Functional role of CD34 family in hematopoiesis

本研究的目的是确定Podocalysin(PODXL)在调节 粒细胞生成。PODXL是一种细胞表面跨膜蛋白，属于CD34家族蛋白。我们的 初步数据显示，Podocalyx在髓系祖细胞中表达，并由 粒细胞集落刺激因子(G-CSF)，是调节粒细胞生成的主要细胞因子。为了破译 Podocalysin在G-CSF诱导的粒细胞生成中的作用，我们建立了PODXL-FLOX/FLOX小鼠模型。 用Vav1-CRE驱动的小鼠选择性地删除包括髓系在内的造血细胞中的PODXL。 在PODXL条件性基因敲除(CKO)小鼠中，G-CSF刺激导致显著升高的 外周血(PB)中性粒细胞。G-CSF诱导的粒细胞生成，也称为需求适应或反应性 粒细胞生成，是在后遗症等条件下满足中性粒细胞需求的主要途径之一 导致中性粒细胞减少症的化疗或电离辐射。此外，G-CSF诱导的粒细胞生成对 在感染期间消除炎症，通常被称为紧急粒细胞生成。G-水平升高 缺乏PODXL的脑脊液诱导的中性粒细胞暗示PODXL在反应性反应中的负调节作用 粒细胞生成。用G-CSF攻击的PODXL-CKO小鼠表现出明显的粒细胞水平升高。 骨髓中的单核细胞祖细胞(GMP)和未成熟中性粒细胞(BM)。有趣的是，我们发现 未成熟的中性粒细胞水平很可能伴随着成熟中性粒细胞的显著减少。 由于加速释放到PB。值得注意的是，缺乏PODXL的PB中性粒细胞显著增加 迁移能力。为了询问PODXL的作用机制，免疫共沉淀加质量 采用LC-MS/MS方法对G-CSF攻击小鼠的骨髓祖细胞进行检测。 Rap1a是一个RAS相关的小GTP酶，是PODXL主要的共检索伙伴。在骨髓中 造血祖细胞PODXL-CKO诱导RAP1aGTP和RAP1aGTP的G-CSF活性增强 抑制PODXL-CKO中性粒细胞迁移。重要的是，G-CSF诱导的PODXL缺陷性中性粒细胞 表现出更强的吞噬活性。因此，我们假设PODXL在 粒细胞集落刺激因子诱导反应性粒细胞生成PODXL对GMP的增殖和分化作用 和未成熟的中性粒细胞，PODXL-Rap1是一个信号轴，通过自外向内调节中性粒细胞的运输 和由内而外的信号。因此，具体目标是：目标1：表征Podocalysin的功能作用 在G-CSF诱导的反应性粒细胞生成过程中。目标2：确定Podocalysin-Rap1a轴在 中性粒细胞在反应性粒细胞生成过程中的运输。拟议的研究将在#年显著推进这一领域。 了解抑制紧急粒细胞生成的关键调控机制。更进一步，成功了 拟议研究的完成可能导致治疗感染性疾病引起的中性粒细胞减少症的新的药物靶点。 疾病、化疗、电离辐射和自身免疫性疾病。

The goal of this research is to define the role of Podocalyxin (PODXL) in the regulation of granulopoiesis. PODXL is a cell surface transmembrane protein belonging to the CD34 family of proteins. Our preliminary data show that Podocalyxin is expressed in myeloid progenitors and is specifically induced by granulocyte colony stimulating factor (G-CSF), the principal cytokine regulating granulopoiesis. To decipher the role of Podocalyxin in G-CSF-induced granulopoiesis, we have generated a Podxl-flox/flox mouse model. Vav1-Cre driver mice were used to selectively delete Podxl in hematopoietic cells, including myeloid lineages. G-CSF challenge in Podxl–conditional knockout (cKO) mice resulted in significantly elevated levels of peripheral blood (PB) neutrophils. G-CSF-induced granulopoiesis, also known as demand-adapted or reactive granulopoiesis, is one of the principal pathways to meet neutrophil requirements under conditions such as post chemotherapy or ionizing radiation that cause neutropenia. Further, G-CSF-induced granulopoiesis is critical to resolve inflammation during infections, often referred to as emergency granulopoiesis. Elevated levels of G- CSF-induced neutrophils in the absence of PODXL imply a negative regulatory role for PODXL in reactive granulopoiesis. Podxl-cKO mice challenged with G-CSF exhibited significantly elevated levels of granulocyte- monocyte progenitors (GMP) and immature neutrophils in bone marrow (BM). Interestingly, we found that high levels of immature neutrophils were concomitant with a significant reduction of mature neutrophils, most likely due to accelerated release to PB. Notably, Podxl- deficient PB neutrophils showed significantly heightened migration abilities. To interrogate Podxl's mechanisms of action, a co-immunoprecipitation plus mass spectrometry (LC-MS/MS) approach was applied using myeloid progenitors from G-CSF-challenged mice. Rap1a, a Ras-related small GTPase, was a predominant co-retrieved Podxl partner. In bone marrow hematopoietic progenitor cells, Podxl-cKO led to heightened G-CSF activation of Rap1aGTP, and Rap1aGTP inhibition attenuated Podxl-cKO neutrophil migration. Importantly, G-CSF-induced Podxl deficient neutrophils exhibited increased phagocytotic activity. Thus, we hypothesize that PODXL plays a negative regulatory role in G-CSF-induced reactive granulopoiesis where Podxl exerts proliferation and differentiation effects at GMPs and immature neutrophils, and Podxl-Rap1 is a signaling axis that regulates neutrophil trafficking via outside-in and inside-out signaling. Thus the specific aims are: Aim 1: To characterize the functional role of Podocalyxin during G-CSF-induced reactive granulopoiesis. Aim 2: To define the role of the Podocalyxin-Rap1a axis in neutrophil trafficking during reactive granulopoiesis. The proposed studies will significantly advance the field in understanding the critical regulatory mechanism that restrains emergency granulopoiesis. Further, successful completion of the proposed studies may lead to novel drug targets to treat neutropenia resulting from infectious diseases, chemotherapy, ionizing radiation and autoimmune disorders.