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At least 127 records · Page 7Linked to original sources
PubMed1997

Osteoclast development from hematopoietic stem cells: apparent divergence of the osteoclast lineage prior to macrophage commitment.

To further clarify the progression of osteoclast development, the relationship of clonogenic osteoclast progenitors (CFU-O) to macrophage or more primitive progenitors was examined. Serum-free culture supernatant of a tumor clone (CESJ) was used as a source of an osteoclast colony stimulating factor (O-CSF). CFU-O-derived colonies were identified by their characteristic positive staining for tartrate resistant acid phosphatase (TRAPase). The effect of macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) on osteoclast progenitors was examined by pre-culturing mouse bone marrow (BM) cells in agar medium containing M-CSF or SCF and overlaying CESJ medium 0-7 days later. The number of TRAPase+ colonies decreased while TRAP- macrophage colonies increased in M-CSF pre-cultures as overlays of CESJ medium were delayed. On the other hand, TRAPase+ and mixed colonies persisted in SCF pre-cultures with CESJ medium overlays. Conversely, all colonies were TRAPase+ and no macrophage colonies developed in O-CSF pre-cultures overlaid with M-CSF. CFU-O, but not CFU-M, survived 7 days without exogenous CSFs in agar medium. In fractionated BM, the majority (> 99%) of CFU-O were in the c-kit positive population; however, a specific antibody to SCF did not affect O-CSF-induced TRAPase+ colony formation, suggesting the proliferation and differentiation of osteoclast progenitors are independent of c-kit-SCF interactions. These studies provide further experimental evidence to support the concept that O-CSF acts on progenitors in earlier stages of development, supporting their differentiation into the osteoclast lineage prior to macrophage commitment.

Animals↗
PubMed1981

Osteoclast formation in vitro from bone marrow mononuclear cells in osteoclast-free bone.

Recent evidence points to the fact that osteoclasts are derived from mononuclear cells of hematopoietic bone marrow. In this study we have examined the formation of osteoclasts from mononuclear cells in vitro. The mononuclear cells were isolated after 7 days from cultures of mouse bone marrow cells. The isolated cells were co-cultured with osteoclast-free, fetal-mouse calvaria. After 10 to 14 days of co-culture, multinucleated cells which have all the characteristics of osteoclasts were found in juxtaposition to seams of woven bone. These data strongly suggest that bone marrow mononuclear cells, when suitably induced, can give rise to osteoclasts in vitro.

Animals↗
PubMed1988

Bone resorption by isolated chick osteoclasts in culture is stimulated by murine spleen cell supernatant fluids (osteoclast-activating factor) and inhibited by calcitonin and prostaglandin E2.

The question of whether any of the agents known to activate bone resorption in vivo or in organ cultures acts directly on the osteoclast or via intermediate target cells that secondarily secrete locally paracrine factors is important for our understanding of bone remodeling. In an attempt to clarify this issue for some of the agents, we have taken advantage of the recent progress in obtaining and culturing relatively pure populations of osteoclasts. We performed an in vitro bone-resorbing assay in which isolated and partially purified chick osteoclasts were cultured on devitalized, paired and standardized bone disks prepared from rat calvaria prelabeled with both 45Ca and 3H-proline. Some of the isolated osteoclasts attached to the devitalized bone matrix, formed a ruffled border, and acidified the bone-resorbing compartment that they established with the matrix, thereby indicating that they resorbed bone in a physiologic manner. Salmon calcitonin added to these cultures (0.3 U/ml = 60 ng/ml) and prostaglandin E2 (PGE2) (10(-6) M) inhibited both basal and stimulated 45Ca and 3H-proline release. Neither parathyroid hormone (PTH) 1-34 (1 U/ml), 1,25-(OH)2-D3 (10(-8) and 10(-9) M), nor interleukin 1 (IL-1) (purified from P388D1 macrophage culture supernatant fluids or recombinant murine IL-1-alpha) (100 ng/ml) stimulated bone resorption in these cultures. In contrast, supernatant fluids from concanavalin A (Con-A)-activated murine spleen cell cultures (murine osteoclast-activating factor; OAF) consistently and significantly induced a 3- to 5-fold stimulation of bone resorption in this system.

Animals↗
PubMed2006

Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP-1 and RANTES.

