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PubMed1993

Integrin receptor-mediated mobilisation of intranuclear calcium in rat osteoclasts.

Cell-matrix interactions have been shown to play an important role in regulating cell function and behaviour. In bone, where calcified matrix formation and resorption events are required to be in dynamic equilibrium, regulation of adhesive interactions between bone cells and their matrix is critical. The present study focuses on the osteoclast, the bone resorbing cell, as well as integrins, which are cell surface adhesion receptors that mediate osteoclast attachment to bone matrix. In osteoclasts, the most abundant integrin receptor is the vitronectin receptor (VNR, alpha v beta 3). The objective of the study was to investigate changes in intracellular calcium, a regulator of osteoclast function, following addition of peptides that bind integrins, in particular the alpha v beta 3 form of the vitronectin receptor (VNR), which is highly expressed in osteoclasts. The study demonstrated a unique spatial localisation of the calcium signal in response to cell membrane receptor occupancy by integrin ligands in rat osteoclasts. Addition of peptides with the Arg-Gly-Asp (RGD) sequence such as BSP-IIA, GRGDSP and GRGDS to rat osteoclasts evoked an immediate increase in free calcium ion concentration [Ca2+]i, localised to the nuclei and to the thin cytoplasmic skirt. These responses were inhibited by F11, a monoclonal antibody to the rat integrin beta 3 chain, as well as echistatin, a snake venom shown to colocalise with the alpha v chain in osteoclasts, suggesting that the calcium signal is mediated by the alpha v beta 3 form of VNR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

The small GTP-binding protein, rho p21, is involved in bone resorption by regulating cytoskeletal organization in osteoclasts.

Rho protein (rho p21), a p21ras-related small guanine nucleotide binding protein, regulates cytoskeletal organization in a number of different types of cells. Evidence has indicated that Clostridium botulinum-derived ADP-ribosyltransferase (C3 exoenzyme) specifically ADP-ribosylates rho p21 at Asn41 and renders it functionally inactive. In this study, we examined the involvement of rho p21 in osteoclastic bone resorption using the C3 exoenzyme. When osteoclast-like multinucleated cells obtained from cocultures of mouse osteoblastic cells and bone marrow cells were placed on dentine slices, they formed ringed structures of podosomes containing F-actin (corresponding to the clear zone) within 8 hours. Many resorption pits were formed on dentine slices after culture for 24 hours. The C3 exoenzyme at 0.15-10 micrograms/ml added to the culture medium disrupted the ringed structure of podosomes in osteoclast-like cells in a dose-dependent manner. Correspondingly, pit formation by osteoclast-like cells on dentine slices was dose-dependently inhibited also by adding the C3 exoenzyme. Microinjection of the C3 exoenzyme into osteoclast-like cells placed on culture dishes completely disrupted the ringed podosome structure within 20 minutes. The amount of the rho p21 which was ADP-ribosylated by the C3 exoenzyme in vitro was much greater in purified osteoclast-like cells than in osteoblastic cells. Prior exposure of the purified osteoclast-like cell preparation to the C3 exoenzyme in vivo markedly decreased the amount of unribosylated rho p21. This indicated that the C3 exoenzyme incorporated into osteoclast-like cells effectively ADP-ribosylates rho p21 in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1997

Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts.

The osteoclasts are the cells responsible for bone resorption. Matrix metalloproteinases (MMPs) appear crucial for this process. To identify possible MMP expression in osteoclasts, we amplified osteoclast cDNA fragments having homology with MMP genes, and used them as a probe to screen a rabbit osteoclast cDNA library. We obtained a cDNA of 1,972 bp encoding a polypeptide of 582 amino acids that showed more than 92% identity to human, mouse, and rat membrane-type 1 MMP (MT1-MMP), a cell surface proteinase believed to trigger cancer cell invasion. By northern blotting, MT1-MMP was found to be highly expressed in purified osteoclasts when compared with alveolar macrophages and bone stromal cells, as well as with various tissues. In situ hybridization on bone sections showed that MT1-MMP is expressed also in osteoclasts in vivo. Antibodies recognizing MT1-MMP reacted with specific plasma membrane areas corresponding to lamellipodia and podosomes involved, respectively, in migratory and attachment activities of the osteoclasts. These observations highlight how cells might bring MT1-MMP into contact with focal points of the extracellular matrix, and are compatible with a role of MT1-MMP in migratory and attachment activities of the osteoclast.

