人CD2AP基因启动子的鉴定及其功能研究
摘要
目的:
鉴定与分析人CD2AP(CD2-associated protein,CD2AP)基因启动子的结构与功能,研究其调控的分子机制,为探讨CD2AP在相关肾脏病发病中的作用积累基础研究。
方法:
采用5'-RACE法确定人CD2AP基因的转录起始位点,利用PCR进行逐段缺失突变,克隆其5'侧翼DNA序列,构建一系列荧光素酶报告载体,转染人肾小管上皮细胞等细胞系,确定CD2AP启动子的核苷酸范围。利用点突变技术、凝胶迁移率改变试验、染色质免疫沉淀、小RNA干扰和基因过表达等实验,分析CD2AP启动子区的转录因子结合位点。观察过氧化氢(H2O2)模拟氧化应激状态下CD2AP启动子的活性变化,以及血管内皮生长因子对CD2AP启动子活性的影响。转染CD2AP启动子荧光素酶报告质粒至人胚肾293细胞,研究呼吸道合胞病毒感染对CD2AP启动子活性的影响与机制。
结果:
第一部分:确定人CD2AP基因启动子位于转录起始位点上游-558bp至-1bp之间,一个CREB和两个SP1转录因子结合位点维持其基本转录活性。
第二部分:证实在人肾小管上皮细胞中,表皮生长因子(epidermal growth factor,EGF)可以募集AP-1家族的JunD和c-fos成员形成复合体与CD2AP启动子区的一个AP-1样位点(5-TGAGCTCA-3)相结合,激活CD2AP启动子的转录活性,并对血管紧张素Ⅱ诱导的人肾小管上皮细胞凋亡产生拮抗作用。
第三部分:证实在氧化应激状态下,CD2AP启动子活性下调,CD2AP与F-actin在细胞内的共定位减少。而低剂量的血管内皮生长因子可以拮抗H2O2诱导的5CD2AP启动子活性下调。
第四部分:证实呼吸道合胞病毒感染早期激活CD2AP启动子活性,晚期则抑制其活化。AP-1家族的JunD参与呼吸道合胞病毒感染早期CD2AP启动子的活性调节。而呼吸道合胞病毒感染晚期CD2AP启动子活性的抑制至少部分依赖于RIG-1/MAVS信号的传导。
结论:
人CD2AP基因启动子位于转录起始位点上游-558至-1bp处,转录因子CREB和SP1维持其基本转录活性。EGF/AP-1/CD2AP启动子是一个促人肾小管上皮细胞生存的信号途径。低剂量的血管内皮生长因子拮抗氧化应激诱导的CD2AP启动子活性下调,可能是其肾脏保护作用的原因之一。呼吸道合胞病毒感染对CD2AP启动子活性的调节有双向作用。呼吸道合胞病毒感染后期造成的CD2AP启动子转录活性显著下调,可能是其产生肾脏损害的机制之一。
The human CD2-associated protein (CD2AP) is involved in several molecular signaling pathways and is an important factor responsible for nephrotic syndrome. Here we report the identification of the transcription start point and promoter region of the human CD2AP gene in renal tubular epithelial cells. With luciferase assays and deletion analysis, we found that the region between -558 and -1 bp ahead of the transcription start point is indispensable for the promoter activity of the human CD2AP gene. A CREB site and two SP1 sites were essential for maintaining the basal transcriptional activity of the human CD2AP promoter. Overexpression of phosphorylated CREB and SP1 transactivated the human CD2AP promoter, whereas small interfering RNA-mediated blockage of CREB and SP1 genes expressions inhibited markedly its activity. These findings provide the first analysis of the human CD2AP gene promoter and demonstrate that not only CREB but also SP1 plays a critical role in regulating basal CD2AP promoter activity in renal tubular epithelial cells.
CD2AP plays a critical role in the maintenance of the kidney filtration barrier. In this study, we showed that epidermal growth factor (EGF) led to an increase of the CD2AP protein and mRNA in the human renal proximal tubular epithelial cell line HK-2 cells, which was due to the elevation of CD2AP promoter activity. Upon deletion and mutation analysis, electrophoretic mobility shift assays and chromatin immunoprecipitation, an AP-1-like element within CD2AP promoter was characterized, by which EGF recruited c-fos and JunD, two components of AP-1, to the human CD2AP gene promoter and suppressed angiotensin II-induced apoptosis in HK-2 cells. Specific siRNA was synthesized to knock down the human CD2AP gene in HK-2 cells. We found that CD2AP deficiency attenuated the inhibitory effects of EGF and predisposed the renal tubular epithelial cells to undergo angiotensin II-induced apoptosis. Furthermore, EGF induced increases of CD2AP protein and mRNA expressions in HK-2 cells were significantly inhibited by the transfection of dominant negative JunD or c-fos vector, which was in parallel with a marked reduction of antiapoptotic effect of EGF. These results indicated that the antiapoptotic effect of EGF/CD2AP signal transduction was mediated by JunD and c-fos, at least partially. Therefore we defined a new EGF/AP-1/CD2AP mediated cell-survival signaling in the human renal proximal tubular epithelial cell.
The activity of CD2AP promoter was markedly decreased under oxidative stress conditions. Low-dose VEGF antagonized oxidative stress-induced down-regulation of CD2AP promoter activity in human embryonic kidney (HEK293) cells. At the early time of respiratory syncytial virus infection, the activity of CD2AP promoter was enhanced significantly, however, which gradually diminished in the later time of RSV infection. JunD and antiviral RIG-1/MAVS signaling were involved in the regulation of CD2AP promoter activity upon RSV infection.
This study might be useful to clarify the molecular mechanisms responsible for CD2AP associated kidney diseases.
