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Seikagaku. The Journal of Japanese Biochemical Society 05/1999; 71(4):241-53. · 0.04 Impact Factor
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K Matsuzaki,
K Katayama,
Y Takahashi,
I Nakamura, N Udagawa,
T Tsurukai,
R Nishinakamura,
Y Toyama,
Y Yabe,
M Hori,
N Takahashi,
T Suda
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ABSTRACT: Subclones of the human osteosarcoma cell line SaOS-2 were established by transfecting with an expression vector containing the human PTH/PTH-related protein (PTHrP) receptor, and their abilities to support osteoclast-like multinucleated cell (OCL) formation were examined in coculture with mouse or human hemopoietic cells. Of four subclones examined, SaOS-2/4 and SaOS-4/3 bound high levels of [125I]-PTH and produced a significant amount of cAMP in response to PTH. OCLs were formed in response to PTH in the cocultures of mouse bone marrow cells with either SaOS-2/4 cells or SaOS-4/3 cells. Human OCLs were also formed in response to PTH in the coculture of SaOS-4/3 cells and human peripheral blood mononuclear cells. Adding dexamethasone together with PTH greatly enhanced PTH-induced human OCL formation. Like mouse OCLs, human OCLs formed in response to PTH were tartrate-resistant acid phosphatase positive, expressed abundant calcitonin receptors and vitronectin receptors, and formed resorption pits on dentine slices. Other osteotropic factors such as 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, and interleukin 6 plus soluble interleukin 6 receptors failed to induce mouse and human OCLs in cocultures with SaOS-4/3 cells. Both mouse and human OCL formation supported by SaOS-4/3 cells were inhibited by either adding an antibody against macrophage-colony stimulating factor or adding granulocyte/macrophage-colony stimulating factor. Thus, it is likely that human and mouse OCL formation supported by SaOS-4/3 cells are similarly regulated. These results indicate that the target cells of PTH for inducing osteoclast formation are osteoblast/stromal cells but not osteoclast progenitor cells in the coculture. This coculture model will be useful for investigating the abnormalities ofosteoclast differentiation and function in human metabolic bone diseases.
Endocrinology 03/1999; 140(2):925-32. · 4.46 Impact Factor
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ABSTRACT: Osteoclasts are formed in cocultures of mouse calvarial cells and hematopoietic cells in the presence of osteotropic factors such as 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], parathyroid hormone (PTH) and prostaglandin E2 (PGE2). We isolated osteoclast precursors (OCPs) from the coculture and examined their characteristics. After coculture for 7 days of mouse calvarial cells and bone marrow cells in the absence of osteotropic factors, hematopoietic cells were recovered and applied to a Sephadex G-10 column. Cells which passed through the column were collected as OCPs. When OCPs were cultured on calvarial cell layers in the presence of 1alpha,25(OH)2D3, tartrate-resistant acid phosphatase (TRAP)-positive cells first appeared within 24 h, and their number increased thereafter. OCPs also differentiated into TRAP-positive cells within 48 h on the calvarial cell layer which had been pretreated with either 1alpha,25(OH)2D3, PTH, or PGE2. Autoradiography using [125I]-labeled calcitonin showed that TRAP-positive cells formed on the calvarial cell layer expressed calcitonin receptors. Direct contact between OCPs and calvarial cells was required for the differentiation of OCPs into TRAP-positive cells. Flow cytometric analysis revealed that OCPs were positive for Mac-1, Mac-2, and Gr-1 but negative for F4/80, B220 and CD3e. Calvarial cells obtained from macrophage-colony stimulating factor (M-CSF)-deficient osteopetrotic (op/op) mice did not support OCP formation. A cell preparation disaggregated from long bones of newborn mice contained OCPs that differentiated into TRAP-positive cells on calvarial cells within 48 h, but cell preparations of freshly isolated bone marrow cells and alveolar macrophages did not. These results suggest that OCPs are specific cells which are formed only in the bone microenvironment and that OCPs recognize a signal(s) expressed by stromal cells in response to osteotropic factors and differentiate into osteoclasts.
