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

• 批准号: 8046474
• 负责人: Wande Li
• 金额: $ 35.84万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8046474
• 关键词: 5' Flanking Region; Affect; Animal Model; Atherosclerosis; Binding Proteins; Biological; Cadmium; Cell Nucleus; Cell model; Cells; Chronic; Cigarette smoke-induced emphysema; Collagen; Copper; DNA; Disease; Dose; Down-Regulation; Elastin; Elements; Enzymes; Epigenetic Process; Exhibits; Extracellular Matrix; Fibroblasts; Fibrosis; Gene Silencing; Genes; Genetic Transcription; Goals; Half-Life; Health; Heavy Metals; Histone H1; Histone H1(s); Homeostasis; Human; Indium; Industry; Ions; Lung; Lung diseases; Lysine; Malignant Neoplasms; Malignant neoplasm of lung; Messenger RNA; Metals; Methylation; Modeling; Molecular; Morphogenesis; Occupational; Organ; Outcome; Oxidation-Reduction; Pathogenesis; Pathology; Phenotype; Physiology; Play; Polyadenylation; Process; Prophylactic treatment; Protein-Lysine 6-Oxidase; Proteins; Proto-Oncogenes; Pulmonary Emphysema; RNA Polymerase II; Rattus; Regulation; Research; Resistance; Role; Source; Testing; Therapeutic; Tobacco; Transcript; Transcription Initiation; Transcriptional Regulation; Tumor Suppressor Proteins; Wound Healing; base; carcinogenesis; catalyst; cell transformation; cigarette smoking; exposed human population; extracellular; lung basal segment; lung tumorigenesis; mRNA Decay; mRNA Precursor; mRNA Stability; metalloenzyme; promoter; response; transcription factor; treatment strategy

DESCRIPTION (provided by applicant): Lysyl oxidase (LO), a copper-(Cu) dependent enzyme, oxidizes peptidyl lysine residues in substrates, e.g., collagen, elastin and histone H1, essential for organization and stabilization of the extracellular matrix (ECM) and the cell nucleus. This enzyme has been identified as a tumor suppressor, for example, inhibiting transforming activity of ras, a proto oncogene. Thus, LO as an intra- and extracellular effector plays a critical role in human physiology and pathology. Chronic exposure of humans to cadmium (Cd), a heavy metal, either from occupational contamination or from cigarette smoke, induces emphysema and lung cancers. However, the mechanisms for Cd-elicited lung pathology remain poorly understood. LO as a metalloenzyme is susceptible to changes in cellular metal homeostasis. Previous studies by this lab investigating the phenotype change from Cd sensitive to Cd resistant of rat lung fibroblasts (RFL6) illustrated downregulation of LO by Cd at mRNA, protein and catalytic levels. Continuing studies further indicated that RFL6 cells in response to Cd displayed inhibition of LO transcription initiation and enhancement of LO mRNA decay both collectively contributing to decreased levels of steady-state LO mRNAs. These findings have led to a hypothesis that transcriptional control and regulation of the LO gene are critical targets for Cd insult and silencing of LO gene transcription by Cd is a key molecular basis for lung diseases. The overall goal of the proposed research is to test this hypothesis by achieving following specific aims: 1) to identify mechanisms for Cd silencing of the LO gene at the transcriptional level by examining Cd modulation of RNA polymerase II- directed LO pre-mRNA synthesis and processing, and of the LO promoter activation regulated by the core promoter; 2) to identify mechanisms for Cd silencing of the LO gene at the transcriptional level by examining Cd modulation of the LO promoter activation regulated by metal and redox-sensitive transcription factors and their cognate cis-elements, and determining LO promoter methylation to provide evidence for Cd epigenetic damage to LO DNA; 3) to identify mechanisms for Cd silencing of the LO gene at the posttranscriptional level by examining Cd effects on the 5'-capping and 3'-polyadenylation status of LO mRNA, and assessing Cd sensitive, LO mRNA stability-related cis-elements in the 3'-untranslation region and their corresponding binding proteins; and 4) to investigate biological consequences of Cd silencing of the LO gene in cell and animal models by examining effects of altered LO expression by Cd on substrate promoter activation and cell transformation in the cell model and assessing the active status of the major LO transcriptional and posttranscriptional machineries as well as aberrant methylation of the LO gene promoter in emphysematous and carcinogenic lungs of rats receiving Cd by chronic administration. The outcomes of the proposed research are expected to enhance our understanding of mechanisms of LO gene silence by Cd providing the basis for developing prophylaxis and treatment strategies for Cd-related lung diseases. PUBLIC HEALTH RELEVANCE: Lysyl oxidase (LO) is a key copper (Cu)-dependent enzyme existing in the extracellular matrix (ECM) and the cell nucleus critical for organ morphogenesis, tissue repair and anti- tumorigenesis of the lung. Previous and preliminary studies by this lab have indicated that cadmium (Cd), a toxic and carcinogenic heavy metal, induced LO gene silencing in rat lung fibroblasts. The proposed research extends previous findings and aims at investigating molecular mechanisms for LO gene silence by Cd in cell and animal models providing the basis for developing protective and therapeutic strategies for Cd-related lung diseases such as emphysema and cancers.

