OXYGEN SENSING PATHWAY IN ERYTHROPOIETIN PRODUCTION

本提案的目的是了解信号转导 负责低氧诱导的促红细胞生成素（Epo）产生的途径。 Hep 3B细胞系先前已经显示出调节Epo的产生， 对缺氧的生理反应。 它也被用于 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。 加入IL-1 α，IL-1 β或TNF-α导致缺氧的剂量依赖性抑制- 诱导Epo生成。 相反，向缺氧的Hep 3B中加入IL/6， 细胞导致剂量依赖性的进一步刺激缺氧诱导的 促红细胞生成素生产。 北方印迹分析表明，这些细胞因子 主要在mRNA水平上影响Epo的产生。 在这项提案中， 申请人计划研究 炎性细胞因子对缺氧诱导的Epo有影响 生产 将进行实验以表征顺式和 反式元件，参与转录和后 这些细胞因子介导的事件的转录调节。 这项工作 将涉及广泛的分析5'和3'侧翼区的基因， Epo基因。 所采用的方法包括功能测定（核运行， 关闭转录测定，瞬时表达测定，体外RNA 降解分析）和结构分析（DNA和RNA迁移率变化 分析、体外DNA酶I足迹分析、蛋白质纯化和 表征）。 申请人将检验以下假设： 鸟苷酸环化酶是血红素蛋白氧传感器， 缺氧诱导的Epo生成增加。 为了解开 缺氧诱导的信号转导通路，磷酸化的变化， 细胞溶质和膜蛋白的研究。 氧气不同 从绝大多数外部细胞信号中分离出来， 不依赖于受体结合而容易扩散到细胞中的分子。 因此，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。 研究的分子基础， 所观察到的这些细胞因子对缺氧诱导的Epo产生的影响可能 提供有关氧传感器分子性质的详细信息。 此外，更好地了解分子机制， 对促红细胞生成素生产的调控可以增强我们对 慢性病贫血的发病机制和急性贫血的性质 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.