Amino Acid Sequence↗

PubMed2002

Localization and possible role of two different alpha v beta 3 integrin conformations in resting and resorbing osteoclasts.

Integrins are membrane receptors that mediate interactions between cells and the extracellular matrix. We recently showed that the osteoclast integrin alpha(v)beta(3) exists in two different conformations, so-called 'basal' and 'activated', with each exhibiting a distinct function. In this study we demonstrate that, in non-resorbing osteoclasts, the 'activated' form of alpha(v)beta(3) accumulates in the motile areas of the plasma membrane. During bone resorption this conformation is prevalent in the ruffled membrane, whereas the 'basal' form of alpha(v)beta(3) is also present in the sealing zone. Moreover, hepatocyte growth factor (HGF) and macrophage colony stimulating factor (M-CSF), two molecules involved in osteoclastogenesis and osteoclast survival, modulate alpha(v)beta(3) conformation in vitro. Preincubation with HGF or M-CSF induces a shift of conformation of alpha(v)beta(3) in primary human osteoclasts (OCs) and in the osteoclast-like cell line (GCT 23). Activated integrin promotes osteoclast migration to the alpha(v)beta(3) ligand osteopontin and enhances bone resorption. Thus, HGF and M-CSF modulate the alpha(v)beta(3) conformational states required for osteoclast polarization and resorption. The capacity of growth factors to alter the affinity of alpha(v)beta(3) toward its ligands offers a potential explanation for the diverse responses of osteoclasts to the same ligand.

Antibodies, Monoclonal↗

PubMed1992

The osteoclast proton pump differs in its pharmacology and catalytic subunits from other vacuolar H(+)-ATPases.

Osteoclasts are multinucleated cells derived from the mononuclear phagocyte system in the hematopoietic bone marrow. Their function is to resorb bone during skeletal growth and remodeling. They perform this function by acidifying an enclosed extracellular space, the bone resorbing compartment. Analysis of proton transport by inside-out vesicles derived from highly purified chicken osteoclast membranes has revealed the presence of a novel type of multisubunit vacuolar-like H(+)-ATPase. Unlike H(+)-ATPases derived from any other cell type or organelle, proton transport and ATPase activity in osteoclast vesicles are sensitive to two classes of inhibitors, namely V-ATPase inhibitors [N-ethyl-maleimide (NEM) and bafilomycin A1] and vanadate (IC50 100 mumol l-1), an inhibitor previously found to affect only P-ATPases. The osteoclast V-ATPase morphologically resembles vacuolar proton pumps and contains several vacuolar-like subunits (115 x 10(3), 39 x 10(3) and 16 x 10(3)M(r)), demonstrated by Western blot analysis. Subunits A and B of the catalytic domain of the enzyme, however, differ from that of other V-ATPases. In osteoclasts, subunit A has an M(r) of 63 x 10(3) instead of 67 x 10(3)-70 x 10(3); in contrast, monocytes, macrophages and kidney microsomes, which contain a vanadate-insensitive H(+)-ATPase, express the classical subunit A (70 x 10(3)M(r)). Moreover, two types of 57 x 10(3)-60 x 10(3)M(r) B subunits are also found: they are differentially recognized by antibodies and one is expressed predominantly in osteoclasts and the other in bone marrow cells and in kidney microsomes. Preliminary cloning data have indicated that the B subunit expressed in osteoclasts may be similar to the brain isoform. The osteoclast proton pump may, therefore, constitute a novel class of V-ATPase, with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme.

Adenosine Triphosphatases↗

PubMed2004

Quercetin suppresses bone resorption by inhibiting the differentiation and activation of osteoclasts.

