Signaling mechanisms by which mitochondria regulates fibrosis in the lung

 描述（由申请方提供）：尽管特发性肺纤维化（IPF）是一种病因不明的致死性和进行性肺部疾病，其患病率随着年龄的增长而显著增加，但几乎没有新的病理生物学模型出现。肺泡富含II型肺泡上皮细胞（AECII）的重复损伤似乎是导致纤维化的关键触发事件，其与促纤维化细胞因子的分泌和增加的细胞凋亡有关。尽管AECII含有约50%的肺线粒体质量，但线粒体在IPF病理生物学中的作用尚不清楚。我们最近发现，来自人IPF肺的AECII具有与参与线粒体稳态的关键保护蛋白PTEN诱导的推定激酶1（PINK 1）的极低表达相关的畸形和功能障碍线粒体的积累（J. Clin. Invest 2015）。PINK 1的低表达与ER应激和衰老相关，导致细胞凋亡和纤维化的易感性增加。然而，没有关于ER应激如何调节PINK 1表达以及PINK 1缺失如何激活促纤维化反应的信息。我们的新的初步研究表明i）ER应激诱导ATF 3，一种转录抑制因子，其降低PINK 1水平导致线粒体通透性转换孔（mPTP）打开，从而导致线粒体去极化、凋亡和mtDNA泄漏，这是一种诱导TGF-β表达的损伤相关分子模式（DAMP），和iii）PINK 1通过与腺嘌呤核苷酸转运蛋白2/3（ANT 2/3）相互作用抑制mPTP开放，参与线粒体孔开放负调节PINK 1基因表达，ii）基于这些观察，我们假设ER应激在转录上抑制AECII中不可缺少的线粒体分子PINK 1的表达，从而通过mPTP的开放、细胞凋亡和诱导促纤维化反应的线粒体DAMP的释放导致线粒体功能障碍。为了验证这一假设，我们提出：（1）确定ER应激是否诱导转录沉默PINK 1基因表达的ATF 3，从而促进AECII中的线粒体功能障碍（目的1）。在这里，我们将使用AECII与特定的缺失和过表达的ATF 3来分析其对PINK 1 mRNA合成的贡献及其损害线粒体稳态的能力，和ATF 3敲除小鼠来研究它们对肺纤维化的易感性。此外，我们将询问PINK 1启动子，以确定由ATF 3调控的核心顺式作用元件。(2)研究PINK 1缺陷是否改变了线粒体膜通透性，从而驱动细胞凋亡和促进促纤维化反应。我们推测PINK 1通过一种新的相互作用抑制了ANT 2/3，后者促进了AECII中线粒体膜的通透性。我们将评估PINK 1-ANT 2/3相互作用如何在限制mPTP开放、减少凋亡、限制线粒体DAMP释放和诱导TGF-β表达中发挥重要作用。此外，我们将分析PINK 1的新底物是否会放大其催化活性，对PINK 1缺陷相关的线粒体损伤和肺纤维化具有预防和/或治疗作用。总的来说，这些研究提出了一个独特的分子模型，将PINK 1缺陷与线粒体功能障碍联系起来，从而导致DAMP的细胞外释放，以及促纤维化反应，为确定改善IPF纤维化严重程度的新治疗靶点奠定了基础。

 DESCRIPTION (provided by applicant): Despite the fact that Idiopathic Pulmonary Fibrosis (IPF) is a fatal and progressive lung disease of unknown etiology whose prevalence dramatically increases with age, few new pathobiologic models have emerged. Repetitive injury of mitochondrial-enriched type II alveolar epithelial cells (AECII) appears to be a key triggering event that leads to fibrosis, linked to secretion of pro- fibrotic cytokines, and increased apoptosis. Although AECII containing approximately 50% of the lung mitochondrial mass, the role of mitochondria in IPF pathobiology is unknown. We recently discovered that AECII from human IPF lung have accumulation of dysmorphic and dysfunctional mitochondria associated with very low expression of the crucial protective protein involved in mitochondrial homeostasis, PTEN-induced putative kinase 1 (PINK1) (J. Clin. Invest 2015). Low expression of PINK1 is associated with ER stress and aging, leading to increased susceptibility to cell apoptosis and fibrosis. However, no information is available how ER stress regulates PINK1 expression and how loss of PINK1 activates pro-fibrotic responses. Our novel preliminary studies suggest i) that the ER stress induces ATF3, a transcriptional repressor that reduced PINK1 levels leads to opening of the mitochondria permeability transition pore (mPTP) that sequentially leads to mitochondrial depolarization, apoptosis and leakage of mtDNA, a damage associated molecular pattern (DAMP) that induces TGF-β expression, and iii) PINK1 inhibits mPTP opening by interaction with the adenine nucleotide transporter 2/3 (ANT2/3), involved in mitochondrial pore opening negatively regulates PINK1 gene expression, ii) Based on these observations, we hypothesize that ER stress transcriptionally inhibits expression of the indispensable mitochondrial molecule, PINK1, in AECII thereby leading to mitochondrial dysfunction, via opening of the mPTP, apoptosis, and release of a mitochondrial DAMP that induces pro-fibrotic responses. To test this hypothesis, we propose: (1) To determine if ER stress induces ATF3 that transcriptionally silences PINK1 gene expression that promotes mitochondrial dysfunction in the AECII (Aim 1). Here, we will use AECII with specific deletion and overexpression of ATF3 to analyze its contribution to PINK1 mRNA synthesis and its ability to impair mitochondrial homeostasis, and ATF3 knockout mice to study their susceptibility to lung fibrosis. In addition, we will interrogate the PINK1 promoter to determine core cis-acting elements regulated by ATF3. (2) To investigate if PINK1 deficiency alters the mitochondrial membrane permeability that drives apoptosis and promotion of pro-fibrotic responses. We hypothesize that PINK1 through a novel interaction represses ANT2/3, the latter promotes mitochondrial membrane permeability in AECII. We will evaluate how PINK1-ANT2/3 interaction is essential in limiting the opening of the mPTP, reducing apoptosis, restricting release of mitochondrial DAMPs, and inducing TGF-β expression. In addition, we will analyze whether a neo-substrate for PINK1 that amplifies its catalytic activity will have a preventive and/or therapeutic effect against PINK1 deficiency-associated mitochondria damage and lung fibrosis. Overall, these studies bring forth a unique molecular model linking PINK1-deficiency to mitochondrial dysfunction, ensuing extracellular release of DAMPs, and the profibrotic response that sets the stage for identifying novel therapeutic targets in ameliorating severity of fibrosis in IPF.