Journal of Cellular Physiology 11/1998; 177(1):26-35. · 3.87 Impact Factor
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ABSTRACT: Skeletal growth is the net product of coordinated bone formation and resorption. Insulin is known to stimulate bone formation by actions on osteoblasts. It is not known whether insulin receptors are present on osteoclasts, or whether insulin regulates osteoclastic function. We present here immunocytochemical evidence of insulin receptor expression by mature mono- and multinucleated murine osteoclast-like cells generated in vitro, and in primary neonatal rat and mouse osteoclasts. Radiolabeled studies indicated that progressive enrichment of osteoclast-like cells in coculture was associated with increased insulin binding. When osteoclast-like cells generated in vitro were plated onto dentine slices, insulin dose-dependently inhibited pit formation by up to 80%, suggesting a role for insulin in osteoclast function. These data are consistent with an effect of insulin on bone resorption in addition to those previously recognized on bone formation, actions that together result in net bone growth.
Bone 10/1998; 23(3):181-6. · 4.02 Impact Factor
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ABSTRACT: Osteopontin (OP) is a highly phosphorylated bone matrix protein and contains the RGD cell-binding motif, which mediates cell adhesion through integrin receptors that include alpha(v)beta3. Casein kinase 2 (CK2) is a factor-independent serine/threonine kinase, which may be the predominant physiologically relevant kinase for OP phosphorylation. This study was designed to examine the effects of unphosphorylated recombinant rat OP, and CK2-phosphorylated OP (P-OP), on the adhesion and function of mouse osteoclasts (OC) and osteoblast-like cells (UMR 201-10B and UMR 106-06) in vitro. OP significantly increased OC adhesion compared to plastic alone, and cell attachment was further increased at least twofold on OP phosphorylated with CK2. Attachment was dependent on the integrity of the RGD domain and was completely abolished in the presence of 1 mM RGD peptide. Neither CK2 phosphorylation of mutant OP, in which the RGD was converted to RGE or RAD, nor protein kinase C (PKC) phosphorylation of wild-type OP enhanced OC attachment. An antibody to the beta3 integrin subunit, but not anti-mouse CD44 antibody, specifically blocked the proportion of attachment due to phosphorylation of OP. Actin ring formation in OC was increased by plating cells onto OP, with no further increase by phosphorylation. Both OP and CK2-phosphorylated OP enhanced attachment of the two osteoblastic cell lines, compared to plastic, but in contrast to OCs, there was no significant difference with phosphorylation. Osteoblast attachment was totally blocked by 1 mM RGD peptide, but was not influenced by the beta3 integrin antibody. Plating of UMR 201-10B cells onto OP further increased retinoic acid-induced alkaline phosphatase expression. The results suggest that specific phosphorylation of OP is important for interaction with OCs, compared with osteoblastic cells, and that alternative integrins may be important in the interaction between osteoblastic cells and OP compared with OCs.
Journal of Cellular Physiology 08/1998; 176(1):179-87. · 3.87 Impact Factor
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K Tsukii,
N Shima,
S Mochizuki,
K Yamaguchi,
M Kinosaki,
K Yano,
O Shibata, N Udagawa,
H Yasuda,
T Suda,
K Higashio
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ABSTRACT: Osteoclast differentiation factor (ODF), a ligand for osteoprotegerin (OPG)/osteoclastogenesis-inhibitory factor (OCIF), induces osteoclast-like cell formation in vitro. To elucidate the role of ODF in the microenvironment of bone, we examined effects of ODF, OPG/OCIF, and anti-ODF polyclonal antibody on bone resorption using a fetal mouse long bone culture system. A genetically engineered soluble-form ODF (sODF) elicited bone resorption in a concentration-dependent manner and OPG/OCIF blocked the bone resorption. Anti-ODF polyclonal antibody, which neutralizes ODF activity, negated bone resorption induced by 1 alpha,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2. OPG/OCIF also abolished bone-resorbing activity elicited by these bone-resorbing agents. Interleukin 1 alpha-stimulated bone resorption was also significantly suppressed by anti-ODF polyclonal antibody and OPG/OCIF. Thus, we conclude that ODF plays a critical role in bone resorption in the microenvironment of bone.