Although quercetin has suppressed bone resorption in several animal studies, its target cells and the mechanism of its action related to bone resorption has not been fully elucidated. We investigated the effect of quercetin on the differentiation and activation of osteoclasts. We used cocultures of mouse spleen cells and ST2 cells, and cultures of osteoclast progenitor cells [M-CSF-dependent (MD) cells from mouse bone marrow and murine monocytic RAW 264 (RAW) cells]. Quercetin dose-dependently inhibited osteoclast-like (OCL) cell formation at 2-5 microM concentration in both the coculture and MD cell culture. Quercetin inhibited the increase of tartrate-resistant acid phosphatase (TRAP) activity of mononuclear preosteoclasts (pOCs) induced by receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) in both MD and RAW cell cultures. Quercetin reversely induced the disruption of actin rings in OCLs. Quercetin also suppressed both pit formation induced by osteoclasts on dentine slices and PTH-stimulated (45)Ca release in mouse long bone cultures. These results suggest that osteoclast progenitors as well as mature osteoclasts, are quercetin's target cells in relation to bone resorption, and that quercetin's suppressive effect on bone resorption results from both its inhibitory effect on the differentiation of osteoclast progenitor cells into pOCs and from its disruptive effect on actin rings in mature osteoclasts.

Acid Phosphatase↗

PubMed1997

[Techniques for purification of rabbit osteoclasts and analysis of their functions].

In the process of bone remodeling or modeling, the balance between bone formation and bone resorption maintains normality of function and structures of bone. Major bone-resorbing cells, osteoclasts, are terminally differentiated from hematopoietic stem cells and multinucleate cells. However, direct effects of osteotropic factors on osteoclast function have been unclear. Attempts to obtain isolated mammalian osteoclasts of high purity have been unsuccessful so far. Initially we succeeded in isolating osteoclasts of high purity using tissue culture dishes because of their high affinity for the tissue culture dishes, but the shortcoming of this method is that it was impossible to detach osteoclasts from the dishes. Therefore, we could not estimate bone-resorbing activity of mature osteoclasts on mineralizing substratum without any influence of bone-cells other than osteoclasts. Recently we developed a method to avoid the defect in the above-described method by the use of collagen gel. Our new method may shed new light on our understanding of the cellular and molecular mechanisms of osteoclasts.

Acid Phosphatase↗

PubMed2000

Human bone-derived cells support formation of human osteoclasts from arthroplasty-derived cells in vitro.

Mononuclear osteoclast precursors are present in the wear-particle-associated macrophage infiltrate found in the membrane surrounding loose implants. These cells are capable of differentiating into osteoclastic bone-resorbing cells when co-cultured with the rat osteoblast-like cell line, UMR 106, in the presence of 1,25(OH)2 vitamin D3. In order to develop an in vitro model of osteoclast differentiation which more closely parallels the cellular microenvironment at the bone-implant interface in situ, we determined whether osteoblast-like human bone-derived cells were capable of supporting the differentiation of osteoclasts from arthroplasty-derived cells and analysed the humoral conditions required for this to occur. Long-term co-culture of arthroplasty-derived cells and human trabecular-bone-derived cells (HBDCs) resulted in the formation of numerous tartrate-resistant-acid-phosphatase (TRAP) and vitronectin-receptor (VNR)-positive multinucleated cells capable of extensive resorption of lacunar bone. The addition of 1,25(OH)2 vitamin D3 was not required for the formation of osteoclasts and bone resorption. During the formation there was release of substantial levels of M-CSF and PGE2. Exogenous PGE2 (10(-8) to 10(-6) M) was found to stimulate strongly the resorption of osteoclastic bone. Our study has shown that HBDCs are capable of supporting the formation of osteoclasts from mononuclear phagocyte precursors present in the periprosthetic tissues surrounding a loose implant. The release of M-CSF and PGE2 by activated cells at the bone-implant interface may be important for the formation of osteoclasts at sites of pathological bone resorption associated with aseptic loosening.

Acid Phosphatase↗

PubMed2003

Activation of protease-activated receptor-2 leads to inhibition of osteoclast differentiation.

UNLABELLED: PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption. INTRODUCTION: Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the "tethered ligand" created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated. MATERIALS AND METHODS: Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR. RESULTS: RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3-induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells. CONCLUSION: These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation.

Animals↗

PubMed2004

Osteoclast deficiency results in disorganized matrix, reduced mineralization, and abnormal osteoblast behavior in developing bone.

