Lung Lysyl Oxidase Regulation by Metal Ion Homeostasis

描述（由申请人提供）：赖氨酸氧化酶（LO）是一种依赖铜（Cu）的酶，可氧化底物中的肽基赖氨酸残基，例如胶原蛋白、弹性蛋白和组蛋白H1，对细胞外基质（ECM）和细胞核的组织和稳定至关重要。该酶已被确定为肿瘤抑制因子，例如，抑制原癌基因ras的转化活性。因此，LO作为细胞内和细胞外的效应物在人体生理和病理中起着至关重要的作用。人类长期暴露于镉这种重金属，无论是来自职业污染还是来自香烟烟雾，都会诱发肺气肿和肺癌。然而，cd诱发肺部病理的机制仍然知之甚少。作为一种金属酶，LO易受细胞金属稳态变化的影响。本实验室之前对大鼠肺成纤维细胞（RFL6）从Cd敏感到Cd抗性表型变化的研究表明，Cd在mRNA、蛋白质和催化水平上下调了LO。持续的研究进一步表明，RFL6细胞对Cd的反应表现出LO转录起始的抑制和LO mRNA衰变的增强，这两者共同导致了稳态LO mRNA水平的下降。这些发现导致了一种假设，即LO基因的转录控制和调控是Cd侵害的关键靶点，Cd沉默LO基因转录是肺部疾病的关键分子基础。本研究的总体目标是通过实现以下具体目标来验证这一假设：1)通过检测Cd对RNA聚合酶II引导的LO前mrna合成和加工的调节，以及由核心启动子调节的LO启动子激活的调节，确定LO基因在转录水平上的Cd沉默机制；2)通过研究金属和氧化还原敏感转录因子及其同源顺式元件对LO启动子激活的Cd调节，以及LO启动子甲基化，在转录水平上确定LO基因的Cd沉默机制，为Cd对LO DNA的表观遗传损伤提供证据；3)通过检测Cd对LO mRNA的5'-capping和3'-聚腺苷化状态的影响，并评估Cd敏感、LO mRNA稳定性相关的3'-非翻译区顺式元件及其相应的结合蛋白，在转录后水平上确定LO基因Cd沉默的机制；4)通过在细胞模型中检测Cd改变LO表达对底物启动子激活和细胞转化的影响，以及在慢性给药Cd的大鼠肺气肿和致癌肺中评估LO主要转录和转录后机制的活性状态以及LO基因启动子的异常甲基化，研究Cd沉默LO基因在细胞和动物模型中的生物学后果。本研究结果有望加深我们对Cd致LO基因沉默机制的理解，为制定Cd相关肺部疾病的预防和治疗策略提供依据。公共卫生相关性：赖氨酸氧化酶（LO）是一种存在于细胞外基质（ECM）和细胞核中的关键铜（Cu）依赖酶，对肺器官形态发生、组织修复和抗肿瘤发生至关重要。本实验室前期和初步研究表明，镉（Cd）是一种有毒和致癌的重金属，可诱导大鼠肺成纤维细胞中LO基因沉默。本研究旨在探索Cd在细胞和动物模型中导致LO基因沉默的分子机制，为制定与Cd相关的肺部疾病（如肺气肿和癌症）的保护和治疗策略提供基础。

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.