Chemokines MCP-1 and RANTES are induced when authentic bone resorbing human osteoclasts differentiate from monocyte precursors in vitro. In addition, MCP-1 and RANTES can stimulate the differentiation of cells with the visual appearance of osteoclasts, being multinuclear and positive for tartrate resistance acid phosphatase (TRAP +). We show here that MIP1alpha is also potently induced by RANKL during human osteoclast differentiation and that this chemokine also induces the formation of TRAP + multinucleated cells in the absence of RANKL. MIP1alpha was able to overcome the potent inhibition of GM-CSF on osteoclast differentiation, permitting the cells to pass through to TRAP + multinuclear cells, however these were unable to form resorption pits. Chemokine receptors CCR2b and CCR4 were potently induced by RANKL (12.6- and 49-fold, P = 4.0 x 10(-7) and 4.0 x 10(-8), respectively), while CCR1 and CCR5 were not regulated. Chemokine treatment in the absence of RANKL also induced MCP-1, RANTES and MIP1alpha. Unexpectedly, treatment with MCP-1 in the absence of RANKL resulted in 458-fold induction of CCR4 (P = 1.0 x 10(-10)), while RANTES treatment resulted in twofold repression (P = 1.0 x 10(-4)). Since CCR2b and CCR4 are MCP-1 receptors, these data support the existence of an MCP-1 autocrine loop in human osteoclasts differentiated using RANKL.

Acid Phosphatase↗
PubMed1999

Basic fibroblast growth factor induces osteoclast formation by reciprocally regulating the production of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in mouse osteoblastic cells.

Basic fibroblast growth factor (bFGF) induced osteoclast formation in co-cultures of mouse spleen cells and osteoblasts. Osteoclastogenesis inhibitory factor (OCIF) and a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, abolished bFGF-induced osteoclast formation. bFGF did not affect spleen cells, but it did affect osteoblasts, to stimulate osteoclast formation. Northern blot analysis revealed that bFGF up-regulated the expression of osteoclast differentiation factor (ODF) and COX-2 and down-regulated the expression of OCIF in primary osteoblastic cells. NS-398 abolished the increase of ODF mRNA, but it had no effect on the decrease of OCIF mRNA. NS-398 suppressed the binding of (125)I-labeled OCIF to osteoblastic cells treated with bFGF. Enzyme-linked immunosorbent assay showed that bFGF inhibited OCIF production by osteoblastic cells, and the inhibition was not affected by NS-398. We conclude that bFGF induces osteoclast formation by stimulating ODF production through COX-2-mediated prostaglandin synthesis and by suppressing OCIF production through a mechanism independent of prostaglandin synthesis.

Animals↗
PubMed1990

Modularity of osteoclast behaviour and "mode-specific" inhibition of osteoclast function.

This study is part of an attempt to understand the role of specific cellular activities in the bone resorptive process. Experiments were performed whereby known pharmacological agents were used to inhibit individual modes of osteoclastic activity, such as motility and secretion. The effects of such treatments on bone resorption were assessed by quantitative scanning electron microscopy. The compounds included colchicine, which was used to inhibit osteoclast motility; molybdate ions which were used to selectively inhibit the catalytic activity of secreted acid phosphatase, and omeprazole which was employed to inhibit the secretion of hydrogen ions. All compounds inhibited osteoclastic bone resorption, but singularly affected defined modes of activity. These findings suggest that each mode of osteoclastic activity is essential for the bone resorptive process, and that "mode-specific" inhibition may provide a means whereby excessive activity of the osteoclast can be regulated in disease.

Acid Phosphatase↗
PubMed1979

Comparison of the effects of 1,25-dihydroxycholecalciferol, prostaglandin E2, and osteoclast-activating factor with parathyroid hormone on the ultrastructure of osteoclasts in cultured long bones of fetal rats.

The effects of 1,25-dihydroxy vitamin D3 [1,25(OH)2D3], prostaglandin (PGE2), and osteoclast-activating factor (OAF) on the size of osteoclasts, nuclei, ruffled borders, and clear zones in cultured long bones of fetal rats were quantitated. In addition, the number of osteoclasts in the bones was counted and the release of calcium from the bone into the culture medium was determined. These data were compared with the corresponding effects of parathyroid hormone (PTH). All agents tested increased the size of the ruffled borders significantly after 3 h, the size of the clear zones after 12 h, and the size of the cells after 12-24 h. No important differences in sizes were noticed between the agents tested or between the agents and PTH. The number of osteoclasts was increased after 24 h of treatment with PTH, but not after the other agents. Calcium release was significantly increased for all agents between 12 and 24 h. It is concluded that bone resorption by 1,25(OH)2D3, OAF, and PGE2 is mediated primarily by increased activity of existing osteoclasts similar to PTH activation.

