转录激活因子3通过调控活化T细胞核因子1介导足细胞损伤被引量：1

Actvating transcription factor 3 modulates nuclear factor of activated T-cells cytoplasmic 1 incuced podocyte injury

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摘要目的:探讨辅助转录因子——转录激活因子3(activating transcription factor 3,ATF3)调控活化T细胞核因子1(nuclear factor of activated T-cells cytoplasmic 1,NFATc1)在足细胞损伤中的作用。方法:(1)通过免疫荧光染色及激光共聚焦显微镜,观察ATF3在不同肾小球疾病患者足细胞中的表达,并通过Western印迹验证不同肾小球疾病患者肾组织ATF3的表达情况;(2)体外培养小鼠永生化足细胞,用脂多糖(LPS)100μg/ml和ionomycin 2μmol/L分别刺激足细胞0h、1h、2h、4h、6h后,采用实时荧光定量PCR(RT-PCR)和Western印迹检测ATF3 mRNA和蛋白的表达;(3)足细胞转染ATF3 siRNA后,通过Annexin V-FITC/PI双染检测细胞凋亡情况,RTPCR和Western印迹检测凋亡相关标记物BAX、Bcl-2的表达,Western印迹检测足细胞标记蛋白podocin的表达;(4)通过Western印迹、免疫荧光染色、激光共聚焦观察在LPS和Ionomycin刺激下细胞核内ATF3表达情况;(5)通过免疫染色质共沉淀(ChIP)实验观察ATF3与核转录因子NFATc1启动子之间的关系,并通过RT-PCR和Western印迹检测足细胞沉默ATF3后对NFATc1的mRNA及蛋白表达的影响。结果:(1)与正常肾组织相比,肾小球疾病患者肾组织足细胞ATF3表达明显增多;(2)用LPS和Ionomycin刺激足细胞,2h后ATF3的表达增高最明显,随后降低,到6h基本恢复正常;(3)沉默ATF3基因后,细胞凋亡率减少,凋亡相关标记物BAX下调,Bcl-2上调,并部分逆转足细胞标记蛋白podocin的下调;(4)在损伤刺激后,细胞核内ATF3表达增多;(5)ChIP实验显示ATF3与核转录因子NFATc1的启动子区域有结合,在Ionomycin刺激后,结合量明显增加,且沉默ATF3基因后,NFATc1表达减少。结论:辅助转录因子ATF3参与NFATc1介导的足细胞损伤,即通过结合NFATc1启动子,增强NFATc1表达,促进足细胞损伤。 Objective: To explore the role of transcriptional coactivator-activating transcription factor 3( ATF3) in the nuclear factor of activated T-cells cytoplasmic 1( NFATc1) induced podocyte injury. Methodology:( 1) The expression of ATF3 in the glomeruli of proteinuric patients( MCD,FSGS or DN) were observed by laser confocal microscopy and Western blotting;( 2) The conditionally immortalized mouse podocyte cell line was cultured in vitro and exposed to LPS( 100 μg/ml) or ionomycin( 2 μmol/L) for different times. Real-time quantity PCR and Western blotting were used to analyze the expression of ATF3;( 3) Cell apoptosis in ATF3 knockdown podocytes was observed by flow cytometry. Real-time quantity PCR and Western blotting were used to analyze the expression of BAX and Bcl-2,and podocin expression in ATF3 knockdown podocytes was analyzed using Western blotting;( 4) Western blotting and immunofluorescent staining were used to evaluate the change of nuclear localization of ATF3;( 5) A chromatin immunoprecipitation assay( ChIP assay) was performed to confirm the potential ATF3 binding sites in the NFATc1 promoter region. Results:( 1) The expression of ATF3 was all elevated in podocytes from MCD patients,FSGS or DN.( 2) ATF3 mRNA and protein increased in LPS or Ionomycin-treated podocytes for 1,2,4 hours. Western blot analysis demonstrated that ATF3 activation peaked at 2 hours and diminished 6 hours after LPS or ionomycin treatment.( 3) After the knockdown of ATF3,the apoptosis ratio reduced,BAX mRNA and protein was down-regulated,Bcl-2 mRNA and protein was up-regulated,podocin protein increased and recovered to a nearly normal expression.( 4) After injurystimulation,nuclear localization of ATF3 increased.( 5) ChIP assay demonstrated ATF3 were binding to the NFATc1 promoter region. When ionomycin-treated,more chromatin immunoprecipitated by ATF3 antibodies was observed. In ATF3 knockdown podocytes,reduced NFATc1 mRNA and protein expression were observed by Real-time quantity PCR and Western blotting. Conclusion: ATF3,a