【摘要】 目的本研究旨在阐明核转录因子C/EBPβ和NFκB是否协同作用影响高渗状态下COX2的基因表达。方法体外培养肾髓间质细胞(RMICs),通过病毒转导或质粒转染技术将体外构建的COX2基因突变载体转入培养细胞,经高渗培养不同时间后,采用免疫印迹、荧光素酶报告基因活性检测等方法观察培养细胞COX2蛋白表达及活性的改变,同时应用染色质免疫沉淀分析法观察COX2基因与C/EBPβ、NFκB结合能力的改变。结果免疫印迹显示,高渗刺激能明显提高对照组COX2蛋白表达,应用突变序列IκBm阻断NFκB活性后COX2表达明显下降,应用突变序列C/EBP-βP20阻断C/EBPβ亦能显著降低COX2表达,但同时运用突变序列C/EBP-βP20和IκBm无法使COX2表达进一步下降。然而,将野生型、含NFκB或C/EBPβ结合位点突变的COX2启动子萤火虫荧光素酶报告基因的重组质粒转染至RMICs,高渗培养24h后显示:高渗刺激能显著增加野生型组COX2荧光素酶的活性,NFκB位点突变无法阻断该作用,而C/EBPβ结合位点突变却能完全抑制COX2高活性。进一步应用染色质免疫沉淀分析研究显示,高渗刺激能显著增加NFκB(P65)或C/EBPβ与COX2基因的结合,并呈时间依赖性;NFκB结合位点突变不能消除高渗诱导的NFκB(P65)与COX2基因的结合增强,但在NFκB和C/EBPβ结合位点同时突变组该作用被阻断。结论在NFκB活化COX2基因转录过程中需要另一个核转录因子C/EBPβ的参与,C/EBPβ途径在高渗诱导肾脏内髓间质细胞表达COX2过程中具有重要作用。更多还原

【Abstract】 Purpose To examine whether the two transcription factors NFκB and C/EBPβ could cooperatively induce the COX2 expression following hypertonic stress. Methods Recombinant DNA with COX2 gene mutants were introduced to cultured rabbit and mouse RMICs by virus transduction or plasmid transfection respectively.After exposed to hypertonic medium for indicated periods of time,immunoblotting and luciferase activity determination were utilized to detect cultured RMICs COX2 reporter activity and their protein expression.Meanwhile chromatin immunoprecipitation(ChIP) assay was used to observe the effect of hypertonicity on binding of C/EBPβ、NFκB to COX2 promotor in RMICs in vitro. Results Immunoblotting demonstrated that hypertonicity can significantly increase COX2 protein expression in control group.The tonicity-induced COX2 expression was significantly suppressed by mutant IκB(IκBm) or C/EBPβ-P20,but no additional suppression was observed when both NFκB and C/EBPβ were simultaneously blocked by IκBm and C/EBPβ-P20.Hypertonic stress could significantly increase the COX2-firefly luciferase activity in wild-type group and mutation of NFκB binding site failed to abolish this effect,but mutation of C/EBPβ binding site could completely abolish the COX2-luciferase hyperactivity.ChIP assay demonstrated that hypertonic stress could enhance the binding of NFκB or C/EBPβ to COX2 promotor in a time dependent manner;Mutation of NFκB binding site fail to abolish the enhancement on binding of NFκB(P65) to COX2 promotor by hypertonicity,but this effect can be blocked with mutation of both NFκB and C/EBPβ binding site. Conclusions C/EBPβ is required for transcriptional activation of COX2 by NFκB.C/EBPβ pathway plays a dominant role in induction of RMIC COX2 by hypertonicity.更多还原