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 细胞的生长和存活取决于来自不同微环境的线索，例如，利基市场。涉及细胞因子、趋化因子和粘附分子等的生态位特异性信号也影响 BM 中发育中 B 细胞的定位和迁移。我们初步研究的主要发现是，CD19-Cre Fak fl/fl 小鼠中的 Fak 缺失会导致 BM 中祖 B 细胞发育受损，并导致 B 细胞前体进入外周循环。在此应用中，我们假设 FAK 作为生态位诱导的信号转导调节祖 B 细胞命运的整合器发挥作用。提出了三个目标来检验这一假设。目标 1 将定义 FAK 如何调节祖 B 细胞的生长和存活。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都将用于检查 FAK 在祖 B 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 功能，将定义 FAK 在调节祖 B 细胞生长和存活的信号通路中的作用。我们将扩展我们对 CD19-Cre Fak fl/fl 小鼠的研究结果，并确定 Fak 缺失对 mb1-Cre EYFP 小鼠中祖 B 细胞发育的影响，因为它们从早期 pro-B 细胞阶段开始表现出更高的 Cre 介导的缺失效率。目标 2 将定义 FAK 如何调节 BM 内不同解剖位置中祖 B 细胞群的运动和分布。对冷冻保存的股骨进行激光扫描细胞术 (LSC) 分析将量化并绘制骨干骨干（骨内与中央髓质区域）和干骺端中 Fak wt 和 Fak-/- 祖 B 细胞群的形态位置。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观察到的 Fak-/- 祖 B 细胞运动和 BM 分布变化的机制。目标 3：明确 FAK 如何调节祖 B 细胞与特定生态位细胞类型的关联。生态位细胞，例如将通过股骨纵向切片的免疫组织学鉴定成骨细胞、血管内皮细胞、IL-7 产生细胞和富含 CXCL12 的网状 (CAR) 细胞。 LSC 与祖 B 和小生境细胞表面抗原的荧光免疫染色相结合，将客观地量化 Fak wt 和 Fak -/- 祖 B 细胞与整个 BM 腔中每种小生境细胞类型密切相关的相对频率。在 LSC 分析之前，将通过用 CXCR4 拮抗剂（例如 AMD3100）处理 Fak wt 和 Fak -/- 小鼠来评估 CXCR4-FAK 通路在介导祖 B 细胞在特定 BM 区室中的重要性。更好地定义控制 B 细胞发育和功能的环境信号将对 B 细胞疾病（包括白血病和免疫缺陷）的发病机制和治疗研究具有重要意义。 公共健康相关性：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器。因此，这里提出的研究将为以前未被认识的 B 细胞白血病和免疫缺陷综合征的发病机制提供重要的见解。