Showing papers on "Tartrate-resistant acid phosphatase published in 2000"
TL;DR: It is demonstrated that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system.
Abstract: Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases.
1,224 citations
TL;DR: Findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes and may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.
Abstract: High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG−/2 mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG−/− mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG−/− mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-κB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG−/− mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.
547 citations
TL;DR: Serum TRAP 5b activity decreased significantly after 6 months of hormone replacement therapy (HRT) of postmenopausal women compared with the change observed in post menopausal women receiving placebo, but no significant differences were observed between the changes in the placebo and HRT groups in total serum TRAP amount.
Abstract: Human serum contains two forms of tartrate-resistant acid phosphatase (TRAP), 5a and 5b. Of these, 5a contains sialic acid and 5b does not. We show here that antigenic properties and pH optimum of TRAP purified from human osteoclasts are identical to those of serum TRAP 5b and completely different from those of serum TRAP 5a, suggesting that 5b would be derived from osteoclasts and 5a from some other source. We developed a novel immunoassay specific for 5b using a monoclonal antibody O1A as capture antibody. O1A did not bind acid phosphatase derived from platelets and erythrocytes. Western analysis showed that O1A was specific for TRAP in both human bone and serum. We measured bound TRAP activity at pH 6.1, where 5b is highly active and 5a almost completely inactive. The immunoassay detected more than 90% of the initial TRAP 5b activity after 8-h incubation of serum samples at 25 degrees C and after 3 days incubation at 4 degrees C. Serum TRAP 5b activity decreased significantly after 6 months of hormone replacement therapy (HRT) of postmenopausal women compared with the change observed in postmenopausal women receiving placebo (p < 0.0001). Instead, no significant differences were observed between the changes in the placebo and HRT groups in total serum TRAP amount. These results show that serum TRAP 5b is a specific and sensitive marker for monitoring antiresorptive treatment. Instead, total serum TRAP cannot be used for that purpose. These findings may turn out to be a significant improvement in using serum TRAP as a resorption marker.
410 citations
TL;DR: The view that TRAP, like several other hydrolases, is synthesized as a relatively inactive proen enzyme, and cleavage is the physiological mechanism of proenzyme activation in osteoclasts is put forth.
210 citations
TL;DR: Measurements of the bone formation rate suggest that the animals compensate for the increased resorption by increasing bone synthesis, which partly ameliorates the phenotype, and provides evidence that inclusion of an irrelevant enhancer does not necessarily override a tissue‐specific promoter.
Abstract: Tartrate-resistant acid phosphatase (TRAP) is a secreted product of osteoclasts and a lysosomal hydrolase of some tissue macrophages. To determine whether TRAP expression is rate-limiting in bone resorption, we overexpressed TRAP in transgenic mice by introducing additional copies of the TRAP gene that contained the SV40 enhancer. In multiple independent mouse lines, the transgene gave a copy number-dependent increase in TRAP mRNA levels and TRAP activity in osteoclasts, macrophages, serum, and other sites of normal low-level expression (notably, liver parenchymal cells, kidney mesangial cells, and pancreatic secretory acinar cells). Transgenic mice had decreased trabecular bone consistent with mild osteoporosis. Measurements of the bone formation rate suggest that the animals compensate for the increased resorption by increasing bone synthesis, which partly ameliorates the phenotype. These mice provide evidence that inclusion of an irrelevant enhancer does not necessarily override a tissue-specific promoter.
177 citations
TL;DR: Investigating its expression and activity in mouse tissues demonstrated that TRAP is a lysosomal enzyme that is found in diverse murine tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages, as previously shown.
