Mechanisms of Asbestos-Induced Alveolar Epithelial Cell Injury

 描述(由申请人提供)： 石棉导致石棉肺(石棉引起的肺纤维化)和恶性肿瘤(肺癌和间皮瘤)，其机制尚未完全阐明。肺泡上皮细胞(AEC)损伤和修复的程度是石棉等毒素致纤维化潜力的关键决定因素。Sirtuin 3(sirtuin 3，SIRT3)是线粒体的主要脱乙酰基酶，调节线粒体代谢，使线粒体活性氧(ROS)解毒。我们发现，线粒体ROS通过P53和线粒体调节的死亡途径来介导石棉诱导的AEC DNA损伤和凋亡，以及线粒体8-氧鸟嘌呤-DNA糖基酶1(mt-hOGG1)通过保护线粒体乌头酸酶(ACO-2)来阻止ROS诱导的AEC凋亡的新机制，从而防止线粒体DNA(MtDNA)损伤。与野生型(WT)小鼠相比，我们发现Ogg1-/-小鼠增加了石棉诱导的肺纤维化，部分原因是ACO-2水平降低和mtDNA损伤增加导致肺泡II型(AT2)细胞凋亡。SIRT3控制包括OGG1、ACO-2等线粒体蛋白的功能，在神经细胞氧化应激的背景下可以增强线粒体DNA修复。我们的初步研究表明，石棉降低AEC SIRT3的表达；SIRT3缺乏促进石棉诱导的AEC mtDNA损伤、细胞凋亡和肺纤维化；SIRT3增强表达(EE)减轻氧化剂诱导的AEC ACO-2耗竭、mtDNA损伤和细胞凋亡。我们推测，AEC mtDNA是一个关键靶点，通过SIRT3调节机制整合了暴露于石棉后的细胞生存/死亡信号。假设：SIRT3通过保留mt-OGG1/ACO-2功能和减少线粒体功能障碍来减轻石棉诱导的AEC mtDNA损伤，这对限制细胞凋亡和肺纤维化至关重要。我们将在未来4年研究的具体目标包括：(1)确定SIRT3缺乏是否由于OGG1和/或指导蛋白质功能的ACO-2乙酰化的改变而促进AEC线粒体DNA损伤和内源性细胞凋亡。我们将评估SIRT3缺乏对OGG1和ACO-2乙酰化、线粒体DNA损伤和细胞凋亡的影响。SIRT3-/-和肺上皮细胞特异性SIRT3-/-小鼠将被用来评估SIRT3缺乏是否会增加石棉和博莱霉素诱导的AEC MnSOD、OGG1和ACO-2乙酰化、线粒体DNA损伤、细胞凋亡和肺纤维化。(2)确定SIRT3增强表达(EE)是否能阻止石棉诱导的AEC线粒体蛋白乙酰化(TOTAL、OGG1、ACO-2、MnSOD)、线粒体DNA损伤以及OGG1/ACO-2改变引起的细胞凋亡。我们将在体外使用药理学(白藜芦醇/长春花素)和遗传方法相结合的方法。我们将使用可供我们使用的SIRT3-EE小鼠和我们将培育的肺上皮细胞特异性SIRT3小鼠 评估SIRT3是否通过阻止AEC线粒体蛋白乙酰化(OGG1和ACO-2)、ACO-2耗竭、线粒体DNA损伤和细胞凋亡来减轻石棉和博莱霉素诱导的肺纤维化。(3)确定线粒体hOGG1-EE是否能减轻石棉暴露后SIRT3缺乏症的有害影响。使用过表达mt-Ogg1和SIRT3缺失的MLE-12细胞的体外研究将用于评估石棉诱导的线粒体蛋白乙酰化(TOTAL、OGG1、ACO-2和MnSOD)、ACO-2水平、mtDNA损伤和内在凋亡。我们还将使用线粒体hOgg1-EE小鼠，在没有和存在SIRT3沉默的情况下，确定mt-hOGG1-EE是否通过在减少细胞凋亡的同时保持AEC ACO-2水平和mtDNA来减轻肺纤维化(石棉/博莱霉素)。创新：这些研究将阐明SIRT3在维持AT2细胞线粒体DNA完整性方面的重要性，这对于预防AT2细胞凋亡和肺纤维化至关重要。建议的研究将促进我们对肺纤维化的理解，这可能对退伍军人中存在的更常见的肺部疾病(即IPF和肺癌)具有更广泛的影响，需要有效的治疗。

 DESCRIPTION (provided by applicant): Asbestos causes asbestosis (pulmonary fibrosis due to asbestos) and malignancies (lung cancer and mesothelioma) by mechanisms that are not fully elucidated. The extent of alveolar epithelial cell (AEC) injury and repair are critical determinants of the fibrogenic potential of toxins, such as asbestos. Sirtuin 3 (SIRT3), the major mitochondrial deactylase, regulates mitochondrial metabolism that detoxifies mitochondrial reactive oxygen species (ROS). We have shown that mitochondrial ROS mediate asbestos-induced AEC DNA damage and apoptosis by a p53- and mitochondria-regulated death pathway as well as a novel mechanism by which mitochondrial human 8-oxoguanine-DNA glycosylase 1 (mt-hOGG1) prevents ROS-induced AEC apoptosis by preserving mitochondrial aconitase (ACO-2), which in turn prevents mitochondrial DNA (mtDNA) damage. Compared to wild type (WT) mice, we showed that Ogg1-/- mice have increased asbestos-induced lung fibrosis due in part to alveolar type II (AT2) cell apoptosis from reduced ACO-2 levels and increased mtDNA damage. SIRT3, which controls the function of mitochondrial proteins including OGG1, ACO-2, and others, can augment mtDNA repair in the setting of oxidative stress in nerve cells. Our preliminary studies