Animals↗
PubMed1999

Etidronate (EHDP) inhibits osteoclastic-bone resorption, promotes apoptosis and disrupts actin rings in isolate-mature osteoclasts.

Bisphosphonates, therapeutic reagents against tumoral bone diseases (Paget's disease or osteoporosis), are potent inhibitors of bone resorption. The mechanisms by which they directly act on mature osteoclasts remain unclear. Using a recently developed technique for isolation of highly purified mammalian mature osteoclasts, we demonstrated that etidronate [ethane-1-hydroxy-1,1-diphosphonate (EHDP), 1-hydroxy-1,1-ethylidenebisphosphonate], inhibited directly osteoclastic bone-resorbing activity by pit assay. In addition, EHDP also directly induced apoptosis and disrupted actin rings in osteoclasts. The data support previous data on non-purified osteoclasts and results in vivo.

Actins↗
PubMed1991

Osteoblast-osteoclast relationships in bone resorption: osteoblasts enhance osteoclast activity in a serum-free co-culture system.

Osteoblast-osteoclast relationships in bone resorption are unclear. We investigated whether osteoblasts constitutively influence osteoclast activity. We employed a serum-free co-culture system in which chicken osteoclasts and chick calvaria or, alternatively, isolated chick osteoblasts were cultured in two different compartments separated by a 0.45 micron porous membrane permeable to soluble molecules. Osteoclastic bone resorption, evaluated by release of 3H-proline from prelabeled bone fragments, was significantly enhanced by bone cells resident in the calvaria, as well as by isolated osteoblasts. Stimulation was specific, since periosteal cells, or skin fibroblasts, failed to mimic osteoblast activity. Conditioned medium from osteoblast cultures stimulated osteoclast function in a similar manner, indicating that paracrine signals, capable of crossing the porous membrane separating the two compartments, are released by the bone forming cells.

Alkaline Phosphatase↗
PubMed2004

Akt1/Akt2 and mammalian target of rapamycin/Bim play critical roles in osteoclast differentiation and survival, respectively, whereas Akt is dispensable for cell survival in isolated osteoclast precursors.

Akt, also known as protein kinase B, is a serine/threonine protein kinase with antiapoptotic activities; also, it is a downstream target of phosphatidylinositol 3-kinase. Here we show that Akt1/Akt2 play a critical role in osteoclast differentiation but not cell survival and that mammalian target of rapamycin (mTOR) and Bim, a pro-apoptotic Bcl-2 family member, are required for cell survival in isolated osteoclast precursors. To investigate the function of Akt1, Akt2, mTOR, and Bim, we employed a retroviral system for delivery of small interfering RNA into cells. Loss of Akt1 and/or Akt2 protein inhibited osteoclast differentiation due to down-regulation of IkappaB-kinase (IKK) alpha/beta activity, phosphorylation of IkappaB-alpha, nuclear translocation of nuclear factor-kappaB (NFkappaB) p50, and NFkappaB p50 DNA-binding activity. Surprisingly, deletion of Akt1 and/or Akt2 protein did not stimulate cleaved caspase-3 activity and failed to promote apoptosis. Conversely, loss of mTOR protein induced apoptosis due to up-regulation of cleaved caspase-3 activity. In addition, we found that mTOR is downstream of phosphatidylinositol 3-kinase (but not Akt) and that macrophage colony-stimulating factor regulates Bim expression through mTOR activation for cell survival. These results demonstrate that Akt1/Akt2 are key elements in osteoclast differentiation and that the macrophage colony-stimulating factor stimulation of mTOR leading to Bim inhibition is essential for cell survival in isolated osteoclast precursors.

Animals↗
PubMed1998

Echistatin inhibits the migration of murine prefusion osteoclasts and the formation of multinucleated osteoclast-like cells.

