Showing papers on "Tartrate-resistant acid phosphatase published in 2002"

Alendronate disturbs vesicular trafficking in osteoclasts.

Abstract: The nitrogen-containing bisphosphonate alendronate inhibits osteoclast-mediated bone resorption through inhibition of the mevalonate pathway. This results in impaired protein prenylation and may affect the function of small GTPases in osteoclasts. Since these proteins are important regulators of vesicle transport in cells, we investigated the possible interference of alendronate with these processes in isolated rat osteoclasts. We show here that alendronate-induced inhibition of bone resorption coincides with accumulation of tartrate-resistant acid phosphatase- and electron dense material-containing tubular vesicles in osteoclasts. Alendronate-induced changes in osteoclasts also included widening of the sealing zone areas and incomplete organization of tight attachments and ruffled borders. Osteoclasts also appeared partially detached from the bone surface, and organic matrix was typically dissolved only at the edges of the resorption pits on alendronate-coated bone slices. In contrast, resorption pits on the control and clodronate-coated bone slices were thoroughly resorbed. Inhibition of bone resorption by alendronate was not, however, related to a decrease in osteoclast number. In conclusion, our findings suggest that alendronate inactivates osteoclasts by mechanisms that impair their intracellular vesicle transport, apoptosis being only a secondary phenomenon to this.

Down-regulation of osteoclast differentiation by daidzein via caspase 3.

Abstract: Phytoestrogens are plant-derived compounds with estrogen-like activity. Phytoestrogen-rich diets may prevent postmenopausal osteoporosis and these molecules maintain bone mass in ovariectomized animals. We compared the effects of the isoflavone daidzein, which has no action on tyrosine kinases, and 17beta-estradiol on the development and activity of osteoclasts in vitro. Nonadherent porcine bone marrow cells were cultured on dentine slices or on culture slides in the presence of 10-8 M of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], with or without 10(-8) M of daidzein, 10(-8) M of 17beta-estradiol for 9-11 days. Multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells that resorbed bone (osteoclasts) developed in the presence of 1,25(OH)2D3. The number of osteoclasts formed in response to 1,25(OH)2D3 was reduced by 58 +/- 8% by daidzein and 52 +/- 5% by estrogen (p < 0.01); these effects were reversed by 10-6 M of ICI 182,780. The area resorbed by mature osteoclasts was reduced by 39 +/- 5% by daidzein and 42 +/- 6% by estradiol (p < 0.01). Both compounds also inhibited the 1,25(OH)2D3-induced differentiation of osteoclast progenitors (mononucleated TRAP+ cells), 53 +/- 8% by daidzein and 50 +/- 7% by estradiol (p < 0.05). Moreover, daidzein and estradiol promoted caspase-8 and caspase-3 cleavage and DNA fragmentation of monocytic bone marrow cells. Caspase-3 cleavage was reversed by 10-8 M of ICI 182,780. Both compounds up-regulated the expression of nuclear estrogen receptors ER-alpha and ER-beta. Thus, daidzein, at the same concentration as 17beta-estradiol, inhibits osteoclast differentiation and activity. This may be caused by, at least in part, greater apoptosis of osteoclast progenitors mediated by ERs.

Cytokines synergistically induce osteoclast differentiation: support by immortalized or normal calvarial cells.

Abstract: Conditionally immortalized murine calvarial (CIMC) cells that support differentiation of precursors into mature osteoclasts were isolated. All six CIMC cell lines supported osteoclast differentiation in response to 1,25-dihydroxyvitamin D(3) or interleukin (IL)-11. CIMC-4 cells also supported osteoclast differentiation in response to tumor necrosis factor (TNF)-alpha, IL-1beta, or IL-6. The resultant multinucleated cells expressed tartrate-resistant acid phosphatase and formed resorption lacunae on mineralized surfaces. CIMC-4 cells, therefore, establish an osteoclast differentiation assay that is responsive to many cytokines and does not rely on isolation of primary stromal support cells. Low concentrations of the cytokines synergistically stimulated differentiation when osteoclast precursors were cocultured with either CIMC-4 cells or primary calvarial cells. Osteoclast differentiation induced by all stimuli other than TNF-alpha was completely blocked by osteoprotegerin, whether the stimulators were examined alone or in combination. Moreover, study of precursors that lack TNF-alpha receptors showed that TNF-alpha induces osteoclast differentiation primarily through direct actions on osteoclast precursors, which is a distinct mechanism from that used by the other bone-resorptive agents examined in this study.