show that asbestos reduces AEC SIRT3 expression; that SIRT3 deficiency promotes asbestos-induced AEC mtDNA damage, apoptosis, and pulmonary fibrosis; and that SIRT3 enforced expression (EE) attenuates oxidant-induced AEC ACO-2 depletion, mtDNA damage, and apoptosis. We reason that AEC mtDNA is a key target that integrates cell survival / death signals following exposure to asbestos by a SIRT3-regulated mechanism. HYPOTHESIS: SIRT3 attenuates asbestos-induced AEC mtDNA damage by preserving mt-OGG1/ ACO-2 function and reducing mitochondrial dysfunction important for limiting apoptosis and lung fibrosis. Our SPECIFIC AIMS that will be examined over the next 4 years include: (1) To determine if SIRT3 deficiency promotes AEC mtDNA damage and intrinsic apoptosis due to alterations in OGG1 and/or ACO-2 acetylation that directs protein function. We will assess the effects of SIRT3 deficiency on acetylation of OGG1 and ACO-2, mtDNA damage, and apoptosis. Sirt3-/- and lung epithelial cell specific Sirt3-/- mice will be used to assess whether SIRT3 deficiency augments asbestos- and bleomycin-induced AEC MnSOD, OGG1 and ACO-2 acetrylation, mtDNA damage, apoptosis, and pulmonary fibrosis. (2) To determine whether SIRT3 enforced expression (EE) prevents asbestos-induced AEC mitochondrial protein acetylation (total, OGG1, ACO-2, MnSOD), mtDNA damage, and apoptosis resulting from altered OGG1 / ACO-2. We will use a combination of pharmacologic (resveratrol / viniferin) and genetic approaches in vitro. We will use Sirt3-EE mice available to us and lung epithelial cell specific Sirt3 mice that we will develop to assess whether SIRT3 attenuates asbestos- and bleomycin-induced lung fibrosis by preventing AEC mitochondrial protein acetylation (OGG1 and ACO-2), ACO-2 depletion, mtDNA damage, and apoptosis. (3) To determine if mitochondrial hOGG1-EE attenuates the deleterious effects of SIRT3 deficiency as occurs following asbestos exposure. In vitro studies using MLE-12 cells that overexpress mt-Ogg1 and are SIRT3 depleted will be used to assess asbestos-induced mitochondrial protein acetylation (total, OGG1, ACO-2, and MnSOD), ACO-2 levels, mtDNA damage, and intrinsic apoptosis. We will also use Mitochondrial hOgg1-EE mice in the absence and presence of SIRT3 silencing to ascertain if mt-hOGG1-EE attenuates lung fibrosis (asbestos / bleomycin) by preserving AEC ACO-2 levels and mtDNA while diminishing apoptosis. Innovation: These studies will elucidate the importance of SIRT3 in maintaining AT2 cell mtDNA integrity crucial for preventing AT2 cell apoptosis and pulmonary fibrosis. The studies proposed will advance our understanding of lung fibrosis that may have broader implications for more common lung disorders (i.e. IPF and lung cancer) present in the veteran population for which effective treatments are needed.