The role of Dock4 in normal and aberrant erythropoiesis

Dock4 在正常和异常红细胞生成中的作用

• 批准号: 8417428
• 负责人: Amit K. Verma
• 金额: $ 42.08万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-26 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8417428
• 关键词: Aberrant DNA Methylation; Actins; Affect; Anemia; Architecture; Biological Models; Blood Cells; Bone Marrow; Categories; Cell Lineage; Cell Shape; Cell Survival; Cell membrane; Cells; Cellular Morphology; Chromosomes; DNA Methylation; Data; Defect; Development; Disease; Down-Regulation; Dysmyelopoietic Syndromes; Elderly; Epigenetic Process; Erythroblasts; Erythrocyte Membrane; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; F-Actin; Fluorescent in Situ Hybridization; Gene Silencing; Genes; Genetic; Genomics; Goals; Guanine; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Hematopoiesis; Hematopoietic; Homeostasis; Human; In Vitro; Knowledge; Laboratories; Lead; Marrow; Mediating; Membrane; Methylation; Modeling; Molecular; Morbidity - disease rate; Morphology; Mutate; Mutation; Osmotic Fragility test; Pathogenesis; Pathway interactions; Patients; Phase; Phosphotransferases; Population; Proteins; Refractory; Regulation; Research; Reticulocytes; Rho-associated kinase; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Skeleton; Sorting - Cell Movement; Staging; Stem cells; TFRC gene; Testing; Work; abstracting; adducin; base; cofactor; cohort; erythroid differentiation; insight; membrane skeleton; novel; promoter; restoration; small hairpin RNA; stem

DESCRIPTION (provided by applicant): Erythropoiesis is tightly controlled at every phase of differentiation. Although much is known in terms of signaling and cell survival during the initial phase, very little is known with respect to signaling during late- stages of erythrocyte membrane remodeling, enucleation and reticulocyte maturation. Moreover, in conditions such as myelodysplastic syndromes (MDS), where anemia is the most common presentation and predominant cause of morbidity, the focus has mainly been to study the early phases of hematopoiesis. In an attempt to uncover genes aberrantly expressed in MDS, we performed an integrative genomic analysis of primary hematopoietic cells from MDS patients. These studies revealed that DOCK4, a guanine exchange factor was significantly under-expressed and hypermethylated in MDS stem and progenitor cells. DOCK4 is an important cofactor for various GTPases and is located on the chromosome 7q segment and is a common deletion in MDS. In addition to aberrant DNA methylation, our preliminary analysis reveals that DOCK4 is mutated in different sets of anemic patients as well, thus implicating it as an important gene in the pathogenesis of anemia. However, there is no knowledge of its functions during normal terminal erythroid development. We have used a dynamic model of human erythropoiesis to demonstrate that DOCK4 is highly expressed during late stages of normal erythropoiesis and knockdown of DOCK4 disrupts the F-actin assembly and alters the osmotic fragility of erythroblasts. Based on these data we hypothesize that DOCK4 is an important signaling intermediate in late-stage erythroblasts that is instrumental in maintaining erythroblast membrane homeostasis. Furthermore we hypothesize that aberrant silencing of DOCK4 contributes to ineffective erythropoiesis seen in MDS. We will test these hypotheses by carrying out the following aims. In Aim 1 we will determine the functional role of DOCK4 in late stages of erythroid differentiation using an in vitro human model that recapitulates all the stages of erythropoiesis. Following knockdown of DOCK4 by lentiviral shRNA, we will examine its impact on cell viability/proliferation, membrane homeostasis, enucleation and reticulocyte maturation. In Aim 2 we will determine the involvement of DOCK4 in key signaling pathways associated with erythropoiesis including its role in activation of downstream Rac and Rap GTPases. Novel downstream effectors will also be determined by phosphoproteomic analysis. In Aim 3 we will determine involvement of DOCK4 in the pathogenesis of anemia associated with MDS using primary erythroid cells isolated from MDS patients. Several approaches such as mutational analysis, promoter DNA methylation and FISH analysis in primary late-stage erythroblasts will be performed. Finally, we will determine whether restoration of DOCK4 expression in MDS erythroblasts will reverse the defects associated with F-actin skeleton and other membrane dynamics. Altogether, these studies will conclusively establish the role of DOCK4 in normal erythropoiesis as well as its significance in the pathogenesis of anemia in MDS. (End of Abstract)

描述（由申请人提供）： 红细胞生成在分化的每个阶段都受到严格控制。虽然在初始阶段的信号传导和细胞存活方面知道很多，但关于 在红细胞膜重塑、去核和网织红细胞成熟的晚期阶段的信号传导。此外，在诸如骨髓增生异常综合征（MDS）的病症中，其中贫血是最常见的表现和发病的主要原因，焦点主要是研究造血的早期阶段。为了发现MDS中异常表达的基因，我们对MDS患者的原代造血细胞进行了整合基因组分析。这些研究表明，DOCK 4，一种鸟嘌呤交换因子，在MDS干细胞和祖细胞中显著低表达和高甲基化。DOCK 4是多种GTP酶的重要辅助因子，位于染色体7 q片段，是MDS中常见的缺失。除了异常的DNA甲基化，我们的初步分析显示，DOCK 4在不同的贫血患者中也发生了突变，从而暗示它是贫血发病机制中的一个重要基因。然而，有没有知识，其功能在正常的终端红细胞发育。我们已经使用了人类红细胞生成的动态模型来证明DOCK 4在正常红细胞生成的晚期阶段高度表达，并且DOCK 4的敲低破坏了F-肌动蛋白组装并改变了成红细胞的渗透脆性。基于这些数据，我们假设DOCK 4是晚期成红细胞中的重要信号传导中间体，其有助于维持成红细胞膜稳态。此外，我们假设DOCK 4的异常沉默有助于MDS中观察到的无效红细胞生成。我们将通过实现以下目标来检验这些假设。在目的1中，我们将使用概括红细胞生成的所有阶段的体外人类模型来确定DOCK 4在红细胞分化的晚期阶段中的功能作用。通过慢病毒shRNA敲低DOCK 4后，我们将研究其对细胞活力/增殖、膜稳态、去核和网织红细胞成熟的影响。在目标2中，我们将确定DOCK 4在与红细胞生成相关的关键信号通路中的参与，包括其在下游Rac和Rap GTP酶激活中的作用。还将通过磷酸蛋白质组学分析确定新的下游效应物。在目的3中，我们将使用从MDS患者分离的原代红系细胞来确定DOCK 4在与MDS相关的贫血的发病机制中的参与。将在初级晚期成红细胞中进行几种方法，如突变分析、启动子DNA甲基化和FISH分析。最后，我们将确定MDS成红细胞中DOCK 4表达的恢复是否会逆转与F-肌动蛋白骨架和其他膜动力学相关的缺陷。总之，这些研究将最终确定DOCK 4在正常红细胞生成中的作用以及其在MDS贫血发病机制中的意义。 (End摘要）