Collagen Oxidation, Myofibroblast Activation and Age-Associated Pulmonary Fibrosis

项目摘要 特发性肺纤维化（IPF）是一种以肺组织瘢痕化为特征的破坏性进行性疾病 含有过量的细胞外基质（ECM）。氧化（氧化还原）环境的变化长期以来一直是 已知伴随IPF，但氧化还原扰动影响IPF发病机制的机制仍然存在 不完全理解。我们的实验室已经发现，一种特殊类型的蛋白质氧化，称为蛋白质 在IPF患者的肺中，谷胱甘肽化增加，并且与肺功能负相关。重要的是， 谷氧还蛋白（GLRX）（一种逆转蛋白质谷胱甘肽化酶）的活性强烈降低 在IPF患者肺中。我们实验室的新研究表明，胶原蛋白1A 1（COL 1）， 纤维化ECM是在IPF中增加的谷胱甘肽化（COL 1-SSG）的靶点。COL 1-的谷胱甘肽化 SSG在C-末端前结构域引起对多种胶原酶降解的部分抗性。我们也 发现COL 1-SSG是成纤维细胞的有效激活剂，并引起氧化信号，促进进一步的 谷胱甘肽化胶原蛋白是ER中产生的最丰富的蛋白质， 组装和加工，需要氧化过程。胶原蛋白有自己的自噬系统， 钙连接蛋白（CANX）和FAM 134 B以通过ER连接的自噬去除异常加工的胶原。自噬 随着年龄的增长而降低，并且CANX和FAM 134 B在IPF患者的肺中均降低。这些集体 观察结果使我们假设，在老化的肺中，ER氧化增加和胶原蛋白减少， 自噬导致COL 1-SSG的分泌，通过激活促进肺纤维化的进展 肌成纤维细胞通过自我传播的刺激，可以减少GLRX。在具体目标1中， 蛋白质二硫键异构酶A3的活性是否在COL 1的氧化折叠中起重要作用，以及 ER氧化还原蛋白1衍生的过氧化氢的伴随增加有助于COL 1-SSG， 与年龄相关的持续性纤维化在具体目标2中，我们将检查COL 1-SSG是否由 纤维化肺的成纤维细胞，以及这是否与ER氧化还原应激和胶原自噬的改变有关。 在具体目标3中，我们将阐明GLRX状态是否影响COL 1-SSG，肌成纤维细胞活化和年龄。 相关的肺纤维化。完成这些利用靶向敲除结合 老年小鼠模型和IPF患者分离的（肌）成纤维细胞将为IPF的治疗提供新的见解。 ECM通过氧化（特别是谷胱甘肽化）失调影响COL 1硬度的机制， 促进肺纤维化的发展。完成工作计划将开始揭示潜在的 异常胶原自噬在IPF中的作用。最后，完成拟议的研究也将进一步阐明 GLRX发挥抗纤维化活性的作用机制，并确定潜在的改善的抗纤维化作用。 纤维化作用的新创建的抗氧化版本的GLRX。

PROJECT SUMMARY Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease characterized by scarred lung tissue containing excessive extracellular matrix (ECM). Changes in the oxidative (redox) environment have long been known to accompany IPF, yet the mechanisms whereby redox perturbations affect IPF pathogenesis remain incompletely understood. Our laboratory has discovered that a specific type of protein oxidation, termed protein glutathionylation, is increased in the lungs of IPF patients and inversely correlates with lung function. Importantly, the activity of glutaredoxin (GLRX), an enzyme that reverses protein glutathionylation, was strongly decreased in IPF patient lungs. New studies from our laboratory showed that collagen 1A1 (COL1), a major component of the fibrotic ECM, is a target for glutathionylation (COL1-SSG) that is increased in IPF. Glutathionylation of COL1- SSG in the c-terminal pro-domain caused partial resistance to degradation by multiple collagenases. We also discovered that COL1-SSG is a potent activator of fibroblasts and causes oxidative signals that promote further glutathionylation. Collagens are the most abundant proteins produced in the ER and are challenging to properly assemble and process, requiring oxidative processes. Collagen has its own autophagy system consisting of calnexin (CANX) and FAM134B to remove aberrantly processed collagen via ER-linked autophagy. Autophagy is decreased in aging, and both CANX and FAM134B are decreased in lungs from IPF patients. These collective observations led us to hypothesize that in the aging lung, increases in ER oxidation and diminished collagen autophagy result in the secretion of COL1-SSG to promote the progression of pulmonary fibrosis by the activation myofibroblasts via a self-propagating stimulus that can be diminished by GLRX. In Specific Aim 1 we will address whether the activity of protein disulfide isomerase A3 which is important in the oxidative folding of COL1, and attendant increases in ER oxidoreductin 1-derived hydrogen peroxide contribute to COL1-SSG and the increases in age-associated persistent fibrosis. In Specific Aim 2 we will examine whether COL1-SSG is secreted by fibroblasts from fibrotic lung and whether this is linked to alterations in ER redox stress and collagen autophagy. In Specific Aim 3 we will elucidate whether GLRX status affects COL1-SSG, myofibroblast activation and age- associated lung fibrosis. Completion of these proposed studies that utilize a combination of targeted knockout models in aged mice and isolated (myo)fibroblasts from patients with IPF will provide novel insights into the mechanisms by which ECM dysregulation via oxidation (specifically glutathionylation) affects COL1 stiffness and promotes the progression of pulmonary fibrosis. Completion of the workplan will begin to unravel the potential role of aberrant collagen autophagy in IPF. Finally, completion of the proposed studies will also further elucidate the mechanisms of action whereby GLRX exerts anti-fibrotic activity and identify the potential improved anti- fibrotic action of a newly created oxidation-resistant version of GLRX.