Osteocytes control myeloid cell proliferation and differentiation through Gsα-dependent and -independent mechanisms

Osteocytes, the bone cells embedded in the mineralized matrix, control bone modeling, and remodeling through direct contact with adjacent cells and via paracrine and endocrine factors that affect cells in the bone marrow microenvironment or distant organs. Osteocytes express numerous G protein-coupled receptors (GPCRs) and thus mice lacking the stimulatory subunit of G-protein (Gsα) in osteocytes (Dmp1-Gsα^KO mice) have abnormal myelopoiesis, osteopenia, and reduced adipose tissue. We previously reported that the severe osteopenia and the changes in adipose tissue present in these mice were mediated by increased sclerostin, which suppress osteoblast functions and promote browning of white adipocytes. Inversely, the myeloproliferation was driven by granulocyte colony-stimulating factor (G-CSF) and administration of neutralizing antibodies against G-CSF only partially restored the myeloproliferation, suggesting that additional osteocyte-derived factors might be involved. We hypothesized that osteocytes secrete Gsα-dependent factor(s) which regulate the myeloid cells proliferation. To identify osteocyte-secreted proteins, we used the osteocytic cell line Ocy454 expressing or lacking Gsα expression (Ocy454-Gsα^cont and Ocy454-Gsα^KO ) to delineate the osteocyte "secretome" and its regulation by Gsα. Here we reported that factors secreted by osteocytes increased the number of myeloid colonies and promoted macrophage proliferation. The proliferation of myeloid cells was further promoted by osteocytes lacking Gsα expression. Myeloid cells can differentiate into bone-resorbing osteoclasts, therefore, we hypothesized that osteocyte-secreted factors might also regulate osteoclastogenesis in a Gsα-dependent manner. Conditioned medium (CM) from Ocy454 (both Gsα^cont and Gsα^KO ) significanlty increased the proliferation of bone marrow mononuclear cells (BMNC) and, at the same time, inhibited their differentiation into mature osteoclasts via a Gsα-dependent mechanism. Proteomics analysis of CM from Ocy454 Gsα^cont and Gsα^KO cells identified neuropilin-1 (Nrp-1) and granulin (Grn) as osteocytic-secreted proteins upregulated in Ocy454-Gsα^KO cells compared to Ocy454-Gsα^cont , whereas semaphorin3A was significantly suppressed. Treatment of Ocy454-Gsα^cont cells with recombinant proteins or knockdown of Nrp-1 and Grn in Ocy454-Gsα^KO cells partially rescued the inhibition of osteoclasts, demonstrating that osteocytes control osteoclasts differentiation through Nrp-1 and Grn which are regulated by Gsα signaling.