描述（由申请人提供）：晶状体氧化酶（LO），铜（CU）依赖性酶，氧化底物中肽基裂解酶残基，例如，胶原蛋白，弹性蛋白和组蛋白H1，对于组织和稳定细胞外基质（ECM）和细胞核的稳定性至关重要。该酶已被确定为肿瘤抑制剂，例如抑制原始癌基因Ras的转化活性。因此，LO作为细胞内和细胞外效应子在人类生理和病理学中起着关键作用。人类长期暴露于镉（CD），一种重金属，无论是从职业污染还是来自香烟烟雾，都会引起肺气肿和肺癌。然而，CD引起的肺病理学的机制仍然鲜为人知。 LO作为金属酶容易受到细胞金属稳态变化的影响。该实验室的先前研究研究了表型从CD敏感到大鼠肺成纤维细胞抗CD的变化（RFL6），说明了在mRNA，蛋白质和催化水平上通过Cd降低LO的下调。继续进行的研究进一步表明，对CD的RFL6细胞表现出对LO转录起始的抑制作用和LO mRNA衰变的增强，既统称均导致稳态LO mRNA水平降低。这些发现导致了一个假设，即LO基因的转录控制和调节是CD损伤和通过CD沉默的关键目标，是肺部疾病的关键分子基础。拟议的研究的总体目标是通过实现以下特定目的来检验这一假设：1）通过检查转录水平的LO基因的CD沉默的机制，通过检查CD调节RNA聚合酶II-定向的LO PRE-MRNA Pre-MRNA合成和处理，以及由核心启动者调节LO启动子激活者的cd调节； 2）通过检查由金属和氧化还原敏感的转录因子调节的LO启动子激活的CD调节，以确定转录水平的CD沉默的机制，并确定LO启动子甲基化以提供对LO DNA的CD表观损害的证据； 3）通过检查CD对LO mRNA的5'Papping和3'-聚乙基化状态的效应，并评估3'- untranslation区域及其相应的结合蛋白中的CD敏感性，与LO mRNA稳定性相关的CIS元素，以确定在转录后水平上CD沉默的机制； 4）通过检查CD通过CD对LO表达的影响对底物启动子激活和细胞模型中的细胞转化的影响，并评估LO转录和转录后机器的活性状态，并评估LO的活跃状态，并评估cd cd型cd cd cd cd cycd的促进者的异常，来研究细胞和动物模型中CD沉默的生物学后果，并评估了主要转录和转录后的活性状态。预计拟议研究的结果有望增强我们对LO基因沉默机制的理解，从而为开发与CD相关的肺部疾病的预防和治疗策略提供了基础。公共卫生相关性：赖氨酸氧化酶（LO）是存在于细胞外基质（ECM）中的关键铜（CU）依赖性酶，而细胞核对于器官形态发生，组织修复和肺的抗肿瘤发生至关重要。该实验室的先前和初步研究表明，镉（CD）是一种有毒和致癌的重金属，诱导了大鼠肺成纤维细胞中的LO基因沉默。拟议的研究扩展了以前的发现，并旨在研究CD和动物模型中CD的LO基因沉默的分子机制，从而为与CD相关的肺部疾病（如肺气肿和癌症）开发保护性和治疗策略提供了基础。