The Bone Marrow-Derived Stromal Cell Lines MC3T3-G2/PA6 and ST2 Support Osteoclast-Like Cell Differentiation in Cocultures with Mouse Spleen Cells

Abstract

After our previous report that osteoclast-like multinucleated cells (MNCs) were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] in cocultures of mouse spleen cells and osteoblast-rich populations freshly isolated from fetal mouse calvariae, we examined whether such primary osteoblast-like cells can be replaced by established cell lines in inducing osteoclast-like cell formation. We first used two clonal cell lines simultaneously established from newborn mouse calvariae. One was the osteoblastic cell line MC3T3-E1, and the other was the preadipose cell line MC3T3-G2/PA6. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive mononuclear cells and MNCs were formed in the cocultures of spleen cells and MC3T3-G2/PA6 cells in the presence of 1 alpha,25-(OH)2D3. Dexamethasone greatly potentiated TRACP-positive MNC formation induced by 1 alpha,25-(OH)2D3, whereas the glucocorticoid alone had no effect on it. In contrast, osteoblastic MC3T3-E1 cells failed to induce TRACP-positive cells in the cocultures. Another bone marrow-derived stromal cell line ST2 also induced TRACP-positive MNC formation in the cocultures in response to 1 alpha,25-(OH)2D3 and dexamethasone. Salmon calcitonin enhanced cAMP production in the cocultures only when TRACP-positive cells were formed. Autoradiographic studies demonstrated that [125I]calcitonin specifically bound to TRACP-positive cells formed in the cocultures. When spleen cells and either MC3T3-G2/PA6 or ST2 cells were cocultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 and dexamethasone, numerous resorption lacunae were formed. These results show that the two bone marrow-derived stromal cell lines can support osteoclast-like cell differentiation in cocultures with spleen cells.

Inhibitory action of bisphosphonates on bone resorption does not involve the regulation of RANKL and OPG expression

“…1,25-(OH) 2 VitD 3 is well known to effectively induce osteoclastogenesis in cocultures of mouse spleen cells and osteoblast-rich populations (Udagawa et al, 1989) and to increase RANKL expression and to decrease OPG expression (Horwood et al, 1998;Murakami et al, 1998;. Consistent with the previous reports, 1,25-(OH) 2 VitD 3 upregulated RANKL expression and downregulated OPG expression in mouse osteoblastic cells ( Figure 1B) and increased RANKL/OPG ratio upto~10 folds.…”

Specific collagen XVIII isoforms promote adipose tissue accrual via mechanisms determining adipocyte number and affect fat deposition

“…For example, it has been shown that Wnt10b-but not Wnt3a-RNA is present at a relatively high level in human mesenchymal precursor cells (27) and preadipocytes (41), and that Wnt10b RNA levels fall quite dramatically in these cell types following addition of adipocytogenic mixtures (43). Conversely, enforced Wnt10b expression in ST2 marrow-derived stromal cells (44) and 3T3-L1 preadipocytes suppresses adipocyte differentiation and lowers Pparγ RNA, even before adipogenesis is induced (41,43). Moreover, high-level Wnt10b expression in transgenic mice reduces both Pparγ expression and WAT development but not adipocyte size (45).…”

Expression and role of mannose receptor/terminal high-mannose type oligosaccharide on osteoclast precursors during osteoclast formation

“…Osteoblasts or stromal cells express RANKL and M-CSF on the surface of cell membranes. A co-culture system of spleen cells with osteoblasts or bone marrow stromal cells has been established to produce osteoclasts (Takahashi et al 1988a, Udagawa et al 1989. RAW264·7 cells express M-CSF continuously, and thus can be clearly differentiated from osteoclast-like cells in the presence of sRANKL alone (Hsu et al 1999).…”