Lung Lysyl Oxidase Regulation by Metal Ion Homeostasis

描述（由申请人提供）：肺细胞外基质（ECM）的结构和功能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酸氧化酶（LO）催化下转化为不溶性纤维聚集体，赖氨酸氧化酶是一种铜[Cu(II)]依赖性酶。这种催化剂氧化这些蛋白质中的赖氨酸残基，产生共价交联，稳定ECM。因此，LO在肺形态发生和组织修复中起核心作用。镉（Cd）是对人类有毒的金属。从环境污染或香烟烟雾中吸入和积累镉可引起金属离子稳态的扰动，这可能是肺部发病的关键机制。初步研究表明，长期Cd暴露后大鼠肺成纤维细胞（RFL6）的Cd抗性（CdR）发展伴随着细胞金属硫蛋白（MT）和谷胱甘肽（GSH）的上调，LO及其胶原和弹性蛋白底物的下调。这些发现导致了与肺气肿发病机制相关的Cd损伤肺ECM机制的假设：在长期暴露于Cd时，肺成纤维细胞上调MT和GSH的合成。这些细胞内硫醇结合Cu离子的亲和力高于Cd，因此严重扰乱了Cu的稳态，限制了其对LO的可用性，并在mRNA，蛋白质和催化水平上导致LO的下调。LO的下调反过来会抑制胶原蛋白和弹性蛋白的交联，有利于它们的不稳定、溶解和最终降解，并干扰它们的修复。由此产生的可溶性胶原蛋白和弹性蛋白可能通过反馈机制抑制其自身的合成，进一步扰乱这些蛋白质合成和降解之间的平衡，破坏ECM，这些事件是肺气肿发展的特征。本文旨在验证这一假设：1)研究Cd干扰CdR-RFL6细胞Cu（ll）稳态的机制；2)在CdR-RFL6细胞的转录、翻译和翻译后水平探讨LO下调的机制；3)探讨LO对CdR-RFL6细胞中胶原蛋白和弹性蛋白底物下调及弹性蛋白修复的影响；4)证实细胞MT和GSH升高、Cu稳态紊乱和LO下调是慢性给药Cd大鼠肺气肿发病的关键机制。本研究的结果有望通过扰乱肺内Cu稳态来确定Cd调控LO基因表达和加工的关键方面，从而增强我们对Cd肺气肿发病机制的分子机制的理解。

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.