The vitronectin receptor alpha(v)beta3 is highly expressed in osteoclasts and was shown to play a critical role in osteoclast function in vivo. The objective of this study was to examine the role of alpha(v)beta3 integrin in osteoclast formation in vitro using the inhibitory disintegrin echistatin, an RGD-containing snake venom. We documented by immunocytochemistry and Northern blot analysis that during murine osteoclast-like cell (OCL) formation in a coculture of mouse osteoblastic MB1.8 cells and bone marrow cells there is increased expression of the alpha(v) and beta3 integrin subunits. Echistatin binds preferentially to the membrane fraction of isolated enriched OCLs (IC50 = 0.6 nM), and this binding is inhibited by vitronectin receptor-blocking polyclonal antibodies. Additionally, cross-linking of radiolabeled echistatin to OCLs, followed by immunoprecipitation with antibodies to vitronectin or fibronectin receptors, shows that alpha(v)beta3 integrin is the predominant receptor for echistatin in this system. In this coculture, echistatin completely inhibits the formation of multinucleated OCLs, but not that of mononuclear prefusion OCLs (pOCs). This inhibition is RGD and dose dependent (IC50 = 0.7 nM). We tested the hypothesis that inhibition of OCL formation may be due to interference with pOC migration and found that echistatin inhibited macrophage colony-stimulating factor-induced migration and fusion of pOCs (IC50 = 1 and 0.6 nM, respectively). Echistatin inhibition of pOCs migration and fusion is also RGD dependent. These results suggest that the integrin alpha(v)beta3 plays a role in pOC migration, which can explain the inhibitory effect of echistatin on multinucleated osteoclast formation in vitro.

Animals↗
PubMed1994

Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. Modulation of osteoclast adhesion in vitro.

The tartrate-resistant acid phosphatase (TRAP) of skeletal osteoclasts was found to partially dephosphorylate the bone matrix phosphoproteins osteopontin (OPN) and bone sialoprotein (BSP). TRAP also partially dephosphorylated metabolically [32P]PO4-labeled OPN as well as BSP, whereas comparable amounts of either alkaline phosphatase or prostatic acid phosphatase, at their respective pH optima, were ineffective, indicating a certain preference of TRAP for these phosphoprotein substrates. It has previously (Flores, M., Norgärd, M., Heinegård, D., Reinholt, F. P., and Andersson, G. (1992) Exp. Cell Res. 201, 526-530) been shown that osteoclasts bind to OPN as well as to BSP coated onto glass. We can now show that the partially dephosphorylated proteins no longer support osteoclast binding. These results indicate that the secretion of TRAP from osteoclasts into the resorption area could exert a regulatory influence on the attachment of the cells to the bone surface. This could imply roles in the development of ruffled borders and/or in the regulation of osteoclast motility on the bone surface.

Acid Phosphatase↗
PubMed2005

Infliximab acts directly on human osteoclast precursors and enhances osteoclast formation induced by receptor activator of nuclear factor kappaB ligand in vitro.

Infliximab is known to protect against the development of joint destruction. In the present study, we sought to determine whether Infliximab acts directly on human osteoclast precursors and influences monocyte-osteoclast differentiation induced by receptor activator of nuclear factor kappaB ligand (RANKL) in vitro. Peripheral blood mononuclear cells (PBMCs) isolated from rheumatoid arthritis (RA) patients and normal controls were cultured in the presence of RANKL and macrophage colony stimulating factor. Infliximab, antihuman tumor necrosis factor alpha (TNFalpha), antihuman TNF soluble receptor p55 (TNFR p55), and antihuman TNF soluble receptor p75 (TNFR p75) antibodies were added. Osteoclast formation was determined by assessing the number of tartrate-resistant acid phosphatase (TRAP) staining cells and the extent of lacunar resorption. Addition of Infliximab resulted in a marked increase in the number of TRAP-positive multinucleated cells (TRAP(+) MNCs) and in the extent of lacunar resorption compared with the control cultures. Antihuman TNFalpha antibody showed the same effect; however, the addition of neither TNFR p55 nor TNFR p75 antibody affected the extent of TRAP(+) MNCs and lacunar resorption. Our results suggest that infliximab acts directly on early osteoclast precursors, and stimulates osteoclast formation and lacunar resorption induced by RANKL in vitro.

