【作者】 杨国斌； 袁国华； 陈硕；

【Author】 Yang Guobin 1,Yuan Guohua 1,Chen Shuo 2(1 State Key Laboratory Breeding Base of Basic Science of Stomatology(Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education(KLOBM),School and Hospital of Stomatology,Wuhan University, Wuhan,China;2 The University of Texas Health Science Center at San Antonio,TX,USA)

【机构】 口腔医学教育部重点实验室(武汉大学)； 武汉大学口腔医院； 武汉大学口腔医学院； 美国德州大学圣安东尼奥分校健康科学中心；

【摘要】 目的:研究成牙本质细胞中BMP2调控DSPP基因转录的具体分子机制。方法:构建含有不同长度DSPP基因启动子片段的荧光素酶报告质粒:p1318、p591和p318,以及DLX3、OSX高表达质粒:pcDNA-DLX3和pcDNA-OSX。使用重组BMP2诱导MD10-F2细胞不同时间段后,western检测细胞内DLX3、OSX和DSP表达。高表达DLX3、OSX以及使用DLX3 SiRNA抑制DLX3后检测DLX3、OSX和DSP的表达。使用双荧光素酶报告基因系统检测DLX3和OSX对DSPP基因启动子的启动活性。使用EMSA和CHIP检测DSPP基因启动子上DLX3和OSX的结合位点。使用蛋白免疫沉淀检测DLX3和OSX是否有相互作用。结果:MD10-F2细胞中,BMP2可诱导DLX3、OSX和DSP表达。高表达DLX3可使OSX、DSP高表达,抑制DLX3表达后可抑制OSX和DSP的表达;高表达OSX仅使DSP高表达,而不影响DLX3表达。DLX3和OSX可以增强p318 DSPP基因启动子活性。在DSPP基因启动子上发现了2个DLX3结合位点(-228～-221,-68～-64)和1个OSX结合位点(-148～-143)。删除DSPP基因启动子上DLX3和OSX结合位点后,可减弱DLX3和OSX对DSPP基因的启动。DLX3和OSX在细胞内有相互作用。结论:在成牙本质细胞中发现了一条新的BMP2调控DSPP的信号通路:BMP2可以诱导DLX3表达,DLX3可诱导OSX表达。DLX3和OSX可以和DSPP启动子上特定结合位点结合,同时DLX3和OSX可以相互作用形成蛋白复合体,启动DSPP基因的转录。更多还原

【Abstract】 Objective:To investigate the molecular mechanisms of BMP2 regulating DSPP transcription in odontoblasts.Methods:Different length DSPP promoter fragment constructs were generated and ligated to a luciferase reporter pGL3-Basic vector:p1318, p591 and p318.DLX3,OSX cDNA were subcloned into pcDNA 3.1 vector:pcDNA-DLX3, pcDNA-OSX.A mouse immortalized preodontoblast cell line MD 10-F2 was used.Cells were induced with BMP2 recombinant protein for different times,and the expression of DLX3,OSX,DSP were detected.Cells were transfected with pcDNA-DLX3,pcDNA-OSX or DLX3 SiRNA,then the expression levels of DLX3,OSX,DSP were detected by western blot.Dual luciferase assay system was used to detect the influence of DLX3,OSX to DSPP promoter activity.EMSA and CHIP were carried out to monitor the protein binding sites on the DSPP promoter.Coimmunoprecipitation was used to detect the protein interaction between DLX3 and OSX.Results:BMP2 induced DLX3,OSX and DSP expression in MD 10-F2 cells.Overexpressin of DLX3 could induce OSX and DSP expression,silencing DLX3 inhibited OSX and DSP expression,and overexpression of OSX induced only DSP expression but not DLX3 expression,indicating OSX is a down stream target of DLX3 and DSPP is a down stream target of DLX3 and OSX.DLX3 and OSX enhanced p318 DSPP promoter activity.Two DLX3 binding sites(-228～-221,-68～-64)and one OSX binding site(-148～-143)were found on DSPP promoter using EMSA and CHIP. Deletion of DLX3 and OSX binding sites could repressed the increasing activities of DLX3 and OSX to DSPP promoter.Using coimmunoprecipitation,the interaction between DLX3 and OSX was identified.Conclusions:A novel activation mechanism of BMP2 regulation of DSPP transcription was found:BMP2 activates DLX3 and DLX3 activates OSX.As transcription factors,DLX3 and OSX could bind to their binding sites in DSPP promoter. Then DLX3 interacts with OSX,forming a protein complex,and finally stimulates DSPP transcription.更多还原