Translational Control of Megakaryocyte and Platelet Function in Sepsis

总结/摘要 在翻译水平上调控基因表达（即，mRNA-蛋白）在正常人中了解甚少。 事实上，炎症性疾病是否改变巨核细胞的翻译反应尚不清楚。 巨核细胞和血小板。也就是说，炎症有助于许多疾病的发病机制， 疾病如败血症和炎性激动剂被认为是mRNA翻译的有效触发剂。这 一项赠款提案将利用人类和小鼠模型系统（体外和体内）和最先进的技术 测序（核糖体足迹法和eIF 4 E富集），以确定翻译控制途径 调节巨核细胞和血小板基因表达-在健康和败血症的情况下。 支持该项目的初步数据表明，哺乳动物雷帕霉素靶蛋白（mTOR）和 MAP激酶相互作用丝氨酸/苏氨酸蛋白激酶1（MNK 1），两种翻译控制途径， 聚集在翻译的起始步骤，调节巨核细胞中的蛋白质合成事件和功能 和血小板。这些初步结果导致了这样的假设，即脓毒症环境将 显著改变巨核细胞和血小板中mTOR和/或MNK 1依赖性翻译， 导致在脓毒症过程中血小板的不适当形成和功能。具体 aim 1将检验脓毒症过程中产生的炎症激动剂会改变蛋白质合成的假设。 巨核细胞中的事件以及血小板以mTOR和MNK 1依赖的方式形成。具体 目的2将确定mTOR或MNK 1的遗传缺失如何调节细胞的离体和体内功能。 血小板在存在或不存在炎症。具体目标3将检验脓毒症 环境激活mTOR和/或MNK 1信号通路，从而通过以下途径增加蛋白质合成： 血小板它还将确定小鼠血小板中mTor和/或Mnk 1的缺失是否会导致不良后果 在败血症的小鼠模型中。这些目标的成功完成将：1）鉴定翻译的mRNA， 在mTOR和/或MNK 1依赖性控制下的巨核细胞，以及这些翻译 信号通路调节血小板生成的最后步骤，2）描绘mTOR和MNK 1的作用 对巨核细胞和血小板在健康和炎症中的功能的影响，3）表征无意的侧- 临床使用的mTOR和MNK 1抑制剂对巨核细胞和血小板的影响，以及4）确定 巨核细胞和血小板中的mTOR或MNK 1调节脓毒症模型的结果。生成的数据将 立即增加我们对翻译控制途径如何改变巨核细胞的理解， 血小板功能在脓毒症中的作用

Summary/Abstract Regulation of gene expression at the translational level (i.e., mRNA-protein) is poorly understood in normal megakaryocytes, and it is virtually unknown if inflammatory diseases alter translational responses in megakaryocytes and platelets. With that said, inflammation contributes to the pathogenesis of numerous diseases such as sepsis, and inflammatory agonists are considered potent triggers of mRNA translation. This grant proposal will utilize human and mouse model systems (in vitro and in vivo) and state-of-the-art sequencing (ribosomal footprinting and eIF4E-enrichment) to determine how translational control pathways regulate megakaryocyte and platelet gene expression – in the setting of health and septic situations. Preliminary data supporting this project demonstrates that the mammalian target of rapamycin (mTOR) and the MAP kinase-interacting serine/threonine-protein kinase 1 (MNK1), two translational control pathways that converge on the initiation step of translation, regulate protein synthetic events and functions in megakaryocytes and platelets. These preliminary results have led to the hypothesis that the septic milieu will significantly modify mTOR and/or MNK1-dependent translation in megakaryocytes and platelets, resulting in inappropriate formation and function of platelets during the course of sepsis. Specific Aim1 will test the hypothesis that inflammatory agonists generated during sepsis will alter protein synthetic events in megakaryocytes and the formation of platelets in an mTOR and MNK1-dependent manner. Specific Aim 2 will determine how genetic deletion of mTOR or MNK1 regulates the ex vivo and in vivo function of platelets in the presence or absence of inflammation. Specific Aim 3 will test the hypothesis that the septic milieu activates the mTOR and/or MNK1 signaling pathways and thereby increases protein synthesis by platelets. It will also determine if deletion of mTor and/or Mnk1 in mouse platelets leads to adverse outcomes in murine models of sepsis. Successful completion of these aims will 1) identify translated mRNAs in megakaryocytes that are under mTOR and/or MNK1-dependent control and whether these translational signaling pathways regulate the final steps of platelet production, 2) delineate the effects of mTOR and MNK1 on functions of megakaryocytes and platelets in health and inflammation, 3) characterize inadvertent side- effects of clinically-used mTOR and MNK1 inhibitors on megakaryocytes and platelets, and 4) determine if mTOR or MNK1 in megakaryocytes and platelets regulate outcomes in models of sepsis. Data generated will immediately increase our understanding of how translational control pathways alter megakaryocyte and platelet functions in the setting of sepsis.