Biochemical and Biophysical Research Communications 06/1998; 246(2):337-41. · 2.48 Impact Factor
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K Matsuzaki, N Udagawa,
N Takahashi,
K Yamaguchi,
H Yasuda,
N Shima,
T Morinaga,
Y Toyama,
Y Yabe,
K Higashio,
T Suda
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ABSTRACT: We have reported that osteoclast differentiation factor (ODF) expressed on the plasma membrane of osteoblasts/ stromal cells is a ligand for osteoclastogenesis inhibitory factor (OCIF). A genetically engineered soluble form of ODF (sODF) induced osteoclast-like multinucleated cells (OCLs) in the presence of M-CSF in mouse spleen cell cultures. Osteoblasts/stromal cells were not required in this process. To elucidate the mechanism of human osteoclastogenesis, human peripheral blood mononuclear cells (PBMCs) were cultured for 7 days with sODF and human M-CSF in the presence or absence of dexamethasone. Treatment of human PBMCs with sODF together with M-CSF induced OCLs, which expressed tartrate-resistant acid phosphatase and vitronectin receptors, produced cAMP in response to calcitonin, and formed resorption pits on dentine slices. OCLs were also formed from the adherent cell population of human PBMCs. Dexamethasone was required for human OCL formation in culture of whole PBMCs but not in culture of the adherent cell population. OCL formation was strongly inhibited by OCIF simultaneously added. These results clearly indicate that like in mouse osteoclastogenesis, ODF is a critical factor for human osteoclastogenesis. The present study also indicates that OCIF acts as a naturally occurring decoy receptor for ODF in inhibiting signal transduction in human osteoclast formation.
Biochemical and Biophysical Research Communications 06/1998; 246(1):199-204. · 2.48 Impact Factor
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H Yasuda,
N Shima,
N Nakagawa,
K Yamaguchi,
M Kinosaki,
S Mochizuki,
A Tomoyasu,
K Yano,
M Goto,
A Murakami,
E Tsuda,
T Morinaga,
K Higashio, N Udagawa,
N Takahashi,
T Suda
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ABSTRACT: Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.
Proceedings of the National Academy of Sciences 03/1998; 95(7):3597-602. · 9.68 Impact Factor
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ABSTRACT: Using immunohistology with two specific antisera raised against N-terminal parathyroid hormone-related protein (PTHrP) and in situ hybridization (riboprobe to common coding exon), evidence is provided for the expression of PTHrP by mouse, rabbit, and human osteoclasts derived from several in vitro and in vivo sources. In cocultures of mouse bone marrow and calvarial cells treated with 1,25-dihydroxyvitamin D3, the generated osteoclasts expressed both PTHrP messenger RNA (mRNA) and protein. In addition, PTHrP was detected in the majority of actively resorbing osteoclasts in sections of newborn and adult mouse long bones. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in osteoclasts at active bone resorption sites as well as in actively synthesizing osteoblasts and bone lining cells. Localization of PTHrP was also demonstrated in osteoclast-like cells of human giant cell tumors from bone. In some of these tumors, a small proportion of the multinucleated cells expressed tartrate resistant acid phosphatase (TRAP), but not PTHrP mRNA or protein. Finally, both mRNA and protein for PTHrP were expressed in osteoclasts in sections of bone or joints from patients with Paget's disease, rheumatoid arthritis, and osteoarthritis. These observations raise the possibility that PTHrP might participate in osteoclast function.
Bone 03/1998; 22(3):189-94. · 4.02 Impact Factor
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ABSTRACT: IL-18 inhibits osteoclast (OCL) formation in vitro independent of IFN-gamma production, and this was abolished by the addition of neutralizing antibodies to GM-CSF. We now establish that IL-18 was unable to inhibit OCL formation in cocultures using GM-CSF-deficient mice (GM-CSF -/-). Reciprocal cocultures using either wild-type osteoblasts with GM-CSF -/- spleen cells or GM-CSF -/- osteoblasts with wild-type spleen cells were examined. Wild-type spleen cells were required to elicit a response to IL-18 indicating that cells of splenic origin were the IL-18 target. As T cells comprise a large proportion of the spleen cell population, the role of T cells in osteoclastogenesis was examined. Total T cells were removed and repleted in various combinations. Addition of wild-type T cells to a GM-CSF -/- coculture restored IL-18 inhibition of osteoclastogenesis. Major subsets of T cells, CD4+ and CD8+, were also individually depleted. Addition of either CD4+ or CD8+ wild-type T cells restored IL-18 action in a GM-CSF -/- background, while IL-18 was ineffective when either CD4+ or CD8+ GM-CSF -/- T cells were added to a wild-type coculture. These results highlight the involvement of T cells in IL-18-induced OCL inhibition and provide evidence for a new OCL inhibitory pathway whereby IL-18 inhibits OCL formation due to action upon T cells promoting the release of GM-CSF, which in turn acts upon OCL precursors.
