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取决于可溶性胶原蛋白和弹性蛋白向不溶性纤维聚集体的转化，该转化由赖氨酰氧化酶（LO）（一种铜[Cu（II）]依赖性酶）催化。该催化剂氧化这些蛋白质内的赖氨酸残基以产生稳定ECM的共价交联。因此，LO在肺形态发生和组织修复中起着重要作用。镉（Cd）是一种对人体有毒的金属。无论是从环境污染或香烟烟雾的镉吸入和积累的肺诱导的金属离子的稳态，这可能是肺的发病机制的一个关键扰动。初步研究表明，长期镉暴露后，大鼠肺成纤维细胞（RFL 6）的镉抗性（CdR）的发展伴随着细胞金属硫蛋白（MT）和谷胱甘肽（GSH）的上调，以及LO及其胶原和弹性蛋白底物的下调。这些发现导致了一个假说的机制，镉损伤肺ECM相关的肺气肿发病机制：在长期暴露于镉，肺成纤维细胞的MT和GSH的合成上调。这些细胞内的硫醇结合铜离子具有比镉更高的亲和力，从而严重扰乱了铜的体内平衡，限制了其对LO的可用性，并有助于LO在mRNA，蛋白质和催化水平的下调，如初步研究所示。LO的下调反过来又会抑制胶原蛋白和弹性蛋白的交联，有利于它们的去稳定化、溶解和最终降解，并干扰它们的修复。所得的溶解的胶原蛋白和弹性蛋白可能通过反馈机制抑制它们自身的合成，进一步干扰这些蛋白质的合成和降解之间的平衡并破坏ECM，这是肺气肿发展的特征性事件。本研究的主要目的是：1）探讨Cd对CdR-RFL 6细胞Cu（II）稳态的影响机制; 2）从转录、翻译和翻译后水平研究LO在CdR-RFL 6细胞中的下调机制; 3）探讨LO在CdR-RFL 6细胞中对胶原和弹性蛋白底物的下调以及对弹性蛋白修复的影响;（4）探讨慢性染镉大鼠肺气肿发病机制中，细胞内MT和GSH升高，Cu稳态紊乱和LO下调的重要机制。拟议的研究结果，预计将确定镉调制LO基因的表达和处理的铜稳态在肺中的扰动的关键方面，从而提高我们对镉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.