鉴定与分析人CD2AP(CD2-associated protein,CD2AP)基因启动子的结构与功能,研究其调控的分子机制,为探讨CD2AP在相关肾脏病发病中的作用积累基础研究。
方法:
采用5'-RACE法确定人CD2AP基因的转录起始位点,利用PCR进行逐段缺失突变,克隆其5'侧翼DNA序列,构建一系列荧光素酶报告载体,转染人肾小管上皮细胞等细胞系,确定CD2AP启动子的核苷酸范围。利用点突变技术、凝胶迁移率改变试验、染色质免疫沉淀、小RNA干扰和基因过表达等实验,分析CD2AP启动子区的转录因子结合位点。观察过氧化氢(H2O2)模拟氧化应激状态下CD2AP启动子的活性变化,以及血管内皮生长因子对CD2AP启动子活性的影响。转染CD2AP启动子荧光素酶报告质粒至人胚肾293细胞,研究呼吸道合胞病毒感染对CD2AP启动子活性的影响与机制。
结果:
第一部分:确定人CD2AP基因启动子位于转录起始位点上游-558bp至-1bp之间,一个CREB和两个SP1转录因子结合位点维持其基本转录活性。
第二部分:证实在人肾小管上皮细胞中,表皮生长因子(epidermal growth factor,EGF)可以募集AP-1家族的JunD和c-fos成员形成复合体与CD2AP启动子区的一个AP-1样位点(5-TGAGCTCA-3)相结合,激活CD2AP启动子的转录活性,并对血管紧张素Ⅱ诱导的人肾小管上皮细胞凋亡产生拮抗作用。
第三部分:证实在氧化应激状态下,CD2AP启动子活性下调,CD2AP与F-actin在细胞内的共定位减少。而低剂量的血管内皮生长因子可以拮抗H2O2诱导的5CD2AP启动子活性下调。
第四部分:证实呼吸道合胞病毒感染早期激活CD2AP启动子活性,晚期则抑制其活化。AP-1家族的JunD参与呼吸道合胞病毒感染早期CD2AP启动子的活性调节。而呼吸道合胞病毒感染晚期CD2AP启动子活性的抑制至少部分依赖于RIG-1/MAVS信号的传导。
结论:
人CD2AP基因启动子位于转录起始位点上游-558至-1bp处,转录因子CREB和SP1维持其基本转录活性。EGF/AP-1/CD2AP启动子是一个促人肾小管上皮细胞生存的信号途径。低剂量的血管内皮生长因子拮抗氧化应激诱导的CD2AP启动子活性下调,可能是其肾脏保护作用的原因之一。呼吸道合胞病毒感染对CD2AP启动子活性的调节有双向作用。呼吸道合胞病毒感染后期造成的CD2AP启动子转录活性显著下调,可能是其产生肾脏损害的机制之一。
The human CD2-associated protein (CD2AP) is involved in several molecular signaling pathways and is an important factor responsible for nephrotic syndrome. Here we report the identification of the transcription start point and promoter region of the human CD2AP gene in renal tubular epithelial cells. With luciferase assays and deletion analysis, we found that the region between -558 and -1 bp ahead of the transcription start point is indispensable for the promoter activity of the human CD2AP gene. A CREB site and two SP1 sites were essential for maintaining the basal transcriptional activity of the human CD2AP promoter. Overexpression of phosphorylated CREB and SP1 transactivated the human CD2AP promoter, whereas small interfering RNA-mediated blockage of CREB and SP1 genes expressions inhibited markedly its activity. These findings provide the first analysis of the human CD2AP gene promoter and demonstrate that not only CREB but also SP1 plays a critical role in regulating basal CD2AP promoter activity in renal tubular epithelial cells.
CD2AP plays a critical role in the maintenance of the kidney filtration barrier. In this study, we showed that epidermal growth factor (EGF) led to an increase of the CD2AP protein and mRNA in the human renal proximal tubular epithelial cell line HK-2 cells, which was due to the elevation of CD2AP promoter activity. Upon deletion and mutation analysis, electrophoretic mobility shift assays and chromatin immunoprecipitation, an AP-1-like element within CD2AP promoter was characterized, by which EGF recruited c-fos and JunD, two components of AP-1, to the human CD2AP gene promoter and suppressed angiotensin II-induced apoptosis in HK-2 cells. Specific siRNA was synthesized to knock down the human CD2AP gene in HK-2 cells. We found that CD2AP deficiency attenuated the inhibitory effects of EGF and predisposed the renal tubular epithelial cells to undergo angiotensin II-induced apoptosis. Furthermore, EGF induced increases of CD2AP protein and mRNA expressions in HK-2 cells were significantly inhibited by the transfection of dominant negative JunD or c-fos vector, which was in parallel with a marked reduction of antiapoptotic effect of EGF. These results indicated that the antiapoptotic effect of EGF/CD2AP signal transduction was mediated by JunD and c-fos, at least partially. Therefore we defined a new EGF/AP-1/CD2AP mediated cell-survival signaling in the human renal proximal tubular epithelial cell.
The activity of CD2AP promoter was markedly decreased under oxidative stress conditions. Low-dose VEGF antagonized oxidative stress-induced down-regulation of CD2AP promoter activity in human embryonic kidney (HEK293) cells. At the early time of respiratory syncytial virus infection, the activity of CD2AP promoter was enhanced significantly, however, which gradually diminished in the later time of RSV infection. JunD and antiviral RIG-1/MAVS signaling were involved in the regulation of CD2AP promoter activity upon RSV infection.
This study might be useful to clarify the molecular mechanisms responsible for CD2AP associated kidney diseases.
引文
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