Collagen Oxidation, Myofibroblast Activation and Age-Associated Pulmonary Fibrosis

项目总结 特发性肺纤维化(IPF)是一种以肺组织疤痕为特征的破坏性、进展性疾病。 含有过量的细胞外基质(ECM)。长期以来，氧化(氧化还原)环境的变化 已知伴随着IPF，但氧化还原扰动影响IPF发病的机制仍然存在 不完全理解。我们实验室发现了一种特殊类型的蛋白质氧化，称为蛋白质 IPF患者肺组织中谷胱甘肽水平升高，且与肺功能呈负相关。重要的是 谷氧还蛋白(GLRX)的活性显著降低，这是一种逆转蛋白质谷胱甘肽基化的酶 在IPF患者的肺组织中。我们实验室的新研究表明，胶原蛋白1A1(COL1)是 纤维化的ECM，是IPF中增加的谷胱甘肽(COL1-SSG)的靶点。Col1-的谷胱甘肽基化反应 C末端前结构域的SSG对多种胶原酶的降解有部分抵抗作用。我们也 发现COL1-SSG是成纤维细胞的有效激活剂，并产生氧化信号，促进进一步 谷胱甘肽基化。胶原蛋白是内质网产生的最丰富的蛋白质，很难正确地 组装和加工，需要氧化过程。胶原蛋白有自己的自噬系统，包括 Calnexin(CanX)和FAM134B通过内质网连接的自噬去除异常加工的胶原蛋白。自噬 在IPF患者肺组织中，CANX和FAM134B均减少。这些集体 观察结果使我们假设，在衰老的肺中，内质网氧化增加，胶原减少 自噬导致Col1-SSG的分泌，通过激活促进肺纤维化的进展 肌成纤维细胞通过一种可被GLRX减弱的自我繁殖刺激。在具体目标1中，我们将解决 在Col1氧化折叠中起重要作用的蛋白质二硫键异构酶A3的活性，以及 伴随着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。在特定目标2中，我们将检测Col1-SSG是否由 来自纤维化肺的成纤维细胞，以及这是否与内质网氧化还原压力和胶原自噬的改变有关。 在特定的目标3中，我们将阐明GLRX状态是否影响COL1-SSG、肌成纤维细胞激活和年龄- 相关的肺纤维化。完成这些拟议的研究，利用有针对性的基因敲除组合 老年小鼠模型和IPF患者分离的(Myo)成纤维细胞将为研究IPF的 细胞外基质通过氧化(特别是谷胱甘肽)调节失调影响CO1硬度和 促进肺纤维化的进展。工作计划的完成将开始揭示这种潜力 异常胶原自噬在特发性肺纤维化中的作用。最后，拟议研究的完成也将进一步阐明 GLRX发挥抗纤维化活性的作用机制及其潜在的改善抗纤维化的作用 一种新创建的抗氧化版本的GLRX的纤维化作用。

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.