].中华医学杂志,2016,96(16):1247-1251.
[9] Sherf BA,Navarro SL,Hannah RR,et al.Dual-LuciferaseTM reporter assay:an advanced co-reporter technology integrating firefly and Renilla luciferase assays[J].Promega Notes,1996,57:2.
[10] 耿德玉,原媛,郭华荣.双荧光素酶报告基因系统的应用研究进展[J].科技资讯,2012(21):1.