UNLABELLED: Studies of the influence of the osteoclast on bone development, in particular on mineralization and the formation of the highly organized lamellar architecture of cortical bone by osteoblasts, have not been reported. We therefore examined the micro- and ultrastructure of the developing bones of osteoclast-deficient CSF-1R-nullizygous mice (Csf1r(-/-) mice). INTRODUCTION: Colony-stimulating factor-1 receptor (CSF-1R)-mediated signaling is critical for osteoclastogenesis. Consequently, the primary defect in osteopetrotic Csf1r(-/-) mice is severe osteoclast deficiency. Csf1r(-/-) mice therefore represent an ideal model system in which to investigate regulation by the osteoclast of osteoblast-mediated bone formation during development. MATERIALS AND METHODS: Bones of developing Csf1r(-/-) mice and their littermate controls were subjected to X-ray analysis, histological examination by light microscopy and transmission electron microscopy, and a three-point bending assay to test their biomechanical strength. Bone mineralization in embryonic and postnatal bones was visualized by double staining with alcian blue and alizarin red. Bone formation by osteoblasts in these mice was also examined by double-calcein labeling and in femoral anlagen transplantation experiments. RESULTS AND CONCLUSIONS: Frequent spontaneous fractures and decreased strength parameters (ultimate load, yield load, and stiffness) in a three-point bending assay showed the biomechanical weakness of long bones in Csf1r(-/-) mice. Histologically, these bones have an expanded epiphyseal chondrocyte region, a poorly formed cortex with disorganized collagen fibrils, and a severely disturbed matrix structure. The mineralization of their bone matrix at secondary sites of ossification is significantly reduced. While individual osteoblasts in Csf1r(-/-) mice have preserved their typical ultrastructure and matrix depositing activity, the layered organization of osteoblasts on the bone-forming surface and the direction of their matrix deposition toward the bone surface have been lost, resulting in their abnormal entrapment by matrix. Moreover, we also found that (1) osteoblasts do not express CSF-1R, (2) the bone defects in Csf1r(-/-) embryos develop later than the development of osteoclasts in normal embryos, and (3) the transplanted Csf1r(-/-) femoral anlagen develop normally in the presence of wildtype osteoclasts. These results suggest that the dramatic bone defects in Csf1r(-/-) mice are caused by a deficiency of the osteoclast-mediated regulation of osteoblasts and that the osteoclast plays an important role in regulating osteoblastic bone formation during development, in particular, in the formation of lamellar bone.

Animals↗

PubMed2005

Identification of multiple osteoclast precursor populations in murine bone marrow.

UNLABELLED: Murine BM was fractionated using a series of hematopoietic markers to characterize its osteoclast progenitor populations. We found that the early osteoclastogenic activity in total BM was recapitulated by a population of cells contained within the CD11b(-/low) CD45R- CD3- CD115high fraction. INTRODUCTION: Osteoclasts are of hematopoietic origin and they have been shown to share the same lineage as macrophages. We further characterized the phenotype of osteoclast progenitor populations in murine bone marrow (BM) by analyzing their cell surface markers. MATERIALS AND METHODS: We used fluorescence-activated cell sorting (FACS) to identify the subsets of BM cells that contained osteoclast progenitors. We fractionated BM according to several markers and cultured the sorted populations for a period of 2-6 days with macrophage-colony stimulating factor (M-CSF) and RANKL. The numbers of multinucleated osteoclast-like cells (OCLs) that formed in the cultures were counted. RESULTS: We found that the CD45R- CD11b(-/low) population recapitulated the early osteoclastogenic activity of total BM. In addition, although previous experiments indicated that osteoclastogenic activity was enriched within the CD45R+ population, we found that highly purified CD45R+ BM was incapable of differentiating into osteoclasts in vitro. We also found that CD45R- CD11b(high) BM cells were an inefficient source of osteoclast progenitors. However, CD11b was transiently upregulated by cells of the CD45R- CD11b(-/low) fraction early (within 24 h) during culture with M-CSF. Finally, further fractionation of BM using CD115 and CD117 showed that, as osteoclast precursor cells matured, they downregulate CD117 but remain CD115+. Curiously, pure populations of CD117- (CD115high) cells isolated fresh from BM have low osteoclastogenic activity in vitro. CONCLUSIONS: We provided a refined analysis of the precise subpopulations of murine BM that are capable of differentiating into OCLs in vitro when treated with M-CSF and RANKL.

Animals↗

PubMed2006

Bone morphogenetic proteins in bone stimulate osteoclasts and osteoblasts during bone development.

