Sirtuins in Lung Aging and Fibrosis

 描述(由申请人提供) 特发性肺纤维化是一种老年性疾病。IPF的发病率和死亡率很高，中位存活率不到三年。IPF的发生率和患病率随着年龄的增长而急剧增加；然而，尽管这种相关性很强，但导致衰老倾向于纤维化疾病的细胞/分子机制尚未阐明。本实验室最近的研究发现，在小鼠肺损伤模型中，一种ROS生成酶NADPH氧化酶-4(NOX4)在介导成纤维细胞(FBS)向肌成纤维细胞(MFbs)分化和体内肺纤维化中起作用。我们的初步研究表明，NOX4的生物学作用可能受到线粒体sirtuin SIRT3表达的调节。细胞衰老和转化生长因子-β1都介导了SIRT3的抑制；我们的初步研究支持SIRT3的表观遗传沉默作用，包括DNA甲基化和组蛋白修饰。SIRT3的表达减少促进了衰老和促纤维化的FB表型。转化生长因子-β1诱导的SIRT3下调与线粒体蛋白的高乙酰化有关，支持线粒体生物能量学的改变在MFbs中的作用。IPF患者的肌成纤维细胞灶中低水平表达SIRT3(通过免疫组织化学方法)，以及从IPF肺分离的体外FBS中。我们开发了一种新的小鼠未消退纤维化的衰老模型；年轻小鼠(2个月)的纤维化在博莱霉素后4个月可通过&gt；50%消退，而老年小鼠(18个月)表现出持续性的纤维化反应。虽然两组小鼠的SIRT3水平在纤维化阶段都有所下降，但年轻小鼠在消退过程中表现出恢复SIRT3水平的能力；相比之下，老年小鼠表现出持续的SIRT3下调。在这项拨款提案中要检验的中心假设是，在衰老的背景下，肺损伤导致持续的、表观遗传调节的SIRT3沉默，导致线粒体功能障碍、MFB衰老和细胞凋亡抵抗，导致随着年龄的增长而持续的纤维化。我们的具体目标是：(1)确定随着细胞衰老和转化生长因子-β-1信号在肺组织中SIRT3表达下调的表观遗传学机制；(2)确定SIRT3在调节线粒体生物能量学、FB衰老和细胞凋亡抵抗中的作用；(3)确定全动物SIRT3基因敲除和/或胶原生成/间充质细胞中SIRT3的条件性基因缺失是否导致(S)幼鼠持续性纤维化。该提案中目标的完成将：(A)阐明通过细胞衰老/衰老来控制SIRT3表达的表观遗传学机制；(B)提供对SIRT3在维持线粒体生物能量学和细胞可塑性/命运中的作用的机械性见解；(C)提供概念证据，证明在年龄相关纤维化的背景下诱导SIRT3促进纤维化的消退，从而揭示一种新的治疗方法，用于治疗诸如IPF等未解决的纤维化疾病。

 DESCRIPTION (provided by applicant) Idiopathic pulmonary fibrosis (IPF) is a disease of aging. IPF carries a high morbidity and mortality, with a median survival rate of less than three years. The incidence and prevalence of IPF increase drastically with age; however, despite this strong association, cellular/molecular mechanisms that account for the aging predilection to fibrotic disease have not been elucidated. Recent studies from our laboratory have identified a reactive oxygen species (ROS)-generating enzyme, NADPH oxidase-4 (Nox4), in mediating differentiation of fibroblasts (Fbs) to myofibroblasts (MFbs), key effectors of fibrogenesis, and in in-vivo lung fibrosis in murine models of lung injury. Our preliminary studies indicate that the biological actions of Nox4 may be modulated by the expression of SIRT3, a mitochondrial sirtuin. Both cellular senescence and TGF-β1 mediate suppression of SIRT3; our preliminary studies support a role for epigenetic silencing of SIRT3 involving both DNA methylation and histone modification. Decreased expression of SIRT3 promotes a senescent and pro-fibrotic Fb phenotype. TGF-β1-induced down-regulation of SIRT3 is associated with hyper-acetylation of mitochondrial proteins, supporting a role for altered mitochondrial bioenergetics in MFbs. Human subjects with IPF express low levels of SIRT3 in myofibroblastic foci (by immunohistochemistry), as well as in ex-vivo Fbs isolated from IPF lungs. We have developed a novel aging model of non-resolving fibrosis in mice; fibrosis in young mice (2 months) resolves by >50% by 4 months post-bleomycin, whereas aged mice (18 months) show persistent fibrotic response. While SIRT3 levels decrease during the fibrogenic phase in both groups, young mice demonstrate a capacity to recover SIRT3 levels during resolution; in contrast, aged mice manifest sustained down-regulation of SIRT3. The central hypothesis to be tested in this grant proposal is that, in the context of aging, lung injury results in sustained, epigenetically-regulated SIRT3 silencing that leads to mitochondrial dysfunction, MFb senescence and apoptosis resistance, leading to persistent fibrosis with aging. Our specific aims are to: (1) determine epigenetic mechanisms for SIRT3 down-regulation with cellular senescence and with TGF-β1 signaling in lung Fbs; (2) determine the role of SIRT3 in regulating mitochondrial bioenergetics, Fb senescence and apoptosis resistance; (3) determine whether whole-animal SIRT3 knockout and/or conditional genetic deletion of SIRT3 in collagen-producing/mesenchymal cells induce(s) persistent fibrosis in young mice. The completion of the Aims in this proposal will: (a) elucidate epigenetic mechanisms that control SIRT3 expression with cellular senescence/aging; (b) provide mechanistic insights into the role of SIRT3 in maintenance of mitochondrial bioenergetics and cellular plasticity/fate; (c) provide proof-of-concept that SIRT3 induction in the context of age-associated fibrosis facilitates fibrosis resolution, uncovering a novel therapeutic approach to non-resolving fibrotic disorders such as IPF.