ADAPTIVE RESISTANCE TO NITRIC OXIDE

一氧化氮（NO）在哺乳动物细胞中由几种NO分解酶产生，包括在炎症期间具有高输出的诱导型形式。 由于NO可能有助于致癌和年龄相关的神经退行性变，我们希望了解哺乳动物细胞如何对抗NO毒性。 我们已经确定了一组12蛋白诱导正常人成纤维细胞暴露于亚毒性水平的NO，包括抗氧化酶血红素加氧酶1（HO-1）。 我们已经表明，HO-1 mRNA的诱导发生通过NO依赖性的变化，其稳定性。 我们将探讨这种调控的机制，并试图确定HO-1的转录后调控的信号转导途径。 其他基因（例如，MAP激酶磷酸酶MKP-1/CL 100）是转录控制的，我们将测试特定的转录调节因子在这种表达中的作用。 我们已经确定了一个适应性耐NO在啮齿动物运动神经元：细胞暴露于低水平的NO诱导HO-1成为耐高得多（通常是细胞毒性）水平的NO。我们将通过识别NO诱导基因和确定其作用，在保护NO介导的毒性在各种细胞类型的这种反应。 HO-1基因敲除小鼠将使我们能够确定HO-1在成纤维细胞，神经元和其他组织中的防御作用。 该项目涉及平行的努力，以遵循一氧化氮损伤的生化标志物（例如，硝基酪氨酸），利用遗传学来建立HO-1在诱导型NO抗性中的作用，阐明调控和信号转导途径，以及鉴定和克隆用于NO抗性的新防御基因。

Nitric oxide (NO) is produced in mammalian cells by several NO synthases, including an inducible form with high output during inflammation. Since NO may contribute to carcinogenesis and age- related neurodegeneration, we wish to understand how mammalian cells counteract NO toxicity. We have identified a group of 12 proteins induced in normal human fibroblasts by exposure to subtoxic levels of NO, including the antioxidant enzyme heme oxygenase 1 (HO-1). We have shown that the induction of HO-1 mRNA occurs via NO-dependent changes in its stability. We will explore the mechanism of this regulation and try to define the signal transduction pathway for post-transcriptional control of HO-1. Other genes (e.g., MAP kinase phosphatase MKP-1/CL100) are controlled transcriptionally, and we will test specific transcription regulators for their roles in this expression. We have identified an adaptive resistance to NO in the rodent motor neurons: cells exposed to low-level NO that induces HO-1 become resistant to much higher (usually cytotoxic) levels of NO. We will characterize this response in various cell types by identifying NO-inducible genes and determining their roles in protection against NO-mediated toxicity. HO-1 knockout mice will allow us to define the defensive role of HO-1 in fibroblasts, neurons, and other tissues. This project involves parallel efforts to follow biochemical markers of nitric oxide damage (e.g., nitrotyrosine), to use genetics to establish the role of HO-1 in inducible resistance to NO, to elucidate regulatory and signal transduction pathways, and to identify and clone novel defense genes for NO resistance.