Lung Lysyl Oxidase Regulation by Metal Ion Homeostasis

描述(申请人提供)：肺细胞外基质(ECM)的结构和功能的完整性在很大程度上依赖于可溶性胶原和弹性蛋白在赖氨酰氧化酶(LO)的催化下转化为不溶的纤维聚集体，赖氨酰氧化酶是一种铜[铜(II)]依赖的酶。这种催化剂氧化这些蛋白质中的赖氨酸残基，产生稳定ECM的共价交联键。因此，LO在肺的形态发生和组织修复中起着核心作用。镉(Cd)是一种对人类有毒的金属。无论是环境污染还是香烟烟雾所致的Cd在肺内的吸入和蓄积均可引起金属离子稳态的紊乱，这可能是肺的发病机制之一。初步研究表明，大鼠肺成纤维细胞(RFL6)长期染镉后，细胞金属硫蛋白(MT)和谷胱甘肽(GSH)表达上调，LO及其胶原和弹性蛋白底物表达下调，从而形成对Cd的抗性。这些发现导致了一种假说，即Cd对肺ECM的损伤与肺气肿的发病机制有关：在长期接触Cd的过程中，肺成纤维细胞上调了MT和GSH的合成。这些细胞内的硫醇以比Cd更高的亲和力结合铜离子，从而严重扰乱了铜的动态平衡，限制了它对LO的有效性，并在mRNA、蛋白质和催化水平上促进了LO的下调。LO的下调反过来会抑制胶原和弹性蛋白的交联，有利于它们的不稳定、增溶和最终降解，并干扰它们的修复。由此产生的溶解的胶原和弹性蛋白可能会通过反馈机制抑制自身的合成，进一步扰乱这些蛋白质的合成和降解之间的平衡，并扰乱ECM，这是肺气肿发展的特征。本研究旨在验证这一假说：1)评估Cd对CDR-RFL6细胞中铜(11)稳态的扰动机制；2)从转录、翻译和翻译后水平研究LO下调CDR-RFL6细胞的机制；3)探讨LO对其胶原和弹性蛋白底物的下调以及对CDR-RFL6细胞弹性蛋白修复的影响；以及4)证明细胞MT和GSH的升高、铜稳态的扰动和LO的下调是慢性染镉大鼠肺气肿发病的关键机制。这项研究的结果有望明确Cd通过扰乱肺内铜的稳态来调节LO基因表达和加工的关键方面，从而加深我们对Cd肺气肿发病机制的理解。

DESCRIPTION (provided by applicant): The structural and functional integrity of the lung extracellular matrix (ECM) is largely dependent on the conversion of soluble collagen and elastin to insoluble, fibrous aggregates catalyzed by lysyl oxidase (LO), a copper [Cu(II)] dependent enzyme. This catalyst oxidizes lysine residues within these proteins to generate covalent cross-linkages stabilizing the ECM. Thus, LO plays a central role in the lung morphogenesis and tissue repair. Cadmium (Cd) is a toxic metal for humans. Inhalation and accumulation by the lung of Cd either from environmental contamination or from cigarette smoke induces perturbations of the metal ion homeostasis, which may be a key mechanism for the pathogenesis of the lung. Preliminary studies showed that development of Cd resistance (CdR) of rat lung fibroblasts (RFL6) following long-term Cd exposure was accompanied by upregulation of cellular metallothionein (MT) and glutathione (GSH), and downregulation of LO and its collagen and elastin substrates. These findings led to a hypothesis for the mechanisms of Cd injury to the lung ECM relevant to emphysema pathogenesis: During long term exposure to Cd, the lung fibroblasts upregulate the synthesis of MT and GSH. These intracellular thiols bind Cu ions with a higher affinity than Cd thus severely perturbing the homeostasis of Cu, limiting its availability to LO, and contributing to the downregulation of LO at the mRNA, protein and catalytic levels as shown in preliminary studies. Downregulation of LO would in turn inhibit the crosslinking of collagen and elastin, favoring their destabilization, solubilization and eventual degradation and interfering with their repair. The resulting solubilized collagen and elastin would inhibit their own synthesis possibly by a feedback mechanism, further disturbing the balance between synthesis and degradation of these proteins and disrupting the ECM, events which are characteristic of the development of emphysema. The following Specific Aims are designed to test this hypothesis: 1) To assess mechanisms of Cd perturbation of Cu(ll) homeostasis in CdR-RFL6 cells; 2) To investigate mechanisms of LO downregulation at transcriptional, translational and posttranslational levels in CdR-RFL6 cells; 3) To explore LO effects on the downregulation of its collagen and elastin substrates and on the elastin repair in CdR-RFL6 cells; and 4) To demonstrate elevation of cellular MT and GSH, perturbation of Cu homeostasis and downregulation of LO as key mechanisms in emphysema pathogenesis of rats receiving Cd by chronic administration. The outcome of the proposed research is expected to define key aspects of Cd modulation of LO gene expression and processing by perturbation of Cu homeostasis in the lung, thus enhancing our understanding of the molecular mechanisms for Cd emphysema pathogenesis.