Abstract: Tartrate-resistant acid phosphatase (TRAP) is a histochemical marker of the osteoclast. It is also characteristic of monohistiocytes, particularly alveolar macrophages, and is associated with diverse pathological conditions, including hairy cell leukemia and AIDS encephalopathy. To study the biology of this enzyme, we investigated its expression and activity in mouse tissues. Confocal fluorescence studies showed that TRAP is localized to the lysosomal compartment of macrophages. In adult mice, high activities of the enzyme were demonstrated in bone, spleen, liver, thymus, and colon, with lower amounts in lung, stomach, skin, brain, and kidney. Trace amounts were detected in testis, muscle, and heart. Expression of TRAP mRNA was investigated in tissue sections by in situ hybridization and protein expression was monitored by histochemical staining or immunohistochemically. TRAP is widely expressed in many tissues, where it is associated with cells principally originating from the bone marrow, including those of osteoclast/macrophage lineage. The cellular distribution of TRAP mRNA and enzyme antigen in the tissues corresponds closely to that of cells staining with an antibody directed to the CD80 (B7) antigen. Therefore, to confirm its putative localization in dendritic cells, isolated bone marrow dendritic cells were matured in culture. These co-stained strongly for TRAP protein and the CD80 antigen. These studies demonstrate that TRAP is a lysosomal enzyme that is found in diverse murine tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages, as previously shown. (J Histochem Cytochem 48:219-227, 2000)
138 citations
TL;DR: The TRAP 5b immunoassay can be used to replace the laborious and time-consuming microscopic counting of osteoclasts in the osteoclast differentiation assay and to test the effects of potential therapeutic agents on osteoc last differentiation, enabling fast screening of large amounts of potentialutic agents.
Abstract: Background: Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) 5b into the circulation. We studied the release of TRAP 5b from osteoclasts using a mouse in vitro osteoclast differentiation assay. Methods: We developed and characterized a polyclonal antiserum in rabbits, using purified human osteoclastic TRAP 5b as antigen. The antiserum was specific for TRAP in Western analysis of mouse osteoclast culture medium and was used to develop an immunoassay. We cultured mouse bone marrow-derived osteoclast precursor cells for 3–7 days with or without clodronate in the presence of vitamin D and analyzed the number of osteoclasts formed and the amount of TRAP 5b activity released into the culture medium. Results: TRAP 5b activity was not secreted from osteoclast precursor cells. Addition of clodronate-containing liposomes decreased in a dose-dependent manner the number of osteoclasts and TRAP 5b activity released in 6-day cultures. The amount of TRAP 5b activity in the medium detected by the immunoassay correlated significantly with the number of osteoclasts formed ( r = 0.94; P Conclusions: The TRAP 5b immunoassay can be used to replace the laborious and time-consuming microscopic counting of osteoclasts in the osteoclast differentiation assay and to test the effects of potential therapeutic agents on osteoclast differentiation, enabling fast screening of large amounts of potential therapeutic agents.
135 citations
TL;DR: There may be a sequential link in tensional force applied on the bone lining cells, up‐regulation of TGF‐β1/OPG, and disappearance of osteoclasts, as suggested in an experimental model of tooth movement in rats.
Abstract: The mechanism controlling the disappearance of osteoclasts from bone surfaces after bone resorption in vivo is largely unknown. This is because there is no suitable experimental system to trace the final fate of osteoclasts. Here, we used an experimental model of tooth movement in rats to show that preexisting osteoclasts disappeared from the bone surface through apoptosis during a force-induced rapid shift from bone resorption to formation. On the distal alveolar bone surface of the maxillary molar in growing rats, many mature osteoclasts were present. When light tensional force was applied to the bone surface through an orthodontic appliance, these preexisting osteoclasts gradually disappeared. One day after the application of force, about 24% of the osteoclasts exhibited apoptotic morphology and the proportion of apoptotic cells was increased to 41% by day 2, then decreased afterward. These changes were undetectable on the control distal alveolar bone surface, which is free from tensional force. As shown by in situ hybridization, a marked increase in transforming growth factor beta1 (TGF-beta1) and osteoprotegerin (OPG) messenger RNA (mRNA) was observed in the stretched cells on the tensioned distal bone surface, simultaneously with the loss of osteoclasts. Both of these factors are known to have a negative effect on osteoclast recruitment and survival. As early as 2 days after force application, some of these stretched cells were identified as cuboidal osteoblasts showing intense signals for both factors. Our data suggest there may be a sequential link in tensional force applied on the bone lining cells, up-regulation of TGF-beta1/OPG, and disappearance of osteoclasts.
