Importance of membrane- or matrix-associated forms of M-CSF and RANKL/ODF in osteoclastogenesis supported by SaOS-4/3 cells expressing recombinant PTH/PTHrP receptors

SaOS-4/3, a subclone of the human osteosarcoma cell line SaOS-2, establishe d by transfecting the human parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor complementary DNA (cDNA), supported osteoclas t formation in response to PTH in coculture with mouse bone marrow cells. O steoclast formation supported by SaOS-4/3 cells was completely inhibited by adding either osteoprotegerin (OPG) or antibodies against human macrophage colony-stimulating factor (M-CSF). Expression of messenger RNAs (mRNAs) fo r receptor activator of NF-kappa B ligand/osteoclast differentiation factor (RANKL/ODF) and both membrane-associated and secreted forms of M-CSF by Sa OS-4/3 cells was up-regulated in response to PTH, SaOS-4/3 cells constituti vely expressed OPG mRNA, expression of which was down-regulated by PTH, To elucidate the mechanism of PTH-induced osteoclastogenesis, SaOS-4/3 cells w ere spot-cultured for 2 h in the center of a culture well and then mouse bo ne marrow cells were uniformly plated over the well. When the spot cocultur e was treated for 6 days with both PTH and M-CSF, osteoclasts were induced exclusively inside the colony of SaOS-4/3 cells, Osteoclasts were formed bo th inside and outside the colony of SaOS-4/3 cells in coculture treated wit h a soluble form of RANKL/ODF (sRANKL/sODF) in the presence of M-CSF. When the spot coculture was treated with sRANKL/sODF, osteoclasts were formed on ly inside the colony of SaOS-4/3 cells. Adding M-CSF alone failed to suppor t osteoclast formation in the spot coculture. PTH-induced osteoclast format ion occurring inside the colony of SaOS-4/3 cells was not affected by the c oncentration of M-CSF in the culture medium. Mouse primary osteoblasts supp orted osteoclast formation in a similar fashion to SaOS-4/3 cells, These fi ndings suggest that the up-regulation of RANKL/ODF expression is an essenti al step for PTH-induced osteoclastogenesis, and membrane- or matrix-associa ted forms of both M-CSF and RANKL/ODF are essentially involved in osteoclas t formation supported by osteoblasts/stromal cells.