Niche-induced Signaling in Progenitor B Cell Development

B 祖细胞发育中生态位诱导的信号转导

• 批准号: 7889153
• 负责人: Leslie Eric Silberstein
• 金额: $ 42.88万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-11 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7889153
• 关键词: AMD3100; Acute Lymphocytic Leukemia; Adhesions; Adhesives; Adoptive Transfer; Antibodies; Apoptosis; B-Cell Development; B-Lymphocyte Subsets; B-Lymphocytes; Behavior; Biological Assay; Blood Circulation; Bone Marrow; C-KIT Gene; CXCL12 gene; CXCR4 gene; Cell Adhesion; Cell Adhesion Molecules; Cell Cycle; Cell division; Cell physiology; Cells; Chronic Lymphocytic Leukemia; Cues; Diaphyses; Disease; Embryo; Exhibits; Extracellular Matrix; Femur; Fibroblasts; Frequencies; Gene Targeting; Growth; Hematopoietic; Immunoglobulin M; Immunologic Deficiency Syndromes; Impairment; In Vitro; Interleukin-7; Label; Laser Scanning Cytometry; Location; Maps; Mediating; Metaphysis; Methods; Methylcellulose; Molecular; Mus; Osteoblasts; PTK2 gene; PTPRC gene; Pathogenesis; Pathway interactions; Peripheral; Play; Population; Positioning Attribute; Proto-Oncogene Protein c-kit; Quantum Dots; Relative (related person); Research; Reticular Cell; Role; Signal Pathway; Signal Transduction; Signaling Protein; Staging; Surface Antigens; Testing; Tissues; Vascular Endothelial Cell; base; cell growth; cell motility; cell type; chemokine; cytokine; in vivo; insight; intravital microscopy; leukemia; migration; multi-photon; neoplastic; novel; progenitor; public health relevance; research study; self-renewal

DESCRIPTION (provided by applicant): In the bone marrow (BM), progenitor B cell growth and survival depend on cues from distinct microenvironments, e.g. niches. Niche specific signals involving among others cytokines, chemokines and adhesion molecules, also influence the localization and migration of developing B cells in BM. The key findings of our preliminary studies are that Fak deletion in CD19-Cre Fak fl/fl mice leads to impairment of progenitor B cell development in BM and egress of pro-B cells into the peripheral circulation. In this application, we hypothesize that FAK functions as an integrator of niche-induced signaling regulating progenitor B cell fate. Three aims are proposed to test this hypothesis. Aim 1 will define how FAK regulates progenitor B cell growth and survival. Both in vitro methylcellulose based colony cell forming cell assays as well as in vivo adoptive transfer experiments will be used to examine the role of FAK in progenitor B cell cell cycle, proliferation, self-renewal, differentiation and apoptosis. To assess FAK function molecularly, the role of FAK in signaling pathways regulating progenitor B cell growth and survival will be defined. We will extend our findings from CD19-Cre Fak fl/fl mice and determine the effect of Fak deletion on progenitor B cell development in mb1- Cre EYFP mice, as they exhibit higher efficiency of Cre-mediated deletion beginning at the early pro-B cell stage. Aim 2 will define how FAK regulates the motility and distribution of progenitor B cell populations in distinct anatomical locations within the BM. Laser scanning cytometry (LSC) analysis of cryopreserved femurs will quantify and map the morphological position(s) of Fak wt and Fak-/- progenitor B cell populations in the diaphysis (endosteal vs. central medullary regions) and metaphyses of the BM. Multi-photon intravital microscopy (MP-IVM) will characterize in vivo the dynamic behavior of Fak wt and Fak-/- progenitor B cell populations and their interactions with adjacent cells in BM. Cell biological assays will be applied towards defining mechanisms for observed changes in Fak-/- progenitor B cell motility and distribution in BM. Aim 3: To define how FAK regulates the association of progenitor B cells with specific niche cell types. Niche cells, e.g. osteoblasts, vascular endothelial cells, IL-7 producing cells and CXCL12-abundant reticular (CAR) cells will be identified by immunohistology on longitudinal sections of femurs. LSC, in conjunction with fluorescent immunostaining of surface antigens for progenitor B and niche cells, will objectively quantify the relative frequency that Fak wt and Fak -/- progenitor B cells are found in close association with each niche cell type throughout the BM cavity. The importance of the CXCR4-FAK pathway in mediating progenitor B cell lodgement in specific BM compartments will be evaluated by treating Fak wt and Fak -/- mice with a CXCR4 antagonist (e.g., AMD3100), prior to LSC analyses. A better definition of environmental signals controlling B cell development and function will have important implications for research in pathogenesis and treatment of B cell disorders including leukemia and immunodeficiency. PUBLIC HEALTH RELEVANCE: FAK is a ubiquitously expressed cell signaling protein, which can regulate cell adhesion, growth and survival. We hypothesize that FAK functions as a key integrator of environmental signals necessary for the growth of normal and neoplastic B cells in the bone marrow. Thus, the research proposed here will provide important insight into previously unrecognized pathogenetic mechanisms of B cell leukemia as well as immunodeficiency syndromes.