119 citations
TL;DR: It is shown that mice OSM, LIF and CT-1 induce osteoclast differentiation and activation, that DEX synergizes with each in this activity, and that mouse OSM induces responses in osteoblasts that are not shown by LIF orCT-1.
99 citations
TL;DR: It is shown that macrophages, isolated from the pseudomembrane surrounding loose arthroplasty components, are capable of differentiating into osteoclastic bone resorbing cells and that OPGL is required for this to occur.
Abstract: OBJECTIVE—Osteoprotegerin ligand (OPGL) is a newly discovered molecule, which is expressed by osteoblasts/bone stromal cells. This ligand and M-CSF are now known to be essential for osteoclast differentiation from marrow and circulating precursors. This study examined whether OPGL and its soluble receptor osteoprotegerin (OPG), influenced osteoclast formation from human arthroplasty derived macrophages, to determine if the effects of OPGL and OPG on these cells could contribute to the osteolysis of aseptic loosening.
METHODS—OPGL (± dexamethasone/M-CSF) was added to cultures of macrophages isolated from the pseudomembrane of loosened hip arthroplasties incubated on glass coverslips and dentine slices. OPG was added to cocultures of arthroplasty derived macrophages and UMR106 osteoblast-like cells. Osteoclast differentiation in long term cultures was assessed by expression of macrophage (CD14) and osteoclast markers (tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and lacunar resorption).
RESULTS—In the absence of osteoblastic cells, the addition of OPGL alone was sufficient to induce differentiation of macrophages (CD14+, TRAP-, VNR-) into TRAP+ and VNR+ multinucleated cells, capable of extensive lacunar resorption. OPG was found to inhibit osteoclast formation by arthroplasty macrophages in a dose dependent manner. OPG (100 ng/ml) more than halved the formation of TRAP+ and VNR+ cells and the extent of lacunar resorption in co-cultures of UMR106 cells and arthroplasty macrophages.
CONCLUSIONS—This study has shown that macrophages, isolated from the pseudomembrane surrounding loose arthroplasty components, are capable of differentiating into osteoclastic bone resorbing cells and that OPGL is required for this to occur. OPG inhibits this process, most probably by interrupting the cell-cell interaction between osteoblasts and mononuclear phagocyte osteoclast precursors present in the pseudomembrane.
76 citations
TL;DR: The results suggest that PPARδ/β may be involved in direct modulation of osteoclastic bone resorption through regulation through their cognate receptors expressed in mature osteoclasts.
TL;DR: Evidence is presented that the change in the adhesion of osteoclasts from the periosteum to the bone surface, resulting in osteoclast activation, is mediated by OPGL.
TL;DR: It is demonstrated that conditioned medium harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures and bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of Mda-231 CM.
TL;DR: If and how bone resorption contributes to the skeletal development of carp, Cyprinus carpio L., a teleost species with ‘normal’ osteocyte-containing (cellular) bone is clarified, and the occurrence of bone remodelling suggest that the endoskeleton of juvenile carp might be involved in mineral metabolism.
