INTRODUCTION: Previous studies have shown that osteocytes, the matrix-embedded cells in bone, control bone modeling and remodeling through direct contact with adjacent cells and via secreted factors that can reach cells in the bone marrow microenvironment (BMM). Osteocytes express several receptors including G protein-coupled receptors (GPCRs) and mice lacking the stimulatory subunit of G-proteins (Gsα) in osteocytes have abnormal myelopoiesis, skeletal abnormalities and reduced adipose tissue. This study aimed at evaluating the effects of osteocyte-secreted factors on myeloid cell proliferation and differentiation in vitro. To investigate cross-talk between osteocytes and the BMM, we established osteocytic cell lines lacking Gsα expression to study the molecular mechanisms by which osteocytes control myeloid cell proliferation and differentiation. METHODS: CRISPR/Cas9 was used to knockout Gsα in the osteocytic cell line Ocy454. Conditioned media (CM) from differentiated Ocy-GsαCtrl and Ocy-GsαKO cells were used to treat myeloid cells and bone marrow mononuclear cells (BMNCs) isolated from long bones of 6-8-week-old C57/BL6 mice. BMNCs were cultured with Macrophage Colony Stimulating Factor (M-CSF), Receptor Activator of Nuclear Factor Kappa β Ligand (RANKL) to induce osteoclast differentiation. Proliferation, TRAP staining, TRAP activity, resorption pit assay, F-actin ring formation and mRNA expression were used to evaluate cell proliferation, differentiation and function of the induced osteoclasts. Proteomics analysis of CM was performed to identify osteocyte-secreted factors capable of controlling myelopoiesis and osteoclastogenesis. RESULTS: Myeloid cells treated with CM from Ocy-GsαKO showed a significant increase in cell proliferation compared to Ocy-GsαCtrl CM and non-treated control. BMNCs treated with CM from Ocy-GsαCtrl and Ocy-GsαKO showed a significant increase in cell proliferation as compared to non-treated control. Osteoclast differentiation was significantly suppressed by CM from Ocy-GsαCtrl and further suppressed by CM from Ocy-GsαKO compared to non-treated control. Osteoclasts exposed to CM from Ocy-GsαKO showed a significant defect in activity and function as compared to cells exposed to CM from Ocy-GsαCtrl and non-treated cells. Osteoclast apoptosis was significantly enhanced by Ocy-GsαCtrl and Ocy-GsαKO CM compared to non-treated control. Among osteocyte secreted factors, we identified neuropilin-1 (NRP-1) as a Gsα-dependent osteocytic factor capable of suppressing osteoclastogenesis. CM from Ocy-GsαKO in which M-CSF was reduced by shRNA demonstrated decrease in BMNCs proliferation, demonstrating that osteocytes are also important sources of this cytokine. CONCLUSIONS: Osteocytes produce several Gsα-dependent and -independent secreted factors capable of supporting myelopoiesis, promoting macrophage proliferation and suppressing osteoclast formation. We identified osteocyte-derived NRP-1 as a novel factor capable of decreasing osteoclastogenesis. In addition, we found that M-CSF secreted by osteocytes is responsible in part for BMNC proliferation. Future studies should focus on determining the role of osteocyte-mediated NRP-1 and other secreted factor(s) in control of myelopoiesis and osteoclastogenesis. / 2020-06-26T00:00:00Z