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起核心作用，是治疗人类贫血的主要药物。与Epo给药相关的两个主要问题是治疗耐药性和不良副作用。治疗抗性通常发生在靶细胞，红系祖细胞的水平。Epo最突出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）。虽然Epo信号已经研究了几十年，但这些重要临床问题的机制基础仍然未知。此外，Epo信号如何促进红系分化的基本问题本身仍然没有解决。十多年来，人们已经知道Epo以某种方式调节红细胞基因转录的主要调节因子加塔-1的功能。在缺乏Epo信号传导的情况下，加塔-1的活化导致细胞死亡而不分化。尽管已经提出了几种用于加塔-1的Epo调节的模型，但尚未建立机制。 这一建议提供了证据，证明Epo通过蛋白激酶D/IIa类组蛋白脱乙酰酶（PKD/HDAC）途径调节加塔-1，建立在以前观察到的加塔-1结合和抑制IIa类HDAC的基础上。在G1 ER和人红系祖细胞中获得的这一证据包括Epo激活PKD和通过PKD抑制阻断Epo驱动的红系分化。进一步的证据包括通过HDAC 5敲低增强红系Epo反应性和通过HDAC抑制剂消除加塔-1对Epo信号传导的依赖性。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磷酸化导致肌原性转录因子MEF 2的不适当激活。因此，PKD的Epo活化可促进加塔-1驱动的红细胞生成和MEF 2驱动的CHF。 在这项提案中，我们将确定的机制Epo激活PKD，检查的EpoR和它的coregulator，c-Kit的结构要求。此外，将分析必需的EpoR下游介质JAK 2和PLC 3对PKD活化的贡献。我们还将研究Epo给药是否诱导体内心肌PKD激活，如果是这样，哪些EpoR相关元素有助于这种信号传导。因此，这些实验将描绘一个新的Epo信号通路的上游组件参与红细胞生成的编程和潜在的诱导CHF。从这些研究中获得的信息将建立一个概念框架，用于探索临床Epo耐药机制和设计逆转Epo耐药的药物。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未解决问题。 本提案是对项目公告“刺激血液学研究：新的努力”（PAS-10-046）的回应。本公告中列出的一个特定主题是“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活化的影响。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信号传导的理解是理解与这些贫血相关的Epo抗性机制的关键第一步。 公共卫生相关性：促红细胞生成素（Epo）对正常的红细胞生成至关重要，并被用于治疗许多贫血患者。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以完全纠正贫血，而不是部分纠正贫血，会增加心血管疾病的风险，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，但对它如何促进红细胞生成以及心脏病的了解相对较少。在这个提议中，一个新的信号通路，可以潜在地解释这两个有益的和有害的影响，Epo的，将被表征。