MECHANISM OF CYTOCHROME P450 GENE EXPRESSION IN THE HYPEROXIC DEVELOPING LUNG

高氧发育肺细胞色素P450基因表达机制

• 批准号: 3779744
• 负责人: THOMAS A HAZINSKI
• 金额: --
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3779744
• 关键词: RNA; antioxidants; autoradiography; cimetidine; complementary DNA; cytochrome P450; deferoxamine; endotoxins; fusion gene; gene deletion mutation; gene expression; hyperoxia; in situ hybridization; northern blottings; nucleic acid hybridization; nucleic acid probes; oxidizing agents; oxygen tension; sheep; superoxide dismutase; tissue /cell culture; tocopherols; transfection

Oxygen exposure increases lung oxidant production and causes acute microvascular injury which can lead to fatal pulmonary edema. Although much is known about the regulation of anti-oxidant defenses, the mechanisms, sites and regulation of oxidant production in the hyperoxic lung are uncertain. In previous work, we defined the physiologic and biochemical changes which occur in the lamb lung during oxygen exposure. We demonstrated that oxygen exposure increases lung cytochrome P450 amount and activity of two potential microsomal oxidant sources, P450 IA1 and IIB1. Treatment of lambs with either of two chemically dissimilar P450 inhibitors significantly reduced hyperoxic lung injury in vivo. Immunocytochemical data indicate that lamb lung endothelial cells contain cytochrome P450 enzymes. These data strongly support the general hypothesis that cytochrome P450-derived oxidants are involved in the pathogenesis of acute pulmonary oxygen toxicity in lambs. The objective of this proposal is to extend our physiologic and biochemical studies by applying the techniques of molecular and cell biology to understand the mechanisms by which oxygen exposure increases lung P450. Preliminary data are presented which indicate that the hyperoxia-induced increases in lung P450 IIB1 and IA1 activities in vivo are preceded by an increase in their respective RNA's. Liver RNA levels for P450 are unaffected by oxygen exposure in vivo. We also demonstrate positive regulation of P450 IA1 by increased oxygen tension using cultured cells which have been stably transfected with a fusion gene containing the full- length mouse P450 IA1 5' flanking region. The proposal outlines a series of experiments in vivo and in cultured lung cells using rodent nucleotide probes to measure changes in steady-state lung P450 RNA levels following treatment with varying concentrations of oxygen, alone and in the presence of agents which influence lung P450 levels. We will also isolate full-length cDNA clones encoding lamb lung P450 genes IA1 and IIB1 and use them to identify their 5' flanking regions in genomic DNA. Then, fusion genes containing the 5' flanking regions linked to reporter genes will be constructed and used in transfection experiments to test the hypothesis that lamb lung P450 gene expression is positively regulated in specific lung cells either directly or indirectly by increases in ambient oxygen tension. We will also construct homologous nucleotide probes and use them to localize P450-specific RNA in situ. These studies will lead to important new insights into the regulation of genes which encode oxidant-producing enzymes in the developing lung and may also serve as a model system to study how small molecules regulate gene expression in higher eukaryotes. In addition, these studies may lead to the development of safe and effective methods to reduce the oxidant stress of oxygen exposure in patients with lung diseases who require treatment with high concentrations of oxygen.

氧气暴露会增加肺部氧化剂的产生并导致急性 微血管损伤可导致致命的肺水肿。 虽然 人们对抗氧化防御的调节了解很多 高氧环境中氧化剂产生的机制、位点和调节 肺不确定。 在之前的工作中，我们定义了生理和 暴露于氧气期间羔羊肺部发生的生化变化。 我们证明氧气暴露会增加肺细胞色素 P450 的量 以及两种潜在微粒体氧化剂来源 P450 IA1 和 P450 IA1 的活性 IIB1。 用两种化学性质不同的 P450 中的任意一种处理羔羊 抑制剂显着减轻体内高氧肺损伤。 免疫细胞化学数据表明，羔羊肺内皮细胞含有 细胞色素 P450 酶。 这些数据有力地支持了一般 假设细胞色素 P450 衍生的氧化剂参与 羔羊急性肺氧中毒的发病机制。 该提案的目的是扩展我们的生理和生化能力 应用分子和细胞生物学技术进行研究 了解氧气暴露增加肺 P450 的机制。 初步数据表明，高氧诱导的 体内肺 P450 IIB1 和 IA1 活性增加之前 各自的RNA增加。 P450 的肝脏 RNA 水平为 不受体内氧气暴露的影响。 我们也展现出积极的一面 使用培养细胞增加氧张力来调节 P450 IA1 已稳定转染含有完整的融合基因 小鼠 P450 IA1 5' 侧翼区域的长度。 该提案概述了一系列体内和培养肺实验 使用啮齿动物核苷酸探针测量稳态变化的细胞 不同浓度的治疗后肺 P450 RNA 水平 氧气，单独使用或存在影响肺 P450 的物质时 水平。 我们还将分离编码羔羊肺的全长 cDNA 克隆 P450 基因 IA1 和 IIB1 并使用它们来识别其 5' 侧翼区域 在基因组DNA中。 然后，包含 5' 侧翼区域的融合基因 将构建与报告基因连接并用于转染的 实验验证了羔羊肺 P450 基因表达的假设 直接或间接地在特定肺细胞中受到正向调节 通过环境氧张力的增加。 我们还将构建同源 核苷酸探针并用它们原位定位 P450 特异性 RNA。 这些研究将为监管带来重要的新见解 编码发育中肺部产生氧化剂的酶的基因，可能 也可作为研究小分子如何调控基因的模型系统 在高等真核生物中表达。 此外，这些研究可能会导致 开发安全有效的方法来减少氧化应激 需要治疗的肺部疾病患者的氧气暴露量 与高浓度的氧气。