Functional role of CD34 family in hematopoiesis

CD34家族在造血中的功能作用

基本信息

批准号：
10009826
负责人：
Pradeep Sathyanarayana
金额：
$ 43万
依托单位：
MAINEHEALTH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-15 至 2020-11-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10009826
关键词：
Ablation Adhesions Antibodies Attenuated Autoimmune Diseases Biological Assay Blood Bone Marrow Bone Marrow Purging Bone Marrow Transplantation CD34 gene Cell Cycle Cell surface Cells Co-Immunoprecipitations Colony Stimulating Factor Activation Communicable Diseases Data Development Disease Dominant-Negative Mutation Emergency Situation Exhibits Family Feedback Flow Cytometry Genes Genetic Transcription Goals Granulocyte Colony-Stimulating Factor Granulopoiesis Hematopoiesis Hematopoietic Hematopoietic stem cells Immune system Infection Inflammation Inflammatory Integral Membrane Protein Integrins Ionizing radiation Knock-out Knockout Mice Lead Lipopolysaccharides Listeria monocytogenes Mass Spectrum Analysis Mediating Metamyelocyte Migration Assay Molecular Molecular Conformation Monomeric GTP-Binding Proteins Mus Myelocyte Myelogenous Myeloid Progenitor Cells Neutropenia Pathway interactions Phagocytes Pharmaceutical Preparations Phenotype Phosphoserine Physiological Processes Play Population Process Progranulocytes Protein Family Proteins Regulation Research Reticulocytes Role STAT3 gene Signal Pathway Signal Transduction Structure System Western Blotting allergic response chemotherapy chromatin immunoprecipitation cytokine gain of function granulocyte granulocyte-monocyte progenitors infectious disease treatment inhibitor/antagonist loss of function migration mouse model mutant myeloblast neutrophil new therapeutic target novel novel strategies peripheral blood phosphoproteomics podocalyxin progenitor response retroviral transduction shear stress small hairpin RNA trafficking

项目摘要

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.

本研究的目的是确定 Podocalyxin (PODXL) 在调节中的作用 PODXL 是一种细胞表面跨膜蛋白，属于 CD34 蛋白家族。初步数据表明，Podocalyxin 在骨髓祖细胞中表达，并由以下物质特异性诱导：粒细胞集落刺激因子（G-CSF），调节粒细胞生成的主要细胞因子。 Podocalyxin 在 G-CSF 诱导的粒细胞生成中的作用，我们生成了 Podxl-flox/flox 小鼠模型。 Vav1-Cre 驱动小鼠用于选择性删除造血细胞（包括骨髓细胞系）中的 Podxl。 Podxl 条件敲除 (cKO) 小鼠中的 G-CSF 攻击导致 G-CSF 水平显着升高外周血 (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。粒细胞生成是在后等条件下满足中性粒细胞需求的主要途径之一此外，G-CSF 诱导的粒细胞生成对于导致中性粒细胞减少症至关重要。解决感染期间的炎症，通常称为紧急粒细胞生成水平升高。在没有 PODXL 的情况下 CSF 诱导的中性粒细胞意味着 PODXL 在反应性中具有负调节作用粒细胞生成。用G-CSF攻击的Podxl-cKO小鼠显示粒细胞水平显着升高。骨髓中的单核细胞祖细胞 (GMP) 和未成熟中性粒细胞 (BM)。未成熟中性粒细胞的水平伴随着成熟中性粒细胞的显着减少，很可能由于加速释放至 PB 值得注意的是，缺乏 Podxl 的 PB 中性粒细胞显示出显着丰富的丰度。探究 Podxl 的作用机制，即免疫共沉淀加质量。使用来自 G-CSF 攻击小鼠的骨髓祖细胞应用光谱测定 (LC-MS/MS) 方法。 Rap1a 是一种与 Ras 相关的小 GTP 酶，是骨髓中主要的共同回收 Podxl 伴侣。造血祖细胞 Podxl-cKO 导致呼吸 G-CSF 激活 Rap1aGTP，以及 Rap1aGTP 抑制减弱了 Podxl-cKO 中性粒细胞迁移，重要的是，G-CSF 诱导的 Podxl 缺陷型中性粒细胞。显示出吞噬活性增加，因此，我们研究 PODXL 在中发挥负调节作用。 G-CSF 诱导的反应性粒细胞生成，其中 Podxl 在 GMP 上发挥增殖和分化作用和未成熟的中性粒细胞，而 Podxl-Rap1 是一个信号轴，通过由外向内调节中性粒细胞运输因此，具体目标是：目标 1：表征 Podocalyxin 的功能作用。目的 2：确定 Podocalyxin-Rap1a 轴在 G-CSF 诱导的反应性粒细胞生成过程中的作用。反应性粒细胞生成过程中的中性粒细胞运输将显着推进该领域的发展。了解抑制紧急粒细胞生成的关键调节机制。完成拟议的研究可能会产生新的药物靶点来治疗感染性中性粒细胞减少症疾病、化疗、电离辐射和自身免疫性疾病。