UNLABELLED: In this study, overexpression of noggin, a BMP antagonist, in developing bone caused significantly decreased osteoclast number as well as bone formation rate, resulting in increased bone mass with immature bone quality. BMP signaling plays important roles in normal bone development and regulation of bone resorption. INTRODUCTION: Bone morphogenetic proteins (BMPs) act on various types of cells. Although involvement of BMP signals in osteoblast differentiation has been studied extensively, the effects of BMPs on osteoclasts have not been widely researched. Consequently, the net effects of BMPs on bone remain unclear. The purpose of this study was to delineate more fully the role of BMPs in skeletal biology. MATERIALS AND METHODS: We generated transgenic mice that express BMP4 or noggin in bone under the control of the 2.3-kb alpha1(I) collagen chain gene (Col1a1) promoter, and analyzed their bone phenotype. We also analyzed bone of transgenic mice expressing BMP4 specifically in cartilage. RESULTS: Mice overexpressing BMP4 in bone developed severe osteopenia with increased osteoclast number. Mice overexpressing noggin, a BMP antagonist, in bone showed increased bone volume associated with decreased bone formation rate and decreased osteoclast number. The noggin-transgenic tibias exhibited reduced periosteal bone formation and reduced resorption of immature bone in marrow spaces, associated with frequent fractures at the diaphysis. Co-culture of primary osteoblasts prepared from noggin-transgenic calvariae and wildtype spleen cells resulted in poor osteoclast formation, which was rescued by addition of recombinant BMP2, suggesting that noggin inhibits osteoclast formation by attenuating BMP activities in noggin-transgenic mice. The expression levels of Rankl were not decreased in primary osteoblasts from noggin transgenic mice. Immunoblot analysis showed increased phosphorylation of Smad1/5/8 in osteoclast precursor cells after 20-minute treatment with BMPs, suggesting that these cells are stimulated by BMPs. Mice overexpressing BMP4 in cartilage had enlarged bones containing thick trabeculae, possibly because of expansion of cartilage anlagen. CONCLUSIONS: Overexpression of noggin in bone revealed that BMP signals regulate bone development through stimulation of osteoblasts and osteoclasts.

Animals↗

PubMed1998

A third-generation bisphosphonate, YM175, inhibits osteoclast formation in murine cocultures by inhibiting proliferation of precursor cells via supporting cell-dependent mechanisms.

The theory that bisphosphonates inhibit osteoclast formation through their effects on osteoblastic cells remains controversial. To confirm the inhibitory effect of bisphosphonates on osteoclast formation and gain some insights into the underlying mechanisms, we examined the effect of disodium dihydrogen (cycloheptylamino)-methylene-bisphosphonate monohydrate (YM175) on osteoclast-like multinucleated cell (OCL) formation in various mouse coculture systems. When different origins of osteoclast precursors (bone marrow, spleen, or nonspecific esterase-positive cells) were cocultured with the same supporting cells (calvarial osteoblasts), YM175 inhibited OCL formation similarly in all cultures. When the same osteoclast precursors (spleen cells) were cocultured with supporting cells of different origin, the results were variable. YM175 inhibited OCL formation almost completely in cocultures with calvarial osteoblasts or osteoblastic cell line KS4, while it did not, or only slightly, inhibit OCL formation in cocultures with stromal cell lines, ST2 or MC3T3-G2/PA6. Temporal addition of YM175 in cocultures of spleen cells with osteoblastic cells revealed that YM175 was effective when it was present at an early phase of the culture period. Consistent with this observation, YM175 in the presence of osteoblastic cells inhibited proliferation of preosteoclastic cells, but did not inhibit the fusion of mononuclear prefusion osteoclasts. In conclusion, the inhibitory effect of YM175 on OCL formation was confirmed in various murine coculture systems, but the effect was dependent on the types of bone-derived cells supporting osteoclastogenesis. The findings suggest that YM175 inhibits osteoclastogenesis by inhibiting the proliferation of osteoclast precursors through its action on supporting cells of osteoblast lineage rather than acting directly on osteoclast precursors.

Animals↗

PubMed2000

Crucial involvement of the EP4 subtype of prostaglandin E receptor in osteoclast formation by proinflammatory cytokines and lipopolysaccharide.

