RTP-801 in Cigarette Smoke-Emphysema

描述(申请人提供)：我们在过去10年的工作发现，香烟烟雾破坏了肺泡结构和分子维持程序，导致肺气肿的发病。目前的应用集中在这样的概念上，即环境应激，如香烟烟雾(CS)和污染物引起的激活“变阻器”分子，控制有害的肺泡间隔细胞反应，包括氧化应激，细胞凋亡和肺泡炎症。根据这一总体范式，我们提出了针对细胞应激反应基因RTP801的机制研究，该基因是在筛选由低氧诱导因子-1a(HIF-a)上调的基因时发现的，并被证明诱导氧化应激和细胞凋亡。RTP801使mTORC1(哺乳动物雷帕霉素复合体1的靶标)失活，mTORC1控制着作为环境应激生理分子传感器的中央细胞信号通路。因此，mTORC整合了生长因子信号、细胞生长和对不利环境条件的生存适应。我们推测，CS引起的氧化应激激活了RTP801的表达，而RTP801抑制了肺泡细胞中的mTOR信号，导致了肺泡细胞的炎症和凋亡。RTP801的上调导致氧化应激触发的动力蛋白轻链分子8(LC8)从I？B释放和核因子？B-激活。我们的初步数据表明，RTP801基因敲除小鼠对香烟烟雾引起的肺部炎症、细胞凋亡和肺气肿具有保护作用。特定目的1将证明应激反应基因RTP801在吸烟诱导的小鼠肺气肿中是必需的。特异性目标2将证明RTP801通过抑制其下游靶分子mTOR而诱导香烟烟雾所致的肺部炎症和细胞凋亡。特异的Aim3将证明RTP801抑制mTOR信号导致依赖氧化应激的动力蛋白轻链分子8(LC8)从I？B移位，导致NF-？B激活。这些目标依赖于翻译和综合方法，包括基因敲除和转基因小鼠，在体和体外操纵RTP801/mTOR/AKT基因表达和信号转导与腺相关病毒或腺病毒基因转导，siRNA或短发夹RNA，针对mTOR和氧化应激的补充药物干预，以及详细的肺泡损伤的形态和细胞评估。我们的建议提出了CS介导的肺气肿肺损伤是由肺泡间隔细胞控制的概念，这是氧化应激和参与细胞应激反应的分子开关相互作用的结果。这种由RTP801介导的相互作用可能构成了先天免疫和获得性免疫的关键调节器，以及生长因子对细胞保护的关键调节器，而生长因子是肺气肿发病的核心。 公共卫生相关性：慢性阻塞性肺疾病(COPD)是造成死亡和发病的第四大原因，具有世界性影响。肺气肿是慢性阻塞性肺疾病的一部分，约有20%-60%的患者患有肺气肿。在肺气肿中，肺实际上消失了，留下了巨大的洞，严重地无法将空气吸入和呼出肺部，肺部获得氧气的能力下降。我们的概念是，环境压力，如香烟烟雾引起的压力，会导致细胞反应的激活，而细胞反应最初是为了保护宿主而设计的。其中一个传感器分子是RTP801。香烟烟雾激活RTP801会激活炎症，导致肺细胞死亡。我们的建议是证明RTP801与吸烟引起的肺气肿有关，以及这一过程中涉及的潜在机制。我们的研究与肺部疾病相关，也与吸烟引起的其他疾病表现相关，例如肌肉损伤导致的体重减轻和心血管疾病。

DESCRIPTION (provided by applicant): Our work over the past 10 years has uncovered that the disruption of alveolar structural and molecular maintenance program by cigarette smoke contributes to the pathogenesis of emphysema. The present application is centered on the concept that environmental stresses, such as caused by cigarette smoke (CS) and pollutants activate "rheostat" molecules that govern harmful alveolar septal cell responses involving oxidative stress, apoptosis, and alveolar inflammation. In line with this overall paradigm, we propose mechanistic studies targeting the cellular stress response gene RTP801, which was discovered in a screening of genes upregulated by hypoxia inducible factor-1a (HIF-a) and shown to induce oxidative stress and apoptosis. RTP801 inactivates mTORC1 (for mammalian target for rapamycin complex 1), which controls a central cell signaling pathway that acts as a physiological molecular sensor of environmental stresses. mTORC therefore integrates growth factor signaling, cell growth, and survival adaptation to adverse environmental conditions. We hypothesize that oxidative stress caused by CS activates RTP801 expression, which suppresses mTOR signaling in alveolar cells, leading to alveolar cell inflammation and apoptosis. Upregulation of RTP801 causes oxidative stress-triggered release of dynein-light chain molecule 8 (LC8) from I?B and NF-?B-activation. Our preliminary data demonstrate that RTP801 knockout mice are protected against lung inflammation, apoptosis, and emphysema caused by cigarette smoke. Specific Aim 1 will demonstrate that the stress response gene RTP801 is required for cigarette smoke- induced emphysema in mice. Specific Aim 2 will demonstrate that RTP801-induced lung inflammation and apoptosis by cigarette smoke are mediated by suppression of its downstream target mTOR. Specific Aim3 will demonstrate that RTP801 suppression of mTOR signaling causes oxidative stress-dependent displacement of the dynein-light chain molecule 8 (LC8) from I?B, leading to activation of NF-?B. These aims rely on a translational and comprehensive approach that includes knockout and transgenic mice, in vivo and in vitro manipulation of RTP801/mTOR/AKT gene expression and signaling with adenoassociated virus or adenovirus gene transduction, and siRNA or short hairpin RNA, complementary pharmacological interventions aimed at mTOR and oxidative stress, and detailed morphometric and cellular assessments of alveolar injury. Our proposal advances the concept that CS-mediated lung injury in emphysema is controlled by alveolar septal cells, as the result of the interaction of oxidative stress and molecular switches involved in cell stress responses. This interaction mediated by RTP801 may constitute a critical modulator of innate and acquired immunity and of cellular protection by growth factors, which are central to the pathogenesis of emphysema. PUBLIC HEALTH RELEVANCE: Chronic obstructive pulmonary diseases (COPD) are the 4th leading cause of mortality and morbidity, with worldwide impact. Emphysema is part of COPD and compromises about 20-60% of patients with the disease. In emphysema, the lung literally disappears, leaving behind large holes, a profound inability to breath air into and out of the lungs, and decrease in the ability of the lungs to get oxygen. Our concept is that environmental stresses, such as those cause by cigarette smoke, cause the activation of cellular responses that are originally designed to protect the host. One of these sensor molecules is RTP801. The activation of RTP801 by cigarette smoke activates inflammation and causes lung cell death. Our proposal is to prove that RTP801 is involved in emphysema caused by cigarette smoke, and the potential mechanisms involved in this process. Our studies have relevance to lung diseases, and also to other manifestations of disease caused by cigarette smoke, such as weigh loss due to muscle damage and cardiovascular disease.