Human Tyr-tRNA Synthetase as a Potent Effector of Megakaryocytopoiesis

 描述(申请人提供)：本项目的目标是了解酪氨酰-tRNA合成酶(YRS)在调节血小板生成中发挥其新的作用的机制，并在此信息的基础上开发治疗血小板减少症的新治疗策略。在初步研究中，我们已经确定YRs具有以前未被认识到的加速巨核细胞发育和血小板生成的特性。这种作用似乎不依赖于血小板生成素(TPO)信号，因为YRS可以支持体外培养的c-MPL-/-(TPO受体敲除)小鼠巨核细胞的增殖和成熟。本申请中提出的研究将建立YRs在调节血小板生成中的新功能的潜在机制。我们还将开发一种新的药物类别的药学方法，这些药物有可能在各种疾病中增强血小板的生成，而在这些疾病中，仅有TPO模拟物是无效的。在目标1中，我们将阐明YRs刺激巨核细胞生成的分子机制。根据初步研究，YRS对巨核细胞生成的影响至少有两种机制：(1)激活单核细胞和细胞因子的产生；(2)扩增Sca1+CD11b+祖细胞，产生造血细胞和血管细胞。我们将确定负责介导YRS效应的细胞和在此过程中靶向的受体(S)。对TLR2-/-和MyD88-/-小鼠的初步检测表明，TLR-MyD88通路参与了TLR-MyD88通路的调控。我们还将关注Sca1+CD11b+祖细胞的独特群体，这些细胞在骨髓细胞培养中通过YRS刺激得到极大的扩增。我们的初步结果提示，YRS诱导的Sca1+CD11b+祖细胞具有向造血细胞和血管平滑肌细胞分化的潜能。我们假设，这些祖细胞的扩张也有助于血小板的生成，要么是通过分化为造血祖细胞，要么是通过在血管壁龛中支持血小板原形成的血管细胞。在目标2中，我们将定义在巨核细胞生成中发挥作用所需的YRs的功能结构域，并使用蛋白质工程来设计具有最佳治疗窗口的YRs变体。在目标3中，我们将使用活体动物模型测试工程YRS作为治疗血小板减少症的潜在药物的有效性。除了在初步研究中采用的急性免疫介导性血小板减少模型外，我们还建立了由低剂量抗体注射、放射或化疗引起的慢性血小板减少模型，以评估YRS在各种情况下的效果，就像在人类病理中可能发生的那样。最后，为了测试血栓副作用的可能性，我们将确定YRS是否影响血小板激活、血小板与白细胞的相互作用以及血栓形成的模型。本申请中提出的研究将描绘一种新的细胞途径，其中YRs有助于调节血小板的产生；并支持具有翻译潜力的创新方法的开发，以改善血小板减少症的治疗。

 DESCRIPTION (provided by applicant): Goal of this project is to understand the mechanisms through which tyrosyl-tRNA synthetase (YRS) exerts its newly recognized role in regulating platelet production and, on the bases of this information, develop a novel therapeutic strategy for the treatment of thrombocytopenia. In preliminary studies we have determined that YRS exhibits the previously unrecognized property of accelerating megakaryocyte development and platelet generation. This effect appears to be independent of thrombopoietin (TPO) signaling because YRS can support the proliferation and maturation of c-Mpl-/- (TPO receptor knock-out) mouse megakaryocytes in vitro. The studies proposed in this application will establish the mechanisms underlying the novel function of YRS in the regulation of platelet production. We will also develop a pharmaceutical approach to a new class of drugs with the potential of enhancing platelet production in various diseases in which TPO mimetics alone are not effective. In aim 1, we will elucidate the molecular mechanisms by which YRS stimulates megakaryocytopoiesis. Based on preliminary studies, the effect of YRS on megakaryocytopoiesis is mediated by at least two mechanisms: (1) activation of monocytic cells and cytokine production, and (2) expansion of Sca1+CD11b+ progenitors that give rise to hematopoietic cells and vascular cells. We will identify the cells responsible to mediate the effect of YRS and the receptor(s) targeted in the process. Involvement of TLR-MyD88 pathway has been shown by preliminary testing of TLR2-/- and MyD88-/- mice. We will also focus on a unique population of Sca1+CD11b+ progenitor cells that are greatly expanded by YRS stimulation in bone marrow cell cultures. Our preliminary result suggested the differentiation potential of YRS-induced Sca1+CD11b+ progenitor cells into hematopoietic cells and vascular smooth muscle cells. We hypothesize that expansion of these progenitor cells also contributes to thrombocytopoiesis either by differentiating into hematopoietic progenitors or vascular cells that support proplatelet formation in the vascular niche. In aim 2, we will define the functional domains of YRS required to exert the effects on megakaryocytopoiesis, and use protein engineering to de- sign YRS variants with an optimal therapeutic window. In Aim 3, we will test the efficacy of engineered YRS as a potential drug for the treatment of thrombocytopenia using in vivo animal models. In addition to the acute immune-mediated thrombocytopenia model employed in preliminary studies, we are generating models of chronic thrombocytopenia induced by low-dose antibody administration, irradiation or chemotherapy to evaluate the effects of YRS in various conditions as may occur in human pathology. Finally, to test the possibility of thrombotic side effects, we will determine whether YRS affects platelet activation, platelet-leukocyte inter- actions and models of thrombosis. The studies proposed in this application will delineate a novel cellular pathway in which YRS contributes to the regulation of platelet production; and support the development of innovative approaches with translational potential for improving the treatment of thrombocytopenia.