Functional role of CD34 family in hematopoiesis

CD34家族在造血中的功能作用

• 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
• 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

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.

这项研究的目的是确定足曲利（PODXL）在调节中的作用 颗粒。 PODXL是属于CD34蛋白质家族的细胞表面跨膜蛋白。我们的 初步数据表明，足曲利蛋白在髓样祖细胞中表达，并由 粒细胞菌落刺激因子（G-CSF），主要的细胞因子控制粒硫代基因。破译 足细胞蛋白在G-CSF诱导的粒状植物中的作用，我们产生了PODXL-FLOX/FLOX小鼠模型。 VAV1-CRE驱动小鼠用于在包括髓样谱系在内的造血细胞中选择性删除PODXL。 PODXL – CONCONITIONAL敲除（CKO）小鼠的G-CSF挑战导致显着升高 外周血（PB）中性粒细胞。 G-CSF诱导的粒子植物，也称为需求适应或反应性 颗粒状是在诸如邮政等条件下满足中性粒细胞要求的主要途径之一 引起中性粒细胞减少症的化学疗法或电离辐射。此外，G-CSF诱导的粒子对 解决感染过程中的炎症，通常称为紧急粒状虫素。 G-的水平升高 在没有PODXL的情况下，CSF诱导的中性粒细胞意味着PODXL在反应性中的负调节作用 颗粒。 PODXL-CKO小鼠面对G-CSF的质疑，暴露于粒细胞的水平显着升高 单核细胞祖细胞（GMP）和骨髓（BM）中未成熟的中性粒细胞。有趣的是，我们发现很高 未成熟的中性粒细胞水平与成熟中性粒细胞显着降低，很可能 由于加速释放到PB。值得注意的是，缺乏PODXL的PB中性粒细胞显着增强 迁移能力。询问PODXL的作用机制，共免疫沉淀加质量 使用来自G-CSF挑战的小鼠的髓样祖细胞应用光谱法（LC-MS/MS）方法。 RAP1A是与RAS相关的小GTPase，是主要的共同退回PODXL伙伴。在骨髓中 造血祖细胞，PODXL-CKO导致RAP1AGTP的G-CSF激活增强和RAP1AGTP 抑制作用减弱了pODXL-CKO中性粒细胞迁移。重要的是，G-CSF诱导的PODXL缺乏中性粒细胞 表现出增加的吞噬活性。这，我们假设PODXL在 G-CSF诱导的反应性颗粒，其中PODXL在GMPS处执行增殖和分化效应 和未成熟的嗜中性粒细胞，PODXL-RAP1是一个信号轴，可通过外部进行调节中性粒细胞运输 和内而外的信号传导。具体目的是：目标1：表征足肠蛋白的功能作用 在G-CSF诱导的反应性颗粒过程中。目的2：定义Pocalyxin-rap1a轴的作用 反应性颗粒虫期间的中性粒细胞运输。拟议的研究将大大推动 了解限制紧急颗粒的关键监管机制。此外，成功 拟议的研究的完成可能会导致新的药物靶标，以治疗感染性的中和 疾病，化学疗法，电离辐射和自身免疫性疾病。