Lung Lysyl Oxidase Regulation by Metal Ion Homeostasis

金属离子稳态对肺赖氨酰氧化酶的调节

基本信息

批准号：
6745614
负责人：
Wande Li
金额：
$ 30.69万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-06-01 至 2008-03-31
项目状态：
已结题

项目摘要

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.

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赖氨酰氧化酶（LO）（一种铜[Cu(II)]催化的可溶性胶原蛋白和弹性蛋白向不溶性纤维聚集体的转化。 )]依赖性酶。该催化剂氧化这些蛋白质内的赖氨酸残基，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核心作用。镉 (Cd) 是一种对人体有毒的金属。环境污染或香烟烟雾中镉的肺部吸入和积累会引起金属离子稳态的扰动，这可能是肺部发病机制的关键机制。初步研究表明，长期暴露于镉后，大鼠肺成纤维细胞（RFL6）出现镉抵抗（CdR），并伴随着细胞金属硫蛋白（MT）和谷胱甘肽（GSH）的上调，以及LO及其胶原蛋白和弹性蛋白底物的下调。这些发现提出了与肺气肿发病机制相关的镉对肺 ECM 损伤机制的假设：在长期暴露于镉的过程中，肺成纤维细胞上调 MT 和 GSH 的合成。这些细胞内硫醇以比 Cd 更高的亲和力结合 Cu 离子，从而严重扰乱 Cu 的稳态，限制其对 LO 的利用，并导致 LO 在 mRNA、蛋白质和催化水平上的下调，如初步研究所示。 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，进一步扰乱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。以下具体目标旨在检验这一假设：1) 评估 Cd 对 CdR-RFL6 细胞中 Cu(II) 稳态的扰动机制； 2) 研究CdR-RFL6细胞中LO在转录、翻译和翻译后水平下调的机制； 3) 探讨LO对其胶原蛋白和弹性蛋白底物的下调以及CdR-RFL6细胞中弹性蛋白修复的影响； 4) 证明细胞 MT 和 GSH 升高、Cu 稳态扰动和 LO 下调是长期服用 Cd 的大鼠肺气肿发病机制的关键机制。这项研究的结果预计将通过扰乱肺部铜稳态来确定镉对 LO 基因表达和加工的调节的关键方面，从而增强我们对镉肺气肿发病机制的理解。