Characterization of a Novel Erythropoietin Signaling Pathway

新型促红细胞生成素信号通路的表征

• 批准号: 8028004
• 负责人: Adam N. Goldfarb
• 金额: $ 30.8万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8028004
• 关键词: Characterization; Novel; Erythropoietin; Signaling; Pathway

DESCRIPTION (provided by applicant): The soluble cytokine erythropoietin (Epo) plays a central role in physiologic erythropoiesis and is a mainstay in treatment of human anemias. Two major problems associated with Epo administration are treatment resistance and adverse side-effects. Treatment resistance usually occurs at the level of the target cells, the erythroid progenitors. The most prominent adverse side-effects of Epo consist of cardiovascular complications, particularly congestive heart failure (CHF). Although Epo signaling has been studied for several decades, the mechanistic basis for these important clinical problems remains unknown. Furthermore, the basic question of how Epo signaling promotes erythroid differentiation remains itself unresolved. It has been known for over a decade that Epo somehow regulates the function of the master regulator of erythroid gene transcription, GATA-1. Activation of GATA-1 in the absence of Epo signaling causes cell death without differentiation. Although several models have been proposed for Epo regulation of GATA-1, no mechanism has been established. This proposal provides evidence for Epo regulation of GATA-1 through a protein kinase D/class IIa histone deacetylase (PKD/HDAC) pathway, building on previous observations of GATA-1 binding and repression by class IIa HDACs. This evidence, obtained in both G1ER and human erythroid progenitors, includes the activation of PKD by Epo and the blockade of Epo-driven erythroid differentiation through PKD inhibition. Further evidence consists of the enhancement of erythroid Epo responsiveness through HDAC5 knockdown and elimination of GATA-1 dependence on Epo signaling by an HDAC inhibitor. Importantly, class IIa HDAC signaling pathways have been implicated in the pathophysiology of cardiac hypertrophy and CHF, wherein class IIa HDAC phosphorylation by PKD or CaMK causes inappropriate activation of the myogenic transcription factor MEF2. Thus Epo activation of PKD may promote both GATA-1-driven erythropoiesis and MEF2-driven CHF. In this proposal, we will determine the mechanism for Epo activation of PKD, examining the structural requirements of the EpoR and its coregulator, c-Kit. In addition, the essential EpoR downstream mediators, JAK2 and PLC3, will be analyzed for their contributions to PKD activation. We will also examine whether Epo adminstration induces PKD activation in vivo in myocardium and, if so, which EpoR-associated elements contribute to this signaling. These experiments will thus delineate the upstream components of a novel Epo signaling pathway involved in programming of erythropoiesis and potentially in induction of CHF. The information gained from these studies will establish a conceptual framework for probing mechanisms of clinical Epo resistance and for design of pharmacologic agents to reverse Epo resistance. Of equal importance, these experiments will mechanistically address the unresolved issue of Epo's association with cardiovascular complications. This proposal is responsive to the program announcement "Stimulating Hematology Investigation: New Endeavors" (PAS-10-046). One specific topic listed in this announcement is "Non-erythroid Expression and Function of Erythropoietin Receptors." A significant component of this project will be devoted to analysis of myocardial PKD activation by Epo administration in mice with wild type and mutant EpoR, as well as in mice with wild type and mutant c-Kit. Another topic listed is "Anemia of Inflammation and Chronic Disease." Improved understanding of normal erythroid EpoR signaling is a critical first step toward understanding the mechanisms of Epo resistance associated with these anemias. PUBLIC HEALTH RELEVANCE: Erythropoietin (Epo) is critical for normal red cell production and is administered as a treatment to numerous patients with anemia. A published, large-scale clinical trial has shown that giving patients sufficient doses of Epo to completely correct anemia, rather than partially correct anemia, confers an increased risk of cardiovascular disease, particularly congestive heart failure (CHF). Despite the tremendous amount of resources invested in Epo therapy and research, relatively little is understood about how it may promote red cell production as well as heart disease. In this proposal, a new signaling pathway, that can potentially explain both the beneficial and deleterious effects of Epo, will be characterized.