Journal Article↗
PubMed1988

Migration and phenotypic transformation of osteoclast precursors into mature osteoclasts: the effect of a bisphosphonate.

Osteoclast-devoid bone explants were cultured together with embryonic liver as a source of osteoclast precursors, but separated from each other by a filter. Cells migrated through the filter toward the calcified matrix and acquired the characteristics of mature, tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts upon contact with the bone explant. Migration and attachment could be visualized separately. Progressive reduction of filter pore size resulted in progressive reduction of resorption because the use of smaller pores made it increasingly difficult for cells to pass. Indeed, the use of 0.22-micron filters, through which no cells can pass, but which still allow full passage of medium, completely blocked the resorption. When migrating cells from fetal liver were arrested for 10 days by using a combination of filters with different pore sizes, the arrested cells showed a tendency to fuse just opposite the mineralized matrix. Furthermore, a great number of the arrested cells expressed the macrophage-specific cell-surface antigen F4/80 and showed acid phosphatase activity, but none of these cells were tartrate resistant. The acquisition of tartrate-resistant acid phosphatase activity upon contact with the bone explant and subsequent resorption of this explant could be prevented by exposure of the system to the bisphosphonate dimethyl-APD (Me2-APD), whereas migration of cells through the filter was not affected. We suggest that the bisphosphonate interferes with a matrix factor that is essential for the attachment and subsequent transformation of the osteoclast precursor into the mature phenotype.

Animals↗
PubMed2003

Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors.

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis.

Active Transport, Cell Nucleus↗
PubMed1993

Interleukin-4 modulates osteoclast differentiation and inhibits the formation of resorption pits in mouse osteoclast cultures.

Interleukin-4 (IL-4) is a well known lymphocyte growth factor, but it may also modulate the activity of other cell types. In the present study we show that IL-4 exerts a potent dose-dependent inhibitory effect on the resorptive activity of mouse bone cells, measured as the number of resorption pits (decreased down to 14% of control, p < 0.001) or the total resorbed area (down to 20% of control, p < 0.001). The results obtained in bone marrow and unfractionated bone cell cultures indicate that such an effect is mainly due to an inhibition of osteoclast precursors differentiation, rather than to a reduced activity of mature osteoclasts, and is not mediated by the IL-4-induced release of other soluble inhibitory factors. Likewise, although IL-4 may stimulate M-CSF expression, its inhibitory effect on osteoclast recruitment was not prevented by anti-M-CSF antibodies, suggesting that it was not mediated by M-CSF. These results point out IL-4 as an potentially important regulatory factor of osteoclast differentiation.

Animals↗
PubMed1996

Bone cells required for osteoclastic resorption but not for osteoclastic differentiation.

It is generally considered that osteoblastic cells are essential for osteoclast formation. We tested the ability of hemopoietic tissue to differentiate osteoclastic characteristics in the absence of osteoblastic cells. We found that large numbers of calcitonin-receptor positive (CTRP) cells can be induced by prostaglandin E2 and 1,25(OH)2 vitamin D3, in cultures of hemopoietic mouse spleen. Moreover, spleen stromal cells were equivalent to bone marrow stromal cells in CTRP-cell induction. The spleen CTRP cells did not resorb bone, but were rapidly induced to full resorptive activity upon osteoblast addition. Thus, bone cells may not be essential for osteoclast formation, but rather are required to activate and regulate the resorptive function of mature osteoclasts.

Animals↗
PubMed1985

On the origin of the osteoclast: the cell surface phenotype of rodent osteoclasts.

The origin and development of the osteoclast is not well defined; although it is derived from a bone marrow stem cell, it is not proven whether the osteoclast progenitor comes from the multipotential hemopoietic stem cell or comprises an entirely separate cell lineage. We have studied the cell lineage relationship of osteoclasts isolated from newborn rodent bone to other bone marrow cell types, in particular the monocyte-macrophage cell line, by the use of cell surface phenotyping. In studies in mouse and rat we failed to detect the expression of markers characteristic of mononuclear phagocytes or other bone marrow cell types, including the hemopoietic tissue restricted common leucocyte antigen (T200). Our findings cast further doubt on the view that osteoclasts arise by fusion of mononuclear phagocytes in a similar fashion to the formation of multinucleate inflammatory giant cells.

Animals↗