Showing papers on "Tartrate-resistant acid phosphatase published in 1999"

A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions.

Abstract: Low phosphorous availability, a common condition of many soils, is known to stimulate phosphatase activity in plants; however, the molecular details of this response remain mostly unknown. We purified and sequenced the N-terminal region of a phosphate starvation induced acid phosphatase (AtACP5) from Arabidopsis thaliana, and cloned its cDNA and the corresponding genomic DNA. The nucleotide sequence of the cDNA predicted that AtACP5 is synthesised as a 338 amino acid-long precursor with a signal peptide. AtACP5 was found to be related to known purple acid phosphatases, especially to mammal type 5 acid phosphatases. Other similarities with purple acid phosphatases, which contain a dinuclear metal centre, include the conservation of all residues involved in metal ligand binding and resistance to tartrate inhibition. In addition, AtACP5, like other type 5 acid phosphatases, displayed peroxidation activity. Northern hybridisation experiments, as well as in situ glucuronidase (GUS) activity assays on transgenic plants harbouring AtACP5:GUS translational fusions, showed that AtACP5 is not only responsive to phosphate starvation but also to ABA and salt stress. It is also expressed in senescent leaves and during oxidative stress induced by H2O2, but not by paraquat or salicylic acid. Given its bifunctionality, as it displays both phosphatase and peroxidation activity, we propose that AtACP5 could be involved in phosphate mobilisation and in the metabolism of reactive oxygen species in stressed or senescent parts of the plant.

Stimulation of Osteoclast Formation by 1,25-Dihydroxyvitamin D Requires Its Binding to Vitamin D Receptor (VDR) in Osteoblastic Cells: Studies Using VDR Knockout Mice

Abstract: Previous studies have shown that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays important roles in the formation of osteoclasts through its actions on osteoblastic cells. We have generated mice lacking vitamin D receptor (VDR) by gene targeting (VDR-/-). These mice had tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and exhibited similar levels of parameters for bone resorption to those in wild type mice. The present studies were undertaken to clarify whether effects of 1,25(OH)2D on osteoclast formation require VDR in osteoblasts, and to examine mechanisms of the formation of osteoclasts without VDR-mediated actions using VDR-/- mice. When wild-type calvarial osteoblasts and spleen cells were co-cultured with 1,25(OH)2D, TRAP-positive osteoclasts were formed regardless of the genotypes of spleen cells. In contrast, when osteoblasts from VDR-/- mice were co-cultured, no osteoclasts could be formed even with wild-type spleen cells. Parathyroid hormone and interleukin-1alpha stimulated osteoclast formation by co-cultures from VDR-/- mice, and the generated osteoclasts showed resorbing activity. These results demonstrate that VDR-mediated actions of 1,25(OH)2D in osteoblasts are essential for osteoclast formation by 1,25(OH)2D, and that functionally intact osteoclasts can be formed without 1,25(OH)2D actions under stimulations by other agents. It is suggested that osteoclastic bone resorption can be maintained without 1,25(OH)2D actions by other stimulatory agents.

Osteoclast markers accumulate on cells developing from human peripheral blood mononuclear precursors.

Abstract: Recent studies show that human osteoclasts develop in vitro from hematopoietic cells; however, special cultures conditions and/or cytokine mobilized peripheral blood are apparently required Here, we report that cells expressing osteoclast markers differentiate from precursors present in nonmobilized peripheral blood mononuclear cells (PBMC), without the addition of stromal cells, growth factors, cytokines or steroids; and characterize their phenotype Three days after establishing high-density PBMC cultures (15 x 10(6) cells/cm2), in serum-containing medium, small adherent colonies of tartrate resistant acid phosphatase positive (TRAP+) cells emerge, amidst massive monocyte cell death These adherent cells have an eccentrically placed, round nucleus, and express low levels of TRAP and sodium fluoride-resistant- alpha-naphthyl-acetate-esterase (NaF-R-NSE) Over the next week, this cell population accumulates phenotypic markers of osteoclasts (vitronectin receptor [VR], calcitonin receptor, TRAP, cathepsin K protein, and mRNA) with increased nuclearity, covering the entire surface by 15 days When cultured on bone, VR+, TRAP+ cells of low multinuclearity appear and cover up to 50% of the surface Resorption lacunae can be observed by day 22 Although these pits are not nearly as numerous as the cells of preosteoclast phenotype, they do represent the activity of a subset of osteoclast-like cells that has achieved osteoclastic maturity under these culture conditions Transcripts for osteoprotegerin ligand (OPGL), an osteoclast differentiation factor (also known as RANKL and TRANCE) are expressed, likely by adherent cells Thus, an adherent population of cells, with preosteoclast/osteoclast phenotypic properties, arises selectively under simple culture conditions from normal PBMC Further characterization of these cells should identify factors involved in the growth, terminal differentiation and activation of osteoclasts

Thiazolidinediones inhibit osteoclast-like cell formation and bone resorption in vitro.

