Jung Y, Wang J, Havens A, et al. failure of hematopoiesis beginning with an early defect in B lymphopoiesis and erythropoiesis(11). Induced osteocyte-deficiency in adult mice also prospects to marked decrease in common lymphoid progenitors and subsequent B cell development(12). osteoblast support of B lymphopoiesis was further augmented by PTH treatment(13) suggesting that this PTH signaling in osteoblastic cells may be a major regulator of B lymphopoiesis. Mice lacking Gs, the stimulatory G protein subunit downstream of G protein-coupled receptors (GPCRs) including PPR, in osteoprogenitors (Osx-GsKO mice) exhibit a dramatically hypoplastic spleen and a specific block in the transition from Prepro B to Pro B cell precursors during B lymphocyte development(21). In contrast, deletion of Gs in mineral-embedded osteocytes did not affect B lymphocytes(22) suggesting that the defective B lymphopoiesis seen UNC1079 in mice with induced osteocyte deficiency(12) is most likely impartial of PTH signaling. We therefore hypothesized that PPR signaling in specific stage(s) UNC1079 of osteoblastic cell differentiation is usually a critical component of the niche regulation of B lymphopoiesis. To test this hypothesis, we generated and examined B lymphopoiesis in mice lacking PPR in osteoprogenitors (Osx-PPRKO), mature osteoblasts (OC-PPRKO), and osteocytes (DMP1-PPRKO). Osx-PPRKO mice developed severe osteopenia and exhibited a specific block in B cell UNC1079 precursor differentiation. By contrast, the OC-PPRKO and DMP1-PPRKO mice did not reveal any effects on B lymphopoiesis. Despite a significant reduction in B cell precursors in BM and severe lymphopenia in peripheral blood, Osx-PPRKO mice display an increased retention of mature B lymphocytes in BM that is due at least in part to overexpression of VCAM1 in Osx+ osteoprogenitors. Taken together, our study demonstrates that PPR signaling in osteoprogenitors but not maturing osteoblasts or osteocytes is essential for regulating B lymphopoiesis and B cell mobilization in BM. MATERIALS AND METHODS Animals Mice lacking PPR in osteoprogenitors were generating by mating PPRfl/fl (23) mice with transgenic mice in which Cre recombinase is usually driven by the Osterix promoter(24). Deletion of PPR in mature osteoblasts and osteocytes was obtained by mating PPRfl/fl mice with mice expressing Cre recombinase driven by Osteocalcin (OC) and DMP1 promoters respectively(22,25). PPRfl/fl (wild-type, WT) littermates were used as controls for all the experiments. Because the presence of Osx-driven Cre recombinase transgene results UNC1079 in mild runting, experiments were also repeated with Osx:Cre-PPR+/+ and PPR+/+ mice as controls. There was no difference in phenotypes between PPRfl/fl and PPR+/+ mice, therefore where applicable we have offered data from PPRfl/fl and Osx:Cre-PPR+/+ mice as controls. Genotyping was performed on genomic DNA obtained from tail UNC1079 biopsies as previously explained(21,26). Cetrorelix Acetate All animals were housed in the Center for Comparative Medicine at the Massachusetts General Hospital and the Comparative Medicine Pavilion in Stanford University or college, and all procedures were approved by the MGH Subcommittee on Research Animal Care or the Stanford Administrative Panel on Laboratory Animal Care. Skeletal Analysis Skeletal DXA and CT analysis was performed as explained in Supplementary methods. Bone chip cell culture Hind limbs were harvested from 3-week aged Osx-Cre:PPRfl/fl and Osx-Cre:PPR+/+ mice. After soft tissue dissection and BM removal by centrifugation(27), bones were minced into small pieces and washed at least 3 times in serum-free MEM medium. Bone chips were then digested in serum-free MEM medium made up of 2 mg/ml Collagenase Type II (Worthington) for 2 hours at 37C and subsequently washed again at least 3 times to remove all the cells in suspension. The resulting bone chips were resuspended in MEM (GIBCO) medium supplemented with 10% warmth.