Journal of Clinical Investigation 03/1998; 101(3):595-603. · 15.39 Impact Factor
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H Murakami,
N Takahashi,
S Tanaka,
I Nakamura, N Udagawa,
S Nakajo,
K Nakaya,
M Abe,
Y Yuda,
F Konno,
A Barbier,
T Suda
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ABSTRACT: Signaling pathways mediated by tyrosine phosphorylation and dephosphorylation have been reported to be involved in the regulation of cytoskeletal organization in osteoclasts, the principal cells responsible for bone resorption. We examined the effects of tiludronate [(4-chlorophenyl)thiomethylene bisphosphonate] on the cytoskeleton and the balance of phosphotyrosine levels in osteoclast-like multinucleated cells (OCLs) formed in cocultures of mouse osteoblastic cells and bone marrow cells. When OCLs were placed on plastic dishes in the presence of 10% fetal bovine serum, they formed a ringed structure of F-actin dots (actin ring) within 2 h. Tiludronate did not inhibit the process of actin ring formation, but it disrupted preformed actin rings in a time- and a dose-dependent manner. Western blot analysis using an antiphosphotyrosine antibody revealed that tyrosine phosphorylation of certain proteins in OCLs was stimulated by tiludronate added to the purified OCLs. Tyrosine kinase activity of the p60c-src immunoprecipitated from cell lysates of the purified OCLs was not affected by tiludronate directly added to the kinase assay. OCL lysates stimulated dephosphorylation of tyrosine-phosphorylated substrates such as phosphoneuroprotein 14 and epidermal growth factor receptors. Like sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, tiludronate dose-dependently inhibited tyrosine dephosphorylation of those substrates induced by OCL lysates. These findings suggest that tiludronate disrupts the preformed actin rings and suppresses bone-resorbing activity by inhibiting protein tyrosine phosphatases in osteoclasts.
Bone 06/1997; 20(5):399-404. · 4.02 Impact Factor
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ABSTRACT: We have established by differential display polymerase chain reaction of mRNA that interleukin (IL)-18 is expressed by osteoblastic stromal cells. The stromal cell populations used for comparison differed in their ability to promote osteoclast-like multinucleated cell (OCL) formation. mRNA for IL-18 was found to be expressed in greater abundance in lines that were unable to support OCL formation than in supportive cells. Recombinant IL-18 was found to inhibit OCL formation in cocultures of osteoblasts and hemopoietic cells of spleen or bone marrow origin. IL-18 inhibited OCL formation in the presence of osteoclastogenic agents including 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-1, and IL-11. The inhibitory effect of IL-18 was limited to the early phase of the cocultures, which coincides with proliferation of hemopoietic precursors. IL-18 has been reported to induce interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) production in T cells, and both agents also inhibit OCL formation in vitro. Neutralizing antibodies to GM-CSF were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to IFN-gamma did not. In cocultures with osteoblasts and spleen cells from IFN-gamma receptor type II-deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-gamma production: IFN-gamma had no effect in these cocultures. Additionally, in cocultures in which spleen cells were derived from receptor-deficient mice and osteoblasts were from wild-type mice and vice versa, we identified that the target cells for IFN-gamma inhibition of OCL formation were the hemopoietic cells. The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM-CSF production and not via IFN-gamma production.