Synovial macrophage-osteoclast differentiation in inflammatory arthritis

Abstract: Background: Pathological bone resorption (marginal erosions and juxta-articular osteoporosis) by osteoclasts commonly occurs in rheumatoid arthritis (RA). Objectives: To define the nature of the mononuclear precursor cells from which osteoclasts are formed in inflamed synovial tissues and to determine the cellular and humoral factors which influence osteoclast differentiation. Method: Macrophage (CD14+), non-macrophage (CD14-), and unsorted (CD14+/CD14-) synovial cell populations from RA and inflammatory/non-inflammatory osteoarthritis (OA) synovium were cultured in the presence of receptor activator for nuclear factor κB ligand (RANKL) and monocyte-colony stimulating factor (M-CSF; in the presence/absence of prostaglandin E2 (PGE2), interleukin 1s (IL1s), tumour necrosis factor α (TNFα), and IL6). Osteoclast differentiation was assessed by expression of tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR), and lacunar resorption. Results: TRAP+ and VNR+ multinucleated cells capable of lacunar resorption were only formed in cultures of CD14+-containing synovial cell populations (that is, CD14+ and CD14+/CD14- cells). No difference in the extent of osteoclast formation was noted in cultures of CD14+ cells isolated from RA, inflammatory OA, and non-inflammatory OA synovium. However, more TRAP+/VNR+ cells and more lacunar resorption was noted in CD14+/CD14- cells from RA and inflammatory OA synovial tissues. The addition of PGE2, IL1s, TNFα, and IL6 did not increase RANKL/M-CSF-induced osteoclast formation and lacunar resorption of both CD14+/CD14- and CD14+ synovial cell populations. Conclusions: Osteoclast precursors in synovial tissues are CD14+ monocyte/macrophages. The increase in osteoclast formation in cultures of CD14+/CD14- compared with CD14+ synovial cells in RA and inflammatory OA points to a role for CD14- cells in promoting osteoclast differentiation and bone resorption in inflamed synovial tissues by a mechanism which does not involve a direct effect of proinflammatory cytokines/prostaglandins on RANKL-induced macrophage-osteoclast differentiation.

Effect of corticosteroids on human osteoclast formation and activity.

Abstract: Chronic corticosteroid treatment is known to induce bone loss and osteoporosis. Osteoclasts are specialised bone-resorbing cells that are formed from mononuclear phagocyte precursors that circulate in the monocyte fraction. In this study we have examined the effect of the synthetic glucocorticoid, dexamethasone, on human osteoclast formation and bone-resorbing activity. Human monocytes were cultured for up to 21 days on glass coverslips and dentine slices, with soluble receptor activator for nuclear factor kappaB ligand (RANKL; 30 ng/ml) and human macrophage-colony stimulating factor (M-CSF; 25 ng/ml) in the presence and absence of dexamethasone (10(-8) M). The addition of dexamethasone over a period of 7 and 14 days of culture of monocytes (during which cell proliferation and differentiation predominantly occurred) resulted in a marked increase in the formation of tartrate-resistant acid phosphatase-positive multinucleated cells and an increase in lacunar resorption. The addition of dexamethasone to monocyte cultures after 14 days (when resorptive activity of osteoclasts had commenced) reduced the extent of lacunar resorption compared with cultures to which no dexamethasone had been added. The addition of dexamethasone to osteoclasts isolated from giant cell tumours of bone significantly inhibited resorption pit formation. Our findings indicate that dexamethasone has a direct effect on osteoclast formation and activity, stimulating the proliferation and differentiation of human osteoclast precursors and inhibiting the bone-resorbing activity of mature osteoclasts.

The immunosuppressant rapamycin, alone or with transforming growth factor-beta, enhances osteoclast differentiation of RAW264.7 monocyte-macrophage cells in the presence of RANK-ligand.