Prostaglandin E2 (PGE2) exerts its effects through the PGE receptor that consists of four subtypes (EP1, EP2, EP3, and EP4). Osteoclast formation in the coculture of primary osteoblastic cells (POB) and bone marrow cells was enhanced more by 11-deoxy-PGE1 (an EP4 and EP2 agonist) than by butaprost (an EP2 agonist) and other agonists, which suggests that EP4 is the main factor in PGE2-induced osteoclast formation. PGE2-induced osteoclast formation was not observed in the coculture of POB from EP4-deficient (EP4 k/o) mice and spleen cells from wild-type (w/t) mice, whereas osteoclasts were formed in the coculture of POB from w/t mice and spleen cells from EP4-k/o mice. In situ hybridization (ISH) showed that EP4 messenger RNA (mRNA) was expressed on osteoblastic cells but not on multinucleated cells (MNCs) in w/t mice. These results indicate that PGE2 enhances osteoclast formation through its EP4 subtype on osteoblasts. Osteoclast formation by interleukin 1alpha (IL-1alpha), tumor necrosis factor alpha (TNF-alpha), basic fibroblast growth factor (bFGF), and lipopolysaccharide (LPS) was hardly observed in the coculture of POB and bone marrow cells, both from EP4-k/o mice, which shows the crucial involvement of PG and the EP4 subtype in osteoclast formation by these molecules. In contrast, osteoclast formation by 1,25-hydroxyvitamin D3 (1,25(OH)2D3) was not impaired and that by parathyroid hormone (PTH) was only partially impaired in EP4-k/o mice, which may be related to the fact that EP4-k/o mice revealed no gross skeletal abnormalities. Because it has been suggested that IL-1alpha, TNF-alpha, bFGF, and LPS are involved in inflammatory bone loss, our work can be expected to contribute to an understanding of the pathophysiology of these conditions.

Animals↗

PubMed2000

The microphthalmia transcription factor regulates expression of the tartrate-resistant acid phosphatase gene during terminal differentiation of osteoclasts.

The defective terminal differentiation of osteoclasts in mice homozygous for the mi allele of the microphthalmia transcription factor (MITF) gene implies that MITF plays a critical role in regulating gene expression during osteoclast ontogeny. To begin addressing the role of this transcription factor in the osteoclast, target genes need to be identified. In the present work, several lines of evidence show that the gene encoding the enzyme tartrate-resistant acid phosphatase (TRAP) is a target of MITF. Analysis of osteoclasts in vivo in the embryonic forelimb showed that MITF and TRAP RNA were coexpressed in a dynamic pattern during the process of endochondral ossification of long bone. Primary osteoclast-like cells (OCLs) produced from mi/mi mutant mice expressed TRAP messenger RNA (mRNA) at 8-fold lower levels than in OCLs derived from normal mice, indicating a direct link between MITF function and TRAP expression. The activity of mouse TRAP promoter-reporter genes was assayed in the primary OCLs by DNA-mediated transfection, and this activity was shown to depend on a conserved sequence (GGTCATGTGAG) located in the proximal promoter. Recombinant MITF protein recognized specifically this conserved sequence element. Expression of a TRAP promoter-green fluorescent protein (GFP) transgene mimicked the expression of the endogenous TRAP gene during differentiation of osteoclast-like cells, and the expression of the transgene was decreased 8-fold when placed into the mutant mi/mi background. These results are consistent with a role for MITF in gene expression during terminal differentiation of the osteoclast and will allow osteoclast-specific mechanisms of gene regulation to be studied in greater detail.

Acid Phosphatase↗

PubMed2000

Bone-resorbing osteoclasts contain gap-junctional connexin-43.

Intercellular gap junctions have been previously described at contact sites between surface osteoblasts, between osteoblasts and underlying osteocytes, and between osteocyte cell processes in the canaliculi. The subunits of gap junction channels are assembled from a family of proteins called connexins. In the present work, we show that rat osteoclasts cultured on bovine bone slices show connexin-43 (Cx43) staining localizing in the plasma membrane of the cells in cell-cell contacts and over the basolateral membrane of osteoclasts. The effect of heptanol, a known gap-junctional inhibitor, was studied using the well-characterized pit formation assay. Heptanol decreased the number and activity of osteoclasts. The proportion of mononuclear tartrate-resistant acid phosphatase (TRAP)-positive cells out of all TRAP-positive cells increased on heptanol treatment, suggesting a defect in the fusion of mononuclear osteoclast precursors to multinucleated mature osteoclasts. Furthermore, the total resorbed area and the number of resorption pits also decreased in the heptanol-treated cultures. These results suggest that gap-junctional Cx43 plays a functional role in osteoclasts and that the blocking of gap junctions decreases both the number and the activity of osteoclasts. This can indicate both a direct communication between multinucleated osteoclasts and mononuclear cells through gap junctions or an indirect effect through gap junctions between osteoblasts.