Journal of Experimental Medicine 04/1997; 185(6):1005-12. · 13.85 Impact Factor
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ABSTRACT: We previously studied regulation of the calcitonin (CT) receptor (CTR) by glucocorticoid (GC) and CT in cultures of mature mouse osteoclast-like cells (OCLs). The present studies were designed to examine the interaction of CT and GC in regulation of the CTR in osteoclasts and the molecular mechanisms involved. Treatment of OCLs with 10(-7) M dexamethasone (Dex) increased the CTR number in a time-dependent manner, whereas treatment with 10(-9) M salmon CT (sCT) reduced CTR number; neither treatment changed receptor affinity. Dex pretreatment somewhat antagonized the CT-induced reduction in [125I]sCT specific binding. Dex increased, and sCT pretreatment decreased, the sCT-responsive adenylate cyclase activity in parallel with the change in receptor binding. Dex treatment resulted in an increase in CTR messenger RNA (mRNA) levels, as assessed by reverse transcription-PCR, indicating that the increased CTR number was mediated by de novo CTR synthesis. This effect was specific to GCs and was not reproduced by mineralocorticoids or sex steroids. Treatment with sCT resulted in a rapid and profound reduction in CTR mRNA expression, and this reductions was somewhat delayed by Dex pretreatment. OCLs were treated with 5,6-dichloro-1 beta-D-ribofuranosyl benzimidazole to enable estimation of the mRNA decay rates in the absence of ongoing transcription. The stability of CTR mRNA was similar to the control value in Dex-treated OCLs, suggesting that the effect of Dex may be due to changes in transcriptional activity. Interestingly, transcriptional inhibition by 5,6-dichloro-1 beta-D-ribofuranosyl benzimidazole abolished the ability of CT to reduce CTR mRNA levels, suggesting that CT may act by increasing the rate of CTR mRNA decay, and that this effect requires ongoing transcription. The 3'-untranslated region of the mouse CTR mRNA contains four copies of the AUUUA motif, as well as other A/U-rich sequences, which have been shown to determine the stability of other mRNA transcripts. The stability results were consistent with the results of the nuclear transcript run-on assay, which indicated that treatment with Dex enhanced the rate of transcription, whereas CT had no effect. These results show that GC and CT influence CTR expression by distinct mechanisms and provide the basis for identification of the cellular factors involved.
Endocrinology 03/1997; 138(2):521-9. · 4.46 Impact Factor
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ABSTRACT: We examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. An immunoelectron microscopic analysis revealed that in the osteoclasts of these mice, no ruffled borders formed, and that vacuolar H+-ATPase (V-ATPase) was present throughout the cytoplasm but not on the apical membranes. The activity of V-ATPase in oc/oc mice was similar to that in normal mice. In normal spleen cell-derived osteoclast-like cells (OCLs), immunoreactivity for V-ATPase was detected in association with Triton X-100-insoluble cellular structure, but not in oc/oc spleen cell-derived OCLs. Moreover, in renal proximal convoluted tubules of oc/oc mice, the basal striation did not form. These results suggest that osteosclerosis in oc/oc mice is possibly due to the dissociation of V-ATPase and cytoskeleton in osteoclasts.
FEBS Letters 02/1997; 401(2-3):207-12. · 3.54 Impact Factor
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ABSTRACT: Interleukin (IL)-11 is a multifunctional cytokine whose role in osteoclast development has not been fully elucidated. We examined IL-11 production by primary osteoblasts and the effects of rat monoclonal anti-mouse glycoprotein 130 (gp130) antibody on osteoclast formation, using a coculture of mouse osteoblasts and bone marrow cells. IL-1, TNF alpha, PGE2, parathyroid hormone (PTH) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) similarly induced production of IL-11 by osteoblasts, but IL-6, IL-4, and TGF beta did not. Primary osteoblasts constitutively expressed mRNAs for both IL-11 receptor (IL-11R alpha) and gp130. Osteotropic factors did not modulate IL-11R alpha mRNA at 24 h, but steady-state gp130 mRNA expression in osteoblasts was upregulated by 1 alpha,25(OH)2D3, PTH, or IL-1. In cocultures, the formation of multinucleated osteoclast-like cells (OCLs) in response to IL-11, or IL-6 together with its soluble IL-6 receptor was dose-dependently inhibited by rat monoclonal anti-mouse gp130 antibody. Furthermore, adding anti-gp130 antibody abolished OCL formation induced by IL-1, and partially inhibited OCL formation induced by PGE2, PTH, or 1 alpha,25(OH)2D3. During osteoclast formation in marrow cultures, a sequential relationship existed between the expression of calcitonin receptor mRNA and IL-11R alpha mRNA. Osteoblasts as well as OCLs expressed transcripts for IL-11R alpha, as indicated by RT-PCR analysis and in situ hybridization. These results suggest a central role of gp130-coupled cytokines, especially IL-11, in osteoclast development. Since osteoblasts and mature osteoclasts expressed IL-11R alpha mRNA, both bone-forming and bone-resorbing cells are potential targets of IL-11.
