OXYGEN SENSING PATHWAY IN ERYTHROPOIETIN PRODUCTION

这项建议的目的是为了了解信号转导 低氧诱导促红细胞生成素(EPO)产生的途径。 此前已有研究表明，Hep3B细胞系在体内调节EPO的产生 对低氧作出反应的一种生理方式。它也被用来 广泛作为模型系统来研究炎症的影响 急性期反应中的细胞因子。IL-1α、IL-1的添加 或肿瘤坏死因子-α导致剂量依赖的缺氧抑制- 诱导产生促红细胞生成素。相反，在缺氧的Hep3B中添加IL/6 细胞对低氧诱导的进一步刺激呈剂量依赖性 EPO生产。Northern印迹分析表明，这些细胞因子 主要在基因水平上影响EPO的产生。在这份提案中， 申请者计划研究细胞的分子机制 炎性细胞因子在低氧诱导促红细胞生成素中的作用 制作。将进行实验，以表征配置项和 参与转录和后转录的反式元件- 这些细胞因子介导的事件的转录调控。这部作品 将涉及对5‘和3’侧翼区域的广泛分析 EPO基因。采用的方法包括功能分析(核运行-- 非转录分析、瞬时表达分析、体外RNA 降解分析)和结构分析(DNA和RNA迁移率变化 分析，体外DNase I足迹分析，蛋白质纯化和 表征)。申请者将检验可溶物质的假设 鸟苷环化酶的形式是血红素蛋白氧感受器，它介导 低氧诱导促红细胞生成素生成增加。在努力解开 低氧诱导的信号转导通路，磷酸化的变化 胞质蛋白和膜蛋白的含量将被研究。氧气不同 通过成为无处不在的 容易扩散到细胞中的分子，不受受体结合的影响。 相应地，从氧传感器到基因的信号转导途径 监管可能与经典的监管有很大不同 配体-受体结合。分子生物学基础的研究进展 观察到这些细胞因子对缺氧诱导的EPO产生的影响可能 提供有关氧传感器的分子性质的详细信息。 此外，对分子机制的更好理解 调节促红细胞生成素的产生可能会加深我们对 慢性病贫血的发病机制及急性贫血的性质 相位响应。

The objective of this proposal is to understand the signal transduction pathway responsible for hypoxia-induced erythropoietin (Epo) production. The Hep3B cell line has previously been shown to regulate Epo production in a physiologic manner in response to hypoxia. It has also been used extensively as a model system to study the effects of inflammatory cytokines in the acute phase response. The addition of IL-1 alpha, IL-1 beta, or TNF-alpha results in a dose-dependent inhibition of hypoxia- induced Epo production. In contrast, the addition of IL/6 to hypoxic Hep3B cells results in a dose-dependent further stimulation of hypoxia-induced Epo production. Northern blot analyses indicate that these cytokines affect Epo production primarily at the mRNA level. In this proposal the applicant plans to investigate the molecular mechanisms by which the inflammatory cytokines exert their effects on hypoxia-induced Epo production. Experiments will be performed to characterize the cis and trans elements which are involved in the transcriptional and post- transcriptional regulation of these cytokine mediated events. This work will involve extensive analysis of the 5' and 3' flanking regions of the Epo gene. Methods to be employed include functional assays (nuclear run- off transcription assays, transient expression assays, in vitro RNA degradation analyses) and structural analyses (DNA and RNA mobility shift assays, in vitro DNAse I footprint analysis, protein purification and characterization). The applicant will test the hypothesis that the soluble form of guanylate cyclase is the heme protein oxygen sensor which mediates the hypoxia-induced increase in Epo production. In an effort to unravel the hypoxia-induced signal transduction pathway, changes in phosphorylation of cytosolic and membrane proteins will be investigated. Oxygen differs from the great majority of external cell signals by being a ubiquitous molecule that diffuses readily into cells independent of receptor binding. Accordingly, the signal transduction pathway from oxygen sensor to gene regulation may differ significantly from these associated with classic ligand-receptor binding. Investigation of the molecular basis for the observed effects of these cytokines on hypoxia-induced Epo production may provide detailed information on the molecular nature of the oxygen sensor. In addition, a better understanding of the molecular mechanisms governing the regulation of Epo production may enhance our understanding of the pathogenesis of the anemia of chronic disease and the nature of the acute phase response.