Abstract: Summary
The present study considers the important role of bone resorption for bone growth in general, and aims to clarify if and how bone resorption contributes to the skeletal development of carp, Cyprinus carpio L., a teleost species with ‘normal’ osteocyte-containing (cellular) bone. To ensure the identification of osteoclasts and sites of bone resorption independently from the morphology of the bony cells, bones were studied by histological procedures, and by demonstration of the enzymes which serve as osteoclast markers, viz. tartrate resistant acid phosphatase (TRAP), ATPase and a vacuolar proton pump. Two types of bone-resorbing cells were observed in juvenile carp: (1) multinucleated giant cells displaying morphological and biochemical attributes which are known from mammalian osteoclasts; and (b) flat cells which lack a visible ruffled border and for which identification requires the performance of enzyme histochemical procedures. Bone resorption performed by osteoclasts mainly occurs at endosteal bone surfaces. To a lesser extent, bone resorption also takes place at periosteal bone surfaces, but without an apparent connection to bone growth. The latter observation, and the occurrence of bone remodelling, suggest that the endoskeleton of juvenile carp might be involved in mineral metabolism. Morphological differences and biochemical similarities to bone resorption in teleosts with acellular bone are discussed.
TL;DR: An ontogenetic relationship between macrophages, DC and osteoclasts is concluded and the data suggest a transdifferentiation between Mo and osteoblasts.
TL;DR: The findings indicate that abundant TRAP expression is not confined to osteoclasts in bone, but occurs in diverse tissues harbouring cells of bone marrow origin, including dendritic cells and other cells belonging to the osteoclast/macrophage lineage.
Abstract: Tartrate-resistant acid phosphatase (TRAP, Acp 5) is considered to be a marker of the osteoclast and studies using ‘knockout’ mice have demonstrated that TRAP is critical for normal development of the skeleton. To investigate the distribution of TRAP in the mammalian embryo, cryostat sections of 18 d murine fetuses were examined by in situ hybridisation, immunohistochemistry and histochemical reactions in situ. Abundant expression of TRAP mRNA was observed in the skin and epithelial surfaces of the tongue, oropharynx and gastrointestinal tract including the colon, as well as the thymus, ossifying skeleton and dental papillae. TRAP protein was identified at the same sites, but the level of expression in the different tissues did not always correlate with apparent enzyme activity. The findings indicate that abundant TRAP expression is not confined to osteoclasts in bone, but occurs in diverse tissues harbouring cells of bone marrow origin, including dendritic cells and other cells belonging to the osteoclast/macrophage lineage.
TL;DR: Results indicate that the various evaluation techniques performed on undecalcified sections, permit us to evaluate the response of human bone marrow cells in HAP implanted into mice.
TL;DR: The data suggest that chronic hypocalcemia affects both bone formation and resorption in rats, with a significant increase in all its variables.
Abstract: Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal-metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell-matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone-calcified cartilage interface in some cases were thickened but generally did not appear affected at bone-bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption.
TL;DR: Rescarch on the biology of TRAP has been intensive and has led to considerable progress on a number of fronts, including the cloning of the gene, but further studies are, however, still required to determine the role ofTRAP in vivo.
Abstract: Tartrate-resistant acid phosphatase (TRAP) is a member of the ubiquitously expressed enzyme family of the acid phosphatases. Nearly 30 years ago, TRAP became known to hematologists as cytochemical marker enzyme of hairy cell leukemia. Physiologically, TRAP is primarily a cytochemical marker of macrophages, osteoclasts and dendritic cells. TRAP is localized intracellularly in the lysosomal compartment. Recent data suggest also secretion of TRAP by some cell types, in particular by osteoclasts. Human, mouse and rat TRAP are biochemically well characterized. While the complete genomic sequence of TRAP has been elucidated, only limited information on the genetic details of the gene and its regulation is available. It appears that the intracellular iron content is involved in the regulation of the enzyme. The physiological substrates for this enzyme have not been identified yet and consequently the functional role of TRAP remains completely unknown, though some hypotheses have been forwarded, e.g. involvement in bone resorption and iron homeostasis (transport, metabolism). Taken together, research on the biology of TRAP has been intensive and has led to considerable progress on a number of fronts, including the cloning of the gene. Further studies are, however, still required to determine the role of TRAP in vivo.
TL;DR: Increased TRAP activity in ESRD was due to increased osteoclastic isoform 5b and related to bone turnover and increased TRAP protein in RA was suspected, but not proven, to be isoforms 5a and not related toBone turnover.
