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化和成熟的基本分子机制，这对于成年巨核细胞（Mk）有效形成血小板至关重要。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，经历倍性的逐步增加，这一过程称为核内有丝分裂，导致大的多小叶，多倍体核。在骨髓增生异常疾病（MDS）和其他形式的骨髓衰竭，低倍性巨核细胞往往占主导地位。我的实验室已经确定了MKL 1/SRF信号转导通路和多倍化之间的联系。血清反应因子（Serum Response Factor，SRF）是一种普遍存在的调节细胞因子相关基因的转录因子。转录辅因子MKL 1结合并激活SRF蛋白。我们已经表明，MKL 1的表达上调Mk成熟过程中，MKL 1是必不可少的正常Mk多倍化。此外，我们已经表明，KO的SRF在Mk谱系导致更严重的表型比MKL 1 KO小鼠血小板减少，并显着降低倍性的Mk异常核和细胞质超微结构通过电子显微镜。先前使用延时显微镜观察内有丝分裂Mk的研究表明，细胞从2N进展到4 N的初始内有丝分裂分裂事件是由于后期胞质分裂失败而发生的，而后期的内有丝分裂事件（4 N到8 N，8 N到16 N等）高达128 N没有显示出显著的卵裂沟形成。GEF-H1和ECT 2，两个鸟嘌呤交换因子，是必不可少的激活和招聘RhoA的分裂沟完成胞质分裂，必须下调顺序Mk进行多倍化。将GEFH 1调节、MKL 1和多倍化联系起来，我们现在已经确定MKL 1诱导GEF-H1下调，并且shRNA介导的GEF-H1敲低挽救了MKL-/-巨核细胞中的倍性缺陷。为了更好地阐明MKL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MKL 1调节GEFH 1表达以促进Mk多倍化的遗传机制; 2）确定肌动蛋白动力学在MKL 1定位和转录激活中的作用; 3）确定MKL 1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，本发明还提供了一种方法， 所获得的数据将揭示MKL 1的分子调节和功能，MKL 1是RhoA/SRF途径中的转录辅因子，其在骨髓增生异常（MDS）尤其是与5 q-综合征相关的骨髓增生异常（MDS）中失调。

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.