Mechanisms of megakaryocyte maturation

描述(申请人提供)：这项建议集中于确定巨核细胞多倍化和成熟的基本分子机制，这是成人巨核细胞有效形成血小板所必需的。在严格控制的分化过程中，二倍体巨核细胞通过重复DNA复制而不进行细胞分裂而逐渐增加倍性，这一过程称为内丝分裂，导致大的多叶、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，低倍体巨核细胞往往占主导地位。我的实验室已经确定了MKL1/SRF信号转导通路与多倍化之间的联系。SRF(血清反应因子)是一种普遍存在的转录因子，调节细胞骨架相关基因。转录辅因子MKL1结合并激活SRF蛋白。我们已经证明，MKL1在Mk成熟过程中表达上调，并且MKL1是正常Mk多倍化所必需的。此外，我们还发现，在Mk系中，SRF的KO导致比MKL1 KO小鼠更严重的表型，在电子显微镜下，血小板减少，Mk倍体显著减少，核质超微结构异常。以前用时间推移显微镜观察内丝分裂Mk的研究表明，细胞从2N进展到4N的最初内丝分裂事件是由于胞质分裂后期的失败而发生的，而后来的内丝分裂事件(4N到8N，8N到16N等)。在128N以下，没有明显的解理沟形成。对RhoA的激活和募集到分裂沟以完成胞质分裂至关重要的两个鸟嘌呤交换因子-Gf-H1和ECT2，必须依次下调，才能使Mk发生多倍化。将GEFH1调控、MKL1和多倍化联系起来，我们现在已经确定MKL1诱导了环境基金-H1的下调，并且shRNA介导的环境基金-H1基因敲除可以挽救MKL-/-巨核细胞的倍体缺陷。为了更好地阐明MKL1促进巨核细胞多倍化和成熟的生物学机制，我们建议：1)确定MKL1调节GEFH1表达促进MK多倍化的遗传机制；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正常巨噬细胞形成的调节机制，而正常巨噬细胞形成是血小板形成和功能的关键。此外, 这些数据将揭示MKL1的分子调控和功能，MKL1是RhoA/SRF途径中的一个转录辅助因子，在骨髓发育不良症(MDS)尤其是与5q综合征相关的MKL1中调节失调。

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.