Animals↗

PubMed2001

Tissue inhibitor of metalloproteinases 1 and 2 directly stimulate the bone-resorbing activity of isolated mature osteoclasts.

Tissue inhibitor metalloproteinases 1 (TIMP-1) and 2 have been reported to inhibit bone resorption. However, here, we report the direct action of both TIMP-1 and TIMP-2 on isolated rabbit mature osteoclasts to stimulate their bone-resorbing activity at significantly lower concentrations (approximately ng/ml) than those (approximately microg/ml) required for the inhibition of bone resorption. The cell population used in this study consisted of a mature osteoclast population with >95% purity. TIMP-1 (approximately 50 ng/ml) and TIMP-2 (approximately 8-10 ng/ml) increased the pit area excavated by the isolated mature osteoclasts. The stimulatory effects of TIMPs were abolished by simultaneous addition of anti-TIMP antibodies. At higher concentrations, the stimulation of bone resorption decreased reversely to the control level. The magnitude of the stimulatory effect of TIMP-2 was more than that of TIMP-1. Metalloproteinase inhibitors such as BE16627B and R94138 could not replace TIMPs with respect to the bone-resorbing activity, suggesting that the osteoclast-stimulating activity of TIMPs was independent of the inhibitory activity on matrix metalloproteinases (MMPs). TIMPs stimulated tyrosine phosphorylation of cellular proteins in the isolated mature osteoclasts. Both herbimycin A, an inhibitor of tyrosine kinases, and PD98059 and U0126, inhibitors of mitogen-activated protein kinase (MAPK), completely blocked the TIMP-induced stimulation of osteoclastic bone-resorbing activity. On the plasma membrane of osteoclasts, some TIMP-2-binding proteins were detected by a cross-linking experiment. These findings show that TIMPs directly stimulate the bone-resorbing activity of isolated mature osteoclasts at their physiological concentrations and that the stimulatory action of TIMPs is likely to be independent of their activities as inhibitors of MMPs.

Acetamides↗

PubMed2003

Effects of cyclosporine on osteoclast activity: inhibition of calcineurin activity with minimal effects on bone resorption and acid transport activity.

Cyclosporine results in rapid and profound bone loss in transplant patients, an effect ascribed to osteoclasts. Cyclosporine, complexed with the appropriate immunophilin, inhibits calcineurin (the calcium/calmodulin dependent serine/threonine phosphatase) activity. We tested the hypothesis that cyclosporine inhibits calcineurin activity in osteoclasts, resulting in stimulation of osteoclast activity. We compared the effects of cyclosporine A and the calmodulin antagonist, tamoxifen, on bone resorption by avian osteoclasts. Tamoxifen inhibits bone resorption approximately 60%, whereas cyclosporine A only inhibited bone resorption 12%. One-hour treatment with 100 nM cyclosporine inhibited osteoclast calcineurin activity 70% in whole cell lysates, whereas 10 microM tamoxifen only inhibited calcineurin activity 25%. We compared the effects of cyclosporine A and tamoxifen on acid transport activity in isolated membrane vesicles and in isolated membrane vesicles obtained from osteoclasts treated with cyclosporine A or tamoxifen under conditions that inhibit calcineurin activity. Direct addition of cyclosporine A in the acid transport assay, or pretreatment of cells with cyclosporine A followed by membrane isolation, had no effect on acid transport activity in membrane vesicles. In contrast, direct addition of tamoxifen to membranes inhibits acid transport activity, an effect that can be prevented by addition of exogenous calmodulin. Furthermore, acid transport activity was also inhibited in membrane vesicles isolated from cells treated with tamoxifen. In conclusion, cyclosporine A inhibits osteoclast calcineurin activity; however, calcineurin inhibition does not correspond to a significant effect on acid transport activity in isolated membrane vesicles or bone resorption by osteoclasts.

Acids↗