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.
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.
|Ciuculescu, Marioara F; Park, Shin-Young; Canty, Kimberly et al. (2015) Perivascular deletion of murine Rac reverses the ratio of marrow arterioles and sinusoid vessels and alters hematopoiesis in vivo. Blood 125:3105-13|
|Park, Shin-Young; Wolfram, Peter; Canty, Kimberly et al. (2013) Focal adhesion kinase regulates the localization and retention of pro-B cells in bone marrow microenvironments. J Immunol 190:1094-102|
|Nombela-Arrieta, César; Pivarnik, Gregory; Winkel, Beatrice et al. (2013) Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nat Cell Biol 15:533-43|
|Nombela-Arrieta, César; Ritz, Jerome; Silberstein, Leslie E (2011) The elusive nature and function of mesenchymal stem cells. Nat Rev Mol Cell Biol 12:126-31|