Abstract: Immunosuppressant therapy is known to cause bone loss. Since this may partly result from direct effects on osteoclast development, we investigated whether cyclosporin A (CsA), rapamycin, or FK506 affect osteoclastic differentiation of RAW264.7 monocytic cells induced by RANK-ligand (RANKL). Furthermore, since the rapamycin receptor protein binds transforming growth factor beta (TGF-beta) receptors, and TGF-beta enhances osteoclastogenesis induced by RANKL, we also examined potential synergistic effects of rapamycin and TGF-beta1. Rapamycin inhibited cell proliferation and stimulated tartrate-resistant acid phosphatase (TRAP) activity of RAW cells in a dose-dependent manner. At the optimal concentration of 10 ng/ml, it increased the number of TRAP+ multinucleated cells (MNC) more than 20-fold and enhanced the expression of TRAP and calcitonin receptor (CTR) mRNAs 2.1- and 10-fold, respectively. CsA, at 125-2000 ng/ml, similarly inhibited proliferation, but at high doses (1000-2000 ng/ml) it decreased TRAP activity, TRAP+MNC formation, and the expression of TRAP and CTR mRNAs. FK506 had no effect on cell proliferation or TRAP activity at concentrations up to 2000 ng/ml; however, like CsA, 1000 ng/ml FK506 inhibited TRAP+MNC formation and the expression of TRAP and CTR mRNAs. The combination of rapamycin (10 ng/ml) and TGF-beta1 (1 ng/ml) increased TRAP+MNC 3.1- and 6.9-fold as compared with rapamycin or TGF-beta1 alone, respectively, and enhanced CTR mRNA expression induced by TGF-beta1 by 1.9-fold. Rapamycin also increased osteoclastic resorption activity by 6.5-fold compared with control, and this was enhanced further by the addition of TGF-beta by 3-fold, compared with rapamycin alone. These data thus indicate that rapamycin, alone or in synergy with TGF-beta, directly enhances osteoclastogenesis and may affect bone metabolism in vivo after long-term use.

Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells

Abstract: Background: It has previously been shown that many osteoclast precursors are included in the granulation tissue within the pseudocapsule obtained at revision arthroplasty from hips with osteolysis. In vitro culture of only cells isolated from the granulation tissue has been previously shown to generate many mature osteoclasts. Objective: To investigate the presence or otherwise of supporting cells, similar to stromal cells, which differentiate osteoclasts within the granulation tissue. Methods: Cells isolated from the granulation tissue were cultured alone, and after four weeks fibroblast-like cells (granulation fibroblasts) remained. Rat non-adherent bone marrow cells (NA-BMCs) were co-cultured with the granulation fibroblasts with or without 1α,25(OH)2D3 (10-8 M) or heat treated ROS 17/2.8 cell conditioned medium (ht ROSCM), or both. Multinucleated cells (MNCs), which formed, were assessed by biochemical and functional characterisation of osteoclasts. Receptor activator of NFκB ligand (RANKL) was investigated by immunohistochemistry. Results: Co-culture of NA-BMCs and granulation fibroblasts caused the formation of tartrate resistant acid phosphatase (TRAP) positive MNCs, which had the calcitonin receptor (CTR), the Kat-1 antigen, which is specific to the surface of rat osteoclasts, and the ability to form pits in the presence of both 1α,25(OH)2D3 and ht ROSCM or in the presence of just ht ROSCM. RANKL was detected in fibroblast-like cells in the granulation tissue. Conclusion: These data suggest that granulation fibroblasts support osteoclast differentiation, as do osteoblasts/stromal cells, and may play a part in aseptic loosening.

Rescue of the osteopetrotic defect in op/op mice by osteoblast-specific targeting of soluble colony-stimulating factor-1.

Abstract: Soluble colony-stimulating factor-1 (sCSF-1) and membrane bound CSF-1 are synthesized by osteoblasts and stromal cells. However, the precise role of each form in osteoclastogenesis is unclear. In the op/op mouse, absence of osteoblast-derived CSF-1 leads to decreased osteoclasts and osteopetrosis. To determine whether sCSF-1 gene replacement can cure the osteopetrotic defect, we took advantage of the osteoblast specificity of the osteocalcin promoter to selectively express sCSF-1 in the bone of op/op mice. Transgenic mice harboring the human sCSF-1 cDNA under the control of the osteocalcin promoter were generated and cross-bred with heterozygous op/wt mice to establish op/op mutants expressing the transgene (op/opT). The op/op genotype and transgene expression were confirmed by PCR and Southern blot analysis, respectively. High levels of human sCSF-1 protein were selectively expressed in bone. At 2(1/2) wk, op/opT mice showed normal growth and tooth eruption. Femurs removed at 5 and 14 wk were analyzed by peripheral quantitative computed tomography and histomorphometry. The abnormal bone mineral density, cancellous bone volume, and growth plate width observed in op/op mice was completely reversed in op/opT mice by 5 wk, and this effect persisted at 14 wk, with measurements comparable with wt/wt mice at each time point. Correction of the skeletal abnormalities in the 5-wk-old op/opT mice correlated with a marked increase in the total osteoclast number, and their number per millimeter of bone surface compared with that of op/op mutants. Osteoclast number was maintained at 14 wk in op/opT mice and morphologically resembled wt/wt osteoclasts. These results indicate that sCSF-1 is sufficient to drive normal osteoclast development and that the osteocalcin promoter provides an efficient tool for delivery of exogenous genes to the bone. Moreover, targeting sCSF-1 to osteoblasts in the bone microenvironment may be a potentially useful therapeutic modality for treating bone disorders.

