Molecular clock dysfunction in lung cellular senescence by environmental tobacco smoke

摘要 环境烟草烟雾(ETS)或“二手烟”暴露会导致应激诱发的不良反应 结果包括细胞衰老和与系统性肺炎相关的肺部毒理学效应 呼吸道疾病中的损伤和炎症。我们最近报道了ETS扰乱了生物钟 功能，诱导氧化应激，刺激异常的炎症反应。Rev-erbα是一个关键 分子时钟的组成部分，它调节核心时钟基因的表达，促进炎症和 支持衰老的调解人。我们的初步数据表明，ETS介导的细胞衰老 依赖于肺部时钟基因核受体REV-ERB的异常活动。我们的预赛 数据进一步显示，分子时钟缺陷的REV-ERBα的dna修复和炎症反应受损 KO小鼠，提示时钟蛋白REV-ERB参与调节DNA损伤/修复。 此外，REV-ERBα激动剂可降低ETS诱导的促炎和促衰老介质水平。 然而，分子时钟功能障碍在慢性ETS介导的DNA损伤/修复中的作用 衰老，毒理作用尚不清楚。Sirtuin 1(SIRT1)是一种蛋白质/组蛋白脱乙酰酶。 被ETS还原，导致细胞衰老、氧化应激和异常炎症反应。我们的 初步数据表明，SIRT1调控REV-ERBα丰度。然而，目前尚不清楚SIRT1是否 通过REV-ERBα依赖机制调节肺细胞衰老和炎症反应。 我们假设ETS扰乱了分子时钟功能，特别是REV-ERBα丰度，导致 DNA损伤通过SIRT1依赖的肺毒性机制启动细胞衰老。我们 建议通过确定内毒素诱导的REV-ERB中断对细胞的影响来检验这一假设 基于以下三项的小鼠模型的衰老、DNA损伤/修复和毒理学反应 目的：目的1：确定ETS介导的REV-1基因破坏的分子机制。 Erb分子时钟功能导致细胞衰老及衰老相关炎症 表型(SASP)；目的2：确定REV-ERB通过非同源末端参与DNA修复 在ETS暴露后的细胞衰老过程中加入(NHEJ)；以及目标3：确定SIRT1在 调节ETS后REV-ERBDNA和损伤诱导的肺细胞衰老。 这一提议的结果将揭开分子钟在调节DNA损伤中的作用。 在外源生物对ETS暴露的反应中，肺细胞通过依赖SIRT1的机制衰老。 反过来，我们的发现将对药物疗法的发展具有巨大的翻译潜力。 靶向分子时钟功能改善肺细胞衰老和DNA损伤的实验研究 长期接触ETS。

SUMMARY Environmental tobacco smoke (ETS) or “secondhand smoke” exposure leads to stress-induced adverse outcomes including cellular senescence and toxicological effects on the lungs associated with systemic injury and inflammation in airway disorders. We have recently reported that ETS disrupts circadian clock function, induces oxidative stress and stimulates abnormal inflammatory responses. REV-ERBα is a critical component of the molecular clock, which regulates the expression of core clock genes, pro-inflammatory and pro-senescent mediators. Our preliminary data suggest that ETS-mediated cellular senescence is dependent on irregular activity of the clock gene nuclear receptor REV-ERB in the lungs. Our preliminary data further show an impaired DNA repair and inflammatory response in molecular clock deficient REV-ERBα KO mice, suggesting the involvement of the clock protein REV-ERB in regulating DNA damage/repair. Further, REV-ERBα agonists reduce ETS-induced levels of pro-inflammatory and pro-senescent mediators. However, the role of molecular clock dysfunction in chronic ETS-mediated DNA damage/repair, cellular senescence, and toxicological effects remains unknown. Sirtuin 1 (SIRT1), a protein/histone deacetylase, is reduced by ETS, resulting in cellular senescence, oxidative stress and abnormal inflammatory responses. Our preliminary data show that SIRT1 regulates REV-ERBα abundance. However, it is not known whether SIRT1 regulates lung cellular senescence and inflammatory responses through a REV-ERBα-dependent mechanism. We hypothesize that ETS disrupts molecular clock function, specifically REV-ERBα abundance, resulting in DNA damage-initiated cellular senescence through a SIRT1-dependent mechanism in pulmonary toxicity. We propose to test this hypothesis by determining the impact of ETS-induced REV-ERBdisruption on cellular senescence, DNA damage/repair, and toxicological responses in a mouse model based on the following three Aims: Aim 1: Determine the molecular mechanism through which ETS-mediated disruption of REV- ERBmolecular clock function leads to cellular senescence and senescence-associated inflammatory phenotype (SASP); Aim 2: Determine the involvement of REV-ERB in DNA repair by non-homologous end joining (NHEJ) during cellular senescence following ETS exposure; and Aim 3: Determine the role of SIRT1 in regulating REV-ERB and DNA damage-induced lung cellular senescence following ETS. The outcome of this proposal will unravel the role of the molecular clock in regulating DNA damage-induced lung cellular senescence via a SIRT1-dependent mechanism during the xenobiotic response to ETS exposure. In turn, our findings will have great translational potential for the development of pharmacological therapies based on targeting molecular clock function to ameliorate lung cellular senescence and DNA damage following chronic exposure to ETS.