TL;DR: The results suggest that MMP-13 plays an important role in morphological changes in the rat femur, at least during the third and fourth week after birth, and that M MP-13 itself is involved in the interaction between osteoblastic and TRAP-positive cells.
Abstract: The interepiphyseal region between the greater trochanter and the capital femoral epiphysis and the medioproximal portion of the femoral neck exhibit extensive morphological changes during the first 4 weeks after birth in rats. Previous reports show that matrix metalloproteinase-13 (MMP-13, rat collagenase) mRNA is expressed in bone and cartilage during embryonal development and fracture healing. We examined MMP-13 mRNA expression and compared it with the distribution of osteopontin and osteocalcine mRNA in the femoral neck. Moreover, we examined histomorphometric analysis in the femoral neck where the morphology changes rapidly. Histomorphometric analysis of the 4-week-old rat femoral neck showed a high rate of bone formation and resorption in the region where shape changed rapidly. Osteopontin mRNA was expressed diffusely along the endosteum. In contrast, MMP-13 mRNA expression was restricted to the medial endosteal portion near the cartilage-bone interface of the femoral neck in 15- and 28-day-old rats and in the deepest endosteal interepiphyseal region of 15-day-old rats. MMP-13 mRNA-expressing osteoblastic cells were also expressing osteopontin but not osteocalcin mRNA. MMP-13 mRNA-expressing cells differ from tartrate-resistant acid phosphatase (TRAP)-positive cells, and MMP-13 mRNA-positive cells are located adjacent to TRAP-positive cells. The results of the site- and cell-specific expression of MMP-13, taken together with its enzymatic property, suggest that MMP-13 plays an important role in morphological changes in the rat femur, at least during the third and fourth week after birth, and that MMP-13 itself is involved in the interaction between osteoblastic and TRAP-positive cells.
TL;DR: Estrogen deficiency results in an increase in ICAM-1 expression on osteoclast precursors, which may be one of the mechanisms underlying bone loss following the menopause or ovariectomy.
Abstract: Objectives Estrogen deficiency caused by the menopause or ovariectomy leads to stimulation of osteoclastogenesis. The adhesion molecules, leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), are necessary for osteoclast formation. In this study, the expression of LFA-1 and ICAM-1 on osteoclast precursors during osteoclast differentiation, and the involvement of ovariectomy in the expression, were investigated.Methods Spleen cells isolated from normal or ovariectomized (OVX) mice were co-cultured with TMS14, stromal cells derived from mouse bone marrow, in the absence or presence of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) for 7 days. On days 3, 5 and 7 of culture, the expression of LFA-1 and ICAM-1 on osteoclast precursors was quantitated using indirect immunofluorescence and confocal laser cytometry, and, on day 7, the number of formed osteoclasts was measured by tartrate-resistant acid phosphatase (TRAP) stain.Results The level of ICAM-1 expression on osteoclas...
TL;DR: In this article, the authors developed an in vitro model of osteoclast differentiation which more closely parallels the cellular micro-environment at the bone-implant interface in situ, 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.
Abstract: 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.
TL;DR: Some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5Cl and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.
TL;DR: Serum creatine kinase-BB was evaluated in relation to known osteoclastic pathophysiology in two human types of autosomal dominant osteopetrosis at baseline and after stimulation with triiodothyronine and in four different rodent mutations, finding that the isoenzyme does not seem to be a valuable screening marker for osteopeterosis.
