KLF4对内毒素所致IL-6基因表达的调控及机制研究
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摘要
脓毒症(sepsis)是由感染引起的全身炎症反应综合征(systemic inflammatoryresponse syndrome,SIRS)。虽然其发生机制尚未完全明确,但是革兰氏阴性细菌胞壁上的脂多糖(LPS)启动体内免疫系统在脓毒症发病中的重要性已经受到广泛关注。LPS进入体内可以激活多条信号通路,直接或间接激活炎症相关转录因子,可以导致大量下游炎症介质基因的过度表达或抑制表达。IL-6是一种重要的炎症介质,在LPS刺激下可以过度表达。LPS与Toll样受体、CD14和MD-2组成的复合物结合,然后激活NF-κB信号转导通路。活化的NF-κB进入核内,激活IL-6的表达。
Kruppel样因子4(Kruppel-like factor 4 or Gut Kruppel-like factor,KLF4/GKLF)是Sp/Kruppel样锌指转录因子家族的成员之一。KLF4能够与下游基因启动子区的GC盒,CACCC盒和基础转录元件等三种结合元件相结合,从而直接调控这些基因的表达。KLF4通过这种调控作用在表皮增殖分化、抑制结肠癌、膀胱癌等恶性肿瘤发生发展中发挥重要作用。本实验室采用cDNA微阵列检测了内毒素休克小鼠肺组织基因表达谱的变化,发现KLF4基因在注射内毒素2小时的小鼠肺组织中表达上调;注射内毒素20小时后,KLF4基因的表达水平进一步增高,达正常水平的10倍以上。研究发现在巨噬细胞中,KLF4能够被IFN-γ、LPS或TNF-α诱导表达;能够通过靶基因启动子区KLF4结合元件以及与NF-κB家族成员p65的相互作用共同诱导iNOS(inducible nitric-oxidesynthase,iNOS)的表达,并通过与smad3的辅助激活子p300的相互作用而抑制smad3应答基因如PAI-I(plasminogen activator inhibitor type-Ⅰ,PAI-Ⅰ)的表达,从而发挥其促炎因子的功能。最近研究表明KLF4可以调节血管内皮细胞的炎症反应,并且在凝血过程中也有重要作用。本实验室证明KLF4在LPS诱导的巨噬细胞的炎症反应中可以直接调控IL-1β、IL-10和后期炎症介质HMGB-1的表达。这些研究表明KLF4在炎症中具有重要作用。
本实验采用KLF4过表达的RAW264.7细胞株,并采用反义寡核苷酸技术建立了内源性KLF4表达抑制的模型。根据生物信息学对炎症介质基因启动子区的分析以及本实验室的工作基础,选取IL-6作为研究对象,观察KLF4对IL-6的基因表达以及释放的影响,并对其调控机制做了初步探讨,获得以下实验结果:(1)LPS可以诱导RAW264.7巨噬细胞KLF4的表达和IL-6的释放(2)KLF4对LPS刺激下IL-6的mRNA水平表达的影响。正常状态下,RAW264.7细胞的IL-6mRNA水平表达很低;LPS刺激后,IL-6的mRNA表达明显升高,而KLF4过表达明显抑制IL-6的mRNA的表达。(3)KLF4对LPS刺激下IL-6的蛋白质水平表达的影响。KLF4过表达可以抑制LPS诱导的IL-6的释放:而KLF4内源性抑制则使这种作用消失。(4)KLF4对IL-6的表达的影响并不是通过与IL-6启动子区的KLF4结合原件直接结合而调控。(5)KLF4对LPS刺激下IL-6的基因启动子转录活性的影响。KLF4可以抑制LPS所致的IL-6基因启动子的转录活性。
上述结果表明,KLF4通过抑制IL-6的表达与释放可能在LPS所致的SIRS中有重要作用。
Sepsis is the systemic inflammatory response syndrome(SIRS) induced by infectibn.It has been emphasized that lipopolysacchride(LPS) plays an important role in the pathogenesis of sepsis by initiating the immune system.Many studies have demonstrated that LPS mediates activation of multiple signal-transduction pathways and some inflammation-related transcriptional factors,and then promotes the expression of multiple target inflammatory genes.However,the particular mechanisms of sepsis are still obscure.IL-6 is an important inflammatory mediator,which can be stimulated by LPS.LPS can activate NF-κB signaling pathway through a complex combination of components which includes Toll-like receptor,CD14 and MD-2.The activated NF-κB could translate into the nucleus,then it activates the expression of IL-6.
