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  • 1
    ISSN: 1432-0827
    Keywords: Osteoclast ; Bone resorption ; Cytochalasin D ; Cytoskeleton
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Summary The osteoclastic cytoskeleton has been demonstrated to be composed of microfilaments. Osteoclastic multinucleated cells were suspended on dentine slices and cultured for 24 hours in the presence or absence of cytochalasin D (CD), a specific and potent inhibitor of actin filament elongation to determine the role of this cytoskeleton. Cultured cells and co-cultured dentine slices were examined ultrastructurally. Unlike those in control cultures without CD, osteoclasts in CD-treated cultures became spherical in shape and lacked microvilli on their basolateral cell surfaces. Most importantly, CD treatment induced a complete disappearance of the ruffled border-clear zone complexes in osteoclasts, which resulted in loss of osteoclast-cytoplasmic polarity. Morphometric analysis of backscattered electron micrographs of co-cultured dentine slices revealed that CD treatment strongly inhibited the formation of resorption lacunae in a dose-dependent manner. These results suggest that the cytoarchitecture, as well as the bone-resorbing function, of the osteoclast is highly regulated by the F-actin-containing microfilamentous cytoskeleton in the ruffled border-clear zone complex.
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  • 2
    ISSN: 1435-5604
    Keywords: Key words: osteoclastogenesis ; osteoclast ; macrophage-colony stimulating factor ; osteoclast precursors ; osteoclast differentiation factor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract: We have isolated osteoclast precursors (OCPs) from cocultures of mouse calvarial cells and bone marrow cells without adding any osteotropic factors. OCPs expressed Mac-1, Mac-2, and Gr-1 antigens but not osteoclast markers such as tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors, and they differentiated into TRAP-positive cells within 48 h on a fixed calvarial cell layer pretreated with osteotropic factors such as 1α,25-dihydroxyvitamin D3. In the present study, we investigated the regulatory mechanisms of OCP formation from hemopoietic cells and TRAP-positive cell formation from OCPs. Calvarial osteoblasts obtained from macrophage-colony stimulating factor (M-CSF) -deficient op/op mice failed to support OCP formation or the differentiation of OCPs into TRAP-positive cells. Both OCP formation and TRAP-positive cell formation supported by osteoblasts were completely inhibited by osteoclastogenesis inhibitory factor (OCIF, also called OPG), which is a decoy receptor of osteoclast differentiation factor (ODF; also called TRANCE, RANKL, and OPGL). When bone marrow cells were cultured for 4 days with soluble ODF (sODF/sRANKL) together with M-CSF, OCPs were formed even in the absence of osteoblasts. When OCPs were treated with sODF/sRANKL and M-CSF in the absence of osteoblasts, they differentiated into TRAP-positive cells within 48 h even in the presence of hydroxyurea. Northern blotting analysis revealed that osteoblasts constitutively expressed a certain level of ODF/RANKL mRNA. These results indicated that M-CSF and sODF/sRANKL produced by osteoblasts are two essential factors for both OCP formation and TRAP-positive osteoclast formation.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 278 (1994), S. 265-271 
    ISSN: 1432-0878
    Keywords: Bone resorption ; Osteoclast ; Vacuolar H+-ATPase ; Immuno-electron microscopy ; Mouse (ddY)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract By means of light- and electron-microscopic immunocytochemistry, we have demonstrated the expression of vacuolar H+-ATPase in mouse osteoclasts. In fully differentiated osteoclasts, intense immunolabeling was observed along the plasma membranes including those of ruffled borders and associated pale vesicles and vacuoles, whereas those of clear zones and basolateral cell surfaces were entirely free of immunoreaction. Specific expression of vacuolar H+-ATPase was also detected over polyribosomes and cisterns of the rough-surfaced endoplasmic reticulum. Multinucleated osteoclastic cells were suspended on dentine slices and cultured for 48 h in the presence or absence of either concanamycin B or bafilomycin A1, specific inhibitors of vacuolar H+-ATPase. Morphometric analysis of co-cultured dentine slices with backscattered electron microscopy revealed that both inhibitors strongly reduced the formation of resorption lacunae in a dose-dependent manner. These results suggest that vacuolar H+-ATPase is produced in the rough-surfaced endoplasmic reticulum, stored in the membrane vesicles, and transported into the ruffled border membranes of osteoclasts, and that this enzyme plays a key role in the creation of an acidic subosteoclastic microenvironment for the demineralization of co-cultered substrates.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 278 (1994), S. 265-271 
    ISSN: 1432-0878