Gender- and age-related differences in osteoclast formation from circulating precursors.

Abstract: A number of bone diseases characterised by excessive osteolysis (e.g. osteoporosis and Paget’s disease) exhibit a marked gender difference in prevalence and are more common in the elderly population. Bone resorption is carried out by osteoclasts, which are formed by fusion of circulating mononuclear precursor cells of haematopoietic origin. In this study, we have determined whether there are gender- and age-related differences in osteoclast formation from circulating precursors. Peripheral blood mononuclear cells (PBMCs) were co-cultured with UMR106 osteoblast-like cells in the presence of macrophage-colony stimulating factor (M-CSF) and 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or cultured alone in the presence of sRANKL (soluble receptor activator of nuclear factor B ligand) and M-CSF. As assessed by the formation of tartrate resistant acid phosphatase (TRAP)positive (TRAP + ) and vitronectin receptor-positive (VNR + ) multinucleated cells (MNCs), there was no

Estrogen Inhibition of PTH-stimulated Osteoclast Formation and Attachment in Vitro: Involvement of Both PKA and PKC

Abstract: Estrogens modulate the catabolic effects of PTH on bone in vivo and in vitro. PTH-stimulated cAMP accumulation in osteoblasts is thought to be linked to increased osteoclastic activity, but the precise mechanism is still unknown. In cocultures of clonal marrow stromal cells (MS1) and normal mouse spleen cells, both 1,25-dihydroxyvitamin D3 and rat PTH (rPTH)-(1-34) can induce the formation of tartrate-resistant acid phosphatase- and calcitonin receptor-positive multinucleated osteoclast-like cells, which can attach to dentine slices and produce resorption pits. In this system, osteoclastogenesis stimulated by PTH, but not by 1,25-dihydroxyvitamin D3, was suppressed by 17beta-E2 (10(-10)-10(-8) M), whereas 17alpha-E2 (10(-8) M) had no effect. Exposure to 10(-8) M 17beta-E2, but not 17alpha-E2, also significantly decreased the PTH-induced attachment of osteoclast-like cells to dentine slices. 17beta-E2 inhibited osteoclast-like cell formation induced by 8-bromo-cAMP (10(-4) M), 12-O-tetradecanoylphorbol 13-acetate (10(-8) M), or rat PTH-(1-34) (10(-7) M) in combination with either rp-adenosine-3',5'-cyclic monophosphorothioate (10(-4) M) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (10(-5) M). 17beta-E2 suppressed the partial stimulation of tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cell formation induced by [Arg(2)]human (h) PTH-(1-34) (10(-7) M) or hPTH-(3-34) (10(-7) M), but not that caused by 10(-7) M hPTH-(53-84). We conclude that estrogens suppress PTH-stimulated osteoclast-like cell formation by blocking both the cAMP-dependent PKA pathway and the PLC-coupled calcium/PKC pathway. In addition to inhibiting formation of osteoclasts and promoting their apoptosis, estrogen may regulate bone resorption by blocking attachment of osteoclasts to bone.

Measurement of tartrate-resistant acid phosphatase and the brain isoenzyme of creatine kinase accurately diagnoses type II autosomal dominant osteopetrosis but does not identify gene carriers.

