Collagen Oxidation, Myofibroblast Activation and Age-Associated Pulmonary Fibrosis

项目总结 特发性肺纤维化(IPF)是一种破坏性的、进行性衰老相关疾病，其特征是 含有过量细胞外基质(ECM)的疤痕肺组织。氧化(氧化还原)的变化 环境早已为人所知，但氧化还原扰动影响IPF的机制 IPF的发病机制尚不完全清楚。我们实验室发现了一种特定类型的蛋白质 氧化，被称为蛋白质谷胱甘肽基化，在IPF患者的肺中增加，并与 肺功能。重要的是，谷氧还蛋白(GLRX)的活性，一种逆转蛋白质谷胱甘肽基化的酶， 在IPF患者肺组织中显著降低。我们实验室的新研究表明，胶原蛋白1A1(COL1)， 纤维化ECM的主要成分是IPF中增加的谷胱甘肽基化(COL1-SSG)的靶点。 C端原结构域中Col1-SSG的谷胱甘肽基化引起对多个 胶原酶。我们还发现，col1-SSG是一种强有力的成纤维细胞激活剂，并导致氧化 促进进一步谷胱甘肽基化的信号。胶原蛋白是内质网产生的最丰富的蛋白质 对适当的组装和加工具有挑战性，需要氧化过程。胶原蛋白有它自己的 由Calnexin(CanX)和FAM134B组成的自噬系统通过 内质网连接的自噬。随着年龄的增长，自噬功能减少，肺组织中的CANX和FAM134B均减少 来自IPF患者。这些集体观察使我们假设，在衰老的肺中，ER增加 氧化和减少胶原自噬导致COL1-SSG的分泌促进进展 通过自繁殖刺激激活肌成纤维细胞而导致肺纤维化，这种刺激可以通过 GLRX。在具体目标1中，我们将讨论蛋白质二硫键异构酶A3的活性是否重要 在Col1的氧化折叠中，以及伴随而来的内质网氧化还原蛋白1衍生的过氧化氢增加 导致CO1-SSG和与年龄相关的持续性纤维化的增加。在具体目标2中，我们将 检查纤维化肺的成纤维细胞是否分泌COL1-SSG，以及这是否与改变有关 内质网氧化还原应激和胶原自噬。在具体目标3中，我们将阐明GLRX状态是否会影响 COL1-SSG、肌成纤维细胞活化与年龄相关性肺纤维化。完成这些拟议的研究 利用老年小鼠的靶向基因敲除模型和患者分离的(Myo)成纤维细胞的组合 将为ECM通过氧化调节失调的机制(特别是 谷胱甘肽)影响CO1僵硬并促进肺纤维化的进展。已完成的 工作计划将开始揭示异常的胶原自噬在IPF中的潜在作用。最后，完成 拟议的研究还将进一步阐明GLRX发挥抗纤维化活性的作用机制。 并确定一种新开发的抗氧化版本GLRX的潜在改善的抗纤维化作用。

PROJECT SUMMARY Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive aging-associated 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.