calcitonin and tartaric-acid

Stromal cells are required for in vitro osteoclast differentiation and maturation. The murine bone marrow stromally derived BMS2 cell line exhibits adipocytic and osteoblastic features as well as the ability to support lymphopoiesis and myelopoiesis. This work examined the ability of the BMS2 cell in either the preadipocyte or adipocyte state to support the formation of osteoclast-like cells. BMS2 cells can be induced to undergo adipogenic differentiation in response to treatment with glucocorticoids or thiazolidinedione compounds. Primary bone marrow cells, enriched for hematopoietic progenitors and depleted of their adherent stromal and macrophage populations, were stimulated with vitamin D3 (vitamin D; 10(-8) M) to undergo osteoclast differentiation and maturation when cocultured with BMS2 cells. In both preadipocyte and adipocyte-enriched BMS2 stromal layers, comparable numbers of tartrate-resistant acid phosphatase-positive osteoclast-like cells, characterized by their response to salmon calcitonin with an increase in cAMP and formation of resorption pits on bovine bone slices, were formed. The gene expression and protein levels of macrophage colony-stimulating factor produced by preadipocyte and adipocyte-rich BMS2 layers were comparable. However, adipocyte-rich stromal layers supported osteoclast-like cell formation longer in culture than preadipocytes, independent of the agent used to induce adipocyte differentiation. These studies demonstrate for the first time that fully differentiated adipocyte stromal cells can support osteoclast-like cell formation and function in vitro.

Topics: Acid Phosphatase; Adipocytes; Animals; Bone Marrow Cells; Calcitonin; Cell Differentiation; Cell Line; Cholecalciferol; Coculture Techniques; Flow Cytometry; Hematopoietic Stem Cells; Macrophage Colony-Stimulating Factor; Mice; Osteoclasts; Stromal Cells; Tartrates; Thiazoles; Thiazolidinediones

Osteoclasts are multinucleated cells which form by fusion of circulating mononuclear hemopoietic precursors. The nature of these precursor cells and the roles bone stromal cells and hormonal factors play in their differentiation to osteoclasts are unknown. We cocultured adherent murine blood monocytes (nonspecific esterase and F4/80 positive; tartrate-resistant acid phosphatase negative) with osteoblastic and fibroblastic stromal cell lines in the presence of 2 x 10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Tartrate-resistant acid phosphatase and calcitonin (CT) receptor-positive osteoclastic cells, which formed numerous resorption pits in vitro, were noted after only 4 days in coculture with UMR106 osteoblast-like cells. Resorption was seen in cocultures to which as few as 100 peripheral blood mononuclear cells had been added. 1,25-(OH)2D3 and contact with live bone stromal cells were absolute requirements for monocyte differentiation into bone-resorbing cells. Both salmon CT (5 IU/ml) and prostaglandin E2 (10(-6) M) significantly inhibited bone resorption. Thus, a significant proportion of the peripheral blood mononuclear cells in the monocyte fraction are capable of differentiating into cells showing the cytochemical and functional characteristics of osteoclasts. The presence of specific hormonal [1,25-(OH)2D3] and bone stromal cell elements is necessary for this process to occur; the resultant resorption can be modulated by known inhibitors of bone resorption, CT and prostaglandin E2.

Topics: Acid Phosphatase; Animals; Bone Resorption; Calcitonin; Cell Differentiation; Culture Techniques; Dinoprostone; Humans; Kinetics; Monocytes; Osteoclasts; Rats; Receptors, Calcitonin; Tartrates; Tumor Cells, Cultured