描述（由申请人提供）：在骨髓（BM）中，祖细胞生长和存活取决于不同微环境的线索，例如利基。涉及细胞因子，趋化因子和粘附分子等涉及的特定特定信号也会影响BM中B细胞的定位和迁移。我们初步研究的主要发现是，CD19-CRE FL/FL小鼠中的FAK缺失会导致BM中祖细胞B细胞发育的损害，并使Pro-B细胞出口到外围循环中。在此应用程序中，我们假设FAK充当了细菌诱导的调节祖细胞B细胞命运的信号传导的积分器。提出了三个目的来检验这一假设。 AIM 1将定义FAK如何调节祖细胞B细胞生长和生存。基于体外甲基纤维素的基于甲基纤维素细胞形成细胞分析以及体内收养转移实验都将用于检查FAK在祖细胞B细胞周期，增殖，自我更新，分化和凋亡中的作用。为了分子评估FAK功能，将定义FAK在调节祖细胞生长和存活的信号通路中的作用。我们将从CD19-CRE FL/FL小鼠中扩展发现，并确定FAK缺失对MB1- CRE EYFP小鼠中祖细胞发育的影响，因为它们在Pro-B早期细胞阶段开始表现出更高的CRE介导的缺失效率。 AIM 2将定义FAK如何调节BM内不同解剖位置中祖细胞群体的运动和分布。冷冻保存的股骨的激光扫描细胞仪（LSC）分析将量化和绘制diaphess（内骨与中央髓质区域）和BM中的FAK WT和FAK - / - 祖细胞B细胞种群的形态位置。多光子插入式显微镜（MP-IVM）将在体内表征FAK WT和FAK - / - 祖细胞B细胞种群的动态行为及其与BM中相邻细胞的相互作用。细胞生物学测定将用于定义的定义机制，以观察到FAK - / - 祖细胞B细胞运动和BM分布的变化。目标3：定义FAK如何调节祖细胞与特定细胞类型的祖细胞的关联。利基细胞，例如成骨细胞，血管内皮细胞，IL-7产生细胞和CXCL12丰富的网状（CAR）细胞将通过免疫组织学在股骨的纵向切片上鉴定。 LSC与祖细胞和小裂细胞的表面抗原的荧光免疫染色结合使用，将客观地量化FAK WT和FAK和FAK - / - 祖细胞B细胞的相对频率与整个BM腔密切相关。在LSC分析之前，CXCR4-FAK途径将通过用CXCR4拮抗剂（例如AMD3100）处理FAK WT和FAK - / - 小鼠，在特定BM室中介导祖细胞B细胞的腔内。控制B细胞发育和功能的环境信号的更好定义将对B细胞疾病（包括白血病和免疫缺陷）的发病和治疗的研究具有重要意义。 公共卫生相关性：FAK是一种普遍表达的细胞信号蛋白，可以调节细胞粘附，生长和生存。我们假设FAK是骨髓中正常和肿瘤B细胞生长所必需的环境信号的关键集成因子。因此，此处提出的研究将为B细胞白血病和免疫缺陷综合征的先前未知的致病机制提供重要的见解。