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.

氧气暴露增加肺氧化剂的产生， 微血管损伤会导致致命的肺水肿 虽然 关于抗氧化防御的调节， 高氧环境中氧化剂产生的机制、部位和调控 肺不确定。 在以前的工作中，我们定义了生理和 在氧暴露期间在羔羊肺中发生的生化变化。 我们证明，氧暴露增加肺细胞色素P450的量， 和两种潜在的微粒体氧化剂源P450 IA 1和 IIB 1. 用两种化学性质不同的P450中的任一种处理羔羊 抑制剂显著降低体内高氧肺损伤。 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衍生的氧化剂参与了 羔羊急性肺氧中毒的发病机制。 这项提案的目的是扩大我们的生理和生化 应用分子和细胞生物学技术， 了解氧气暴露增加肺P450的机制。 初步数据表明，高氧诱导的 在体内肺P450 IIB 1和IA 1活性增加之前， 增加了各自的RNA。 P450的肝脏RNA水平是 不受体内氧气暴露的影响。 我们还展示了积极的 使用培养的细胞通过增加氧张力调节P450 IA 1 其已经用含有完整的- 长度小鼠P450 IA 1 5'侧翼区。 该提案概述了一系列在体内和培养肺中的实验 使用啮齿动物核苷酸探针测量稳态细胞中的变化 用不同浓度的 氧，单独和存在影响肺P450的试剂时 程度. 我们还将分离编码羊肺的全长cDNA克隆， P450基因IA 1和IIB 1的5'端侧翼序列 基因组DNA中。 然后，含有5'侧翼区的融合基因 将构建与报告基因连接的载体并用于转染 实验来检验假设，羔羊肺P450基因表达是 在特定肺细胞中直接或间接正向调节 通过增加周围的氧气压力。 我们还将构建同源 核苷酸探针，并使用它们来定位P450特异性RNA原位。 这些研究将导致重要的新的见解的监管， 在发育中的肺中编码氧化剂产生酶的基因， 也可以作为研究小分子如何调节基因的模型系统。 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 高浓度的氧气。