The presence and biological activity of an Osteoclast Growth Factor (OGF) was investigated in serum-free medium conditioned by periostless fetal rat calvaria in culture. OGF activity was assessed using in vitro systems of fetal rat long bones and adult rat bone marrow cells. Rat calvaria conditioned medium (RCCM) increased the number of osteoclasts in the long bone cultures, partly due to stimulation of progenitor proliferation. RCCM did not exert a direct bone-resorbing activity (45Calcium release assay) on the pre-existing osteoclasts residing in the long bones, but stimulated bone resorption in long term cultures, apparently in an indirect manner by enhancing the number of osteoclasts. In cultures of bone marrow cells isolated from adult rats, RCCM markedly stimulated the formation of mononuclear cells which were positively stained for tartrate-resistant acid phosphatase (TRAP). The osteoclastic nature of the cells was confirmed by specific labeling with 125I-calcitonin. Formation of the TRAP-positive cells was significantly inhibited by salmon calcitonin. CM from fetal rat skin cultures did not display a significant OGF activity. Furthermore, unlike the bone marrow cells, peritoneal macrophages did not respond to RCCM and remained devoid of TRAP activity. Neutralization experiments with a specific antibody to GM-CSF indicated that OGF activity in the RCCM could not be ascribed to this hemopoietic growth factor. Secretion of OGF activity was mainly dependent on protein synthesis as addition of cycloheximide to the calvaria cultures significantly inhibited the secretion of OGF into the medium. G3000 HPLC fractionation of RCCM revealed two major OGF peaks with Mr 14,000 and 70,000. Two subsequent reverse-phase HPLC steps using the lower Mr OGF fraction led to a highly purified OGF fraction. The results of this study further provide evidence that bone tissue produces factor(s) which specifically govern the process of osteoclast development, thus providing information about one of the mechanisms controlling bone resorption.

Topics: Acid Phosphatase; Animals; Antibodies; Bone and Bones; Bone Marrow Cells; Calcitonin; Cell Division; Cells, Cultured; Culture Media; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Osteoclasts; Rats; Tartrates

We have reported that osteoblastic cells are required for differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts. In the present study we examined the pathogenesis of the osteoclast deficiency in osteopetrotic (op/op) mice using a coculture system of spleen cells and osteoblastic cells. When spleen cells obtained from op/op or normal (+/?) littermates of op/+ parent mice were cocultured with osteoblastic cells obtained from calvaria of normal ddy strain mice, numerous tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) were formed in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Most of the TRAP-positive MNCs bound [125I]salmon calcitonin. This suggests that there is no abnormality in the osteoclast progenitors present in the splenic tissues of op/op mice. When osteoblastic cells from +/? littermates were cocultured with normal spleen cells from ddy mice, TRAP-positive MNCs were similarly formed in response to 1 alpha,25(OH)2D3. In contrast, in cocultures of op/op osteoblastic cells with normal spleen cells, no TRAP-positive cells appeared, even in the presence of 1 alpha,25(OH)2D3. The op/op mutation was recently reported to exist in the coding region of the macrophage colony-stimulating factor (M-CSF) gene. Adding M-CSF and 1 alpha,25(OH)2D3 to the coculture with op/op osteoblastic cells induced the appearance of TRAP-positive MNCs with calcitonin receptors. These results clearly indicate that osteoclast deficiency in op/op mice is due to a defect in the local microenvironment in bone, in which M-CSF produced by osteoblastic cells plays a critical role in osteoclast development.

Topics: Acid Phosphatase; Animals; Calcitonin; Calcitriol; Cell Differentiation; Cells, Cultured; Female; Macrophage Colony-Stimulating Factor; Male; Mice; Osteoblasts; Osteoclasts; Osteopetrosis; Spleen; Stem Cells; Tartrates

We developed a co-culture system with mouse spleen cells and osteoblastic cells to examine the role of osteoblasts in osteoclast formation. When mouse spleen cells and osteoblastic cells isolated from fetal mouse calvariae were co-cultured in the presence of 10 nM 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], numerous tartrate-resistant acid phosphate (TRACP)-positive mononuclear and multinucleated cells were formed within 8 days. Neither the same co-cultures without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced TRACP-positive cells. Salmon calcitonin (CT) markedly increased cAMP production in the co-cultures treated with 1 alpha,25(OH)2D3. Autoradiographic studies clearly demonstrated that [125I]-CT specifically bound to the TRACP-positive cells formed in the co-cultures with the vitamin. When spleen cells and osteoblastic cells were co-cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, numerous resorption lacunae were formed on the slices. Neither co-cultures of alveolar macrophages and osteoblastic cells nor those of spleen cells and mouse skin-derived fibroblasts induced TRACP-positive cells even in the presence of 1 alpha,25(OH)2D3. When spleen cells and osteoblastic cells were cultured separately from each other by a membrane filter (0.45 micron), no TRACP-positive cells were formed. These results indicate that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts.

Topics: Acid Phosphatase; Animals; Calcitonin; Calcitriol; Cell Differentiation; Cells, Cultured; Cyclic AMP; Embryo, Mammalian; Mice; Microscopy, Electron, Scanning; Osteoblasts; Osteoclasts; Spleen; Stem Cells; Tartrates