Kruppel-like factor 4(KLF4),a member of Sp1/KLF zinc-finger transcriptional factor family,binds to the binding sites including GC box, CACCC box and basic transcription elements on the promoters of target genes,to regulate the expression of target genes directly.By regulating the expression of the target genes,KLF4 plays important roles in cellular proliferation and differentiation,embryogenesis and the development of carcinoma.However on the expression pattem of KLF4 under LPS treatment,and the roles and mechanisms of KLF4 in regulating the expression of inflammatory genes is not delineated.Our previously research from cDNA microarray assays revealed that the expression level of KLF4 was up-regulated in lung tissues at 2 h,and further increased up to more than 10 times of the normal level at 20 h after LPS injection. KLF4 can be induced by IFN-γ,LPS or TNF-αin macrophages.KLF4 can induce iNOS(inducible nitric-oxide synthase,iNOS)through the promoter region of KLF4.And it can inhibit PAI-Ⅰ(plasminogen activator inhibitor type-Ⅰ,PAI-Ⅰ)expression.Thus it plays a key role in pro-inflammatory activation.Recent study has demonstrated that KLF4 plays an important role in regulating the endothelial cell's inflammatory response.Our studies have demonstrated that KLF4 can regulate IL-1β, IL-10 and post-inflammatory mediators HMGB-1 directly.These results indicate that KLF4 may be an important transcriptional factor participating in the expression regulation of inflammatory mediator genes.
Our study uses RAW264.7 cells over-expresses KLF4,and inhibited the expression of KLF4 gene in RAW264.7 macrophages by transfection of KLF4 antisense oligonucleotides.Then,according to the screening results from bioinformatics and our study,we selected IL-6 to investigate the effect of KLF4 on its expression and release under normal condition and LPS treatment.The results showed that,overexpression of KLF4 could inhibit the expression and release of IL-6 induced by LPS, on the other hand,the inhibition of KLF4 by antisense oligonucleotides could promote the expression and release of IL-6.The results from EMSA demonstrated that KLF4 could not bind to the KLF4 binding sites on the promoter of IL-6 gene.The results from luciferase reporter gene assay demonstrated that KLF4 could inhibit the transcriptional activity of IL-6 gene.
In summary these results show that,KLF4 may play an important role in SIRS by LPS.
Kruppel样因子4(Kruppel-like factor 4 or Gut Kruppel-like factor,KLF4/GKLF)是Sp/Kruppel样锌指转录因子家族的成员之一。KLF4能够与下游基因启动子区的GC盒,CACCC盒和基础转录元件等三种结合元件相结合,从而直接调控这些基因的表达。KLF4通过这种调控作用在表皮增殖分化、抑制结肠癌、膀胱癌等恶性肿瘤发生发展中发挥重要作用。本实验室采用cDNA微阵列检测了内毒素休克小鼠肺组织基因表达谱的变化,发现KLF4基因在注射内毒素2小时的小鼠肺组织中表达上调;注射内毒素20小时后,KLF4基因的表达水平进一步增高,达正常水平的10倍以上。研究发现在巨噬细胞中,KLF4能够被IFN-γ、LPS或TNF-α诱导表达;能够通过靶基因启动子区KLF4结合元件以及与NF-κB家族成员p65的相互作用共同诱导iNOS(inducible nitric-oxidesynthase,iNOS)的表达,并通过与smad3的辅助激活子p300的相互作用而抑制smad3应答基因如PAI-I(plasminogen activator inhibitor type-Ⅰ,PAI-Ⅰ)的表达,从而发挥其促炎因子的功能。最近研究表明KLF4可以调节血管内皮细胞的炎症反应,并且在凝血过程中也有重要作用。本实验室证明KLF4在LPS诱导的巨噬细胞的炎症反应中可以直接调控IL-1β、IL-10和后期炎症介质HMGB-1的表达。这些研究表明KLF4在炎症中具有重要作用。
本实验采用KLF4过表达的RAW264.7细胞株,并采用反义寡核苷酸技术建立了内源性KLF4表达抑制的模型。根据生物信息学对炎症介质基因启动子区的分析以及本实验室的工作基础,选取IL-6作为研究对象,观察KLF4对IL-6的基因表达以及释放的影响,并对其调控机制做了初步探讨,获得以下实验结果:(1)LPS可以诱导RAW264.7巨噬细胞KLF4的表达和IL-6的释放(2)KLF4对LPS刺激下IL-6的mRNA水平表达的影响。正常状态下,RAW264.7细胞的IL-6mRNA水平表达很低;LPS刺激后,IL-6的mRNA表达明显升高,而KLF4过表达明显抑制IL-6的mRNA的表达。(3)KLF4对LPS刺激下IL-6的蛋白质水平表达的影响。KLF4过表达可以抑制LPS诱导的IL-6的释放:而KLF4内源性抑制则使这种作用消失。(4)KLF4对IL-6的表达的影响并不是通过与IL-6启动子区的KLF4结合原件直接结合而调控。(5)KLF4对LPS刺激下IL-6的基因启动子转录活性的影响。KLF4可以抑制LPS所致的IL-6基因启动子的转录活性。
上述结果表明,KLF4通过抑制IL-6的表达与释放可能在LPS所致的SIRS中有重要作用。