Journal of Experimental Medicine 07/1996; 183(6):2581-91. · 13.85 Impact Factor
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ABSTRACT: We previously reported that osteoclast formation in vitro, by coculture of mouse bone marrow and primary osteoblastic cells, occurs in two phases: proliferation of osteoclast progenitors followed by terminal differentiation into mature osteoclasts. Using this coculture system, we examined the effects of c-fos antisense and sense phosphorothioate oligonucleotides on osteoclast development and macrophage differentiation. Treatment with c-fos antisense for the first 4 days of coculture inhibited osteoclast formation in a dose-dependent fashion. However, when c-fos antisense was added during the second phase of coculture (4-6 days), osteoclast formation was unaffected. In contrast, c-fos antisense treatment had no effect on the appearance of F4/80 antigen-positive cells of the macrophage lineage in these cultures or on the induction by colony stimulating factor-1 of macrophage colony formation in cultures of mouse bone marrow cells in agar. Neither osteoclast differentiation nor macrophage appearance was inhibited by adding control c-fos sense in the cocultures. When c-fos antisense was added into an assay of bone resorption by mature osteoclasts, pit formation on dentine slices was unaffected. These results indicate that c-fos plays an important role in the proliferative phase of osteoclast progenitors in osteoclast development, but not in the terminal differentiation phase or in the bone resorbing activity of mature osteoclasts. c-fos antisense specifically inhibited osteoclast formation but had no effect on macrophage development.
Bone 07/1996; 18(6):511-6. · 4.02 Impact Factor
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ABSTRACT: We have reported that short calcitonin (CT) treatment of mature mouse osteoclast-like cells (OCLs) in culture induced prolonged down-regulation of the CT receptor (CTR) and desensitization to CT rechallenge, at the level of adenylate cyclase activity. In this study, we have extended those studies to examine the bone resorbing activity of OCLs pretreated with CT. OCLs, which formed on gelled type I collagen, were pretreated with salmon CT (sCT)(10(-9)M, 1 h) and 24 h later were replated onto plastic dishes or dentine slices after removal from the gel by collagenase digestion. The number and population of either mononuclear or multinuclear OCLs that adhere to either surface was not affected by sCT pretreatment. It was found that OCLs pretreated with sCT regained reduced but significant bone resorbing capacity, which was quantitated as the surface area resorbed by OCLs on dentine slices. However, compared with control, the number of resorption pits produced by sCT- pretreated OCLs was slightly reduced, and the total pit area was decreased by approximately 40-50%. The distribution of individual pit sizes was altered by sCT-pretreatment so that the number of larger pits was predominantly reduced, suggesting that short sCT treatment may produce a long lasting decrease in osteoclast mobility. sCT was able to inhibit bone resorption activity of CT-pretreated OCLs (ED50:10(-13)-10(-12)M). Importantly, the ED50 of sCT inhibition of bone resorption in sCT-pretreated OCLs was approximately 100-fold greater than for control, indicating resistance of the OCLs to CT rechallenge. Consistent with these results, treatment of OCLs with sCT greatly decreased the expression of CTR messenger RNA, whereas no significant effect was observed on the tartrate-resistant acid phosphatase messenger RNA expression, a marker of resorptive capacity of osteoclasts. These results indicate, therefore, that an important component of escape of osteoclastic resorption from CT inhibition is CT resistance of mature osteoclasts, which regain bone resorbing function.
Endocrinology 04/1996; 137(3):1042-8. · 4.46 Impact Factor
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ABSTRACT: We have reported that calcitonin (CT) treatment induced downregulation of the CT receptor (CTR) in mouse osteoclast-like cells (OCLs). Here, we studied the features of homologous down-regulation of the CTR in mature mouse OCLs. Treatment with salmon CT (sCT) and human CT (hCT) reduced [125I]sCT specific binding. The decreased binding after 24 h of CT treatment was associated with a decrease in the cell surface receptor concentration. The extent of CT-induced down-regulation in 24 h was dose-dependent, and the ED50 value was 3.6 +/- 4.1 (mean +/- SD; n = 3) x 10(-13 M for sCT and 4.9 +/- 3.3 x 10(-11) M for hCT. These values were very similar to those for the CT inhibition of the bone-resorbing activity of OCLs. The data suggest that these two distinct actions of CT may be mediated by a common intracellular pathway. Treatment of OCLs with activators of protein kinase A (PKA) mimicked the effect of CT on CTR downregulation, whereas neither activation of protein kinase C nor elevation of intracellular Ca2+ did so. Attenuation of CT-induced CTR down-regulation by the competitive cAMP antagonist, RpcAMP, and high concentrations of H-7, but not by protein kinase C-specific inhibitors (sphingosine, staurosporine, and a lower concentration of H-7), suggested that the PKA pathway is primarily involved in homologous regulation of the CTR. The changes in CTR messenger RNA confirm the findings in binding studies and demonstrate that CT treatment of OCLs results in decreased CTR synthesis through the PKA pathway. The low concentrations of hCT that result in CTR regulation are very close to the physiological range, providing new insights into a dynamic relationship between circulating levels of CT and CTR expression in osteoclasts.