Abstract: Autosomal dominant osteopetrosis type II (ADO2) is typically diagnosed from radiographs, which demonstrate the pathognomonic findings of osteosclerosis and endobone formation. Individuals with ADO2 also have elevated serum levels of tartrate-resistant acid phosphatase (TRAP) and the BB isoenzyme of creatine kinase (CK-BB). In the current study, we tested the utility of these enzymes in making or refuting a diagnosis of ADO2. Furthermore, because ADO2 has incomplete penetrance, we examined whether TRAP and CK-BB were helpful in identifying gene carriers. We studied eight families, measured serum levels of TRAP and CK-BB in 52 affected individuals and 12 obligate gene carriers, and compared their values with age-matched controls. Our results demonstrate that affected patients have significantly elevated levels of both TRAP and CK-BB. In contrast, gene carriers have values that are not different from controls. Furthermore, in our study population, TRAP and CK-BB have a high diagnostic sensitivity and specificity, particularly in children. From this large study of ADO2 patients and carriers, we conclude that: 1) TRAP and CK-BB are significantly elevated in patients with ADO2, 2) obligate carriers cannot be adequately identified by measurement of these analytes, and 3) TRAP and CK-BB are highly sensitive and specific diagnostic tests that can efficiently and effectively screen high-risk individuals who have not had previous radiographic assessment.

Effects of Cyclic Pressure on Bone Marrow Cell Cultures

Abstract: The present in-vitro study used bone marrow cell cultures and investigated the effects of cyclic pressure on osteoclastic bone resorption. Compared to control (cells maintained under static conditions), the number of tartrate resistant acid phosphatase (TRAP)-positive, osteoclastic cells was significantly (p<0.05) lower when, immediately upon harvesting, bone marrow cells were exposed to cyclic pressure (10-40 kPa at 1.0 Hz). In contrast, once precursors in bone marrow cells differentiated into osteoclastic cells under static culture conditions for 7 days, subsequent exposure to the cyclic pressure of interest to the present study did not affect the number of osteoclastic cells. Most important, exposure of bone marrow cells to cyclic pressure for 1 h daily for 7 consecutive days resulted in significantly (p<0.05) lower osteoclastic bone resorption and in lowered mRNA expression for interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-α), cytokines that are known activators of osteoclast function. In addition to unique contributions to osteoclast physiology, the present study provided new evidence of a correlation between mechanical loading and bone homeostasis as well as insight into the molecular mechanisms of bone adaptation to mechanical loading, namely cytokine-mediated control of osteoclast functions.

Serum activities of tartrate-resistant acid phosphatase and bone specific alkaline phosphatase as indices of bone metabolism in the cow.

Abstract: The correlation between the serum hydroxyproline concentration and serum activity levels of TRAP and BALP was examined in 41 cows. The correlated coefficient (r) was 0.6391 for TRAP and 0.3147 for BALP, respectively. Judging from the significant correlation to the serum hydroxyproline concentration, serum TRAP activity is an index for bone metabolism in cows. Serum TRAP activity was therefore measured in 205 healthy cows (2-9 years old) in order to observe the changes in bone resorption with aging and milk production. TRAP levels differed slightly between group A (≤4 yrs) and B (5 yrs≤) at the same stage of lactation. The activity levels rose slightly at the height of lactation stage and during the dry stage.

Transcription From the Tartrate-resistant Acid Phosphatase Promoter Is Negatively Regulated by the Myc Oncoprotein

Abstract: TRAP, a characteristic marker of osteoclast differentiation, is an enzyme that plays an active role in the process of bone resorption. Despite the importance of TRAP in osteoclast biology, the components involved in the transcriptional regulation of this gene are largely unknown. This study investigated the regulation of TRAP transcription by the Myc oncoprotein in three different cell types. A series of nested TRAP promoter deletion constructs were cotransfected into P388D1 murine macrophages and C3H10T1/2 murine embryonic fibroblasts along with a backbone plasmid control or expression plasmids containing v-Myc, c-Myc, or an inactive v-Myc protein construct (delta84/NLS). Both v-Myc and c-Myc negatively regulated transcription from the TRAP promoter in P388D1 and C3H10T1/2 cells, 90% and 50%, respective to cell type and amount of endogenous Myc protein, and delta84/NLS had no effect. The functional Myc-responsive element(s) within the TRAP promoter was localized to a region between -436 and +1 bp, which contains two putative Myc-inhibitory binding sites coincident with an initiator element (Inr) at -116 bp and -18 bp. Conversely, in the HD-11EM chicken v-Myc transformed preosteoclast cell line, the full-length TRAP promoter transcription was increased when endogenous v-Myc levels were decreased in response to pretreatment of these cells with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. This report provides the first evidence of the specific regulation of TRAP at the transcriptional level by Myc, a transcription factor that is normally expressed at relatively high levels in preosteoclasts and other myelomonocytic cells and suggests that Myc plays an active role in suppressing the transcription of a mature osteoclast selective gene.

