ROLE OF NRF2 IN ALVEOLAR EPITHELIAL REGENERATION DURING LUNG REPAIR

肺损伤后肺泡上皮细胞的再生是动态平衡的关键。在受损的情况下 修复 肺泡上皮 ，炎症反应不受控制，肺部炎症和 组织修复通常不会消失。这一应用中的研究将检验损伤的假设 肺 修理 损伤后是GSH/AKT信号失衡的结果 2型肺泡上皮细胞 (AEC2) 受关键的氧化还原调节蛋白Nrf2调控。这种转录因子调节基因 应激刺激下细胞保护和生存所需的表达。 在上一次 资助期内，我们发现g Nrf2基因的全局缺失损害亚致死性高氧性肺损伤后的肺修复 伤害(HLI)。为了进一步确定肺内细胞应激对HLI和修复的贡献，我们 已经培育出Nrf2的小鼠，并发现Nrf2在肺上皮细胞中缺失，但在 内皮细胞或驻留的肺泡巨噬细胞以类似的方式损害HLI的分辨率 与在Nrf2基因缺失的小鼠中观察到的结果相一致，表明肺上皮细胞-Nrf2信号在 调节促分解反应和肺修复。与这个概念一致，我们发现 缺乏Nrf2的原代AEC2(Nrf2-/-AEC2)由于氧化应激和G2/M细胞增殖不良 周期拘禁。有趣的是，外源N-乙酰半胱氨酸减轻了Nrf2-/-AEC2的氧化应激，但 不能促进G2/M期进展。相反，外源GSH缓解了压力，激活了AKT Nrf2-/-AEC2的信号转导和恢复增殖。初步的3D细胞培养实验显示 亚致死剂量Nrf2+/+小鼠AEC2对肺泡球形成的抑制作用 (48-h)与室内空气对应的高氧血症，以及暴露于任何一种环境中的Nrf2-/-小鼠的AEC2 室内空气或高氧形成无序、数量减少的“肺泡球”。NRF2-/-AEC2 添加GSH可以促进肺泡球的形成，但不能有效地促进AEC2/1反式- 差异化。因此，我们假设AEC2特异的Nrf2调节信号对 使平衡向任何一种倾斜，以实现最佳的GSH/AKT依赖的AEC2增殖和 GSH/AKT非依赖性AEC2/1反式分化。我们将使用多种方法来提供 这一假说的机械测试包括使用AEC2-组织特异性功能丧失(NRF2-/- AEC2)和功能增强(Nrf2抑制剂Keap1-/-AEC2)小鼠模型和小分子Nrf2 激活剂。具体的目标是：1)确定Nrf2调控的GSH/AKT- AEC2最佳增殖和AEC2/1转分化机制中的信号传导 2)在体内研究AEC2特异性Nrf2作为促生存和促再生机制的作用 HLI和细菌感染后，以及3)检验HLI或细菌感染后Nrf2激活的假设 感染会加速AEC的修复。高氧血症被广泛用于肺部疾病的治疗。 对于退伍军人来说，但它对患者AEC修复的影响尚不清楚。同样，不正常 细菌感染引起的肺AEC修复是退伍军人的一大健康问题。因此，这些研究 提出了具有重大科学和临床意义的 敬退伍军人。

Alveolar epithelial cell regeneration after lung injury is essential for homeostasis. In case of impaired repair of alveolar epithelium , the inflammatory response is unchecked and lung inflammation and tissue repair do not normally resolve. Studies in this application will test the hypothesis that impaired lung repair after injury is the result of GSH/AKT signaling imbalance in type 2 alveolar epithelial cells (AEC2s) regulated by crucial redox regulatory protein, Nrf2. This transcription factor regulates gene expression required for cyto-protection and survival in response to stressful stimuli. In the previous funding period, we found that g lobal deletion of Nrf2 impairs lung repair after sub-lethal hyperoxic lung injury (HLI). To further determine the contribution of lung resident cellular stress to HLI and repair, we have developed Nrf2 "floxed" mice and found that deletion of Nrf2 in lung epithelium, but not in endothelial cells or resident alveolar macrophages, impaired the resolution of HLI in a manner similar to that observed in Nrf2-null mice, suggesting that lung epithelial-Nrf2 signaling plays a major role in regulation of pro-resolution response and lung repair. Consistent with this notion, we found that primary AEC2s lacking Nrf2 (Nrf2-/-AEC2s) proliferate poorly due to oxidative stress and G2/M cell cycle arrest. Interestingly, exogenous N-acetyl-cysteine mitigated oxidative stress in Nrf2-/-AEC2s, but it could not promote G2/M progression. In contrast, exogenous GSH mitigated stress, activated AKT signaling and restored proliferation in Nrf2-/-AEC2s. Preliminary 3D cell culture experiments showed reduced size of "alveolospheres" formation by AEC2s isolated from Nrf2+/+ mice exposed to sub-lethal (48-h) hyperoxia compared to room air counterparts, and AEC2s from Nrf2-/- mice exposed to either room air or hyperoxia formed disorganized and reduced number of "alveolospheres". Nrf2-/-AEC2s supplemented with GSH exhibited improved alveolosphere formation, but not efficient AEC2/1 trans- differentiation. Thus, we hypothesize that AEC2-specific Nrf2 regulated signaling is essential for tipping the equilibrium towards either for optimal GSH/AKT-dependent AEC2 proliferation and GSH/AKT-independent AEC2/1 trans-differentiation. We will use multiple approaches to provide a mechanistic test of this hypothesis including the use of AEC2-tissue-specific loss-of-function (Nrf2-/- AEC2) and gain-of-function (Nrf2 inhibitor Keap1-/-AEC2) mouse models and small molecule Nrf2 activators. The Specific Aims to be pursued are: 1) to determine the role of Nrf2 regulated GSH/AKT- mediated signaling in the mechanisms of optimal AEC2 proliferation and AEC2/1 trans-differentiation, 2) to address in vivo the role of AEC2-specific Nrf2 as a pro-survival and pro-regenerative mechanism after HLI and bacterial infection, and 3) to test the postulate that Nrf2 activation post-HLI or bacterial infection will accelerate AEC repair. Hyperoxia is widely used in the treatment of pulmonary diseases for veterans, but its effects on AEC repair in patients are not clearly understood. Likewise, abnormal lung AEC repair caused by bacterial infection is a major health concern of veterans. Thus, the studies proposed are of major scientific and clinical importance to veterans.