Egr-1/ATF3乙酰化修饰调控MsPGN肾系膜细胞凋亡的作用与机制

Human mesangial proliferative glomerulonephritis (MsPGN) is a disease with high incidence, and the apoptosis of glomerular mesangial cells (GMCs) in the early stage of MsPGN has been confirmed, but the mechanism of GMCs apoptosis remains unclear. It has been demonstrated that sublytic C5b-9 complex is a trigger for GMCs apoptosis. And our previous studies revealed that glomerular sublytic C5b-9 deposition and GMCs apoptosis were seen in the early stage of rat Thy-1 nephritis (a model of human MsPGN). Given that transcriptional coactivator p300 and transcription factors such as the early growth responsive gene-1 (Egr-1) and activating transcription factor 3 (ATF3) are significantly up-regulated both in the renal tissue of rats with Thy-1 nephritis (in vivo) and in the GMCs stimulated with sublytic C5b-9 complexes (in vitro), and to explore the immunopathologic mechanism of the GMCs apoptosis in Thy-1 nephritis, we attempt to determine the effects of p300, Egr-1 or ATF3 expression including Egr-1 and (or) ATF3 acetylation modification mediated by p300 on GMCs apoptosis induced by sublytic C5b-9 including the relationship of Egr-1 and ATF3. The aim of this study is to elucidate the key factors resulting in GMCs apoptosis in rat Thy-1 nephritis and epigenetic regulatory mechanism, and further to provide a novel clue for preventing and treating human MsPGN.

人类系膜增生性肾炎(MsPGN)发病率高，其早期病变涉及肾小球系膜细胞(GMCs)的凋亡，但其凋亡的机制尚未阐明。亚溶解型 (sublytic) C5b-9是GMCs凋亡的启动原已被确认。我们先前的研究发现，大鼠Thy-1肾炎(MsPGN模型)早期，肾内既可查见sublytic C5b-9的沉积，又能见到GMCs凋亡。鉴于转录辅激活子p300和转录因子Egr-1及ATF3在Thy-1肾炎(体内)和sublytic C5b-9刺激的GMCs (体外)中均明显上调，故为了探讨Thy-1肾炎的免疫病理学机制，本研究拟从体内外探讨由sublytic C5b-9刺激上调的p300、Egr-1、ATF3及Egr-1或ATF3被p300乙酰化修饰后对GMCs凋亡的影响，包括Egr-1与ATF3之间的关系。旨在为揭示Thy-1肾炎GMCs凋亡的关键分子及其表观遗传学调控机制，并为防治MsPGN提供启示。

大鼠Thy-1肾炎（Thy-1N）是研究人类系膜增生性肾炎（MsPGN）常用的动物模型，而肾小球系膜细胞（GMC）凋亡又是Thy-1N大鼠发病早期的一个重要的致病环节。尽管GMC的凋亡常伴有GMC表面亚溶解型（sublytic）C5b-9复合物的沉积，且sublytic C5b-9能启动GMC凋亡已被证实，但其诱导细胞凋亡的分子机制至今尚未完全阐明。本课题的研究发现，不论是Thy-1N大鼠发病早期的肾组织（体内），还是用sublytic C5b-9刺激培养的GMC（体外），其乙酰转移酶p300、转录因子Egr-1、ATF3以及促凋亡蛋白Gadd45基因均显著上调，且升高的峰值与GMC凋亡数量的增加基本一致。进一步体外实验发现，sublytic C5b-9作为启动原能诱导GMC凋亡，其机制与上调了前述几种基因的表达明显相关。深入研究揭示，作为转录因子的Egr-1，受刺激后其表达稍先于转录因子ATF3，ATF3属于Egr-1的下游基因。Egr-1可通过促进ATF3 mRNA的稳定性，进而维持了ATF3的蛋白水平。另外，升高的Egr-1、ATF3还能分别与Gadd45β/γ基因启动子区的相应元件结合，并启动其基因的转录。而在此过程中，由sublytic C5b-9诱导表达的p300可促使ATF3第42位赖氨酸（K42）发生乙酰化修饰，更加增强了ATF3与Gadd45β/γ启动子的结合，最终导致了GMC的凋亡病变（但Egr-1被p300乙酰化的作用未被证实）。再者，体内研究证实，将应用慢病毒（LV）包装的小干扰质粒（LV-shp300，LV-shEgr-1，LV-shATF3和LV-shGadd45β/γ）导入大鼠的肾组织后，其Thy-1N大鼠肾内上述基因的表达水平、GMC凋亡病变、继发增生、细胞外基质（ECM）分泌和尿蛋白的产生均显著下调。综上全部实验提示，Thy-1N大鼠的GMC凋亡确为sublytic C5b-9所触发，而在GMC凋亡过程中，由sublytic C5b-9诱导上调的Egr-1/p300-ATF3/Gadd45通路对此起到了较为关键的作用。这些研究成果为今后探讨和防治人类MsPGN提供了新的启示。