Endocrinology 02/1996; 137(1):312-20. · 4.46 Impact Factor
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ABSTRACT: Chronic immune responses and inflammatory reactions in rheumatoid arthritis (RA) often cause severe destruction of cartilage and bone, but its mechanism is still a matter of controversy. We reported that interleukin-6 (IL-6) alone does not induce osteoclast formation, but soluble interleukin-6 receptors (sIL-6R) triggered the formation in the presence of IL-6 in cocultures of murine osteoblastic cells and bone marrow cells. In this study, we examined the involvement of sIL-6R and IL-6 in joint destruction in patients with RA. Although the frequency of patients having osteoclast-like multinucleated cells in synovium derived from the knee joint was not significantly different between RA (65%) and osteoarthritis (OA) patients (43%), the number of osteoclast-like cells found in the synovium was greater in the former than in the latter. Multinucleated cells obtained from RA synovium expressed the osteoclast-specific phenotype such as tartrate-resistant acid phosphatase, carbonic anhydrase II, vacuolar proton-ATPase and vitronectin receptors at similar levels to those from a human giant cell tumor of bone. The concentration of both IL-6 and sIL-6R was significantly higher in the synovial fluids from patients with RA than with OA. The concentration of IL-6 and sIL-6R correlated well with the roentgenologic grades of joint destruction. Dose-response curves for human IL-6 and human sIL-6R in inducing osteoclast-like cell formation in cocultures indicated that the RA synovial fluids contained sufficient IL-6 and sIL-6R to induce osteoclastogenesis. When synovial fluids from RA and OA patients were added to the cocultures, some of the RA synovial fluids containing high levels of IL-6 and sIL-6R stimulated osteoclast-like cell formation, which was strikingly inhibited by adding anti-IL-6R antibody simultaneously. These results suggest that IL-6 in the RA synovial fluids is at least in part responsible for joint destruction in the presence of sIL-6R through osteoclastogenesis.
Journal of Bone and Mineral Research 02/1996; 11(1):88-95. · 6.37 Impact Factor
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N Udagawa,
N Takahashi,
T Katagiri,
T Tamura,
S Wada,
D M Findlay,
T J Martin,
H Hirota,
T Taga,
T Kishimoto,
T Suda
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ABSTRACT: We reported that interleukin (IL) 6 alone cannot induce osteoclast formation in cocultures of mouse bone marrow and osteoblastic cells, but soluble IL-6 receptor (IL-6R) strikingly triggered osteoclast formation induced by IL-6. In this study, we examined the mechanism of osteoclast formation by IL-6 and related cytokines through the interaction between osteoblastic cells and osteoclast progenitors. When dexamethasone was added to the cocultures, IL-6 could stimulate osteoclast formation without the help of soluble IL-6R. Osteoblastic cells expressed a very low level of IL-6R mRNA, whereas fresh mouse spleen and bone marrow cells, both of which are considered to be osteoclast progenitors, constitutively expressed relatively high levels of IL-6R mRNA. Treatment of osteoblastic cells with dexamethasone induced a marked increase in the expression of IL-6R mRNA. By immunoblotting with antiphosphotyrosine antibody, IL-6 did not tyrosine-phosphorylate a protein with a molecular mass of 130 kD in osteoblastic cells but did so in dexamethasone-pretreated osteoblastic cells. Osteoblastic cells from transgenic mice constitutively expressing human IL-6R could support osteoclast development in the presence of human IL-6 alone in cocultures with normal spleen cells. In contrast, osteoclast progenitors in spleen cells from transgenic mice overexpressing human IL-6R were not able to differentiate into osteoclasts in response to IL-6 in cocultures with normal osteoblastic cells. These results clearly indicate that the ability of IL-6 to induce osteoclast differentiation depends on signal transduction mediated by IL-6R expressed on osteoblastic cells but not on osteoclast progenitors.
Journal of Experimental Medicine 12/1995; 182(5):1461-8. · 13.85 Impact Factor
