Collagen Oxidation, Myofibroblast Activation and Age-Associated Pulmonary Fibrosis

项目摘要 特发性肺纤维化（IPF）是一种破坏性的、进行性的衰老相关疾病，其特征在于 含有过量细胞外基质（ECM）的疤痕肺组织。氧化还原（redox） 长期以来，人们一直知道环境伴随IPF，但氧化还原扰动影响IPF的机制 IPF发病机制仍不完全清楚。我们的实验室发现一种特殊的蛋白质 氧化，称为蛋白谷胱甘肽化，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，一种逆转蛋白谷胱甘肽化的酶， 在IPF患者肺中显著降低。我们实验室的新研究表明，胶原蛋白1A 1（COL 1）， 纤维化ECM的主要成分，是在IPF中增加的谷胱甘肽化（COL 1-SSG）的靶点。 COL 1-SSG在C-末端前结构域的谷胱甘肽化导致对多种降解的部分抗性。 胶原酶我们还发现COL 1-SSG是成纤维细胞的有效激活剂，并引起氧化应激。 促进进一步谷胱甘肽化的信号。胶原蛋白是ER中产生的最丰富的蛋白质， 是具有挑战性的，以适当地组装和处理，需要氧化过程。胶原蛋白有它自己的 由钙连接蛋白（CANX）和FAM 134 B组成的自噬系统，通过 ER连锁自噬。自噬在衰老中减少，并且CANX和FAM 134 B在肺中均减少 IPF患者。这些集体观察使我们假设，在老化的肺中，ER的增加 氧化和减少的胶原自噬导致COL 1-SSG的分泌，以促进进展 肺纤维化通过激活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，我们将讨论蛋白质二硫键异构酶A3的活性， COL 1的氧化折叠，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和年龄相关的持续性纤维化的增加。具体目标2 检查COL 1-SSG是否由来自纤维化肺的成纤维细胞分泌，以及这是否与改变有关。 在内质网氧化还原应激和胶原自噬中的作用。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性肺纤维化。完成这些拟议的研究 其利用老年小鼠中的靶向敲除模型和来自患者的分离的（肌）成纤维细胞的组合 将提供新的见解ECM失调的机制，通过氧化（特别是 谷胱甘肽化）影响COL 1硬度并促进肺纤维化的进展。完成 工作计划将开始揭示异常胶原自噬在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 并确定新创建的抗氧化版本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.