Mechanisms of megakaryocyte maturation

描述（由申请人提供）：本提案的重点是确定巨核细胞多倍体化和成熟的基本分子机制，这是成年巨核细胞（Mk）有效血小板形成的必要条件。在严格控制的分化过程中，二倍体巨核母细胞在没有细胞分裂的情况下，通过重复的DNA复制，逐渐增加倍性，这一过程被称为内膜分裂，产生大的多分叶多倍体细胞核。在骨髓增生异常疾病（MDS）和其他形式的骨髓衰竭中，低倍性巨核细胞通常占主导地位。我的实验室已经确定了MKL1/SRF信号转导途径与多倍体化之间的联系。SRF（血清反应因子）是一种普遍存在的调节细胞骨架相关基因的转录因子。转录辅助因子MKL1结合并激活SRF蛋白。我们已经证明MKL1的表达在Mk成熟过程中上调，并且MKL1对正常的Mk多倍体化至关重要。此外，我们在电镜下发现Mk谱系中SRF的KO比MKL1 KO小鼠的表型更严重，血小板减少更大，Mk倍性显著降低，细胞核和细胞质超微结构异常。先前使用延时显微镜观察内膜分裂Mk的研究表明，细胞从2N到4N的初始内膜分裂事件是由于细胞分裂后期的失败而发生的，而后期的内膜分裂事件（4N到8N， 8N到16n等）直到128N并没有明显的分裂沟形成。GEF-H1和ECT2是两个鸟嘌呤交换因子，对于RhoA的激活和募集到卵裂沟以完成细胞分裂至关重要，它们必须依次下调才能使Mk进行多倍体化。将GEFH1调控、MKL1和多倍体化联系起来，我们现在已经确定MKL1诱导GEF-H1下调，shrna介导的GEF-H1敲低挽救了Mkl-/-巨核细胞的倍性缺陷。为了更好地阐明MKL促进巨核细胞多倍体化和成熟的生物学机制，我们建议：1)确定MKL1调控GEFH1表达促进Mk多倍体化的遗传机制；2)确定肌动蛋白动力学在MKL1定位和转录激活中的作用；3)确定MKL1促进巨核细胞成熟的机制。这些研究将有助于阐明调节正常Mk形成的机制，这对血小板的形成和功能至关重要。此外,

DESCRIPTION (provided by applicant): This proposal is focused on determining the basic molecular mechanisms underlying polyploidization and maturation of megakaryocytes, which are essential for effective platelet formation by adult megakaryocytes (Mk). In a tightly controlle differentiation process, diploid megakaryoblasts undergo a progressive increase in ploidy by repeated DNA replication without cell division, a process termed endomitosis, resulting in large multilobulated, polyploid nuclei. In myelodysplastic diseases (MDS) and other forms of BM failure, low ploidy megakaryoblasts often predominate. My laboratory has identified a link between the MKL1/SRF signal transduction pathway and polyploidization. SRF (Serum Response Factor) is a ubiquitous transcription factor that regulates cytoskeleton-associated genes. The transcriptional cofactor MKL1 binds to and activates the SRF protein. We have shown that MKL1 expression is upregulated during Mk maturation, and that MKL1 is essential for normal Mk polyploidization. In addition, we have shown that KO of SRF in the Mk lineage leads to a more severe phenotype than the MKL1 KO mice with a greater decrease in platelets, and significantly decreased ploidy of Mk with abnormal nuclear and cytoplasmic ultrastructure by electron microscopy. Prior studies using time-lapse microscopy to observe endomitotic Mk suggest that the initial endomitotic cleavage event in which cells progress from 2N to 4N occurs due to failure at late cytokinesis, whereas later endomitotic events (4N to 8N, 8N to16N, etc.) up to 128N do not show significant cleavage furrow formation. GEF-H1 and ECT2, two guanine exchange factors that are essential for activation and recruitment of RhoA to the cleavage furrow for completion of cytokinesis, must be downregulated sequentially for Mk to undergo polyploidization. Linking GEFH1 regulation, MKL1, and polyploidization, we have now determined that MKL1 induces GEF-H1 downregulation, and that shRNA-mediated GEF-H1 knockdown rescues the ploidy defect in Mkl-/- megakaryocytes. In order to better elucidate the biological mechanisms by which MKL promotes megakaryocyte polyploidization and maturation, we propose to 1) Determine the genetic mechanism by which MKL1 regulates GEFH1 expression to promote polyploidization of Mk; 2) Determine the role of actin dynamics in MKL1 localization and transcriptional activation, and 3) Determine the mechanism by which MKL1 promotes megakaryocyte maturation. These studies will help to elucidate the mechanisms regulating normal Mk formation, which is critical for platelet formation and function. In addition, the data obtained will reveal the molecular regulation and functions of MKL1, a transcriptional cofactor in the RhoA/SRF pathway, which is dysregulated in Myelodysplasia (MDS) especially that associated with 5q- syndrome. PUBLIC HEALTH RELEVANCE: Blood stem cells in the bone marrow are responsible for maintaining a population of cells, termed megakaryocytes, which release platelets into the blood. Platelets are needed to prevent bleeding. It is very important that the number of platelets in the blood be regulated. Too few platelets put patients at risk for bleeding, and too many platelets can cause blood clots. The work proposed in this grant focuses on novel findings that have elucidated how the megakaryocytes grow and mature in order to produce normal platelets. The aims of this work are to determine the basic molecular mechanisms this maturation of megakaryocytes, which is essential for effective platelet formation. The results of these studies will help us to understand normal platelet formation and function, which are affected during bone marrow transplantation putting patients at risk for bleeding, and which are abnormal in blood diseases in which megakaryocyte maturation and platelet formation are abnormal including Myelodysplasia. The studies may also lead to improved methods for manufacturing platelets for transfusion medicine.