Mechanisms of Asbestos-Induced Alveolar Epithelial Cell Injury

石棉引起的肺泡上皮细胞损伤的机制

• 批准号: 9136340
• 负责人: DAVID W KAMP
• 金额: --
• 依托单位: JESSE BROWN VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9136340
• 关键词: 8-Oxoguanine DNA Glycosylase; AGTR2 gene; Acetylation; Aconitate Hydratase; Alveolar; Amphiboles; Apoptosis; Asbestos; Asbestosis; Attenuated; Bleomycin; Bronchogenic Carcinoma; Cell Death; Cell Survival; Cells; Cessation of life; Crocidolite Asbestos; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; Disease; Electron Transport; Epithelial Cells; Event; Exposure to; Fiber; Fibrosis; Figs - dietary; Hamman-Rich syndrome; Health; Human; In Vitro; International Union Against Cancer; Lung; Lung diseases; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Mesothelioma; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Molecular; Mus; Neurons; OGG1 gene; Oxidants; Oxidative Stress; Pathogenesis; Pathway interactions; Population; Preparation; Protein Acetylation; Pulmonary Fibrosis; Reactive Oxygen Species; Resveratrol; Role; Signal Transduction; TP53 gene; Toxin; Veterans; Wild Type Mouse; biological adaptation to stress; carcinogenesis; cell injury; effective therapy; genetic approach; in vivo; injury and repair; innovation; insight; mitochondrial dysfunction; mitochondrial metabolism; mouse model; novel; overexpression; prevent; protein function; public health relevance

 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.

 描述（由申请人提供）： 石棉通过尚未完全阐明的机制引起石棉沉滞症（石棉引起的肺纤维化）和恶性肿瘤（肺癌和间皮瘤）。肺泡上皮细胞（AEC）损伤和修复的程度是毒素（如石棉）致纤维化潜力的关键决定因素。Sirtuin 3（SIRT 3）是线粒体主要的脱乙酰酶，调节线粒体代谢，使线粒体活性氧（ROS）解毒。我们已经表明，线粒体活性氧介导石棉诱导的AEC DNA损伤和凋亡的p53和p53调节的死亡途径，以及一种新的机制，线粒体人8-氧代鸟嘌呤-DNA糖基化酶1（mt-hOGG 1）防止ROS诱导的AEC凋亡，通过保存线粒体乌头酸酶（ACO-2），这反过来又防止线粒体DNA（mtDNA）损伤。与野生型（WT）小鼠相比，我们发现Ogg 1-/-小鼠增加石棉诱导的肺纤维化，部分原因是由于肺泡II型（AT 2）细胞凋亡减少ACO-2水平和增加mtDNA损伤。SIRT 3控制线粒体蛋白质包括OGG 1，ACO-2等的功能，可以在神经细胞的氧化应激环境中增强mtDNA修复。我们的初步研究表明，石棉减少AEC SIRT 3表达; SIRT 3缺乏促进石棉诱导的AEC mtDNA损伤，细胞凋亡和肺纤维化; SIRT 3强制表达（EE）减弱氧化剂诱导的AEC ACO-2耗竭，mtDNA损伤和细胞凋亡。我们的理由是AEC mtDNA是一个关键的目标，整合细胞生存/死亡信号暴露于石棉后的SIRT 3调节机制。 假设：SIRT 3通过保护mt-OGG 1/ 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年内研究的具体目标包括：（1）确定SIRT 3缺陷是否会促进AEC mtDNA损伤和内在凋亡，这是由于OGG 1和/或ACO-2乙酰化的改变导致的。我们将评估SIRT 3缺陷对OGG 1和ACO-2乙酰化、mtDNA损伤和细胞凋亡的影响。Sirt 3-/-和肺上皮细胞特异性Sirt 3-/-小鼠将用于评估SIRT 3缺乏是否增强石棉和博来霉素诱导的AEC MnSOD、OGG 1和ACO-2乙酰化、mtDNA损伤、细胞凋亡和肺纤维化。(2)为了确定SIRT 3强制表达（EE）是否可以防止石棉诱导的AEC线粒体蛋白乙酰化（总，OGG 1，ACO-2，MnSOD），mtDNA损伤和OGG 1/ 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/葡萄苷）和遗传学方法的组合。我们将使用可供我们使用的Sirt 3-EE小鼠和我们将开发的肺上皮细胞特异性Sirt 3小鼠， 评估SIRT 3是否通过阻止AEC线粒体蛋白乙酰化（OGG 1和ACO-2）、ACO-2耗竭、mtDNA损伤和凋亡来减弱石棉和博来霉素诱导的肺纤维化。(3)确定线粒体hOGG 1-EE是否减弱石棉暴露后SIRT 3缺乏的有害影响。使用过表达mt-Ogg 1和SIRT 3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（总，OGG 1，ACO-2和MnSOD），ACO-2水平，mtDNA损伤和内在凋亡。我们还将在SIRT 3沉默的存在和不存在下使用线粒体hOGG 1-EE小鼠，以确定mt-hOGG 1-EE是否通过保持AEC ACO-2水平和mtDNA同时减少细胞凋亡来减弱肺纤维化（石棉/博来霉素）。 创新：这些研究将阐明SIRT 3在维持AT 2细胞mtDNA完整性方面的重要性，这对于防止AT 2细胞凋亡和肺纤维化至关重要。拟议的研究将促进我们对肺纤维化的理解，这可能对退伍军人人群中存在的更常见的肺部疾病（即IPF和肺癌）具有更广泛的意义，这些疾病需要有效的治疗。