Lung Lysyl Oxidase Regulation by Metal Ion Homeostasis

描述（由申请人提供）：赖氨酰氧化酶（LO），一种铜（Cu）依赖性酶，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）和细胞核的组织化和稳定化至关重要。该酶已被鉴定为肿瘤抑制剂，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效应物在人体生理和病理中起着关键作用。人类长期暴露于镉（Cd），一种重金属，无论是从职业污染或香烟烟雾，诱发肺气肿和肺癌。然而，镉引起的肺部病理机制仍然知之甚少。LO作为一种金属酶，对细胞内金属稳态的变化很敏感。本实验室前期研究了大鼠肺成纤维细胞（RFL 6）从镉敏感到镉抗性的表型变化，结果表明镉在mRNA、蛋白和催化水平下调LO的表达。进一步的研究表明，RFL 6细胞响应于镉显示抑制LO转录起始和增强LO mRNA衰变两者共同有助于降低稳态LO mRNA的水平。这些研究结果导致了一个假设，即LO基因的转录控制和调节是镉损伤的关键目标，镉对LO基因转录的沉默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指导的LO前体mRNA合成和加工以及由核心启动子调控的LO启动子激活的调节，在转录水平上鉴定Cd沉默LO基因的机制; 2）通过检测Cd对由金属和氧化还原敏感性转录因子及其同源顺式元件调节的LO启动子激活的调节，在转录水平上鉴定Cd沉默LO基因的机制，并测定LO启动子甲基化，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 ′-加帽和3 ′-聚腺苷酸化状态的影响，并评估Cd敏感性，在转录后水平上鉴定Cd沉默LO基因的机制，LO mRNA 3 '端非翻译区稳定性相关顺式元件及其结合蛋白;和4）的方法通过检查Cd改变LO表达对细胞模型中底物启动子激活和细胞转化的影响，并评估主要LO转录和转录后机制的活性状态，研究细胞和动物模型中Cd沉默LO基因的生物学后果，以及慢性镉中毒大鼠肺气肿和致癌肺组织LO基因启动子甲基化异常。本研究的结果有望加深我们对镉致LO基因沉默机制的理解，为制定镉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：赖氨酰氧化酶（Lysyl oxidase，LO）是存在于细胞外基质（extracellular matrix，ECM）和细胞核中的关键铜（Cu）依赖性酶，其对于肺的器官形态发生、组织修复和抗肿瘤发生至关重要。本实验室的前期研究表明，镉（Cd），一种有毒和致癌的重金属，诱导大鼠肺成纤维细胞LO基因沉默。拟议的研究扩展了以前的研究结果，旨在研究细胞和动物模型中镉对LO基因沉默的分子机制，为开发镉相关肺部疾病（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.