Title: Osteocytes Control Macrophages and Osteoclast by Gsα Signaling
Ehab Azab (Presenter)
Yuhei Uda, Boston University
ningyuan Sun, Boston University
Paola Divieti Pajevic, Boston University
Objectives: Previous studies have shown that osteocytes, the matrix-embedded cells in bone, control bone marrow cells through direct contact and via secrete factors that can reach the bone marrow microenvironment (BMM). Osteocytes express several receptors including G protein-coupled receptors (GPCR) and mice lacking Gsα expression in osteocytes have abnormal myelopoiesis. This study aimed to evaluate the effect of osteocytes on macrophages proliferation and differentiation. To investigate the molecular mechanisms by which osteocytes control myelopoiesis, we established an osteocytic cell line lacking Gsα expression to investigate molecular mechanisms by which osteocytes control macrophages proliferation and differentiation.
Methods: The osteocytic cells line Ocy454 was used to knocked out Gsα by CRISPR/Cas9 gene editing. Conditioned medium (CM) from differentiated Ocy-GsαControl and Ocy-GsαKO cells was used to treat bone marrow macrophages (BMMs) isolated from long bones of 6 weeks wild type mice and cultured with Macrophage Colony Stimulating Factor (M-CSF), Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL). Proliferation, TRAP staining, TRAP activity, resorption pit assay, F-actin ring and mRNA expression were used to evaluate cell proliferation, differentiation and function. Proteomics analysis of CM was performed to identify osteocyte secreted factors capable of controlling myelopoiesis and osteoclastogenesis.
Results: BMMs were treated with CM from Ocy-Gsαcontrol or Ocy-GsαKO showed a significant increase in cell proliferation as compared to non- treated control. Osteoclast differentiation was significantly suppressed by CM from Ocy-Gsαcontrol and further suppressed by CM from Ocy-GsαKO compared to non-treated control. Osteoclasts showed a significant defect in activity and function as compared to non-treated cells. Further experiments are ongoing to identify osteocyte-secreted factors.
Conclusions: Osteocytes secrete factor capable of inducing myeloid proliferation and inhibiting osteoclastogenesis and this factor(s) is Gsα-dependent.