Abstract: In mammalian osteopetrosis the different mutations exemplify reduced bone resorption leading to net accumulation of bone. Recently, high blood levels of creatine kinase-BB have been reported in some human forms, suggesting it as a marker of osteopetrosis. In the current study serum creatine kinase-BB was evaluated in relation to known osteoclastic pathophysiology in two human types of autosomal dominant osteopetrosis at baseline and after stimulation with triiodothyronine and in four different rodent mutations. Creatine kinase-BB was increased markedly in Type 2 autosomal dominant osteopetrosis and in the incisors absent rat, both characterized by large numbers of giant osteoclasts, and did not change significantly after stimulation. Although creatine kinase-BB was unchanged in Type 1 autosomal dominant osteopetrosis at baseline and after stimulation, the rodent counterparts characterized by small osteoclasts, microphthalmic and osteopetrotic mice and toothless rats, had significantly decreased levels. Similar differences were observed in both types of autosomal dominant osteopetrosis compared with controls concerning tartrate resistant acid phosphatase. Creatine kinase-BB in mammalian osteopetrosis is related to osteoclastic number and size, where it probably reflects the differentiation and maturation of inactive bone resorbing cells. The isoenzyme does not seem to be a valuable screening marker for osteopetrosis.
DSM1
TL;DR: These results provide the first description of a new iron/hemin-responsive transcriptional regulation in the human TRAP gene.
Abstract: The biochemical properties and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, are well known. In contrast, little is known about the physiology and genie structure of this unique enzyme. In some diseases, like hairy cell leukemia, Gaucher's disease and osteoclastoma, cytochemically detected TRAP expression is used as a disease-associated marker. In order to begin to elucidate the regulation of this gene we generated different deletion constructs of the TRAP 5-flanking region, placed them upstream of the luciferase reporter gene and assayed them for their ability to direct luciferase expression in human 293 cells. Treatment of these cells with the iron-modulating reagents transferrin and hemin causes opposite effects on the TRAP promoter activity. Two regulatory GAGGC tandem repeat sequences (the hemin responsive elements, HRE) within the 5'-flanking region of the human TRAP gene were identified. S...
Journal Article•
TL;DR: No-cGMP pathway is involved in the CsA induced bone loss, and the effects of nitric oxide on bone are biphasic; at high doses, NO increases bone resorption.
Abstract: Treatment with cyclosporin A (CsA) following solid organ transplantations such as heart or liver generally results in bone loss. However, in vitro studies show that CsA inhibits bone resorption. Our previous in vivo animal studies demonstrated that the effects of nitric oxide (NO) on bone are biphasic; at high doses, NO increases bone resorption. In this study, we have examined in an in vitro setting to determine whether the bone loss caused by CsA administration is dependent on the NO-cyclic guanosine monophosphate (cGMP) pathway. Freshly isolated osteoclast-rich neonatal rat long bone marrow cells were added to 100 IM thick dentin sections that had been seeded with neonatal-rat calvarial osteoblasts. These co-cultures were maintained for 48 hrs in a basal medium with CsA (1, 5, and 10 Ig/ml), both alone and with either L-Arginine (NO substrate; 10-3M), L-NAME (NO synthase enzyme inhibitor; 10-4M), or the combination of the two. The cultures were then fixed in cold 95% ethanol and stained with tartrate resistant acid phosphatase (TRAP) to identify osteoclasts and sites of osteoclastic resorption. Preparations were analyzed using an automated histomorphometry software package. Scanning electron microscopy affirmed that the areas identified by light microscopy as resorption sites contained osteoclastic lacunae. CsA inhibited bone resorption dose-dependently. CsA at 10 Ig/ml produced a 90% inhibition of bone resorption (control = 5.5 ± 2.0%; CsA = 0.64 ± 0.09%). L-Arginine reversed this inhibition by 90% (Arg + CsA = 4.23 ± 1.5%; CsA = 0.64 ± 0.09%). The application of NOS inhibitor L-NAME inhibited bone resorption by 87% (Arg + CsA + L-NAME = 0.55 ± 0.14%; Arg + CsA = 4.23 ± 1.5%). We conclude that NO-cGMP pathway is involved in the CsA induced bone loss.
TL;DR: It is suggested that IL‐4 initiates osteoclast development of UG3 cells, although stroma cells were necessary for development of functional osteoclasts, although D3 had only a "supportive” effect on this differentiation.