Tartrate-resistant acid phosphatase: three-dimensional structure and structure-based functional studies : studies on the enzyme using recombinant protein produced by baculovirus expression vector system in insect cells

Abstract: Osteoporosis is a disease characterized by abnormalities in the amount and architectural arrangement of bone tissue, which leads to impaired skeletal strength and increased susceptibility to fractures. Type 5 tartrate-resistant acid phosphatase (TRACP, AcP5) has been suggested to participate directly

A new cytometric method for the immunophenotypic characterization of bone-derived human osteoclasts.

Abstract: Background Osteoclast cell function relates to bone resorption. Isolation and characterization of these cells from in vivo sources remain difficult. The aim of this study was to show the feasibility of using flow cytometry to identify and characterize human mature osteoclasts obtained from bone tissues. Methods Bone femoral heads obtained as discarded surgical material were used. To check the nature of 121F+ (a monoclonal antibody specific for human osteoclasts) cells by flow cytometry, we used laser scanning cytometry to analyze simultaneously the immunophenotype and DNA cell content of osteoclast-like cell-enriched bone samples. Results Results were compared with conventional morphologic and cytochemical studies. The percentage of cells that showed both cytochemical (tartrate-resistant acid phosphatase [TRAP]+) and immunophenotypic (121F+) osteoclast-associated characteristics was very similar (12.5 ± 6.2 versus 14.7 ± 11.7; P = 0.46). Laser scanning cytometry showed that 121F+ cells were bigger (P = 0.04) and they had a higher DNA cell content (P = 0.04) and more nuclei per cell (P = 0.04) than the 121F- cells present in the same sample. Discussion This study relied on the combined use of the 121F+ antibody and different cytometry-based techniques to characterize the osteoclast populations from human bone. Cytometry (Clin. Cytometry) 49:261–266, 2002. © 2002 Wiley-Liss, Inc.

High extracellular calcium affects osteoclastogenesis in mouse bone marrow cell culture.

Abstract: The effects of high extracellular calcium (high Ca) in the local microenvironment on osteoclasts, osteoclast progenitors and stromal cells are not fully understood. We examined high Ca effect on osteoclastogenesis in mouse bone marrow cell culture. Mouse bone marrow cells were cultured for up to 6 days in the medium supplemented with 1, 25(OH)2 vitamin D3 (D3). High Ca treatment at the early stage of culture (the initial 24 hours) reduced the number of tartrate resistant acid phosphatase-positive multinuclear cells (TRAP(+)MNCs). This treatment slightly up-regulated the mRNA expressions of receptor activator of NF-(B ligand (RANKL), RANK and osteoprotegerin (OPG). This inhibitory effect on the formation of TRAP(+)MNCs was recovered by RANKL. In contrast, high Ca treatment at the later stage of osteoclastogenesis (the last 2 days of culture) stimulated the formation of TRAP(+)MNCs, increased RANKL and RANK mRNA expressions and decreased OPG mRNA. High Ca at neither the early nor the later stage of culture affected the total number of adherent cells and the mRNA expression of alkaline phosphatase and osteopontin. In conclusion, high Ca affects osteoclastogenesis in a manner depending on the stage of osteoclastogenesis, which is partly mediated via the RANKL-RANK-OPG regulatory system.

[Gaucher's disease ].

Abstract: Gaucher disease is an uncommon autosomic recessive disorder. The disease is caused by a deficiency in the activity of the lysosomal enzyme glucocerebrosidase which is responsible for the degradation of glucosylceramide, resulting from the breakdown of red and white cell-membranes. In the absence of enzyme glucosylceramide accumulates in the lysosomes of macrophages. This accumulation leads to hepatomegaly, splenomegaly with subsequent haematologic abnormalities (leucopenia, anemia, thrombopenia) and bone manifestations. Three types of Gaucher disease are described: type 1 is the most common, type 2 and 3 are associated with neurologic symptoms. Macrophages are the likely cellular source of biochemical abnormalities: elevated blood level of ferritin, angiotensin converting enzyme, immunoglobulins and haemostasis abnormalities. Lysosomal perturbations lead to increased blood level of tartrate resistant acid phosphatase and chitotriosidase. Enzyme replacement therapy is available in France since 1991. In 2002, 136 patients are treated. The efficacy is overt on the asthenia, organomegaly and haematological manifestations. Bone pains disappear or decrease in intensity, however bone complications may be irreversible justifying treatment initiations before the appearance of lesions that may lead to serious functional impairment.

Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).

Abstract: We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.

Characterization of four monoclonal antibodies to recombinant human tartrate-resistant acid phosphatase.

Abstract: In this study we produced a recombinant human Tartrate-resistant acid phosphatase (TRAP) enzyme from baculovirus-infected insect cells, generated four monoclonal antibodies (MAbs) 15A4, 13B9, 1C6 and 3G7, to the enzyme, and characterized these antibodies. In the human serum and lung specimen, all four antibodies appeared to have a high specificity for native TRAP enzyme in western blot analysis, immunohistochemical analysis and enzyme immunoassay. These antibodies may react with respective conformational determinants, therefore, they may be useful for detection of active TRAP. Only one of the antibodies, 15A4 also reacted with a denatured epitope, therefore, it is suitable for western blot analysis, enzyme immunoassay and for immunohistochemistry in the rat. Taken together, having characterized properties of four monoclonal antibodies against recombinant human TRAP enzyme may be useful for development of TRAP specific immunoassays in pathology and hematology of the bone. They will certainly be of use for the study of biosynthesis, regulation and function of the TRAP enzyme.

Can a determination of tartrate-resistant acid phosphatase predict postmenopausal loss of bone mass?

Abstract: Background A study was carried out over a 24-month interval to determine if an initial measurement of serum tartrate-resistant acid phosphatase would be predictive of bone mass loss quantified by dual-energy X-ray absorptiometry, as total bone mineral content and total bone mineral content corrected for weight. Design Sixty-two women were studied (at onset: mean age 59·7 ± 8·9 years, 10·8 ± 8·8 years since menopause; at conclusion: mean age 61·9 ± 8·8 and 13·0 ± 8·7 since menopause). Results A paired Wilcoxon test showed a small, but significant, increase in weight (P < 0·05) and decrease in height (P < 0·05). Total bone mineral content and total bone mineral content corrected for weight decreased (P < 0·005 and 0·0001, respectively). Serum tartrate-resistant acid phosphatase increased (P < 0·005). Single-regression analysis showed that the per cent bone mass loss observed between the first and second body bone mineral content measurements correlated negatively with the first serum tartrate-resistant acid phosphatase determination (r = −0·62, P < 0·0001). Changes in tartrate-resistant acid phosphatase correlated negatively with changes in total bone mineral content (r = −0·79, P < 0·0001). In a multiple regression analysis of per cent change in bone mass against initially important variables such as age, years since menopause, weight, and tartrate-resistant acid phosphatase, only tartrate-resistant acid phosphatase was significant (P < 0·0001). The sensitivity and specifity of tartrate-resistant acid phosphatase for evaluating bone loss were 86% and 78%, respectively, and the area under the curve was of 0·83 (95% CI 0·71–0·95). Conclusion These results show that a simple measurement of serum tartrate-resistant acid phosphatase can help to predict the potential rate of bone mass loss in women.

Bone biochemical markers and bone mineral density among osteoporosis patients who were seen in a pain clinic

Abstract: Osteoporosis is a common and debilitating disease affecting all bones. Examination of the serum concentration of bone biochemical markers is useful to distinguish the type of osteoporosis and to determine the optimal therapy for each patient. In the present study, four serum markers of bone formation (bone Gla protein; BGP, intact bone Gla protein; iBGP, bone-specific alkaline phosphatase; BAP, carboxy-terminal peptide of type I procollagen; PICP) and a plasma marker of bone resorption (tartrate resistant acid phosphatase; TRAP) were measured in 165 female patients who visited our pain clinic complaining of back and/or leg pain, and who were suspected of having osteoporosis (Japanese osteoporosis criteria II or III) on standard skeletal radiograph. No significant correlation was observed between bone biochemical markers and mineral density, or between biochemical markers and age. The amount of BGP and iBGP determined in patients older than 70 was significantly higher than in patients younger than...