The apoptosis of glomerular mesangial cells (GMCs) in the early phase of rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis (MsPGN), is primarily triggered by sublytic C5b-9. However, the mechanism of GMC apoptosis induced by sublytic C5b-9 remains unclear. In this study, we demonstrate that expressions of TNFR1-associated death domain–containing protein (TRADD) and IFN regulatory factor–1 (IRF-1) were simultaneously upregulated in the renal tissue of Thy-1N rats (in vivo) and in GMCs under sublytic C5b-9 stimulation (in vitro). In vitro, TRADD was confirmed to be a downstream gene of IRF-1, because IRF-1 could bind to TRADD gene promoter to promote its transcription, leading to caspase 8 activation and GMC apoptosis. Increased phosphorylation of p38 MAPK was verified to contribute to IRF-1 and TRADD production and caspase 8 activation, as well as to GMC apoptosis induced by sublytic C5b-9. Furthermore, phosphorylation of MEK kinase 2 (MEKK2) mediated p38 MAPK activation. More importantly, three sites (Ser153/164/239) of MEKK2 phosphorylation were identified and demonstrated to be necessary for p38 MAPK activation. In addition, silencing of renal MEKK2, IRF-1, and TRADD genes or inhibition of p38 MAPK activation in vivo had obvious inhibitory effects on GMC apoptosis, secondary proliferation, and urinary protein secretion in rats with Thy-1N. Collectively, these findings indicate that the cascade axis of MEKK2–p38 MAPK–IRF-1–TRADD–caspase 8 may play an important role in GMC apoptosis following exposure to sublytic C5b-9 in rat Thy-1N.

目的:构建大鼠生长阻滞及DNA损伤诱导基因45(growth arrest and DNA-damage-inducible gene 45,Gadd45)β/γ启动子(全长和截短)荧光素酶报告质粒,并观察在大鼠肾小球系膜细胞(glomerular messangial cells,GMCs)中过表达激活转录因子3(activating transcription factor 3,ATF3)后分别对大鼠Gadd45β/γ基因启动活性的影响.同时筛选其可能的ATF3结合位点.方法:采用PCR技术,将扩增出的大鼠Gadd45β基因启动子全长(-1 105 ～ +236 nt)和Gadd45γ基因启动子全长(-913～+72nt)分别插入到荧光素酶报告基因载体pGL3-basic中,获得Gadd45β/γ基因启动子全长荧光素酶报告质粒(pGL3-Gadd45β/γ-FL),再将pGL3-Gadd45β/γ-FL分别与课题组前期构建的大鼠野生型ATF3过表达质粒(pIRES2/ATF3)共转染GMCs,检测其荧光素酶活性,以确定ATF3对Gadd45β/γ基因的启动作用.另用生物信息学软件预测Gadd45β/γ基因启动子上ATF3潜在的结合位点,并据此构建4个Gadd45β和3个Gadd45γ基因启动子截短片段的荧光素酶报告质粒.将Gadd45β/γ基因启动子全长和各截短片段的荧光素酶报告质粒与pIRES2/ATF3共转染GMCs,再行荧光素酶活性测定,以筛选ATF3的结合位点.结果:菌液PCR及核酸测序证实,大鼠Gadd45β/-γ基因启动子(全长和截短)的荧光素酶报告质粒均构建成功.将pGL3-Gadd45β/γ-FL分别和pIRES2/ATF3共转染GMCs发现,Gadd45β/γ基因启动子活性均显著增加.而将Gadd45β启动子全长及4个截短质粒分别与pIRES2/ATF3共转染GMCs后显示,pGL3-Gadd45β-4的启动活性显著低于pGL3-Gadd45β-FL、pGL3-Gadd45β-1、pGL3-Gadd45β-2和pGL3-Gadd45β-3.提示ATF3可能结合在Gadd45β基因启动子的(-146～+23 nt)区域.同样,将Gadd45γ启动子全长及3个截短质粒分别与pIRES2/ATF3共转染GMCs后显示,pGL3-Gadd45γ-2、pGL3-Gadd45γ-3的启动活性显著低于pGL3-Gadd45γ-FL和pGL3-Gadd45γ-1.提示ATF3可能结合在Gadd45γ基因启动子的(-456～-61 nt)区域,且这段区域可能包含1个以上ATF3结合位点.结论:成功构建了大鼠Gadd45β/γ基因启动子全长及各截短片段荧光素酶报告质粒,并初步确定了ATF3在Gadd45β/γ基因启动子上的结合区域.

The apoptosis of glomerular mesangial cells (GMCs) is considered to be an important contributor to the initiation and development of rat Thy-1 nephritis (Thy-1N) and is accompanied by sublytic C5b-9 deposition. However, the mechanism by which sublytic C5b-9 triggers GMC apoptosis has not been elucidated. In this study, functional and histological examinations were performed on GMCs treated with sublytic C5b-9 (in vitro) and renal tissues of Thy-1N rats (in vivo). The in vitro studies found that sublytic C5b-9 could trigger GMC apoptosis through upregulating Egr-1, ATF3, and Gadd45 expression. Egr-1-mediated post-transcriptional modulation of ATF3, Egr-1/ATF3-enhanced Gadd45 promoter activity, and p300-mediated ATF3 acetylation were all involved in GMC apoptosis. More importantly, the effective binding elements for Egr-1 and ATF3 to Gadd45β/γ promoters and the ATF3 acetylation site were identified. In vivo, silencing renal p300, Egr-1, ATF3, and Gadd45β/γ significantly decreased GMC apoptosis, secondary GMC proliferation, and urinary protein secretion in Thy-1N rats. Together, these findings implicate that sublytic C5b-9-induced activation of Egr-1/p300-ATF3/Gadd45 axis plays a critical role in GMC apoptosis in Thy-1N rats.

目的:构建大鼠趋化因子CCL5基因启动子(全长和截短)荧光素酶报告质粒,检测大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)中过表达Kruppel样转录因子6(Kruppel-like factor 6,KLF6)对CCL5基因启动子活性的影响.同时,筛选KLF6与CCL5基因启动子区的结合位点.方法:采用PCR技术,将扩增出的大鼠CCL5基因启动子全长序列(-1744nt ～-14nt)插入荧光素酶报告基因载体pGL3-basic中,获得CCL5基因启动子全长荧光素酶报告质粒(pGL3-CCL5-FL).然后,将pGL3-CCL5-FL与大鼠野生型KLF6表达质粒(pIRES2/KLF6)共转染GMC,测定其荧光素酶活性.另用生物信息学软件预测CCL5基因启动子上KLF6潜在的结合位点,并据此构建出4个CCL5基因启动子截短的荧光素酶报告质粒(即pGL3-CCL5-1～4).将上述CCL5基因启动子全长和各截短的荧光素酶报告质粒分别与KLF6过表达质粒共转染GMC,再行荧光素酶活性的测定,初筛KLF6可能的结合部位.结果:菌液PCR以及核酸测序结果证实,上述所有启动子荧光素酶报告质粒均构建成功.pGL3-CCL5-FL和pIRES2/KLF6共转染GMC结果显示,CCL5基因启动子的活性显著增强.pGL3-CCL5-FL、pGL3-CCL5-1～4分别与pIRES2/KLF6共转染GMC后发现,pGL3-CCL5-4的启动活性显著降低.提示KLF6可能结合在CCL5基因启动子的-343nt ～-191nt区域.结论:本实验成功构建了大鼠CCL5基因启动子全长及截短荧光素酶报告质粒,并初步筛查出KLF6在CCL5基因启动子上可能的结合部位在-343nt ～-191nt区域.