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, established by transfecting the human parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor complementary DNA (cDNA), supported osteoclast formation in response to PTH in coculture with mouse bone marrow cells. Osteoclast 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) for receptor activator of NF-kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and both membrane-associated and secreted forms of M-CSF by SaOS-4/3 cells was up-regulated in response to PTH. SaOS-4/3 cells constitutively expressed OPG mRNA, expression of which was down-regulated by PTH. To elucidate the mechanism of PTH-induced osteoclastogenesis, SaOS-4/3 cells were spot-cultured for 2 h in the center of a culture well and then mouse bone marrow cells were uniformly plated over the well. When the spot coculture 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 both inside and outside the colony of SaOS-4/3 cells in coculture treated with 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 only inside the colony of SaOS-4/3 cells. Adding M-CSF alone failed to support osteoclast formation in the spot coculture. PTH-induced osteoclast formation occurring inside the colony of SaOS-4/3 cells was not affected by the concentration of M-CSF in the culture medium. Mouse primary osteoblasts supported osteoclast formation in a similar fashion to SaOS-4/3 cells. These findings suggest that the up-regulation of RANKL/ODF expression is an essential step for PTH-induced osteoclastogenesis, and membrane- or matrix-associated forms of both M-CSF and RANKL/ ODF are essentially involved in osteoclast formation supported by osteoblasts/stromal cells.
Itoh
K
K
Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan.
Udagawa
N
N
Matsuzaki
K
K
Takami
M
M
Amano
H
H
Shinki
T
T
Ueno
Y
Y
Takahashi
N
N
Suda
T
T
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D005109:Extracellular Matrix / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D006651:Histocytochemistry; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects; D053244:Osteoprotegerin; D010281:Parathyroid Hormone / Q000494:pharmacology; D053245:RANK Ligand; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D044168:Receptor, Parathyroid Hormone, Type 1; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D018016:Receptors, Parathyroid Hormone / Q000235:genetics / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D010281:Parathyroid Hormone; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D044168:Receptor, Parathyroid Hormone, Type 1; D018160:Receptors, Cytoplasmic and Nuclear; D018016:Receptors, Parathyroid Hormone; D018124:Receptors, Tumor Necrosis Factor; C505686:TNFRSF11A protein, human; C506030:TNFRSF11B protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D016173:Macrophage Colony-Stimulating Factor