描述（由申请人提供）：可溶性细胞因子促红细胞生成素（Epo）在生理性红细胞生成中发挥核心作用，并且是治疗人类贫血的支柱。与 Epo 给药相关的两个主要问题是治疗抵抗和不良副作用。治疗抵抗通常发生在靶细胞（红系祖细胞）水平。 Epo 最显着的不良副作用包括心血管并发症，特别是充血性心力衰竭 (CHF)。尽管 Epo 信号传导已被研究数十年，但这些重要临床问题的机制基础仍然未知。此外，Epo 信号如何促进红细胞分化的基本问题本身仍未解决。十多年来，人们都知道 Epo 以某种方式调节红细胞基因转录主调节因子 GATA-1 的功能。在没有 Epo 信号传导的情况下，GATA-1 的激活会导致细胞死亡而不分化。尽管已经提出了几种 Epo 调节 GATA-1 的模型，但尚未建立机制。 该提案基于之前对 GATA-1 结合和 IIa 类 HDAC 抑制的观察结果，为 Epo 通过蛋白激酶 D/IIa 类组蛋白脱乙酰酶 (PKD/HDAC) 途径调节 GATA-1 提供了证据。这一证据是在 G1ER 和人类红系祖细胞中获得的，包括 Epo 激活 PKD 以及通过抑制 PKD 来阻断 Epo 驱动的红系分化。进一步的证据包括通过 HDAC5 敲低增强红细胞 Epo 反应性，以及通过 HDAC 抑制剂消除 GATA-1 对 Epo 信号传导的依赖。重要的是，IIa类HDAC信号传导途径与心脏肥大和CHF的病理生理学有关，其中PKD或CaMK引起的IIa类HDAC磷酸化导致肌源性转录因子MEF2的不适当激活。因此，PKD 的 Epo 激活可能促进 GATA-1 驱动的红细胞生成和 MEF2 驱动的 CHF。 在本提案中，我们将确定 PKD 的 Epo 激活机制，检查 EpoR 及其核心调节器 c-Kit 的结构要求。此外，还将分析重要的 EpoR 下游介质 JAK2 和 PLC3 对 PKD 激活的贡献。我们还将检查 Epo 给药是否会诱导心肌体内 PKD 激活，如果是的话，哪些 EpoR 相关元件有助于这种信号传导。因此，这些实验将描绘出参与红细胞生成编程并可能诱导 CHF 的新型 Epo 信号通路的上游组件。从这些研究中获得的信息将为探索临床 Epo 耐药机制和设计逆转 Epo 耐药的药物制剂建立概念框架。同样重要的是，这些实验将从机制上解决 EPO 与心血管并发症相关的尚未解决的问题。 该提案是对计划公告“刺激血液学研究：新努力”(PAS-10-046) 的回应。本公告中列出的一个具体主题是“促红细胞生成素受体的非红细胞表达和功能”。该项目的一个重要组成部分将致力于分析野生型和突变型 EpoR 小鼠以及野生型和突变型 c-Kit 小鼠中 Epo 给药对心肌 PKD 的激活。列出的另一个主题是“炎症性贫血和慢性病”。提高对正常红细胞 EpoR 信号传导的了解是了解与这些贫血相关的 Epo 抵抗机制的关键的第一步。 公共健康相关性：促红细胞生成素 (Epo) 对于正常红细胞生成至关重要，并作为许多贫血患者的治疗方法。一项已发表的大规模临床试验表明，给予患者足够剂量的 Epo 来完全纠正贫血，而不是部分纠正贫血，会增加患心血管疾病的风险，特别是充血性心力衰竭 (CHF)。尽管在 EPO 治疗和研究方面投入了大量资源，但人们对其如何促进红细胞生成以及心脏病的了解却相对较少。在该提案中，将描述一种新的信号传导途径，它可以潜在地解释 Epo 的有益和有害作用。