The role of Dock4 in normal and aberrant erythropoiesis

描述(由申请人提供)： 红细胞的生成在分化的每个阶段都受到严格的控制。尽管人们对初始阶段的信号转导和细胞存活知之甚少，但对 红细胞膜重塑、去核和网织红细胞成熟后期的信号转导。此外，在诸如骨髓增生异常综合征(MDS)这样的疾病中，贫血是最常见的表现和发病的主要原因，研究的重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综合基因组分析。这些研究表明，鸟嘌呤交换因子DOCK4在MDS干细胞和祖细胞中显著低表达和高甲基化。DOCK4是多种GTP酶的重要辅因子，位于染色体7q段，是MDS中常见的缺失。除了DNA甲基化异常外，我们的初步分析显示，DOCK4在不同的贫血患者中也发生了突变，因此它可能是贫血发病机制中的一个重要基因。然而，目前尚不清楚其在红系终末发育过程中的功能。我们使用了一个人类红细胞生成的动态模型，证明了DOCK4在正常红细胞生成的后期高表达，并且DOCK4的敲除破坏了F-肌动蛋白的组装，改变了红细胞的渗透脆性。根据这些数据，我们推测DOCK4是晚期红细胞中重要的信号中间产物，有助于维持红细胞膜的动态平衡。此外，我们假设DOCK4的异常沉默有助于MDS中看到的无效的红细胞生成。我们将通过实现以下目标来检验这些假设。在目标1中，我们将使用一个概括了红系形成的所有阶段的体外人体模型来确定DOCK4在红系分化后期的功能作用。在慢病毒shRNA敲除DOCK4后，我们将检测其对细胞存活/增殖、膜动态平衡、去核和网织红细胞成熟的影响。在目标2中，我们将确定DOCK4在与红细胞生成相关的关键信号通路中的作用，包括它在下游Rac和Rap GTP酶激活中的作用。新的下游效应子也将通过磷酸蛋白质组分析来确定。在目标3中，我们将利用从MDS患者分离的原代红系细胞来确定DOCK4在MDS相关贫血的发病机制中的作用。将对原代晚期红细胞进行突变分析、启动子DNA甲基化和FISH分析等几种方法。最后，我们将确定在MDS红细胞中恢复DOCK4的表达是否会逆转与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DOCK4在正常红细胞生成中的作用以及它在MDS贫血发病机制中的意义。 (摘要结束)

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)