    Keywords: Key words: Bone resorption – Osteoclast – Vacuolar H+-ATPase – Immuno-electron microscopy – Mouse (ddY)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. By means of light- and electron-microscopic immunocytochemistry, we have demonstrated the expression of vacuolar H+-ATPase in mouse osteoclasts. In fully differentiated osteoclasts, intense immunolabeling was observed along the plasma membranes including those of ruffled borders and associated pale vesicles and vacuoles, whereas those of clear zones and basolateral cell surfaces were entirely free of immunoreaction. Specific expression of vacuolar H+-ATPase was also detected over polyribosomes and cisterns of the rough-surfaced endoplasmic reticulum. Multinucleated osteoclastic cells were suspended on dentine slices and cultured for 48 h in the presence or absence of either concanamycin B or bafilomycin A1, specific inhibitors of vacuolar H+-ATPase. Morphometric analysis of co-cultured dentine slices with backscattered electron microscopy revealed that both inhibitors strongly reduced the formation of resorption lacunae in a dose-dependent manner. These results suggest that vacuolar H+-ATPase is produced in the rough-surfaced endoplasmic reticulum, stored in the membrane vesicles, and transported into the ruffled border membranes of osteoclasts, and that this enzyme plays a key role in the creation of an acidic subosteoclastic microenvironment for the demineralization of co-cultered substrates.
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  • 5
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Experiments have been carried out to determine the mechanisms involved in the formation of osteoclast-like cells from spleen cells in mice. Osteoclasts were defined as tartrate-resistan. acid phosphatase-positive multinucleated cells (TRACP-positive MNCs) in which specific calcitonin receptors were identified by autdradiography with labeled salmon calcitonin. Furthermore, cultures rich in these cells produced resorption pits when grown on dentine slices. Several clonal cell lines were obtained from fetal mouse calvariae and screened for their ability to induce TRACP-positive MNCs in response to 1α, 25-dihydroxyvitamin D3 “1α,25(OH)2D3” in co-cultures with spleen cells. A cell line, KS-4, was identified with the greatest potency in inducing osteolast-like cell formation in co-culture with spleen cells. The capacity of KS-4 cells to produce this effect was much greater than that of two bone marrow-derived stromal cell lines (MC3T3-G2/PA6 and ST2 cells), which we have previously shown to be effective in this system but to require treatment with dexamethasone in addition to 1α25(OH)2D3 (Udagawa et al.: Endocrinology 125:1805-1813, 1989). Parathyroid hormone (PTH) in creased cAMP production in KS-4 cells, and PTH and interleukin-1α also induced TRACP-positive MNCs in co-cultures with spleen cells. Contact between living KS-4 and spleen cells was necessary for osteoclast formation to take place, since this did not occur when the two populations were separated by a membrane filter, or when the KS-4 cells were killed by fixation. Separate cultures of either spleen cells or KS-4 cells formed no TRACP-positive MNCs. KS-4 cells synthesized predominantly type I collagen, formed bone nodules without added of β-glycerophosphate in a long-term culture, and expressed increasing alkaline phosphatase activity after confluence in culture. These results indicate that the KS-4 cells have properties consistent with progression toward the osteoblast phenotype, and represent a single cell line with the ability to promote osteoclast formation by a contact-requiring process.
    Additional Material: 6 Ill.
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  • 6
    ISSN: 0730-2312
    Keywords: Key words ; osteoclast ; focal adhesion kinase ; tyrosine kinase ; tyrosine phosphorylation ; podosome ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast-like multinucleated cells prepared from co-cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1α,25-dihydroxyvitamin D3. When osteoclast-like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti-phosphotyrosine antibody. Western blot analysis revealed that 115-to 130-kD proteins were tyrosine-phosphorylated in osteoclast-like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115-130 kD was identified as focal adhesion kinase (p125FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti-p 125FAK antibody revealed that p125FAK was mainly localized at the periphery of osteoclast-like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125FAK but also changed the intracellular localization of p125FAK and disrupted a ringed structure of F-actin-containing podosomes in osteoclast-like cells. Antisense oligodeoxynucleotides to p125FAK inhibited dentine resorption by osteoclast-like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125FAK is critical for regulating osteoclast function.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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