Abstract: The formation of multinucleated giant cells (MGCs) from monocytes/macrophages is controlled by various cytokines, the roles of which are not fully understood. Both interleukin (IL)-4 and 1alpha,25(OH)(2) vitamin D(3) (D(3)) are known to induce MGC formation from monocytes/macrophages. D(3) is also known as a stimulator of osteoclast formation in the presence of stroma cells, and IL-4 as an inhibitor. Previously, we showed that IL-4-induced MGCs from monocytes/macrophages expressed tartrate resistant acid phosphatase (TRAP) activity and hydroxyapatite-resorptive activity in the presence of M-CSF without stroma cells. In this study, we examined the effects of D(3) and/or IL-4 on MGC formation and the characteristics of these MGCs using a monoblastic cell line (UG3), to elucidate the involvement of these factors in osteoclast development without stroma cells. D(3)-induced MGCs showed none of the markers of osteoclasts, such as TRAP activity, calcitonin receptor (cal-R) expression, hydroxyapatite-resorptive activity, and bone-resorptive activity. A low concentration of D(3) synergistically stimulated IL-4-induced TRAP-positive MGC formation, whereas a high concentration of D(3) inhibited it. When IL-4 was added on day 7 of the 2-week culture with D(3), TRAP positivity reached maximum. On the other hand, delayed addition of D(3) on day 7 of culture did not increase the TRAP positivity. Although the fusion rate increased during the first week of the 2-week culture in the presence of D(3), it increased further in the second week following the addition of IL-4 on day 7. Furthermore, IL-4-induced, or IL-4- and D(3)-induced MGCs differentiated into functional osteoclasts with bone-resorptive activity following coculture with osteoblastic cells, whereas D(3)-induced MGCs did not acquire bone-resorptive activity even after coculture with osteoblastic cells in the presence of D(3). These findings suggest that IL-4 initiates osteoclast development of UG3 cells, although stroma cells were necessary for development of functional osteoclasts. On the other hand, D(3) had only a "supportive" effect on this differentiation. IL-4 and direct contact with stroma cells may regulate different stages in the multistep process of osteoclastogenesis of UG3 cells.
Journal Article•
01 Jul 2000-Journal of gravitational physiology : a journal of the International Society for Gravitational Physiology
TL;DR: Examining the rabbit primary osteoclasts applied to hypergravity and comparing the mRNA expression of two kinds of osteoclast marker enzymes suggested that hypergravity enhances the expression of TRAP and cathepsin K at the transcription level, but enhances the resorption activity of activated osteoclast.
Abstract: The long-term space flight induces a loss of bone density. However, the mechanism has not been well understood, especially about gravity effect on osteoclast. To elucidate the gravitational effect on osteoclasts, we examined the rabbit primary osteoclasts applied to hypergravity and compared the mRNA expression of two kinds of osteoclast marker enzymes, TRAP (tartrate-resistant acid phosphatase) and cathepsin K at the transcription level. Rabbit osteoclasts isolated according to the modified method of Kakudo et al. were cultured on ivory and exposed to 30 x g for 2 hr or 18 hr by placing the culture tubes in a swinging bucket rotor. Results by RT-PCR suggested that hypergravity enhanced the mRNA expression of both enzymes with different manner; the expression of the TRAP showed a smaller increase, that of the cathepsin K showed a non-monotonous time course with maximum hypergravity effect for 2 hr incubation. Moreover we examined the hypergravity effect on actin ring formation in osteoclasts; however, we got no hypergravity effect on the numbers of activated osteoclasts with actin ring formation. These results might suggest that hypergravity has no effect on the number of osteoclasts with resorption activity, but enhances the resorption activity of activated osteoclasts.
TL;DR: Novel methods to isolate osteoclasts (OC s) and generate osteoclast-like cells (OCL s) from the bone and bone marrow of the equine femur are reported and the validity of using OCL s as a method for studying OC differentiation and activity is confirmed.