Thursday, July 16, 2009

Sloane Robinson Building, 2nd Floor

2.00 – 3.00PM

3.00 – 4.00PM

Focal Adhesion Kinase (FAK) Regulates Progenitor B Cell Growth And Localization in Bone Marrow Niches

Dr. Shin-Young Park, Children’s Hospital Boston and Immune Disease Institute/Harvard Medical School, Boston, MA USA

In the bone marrow (BM), progenitor B cell growth and survival depend on cues from distinct microenvironments, e.g., niches that are defined by cellular components, soluble regulators, and the extracellular matrix (ECM). Niche-specific signals, involving cytokines, chemokines and adhesion molecules, also influence the localization and migration of normally developing B cells in BM. However, the intracellular pathways downstream of these niche-specific signals are not fully understood. Collectively, our previous studies suggest that the CXCR4-FAK-VLA4 pathway is important for sustained adhesive interactions between progenitor B cells and the BM microenvironment. Based on these findings, we hypothesize that FAK may play an important role in niche-induced signaling regulating progenitor B cell fate decisions, e.g., proliferation, differentiation, apoptosis, and migration.

To test the role of FAK function in B lymphopoiesis, we generated CD19-Cre FAK floxed mice in which FAK was conditionally deleted in B lineage cells. FAK deletion led to a selective decrease (30-40%) in the number of BM pro-, pre- and immature B cells. FAK-deleted pro-B cells showed increased cell division followed by an increase in apoptosis (70%) in vitro cultured in semisolid methylcellulose medium with IL-7 alone or with CXCL12. We also found that FAK deletion leads to increased mobilization of pro-B cells to the peripheral blood. Using laser scanning cytometry, we objectively quantified the morphological localization of progenitor and immature/mature B cells in the longitudinal cryo-preserved 5 micron sections of wild type and FAK KO mouse femurs. LSC analysis of B cell subpopulations is validated by identification of the known follicular organization of B220+ cells within the spleen and also by identification of negligible B220+ IgM+ population in the femoral BM cavity of Rag2-deficient mice, which have a maturation defect in V(D)J recombination at pro-B cell stage and therefore have no IgM expression.

We found that niche environments in the metaphyses as well as the diaphysis serve as major sites for B lymphopoiesis in wild-type mice. In the diaphysis, early progenitor B cells (B220 + c-Kit +) localize in the endosteal region while mature (B220+ IgM+) B cells localize in the central medullary region. To examine whether FAK deletion affects localization of progenitor B cells in BM, we examined the distribution of B220 + c-Kit + cells in the femoral BM of FAKFAK-deleted mice. In comparison to wild type (C57BL/6) B220 + c-Kit + cells, lodging of B220 + c-Kit + cells in the metaphyses is significantly impaired in FAKFAK-deleted mice. This impaired lodging of B220 + c-Kit + cells is also observed in the endosteal region of the diaphysis, albeit to a lesser degree. Taken together, we propose that FAK functions as a key intermediary of BM niche-induced signals controlling early B lineage development.

Shin-Young Park¹, Peter Wolfram¹, Brendan Harley¹, John Manis¹, Hilary E Beggs² and Leslie E Silberstein¹. ¹Children's Hospital Boston and Immune Disease Institute/Harvard Medical School, Boston, MA, United States, 02115 and ²Ophthalmology & Physiology, University of California at San Francisco, San Francisco, CA 94143, USA