Lung Lysyl Oxidase Regulation by Metal Ion Homeostasis

项目摘要。赖氨酰氧化酶（LO）是一种铜依赖性酶，氧化肽酰赖氨酸 底物中的残留物，例如，胶原蛋白、弹性蛋白和组蛋白H1，对组织和稳定 细胞外基质（ECM）和细胞核。这种酶已被鉴定为肿瘤抑制因子， 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 效应器在人体生理学和病理学中起着至关重要的作用。人类长期接触镉 (Cd)，一种重金属，无论是来自职业污染还是来自香烟烟雾，都会引起肺气肿， 肺癌然而，镉引起的肺部病理机制仍然知之甚少。作为一个 金属酶易受细胞金属稳态变化的影响。本实验室以前的研究 研究大鼠肺成纤维细胞（RFL 6）从镉敏感到镉抗性的表型变化， 镉在mRNA、蛋白质和催化水平下调LO。持续的研究进一步表明， 镉对RFL 6细胞LO转录起始的抑制和LO mRNA表达的增强 衰减共同导致稳态LO mRNA水平降低。这些发现导致 LO基因的转录控制和调节是Cd损伤的关键靶点， 镉导致LO基因转录的沉默是导致肺部疾病的重要分子基础。的总体目标 本研究拟通过以下具体目标来验证这一假设：1）确定机制 通过检测Cd对RNA聚合酶II的调节，在转录水平上对LO基因进行Cd沉默。 指导LO前mRNA的合成和加工，以及由核心调控的LO启动子激活 启动子; 2）通过检测Cd对LO基因转录水平的影响， 镉对金属和氧化还原敏感性转录因子调控的LO启动子激活的调节， 它们的同源顺式元件，并确定LO启动子甲基化，为镉的表观遗传提供证据 3）在转录后水平上鉴定Cd对LO基因沉默的机制 通过检测镉对LO mRNA的5 '-加帽和3'-聚腺苷酸化状态的影响， 3 '-非翻译区中敏感的、LO mRNA稳定性相关的顺式元件及其相应的结合 研究镉沉默LO基因在细胞和动物中的生物学后果 通过检测镉改变LO表达对底物启动子激活和细胞增殖的影响， 在细胞模型中的转化，并评估主要LO转录和 肺气肿患者LO基因启动子的转录后机制以及异常甲基化 和致癌性的大鼠肺接受镉慢性管理。拟议研究的成果 以期加深我们对镉致LO基因沉默机制的理解，为进一步研究镉致LO基因沉默机制提供基础。 制定镉相关肺部疾病的预防和治疗策略。

PROJECT ABSTRACT. 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.