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B细胞生长和存活取决于来自不同微环境（例如小生境）的线索。尤其涉及细胞因子、趋化因子和粘附分子的小生境特异性信号也影响发育中的B细胞在BM中的定位和迁移。我们初步研究的关键发现是，CD 19-Cre Fak fl/fl小鼠中的Fak缺失导致BM中祖B细胞发育受损和前B细胞流出到外周循环中。在本申请中，我们假设FAK作为小生境诱导的信号调节祖B细胞命运的整合剂发挥功能。提出了三个目标来检验这一假设。目的1将明确FAK如何调节祖B细胞的生长和存活。将使用基于体外甲基纤维素的集落细胞形成细胞测定以及体内过继转移实验来检查FAK在祖B细胞细胞周期、增殖、自我更新、分化和凋亡中的作用。为了从分子水平评估FAK的功能，将确定FAK在调节祖B细胞生长和存活的信号传导途径中的作用。我们将从CD 19-Cre Fak fl/fl小鼠中扩展我们的发现，并确定Fak缺失对mb 1- Cre EYFP小鼠中祖B细胞发育的影响，因为它们在早期前B细胞阶段开始表现出更高的Cre介导的缺失效率。目的2将定义FAK如何调节BM内不同解剖位置中祖B细胞群的运动性和分布。冷冻保存股骨的激光扫描细胞术（LSC）分析将量化和绘制Fak wt和Fak-/-祖B细胞群在BM骨干（骨内膜与中央髓区）和干骺端中的形态学位置。多光子活体显微镜（MP-IVM）将在体内表征Fak wt和Fak-/-祖B细胞群体的动态行为及其与BM中相邻细胞的相互作用。将应用细胞生物学测定来确定观察到的Fak-/-祖B细胞运动性和BM中分布变化的机制。目的3：明确FAK如何调节B祖细胞与特定的小生境细胞类型的关联。将通过免疫组织学在股骨纵切片上鉴定小生境细胞，例如成骨细胞、血管内皮细胞、IL-7产生细胞和富含CXCL 12的网状（CAR）细胞。LSC结合祖B细胞和小生境细胞的表面抗原的荧光免疫染色，将客观地定量发现Fak wt和Fak -/-祖B细胞与整个BM腔中的每种小生境细胞类型密切相关的相对频率。CXCR 4-FAK途径在介导祖B细胞在特定BM区室中沉积中的重要性将通过用CXCR 4拮抗剂（例如，AMD3100）。更好地定义控制B细胞发育和功能的环境信号将对包括白血病和免疫缺陷在内的B细胞疾病的发病机制和治疗的研究具有重要意义。 公共卫生相关性：FAK是一种广泛表达的细胞信号蛋白，可调节细胞粘附、生长和存活。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整合者。因此，本文提出的研究将为以前未被认识的B细胞白血病以及免疫缺陷综合征的发病机制提供重要的见解。