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.

氧气暴露会增加肺氧化剂的产生并导致急性 微血管损伤可能导致致命的肺水肿。 虽然 关于抗氧化剂防御的调节，众所周知 高氧化中氧化剂产生的机理，位点和调节 肺不确定。 在以前的工作中，我们定义了生理学和 氧气暴露期间羔羊肺中发生的生化变化。 我们证明氧气暴露增加了肺细胞色素P450量 和两个潜在的微粒氧化剂源P450 IA1和 IIB1。 用两个化学上不同的P450中的任何一个处理羔羊 抑制剂显着降低了体内高氧肺损伤。 免疫细胞化学数据表明羔羊肺内皮细胞包含 细胞色素P450酶。 这些数据强烈支持一般 假设细胞色素P450衍生的氧化剂参与 羔羊急性肺氧毒性的发病机理。 该建议的目的是扩展我们的生理和生化 通过将分子和细胞生物学技术应用于 了解氧气暴露增加肺P450的机制。 提供了初步数据，表明高氧诱导 在体内肺P450 IIB1和IA1活性的增加之前 增加各自的RNA。 P450的肝脏RNA水平为 不受体内氧气暴露的影响。 我们也表现为积极 使用培养细胞增加氧气张力来调节P450 IA1 用包含全融合基因稳定转染的 长度小鼠P450 IA1 5'侧翼区域。 该提案在体内和培养的肺中概述了一系列实验 使用啮齿动物核苷酸探针测量稳态变化的细胞 治疗后肺P450 RNA水平不同 氧气，单独和影响肺P450的药物的存在 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 将链接到报告基因的基因将被构建并用于转染 测试羔羊肺P450基因表达的假设的实验是 直接或间接地在特定的肺部细胞中进行积极调节 通过增加环境氧张力。 我们还将构建同源 核苷酸探针并使用它们定位原位P450特异性RNA。 这些研究将导致对调节的重要新见解 编码发育中肺中产生氧化剂酶的基因，可能 还用作模型系统，以研究小分子如何调节基因 在较高的真核生物中的表达。 另外，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。