Sepsis is the systemic inflammatory response syndrome(SIRS) induced by infectibn.It has been emphasized that lipopolysacchride(LPS) plays an important role in the pathogenesis of sepsis by initiating the immune system.Many studies have demonstrated that LPS mediates activation of multiple signal-transduction pathways and some inflammation-related transcriptional factors,and then promotes the expression of multiple target inflammatory genes.However,the particular mechanisms of sepsis are still obscure.IL-6 is an important inflammatory mediator,which can be stimulated by LPS.LPS can activate NF-κB signaling pathway through a complex combination of components which includes Toll-like receptor,CD14 and MD-2.The activated NF-κB could translate into the nucleus,then it activates the expression of IL-6.
Kruppel-like factor 4(KLF4),a member of Sp1/KLF zinc-finger transcriptional factor family,binds to the binding sites including GC box, CACCC box and basic transcription elements on the promoters of target genes,to regulate the expression of target genes directly.By regulating the expression of the target genes,KLF4 plays important roles in cellular proliferation and differentiation,embryogenesis and the development of carcinoma.However on the expression pattem of KLF4 under LPS treatment,and the roles and mechanisms of KLF4 in regulating the expression of inflammatory genes is not delineated.Our previously research from cDNA microarray assays revealed that the expression level of KLF4 was up-regulated in lung tissues at 2 h,and further increased up to more than 10 times of the normal level at 20 h after LPS injection. KLF4 can be induced by IFN-γ,LPS or TNF-αin macrophages.KLF4 can induce iNOS(inducible nitric-oxide synthase,iNOS)through the promoter region of KLF4.And it can inhibit PAI-Ⅰ(plasminogen activator inhibitor type-Ⅰ,PAI-Ⅰ)expression.Thus it plays a key role in pro-inflammatory activation.Recent study has demonstrated that KLF4 plays an important role in regulating the endothelial cell's inflammatory response.Our studies have demonstrated that KLF4 can regulate IL-1β, IL-10 and post-inflammatory mediators HMGB-1 directly.These results indicate that KLF4 may be an important transcriptional factor participating in the expression regulation of inflammatory mediator genes.
Our study uses RAW264.7 cells over-expresses KLF4,and inhibited the expression of KLF4 gene in RAW264.7 macrophages by transfection of KLF4 antisense oligonucleotides.Then,according to the screening results from bioinformatics and our study,we selected IL-6 to investigate the effect of KLF4 on its expression and release under normal condition and LPS treatment.The results showed that,overexpression of KLF4 could inhibit the expression and release of IL-6 induced by LPS, on the other hand,the inhibition of KLF4 by antisense oligonucleotides could promote the expression and release of IL-6.The results from EMSA demonstrated that KLF4 could not bind to the KLF4 binding sites on the promoter of IL-6 gene.The results from luciferase reporter gene assay demonstrated that KLF4 could inhibit the transcriptional activity of IL-6 gene.
In summary these results show that,KLF4 may play an important role in SIRS by LPS.
引文
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[2] Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med,2003,348(2):138-150.
[3] Opal SM, Gluck T. Endotoxin as a drug target. Crit Care Med,2003,31(1 Suppl):S57-64.
[4] Alexander WS, Hilton DJ. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu Rev Immunol,2004, 22:503-529.
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