Abstract: Osteoblasts and adipocytes are derived from common bone marrow stromal cells that play crucial roles in the generation of osteoclasts. Activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) induces adipogenic differentiation of stromal cells; however, whether this would affect osteoblast/osteoclast differentiation is unknown. Thus, we examined the effects of the thiazolidinedione (TZD) class of antidiabetic agents that activate PPARgamma on osteoblast/osteoclast differentiation using mouse whole bone marrow cell culture. As reported, all TZDs we tested (troglitazone, pioglitazone, and BRL 49653) markedly increased the number of Oil Red O-positive adipocytes and the expression of adipsin and PPARgamma 2. 1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] did not affect adipogenic differentiation induced by TZDs. TZDs did not affect alkaline phosphatase activity, an early marker of osteoblastic differentiation, despite their marked adipogenic effects. TZDs decreased the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cells induced by 1,25-(OH)2D3 or PTH. Troglitazone dose dependently inhibited basal and 1,25-(OH)2D3- and PTH-induced bone resorption as assessed by pit formation assay. Interleukin-11 blocked the induction by troglitazone of adipogenesis, but had no effect on the inhibition of osteoclast-like cell formation. These results indicate that TZDs are potent inhibitors of bone resorption in vitro. Inhibitory effects of TZDs on osteoclastic bone resorption was not osteotropic factor specific and did not appear to be related to their adipogenic effects. Thus, TZDs may suppress bone resorption in diabetic patients and prevent bone loss.

Expression profiles of mRNAs for osteoblast and osteoclast proteins as indicators of bone loss in mouse immobilization osteopenia model.

Abstract: An experimental mouse model for disuse osteopenia was developed using unilateral cast immobilization. Analysis of the distal femurs and proximal tibias by quantitative histomorphometry revealed significant osteopenia within 10-21 days of immobilization. At 3 weeks, bone loss was also demonstrated with peripheral quantitative computed tomography as diminished bone mineral content and as concomitant reduction in the cross-sectional moment of inertia. These structural and geometrical alterations resulted in decreased strength of the distal femurs tested by cantilever bending. Analysis of the underlying cellular and molecular mechanisms of bone loss revealed a rapid increase in bone resorption within 3 days of immobilization. The mRNA levels for cathepsin K, matrix metalloproteinase-9, and tartrate resistant acid phosphatase were all significantly increased during the 21-day immobilization period, but with different expression profiles. These increases were paralleled by an increased number of osteoclasts as measured by histomorphometry. By day 6 of immobilization, the balance of bone turnover was further shifted toward net bone loss as the mRNA levels for major bone components (type I collagen and osteocalcin) were decreased. In histomorphometric analysis this was observed as reduced rates of mineral apposition and bone formation after 10 days of immobilization. The results of this study demonstrate that immobilization has a dual negative effect on bone turnover involving both depressed bone formation and enhanced bone resorption.

Clinical Significance of Immunoassays for Type-5 Tartrate-resistant Acid Phosphatase

Abstract: Background: Tartrate-resistant acid phosphatase (TRAP; EC [3.1.3.2][1]) is a product of osteoclasts and a biochemical marker of bone resorption rate. However, erythrocytes and platelets contribute to total TRAP activity in serum, reducing the specificity of direct biochemical assays in serum. Osteoclast TRAP is also known as type-5 TRAP and is antigenically unique. Immunoassays are sought to improve the specificity and sensitivity of TRAP as a bone marker. Methods: We developed two colorimetric microplate assays for type-5 TRAP: an enzyme capture immunoassay to measure antibody-bound enzymatic activity, and a two-site immunoassay to measure bound enzyme protein. Both use the same monoclonal antibody (14G6) to capture type-5 TRAP, which permits determination of specific activity of serum TRAP in health and disease. Results: Both TRAP assays were linear from one-tenth to fivefold the mean value in 18 healthy subjects. In these subjects, the mean (SD) TRAP activity was 3.2 (0.54) U/L for the enzyme capture assay and 37 (13) μg/L for the two-site assay. Mean TRAP activity was not significantly increased in 64 patients with endstage renal disease requiring hemodialysis (HD) or 99 unselected patients with rheumatic diseases. By contrast, TRAP protein was increased in both the HD and rheumatic disease groups. The specific activity of TRAP in the 17 of 64 HD sera that had increased TRAP activity (0.088 U/μg) was similar to that in healthy subjects (0.091 U/μg). By contrast, the specific activity of TRAP in the 31 of 99 rheumatic sera with increased TRAP protein (0.035 U/μg) was significantly decreased. Conclusions: Wide sample distributions for TRAP activity in HD patients and TRAP protein in rheumatic disease patients suggest the presence of subpopulations of HD patients with increased TRAP activity and of rheumatic patients with increased TRAP protein. Each assay for TRAP activity and protein may have its own biological significance and clinical applications in specific groups of patients. [1]: pending:yes

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

Abstract: The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2 mM sodium fluoride, whereas for osteoclasts 50–100 mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.

Autocrine Regulation of Osteoclast Formation and Bone Resorption by IL-1α and TNFα

Abstract: Bone resorption is regulated by the cytokines within marrow cells that mediate osteoclast formation and activation. IL-1 and TNF induce bone resorption by stimulating the production of osteoclast-like multinucleated cells and by increasing the bone-resorbing activity of formed osteoclasts. This study was designed to detect IL-1 and TNF in osteoclasts in vitro and to determine whether these cytokines up-regulate osteoclast differentiation and bone resorption. The production of IL-1 alpha, -beta, and TNF alpha, beta in osteoclasts was examined immunohistochemically and by in situ hybridization. In the co-culture of C57BL/6N mouse bone marrow and MC3T3-G2/PA6 cells, a colony of osteoclasts formed, and IL-1 alpha and TNF alpha were detected. However, IL-1 beta and TNF beta were not detected. To investigate the role of IL-1 alpha and TNF alpha from osteoclasts, we enumerated TRAP-positive cells and measured the resorption pit areas in the presence of antibodies against IL-1 alpha and TNF alpha. The addition of antibodies against IL-1 alpha and TNF alpha to the co-culture system decreased the number of TRAP-positive colonies at seven days after incubation (anti-IL-1 alpha, 25.0 +/- 2.3%; anti-TNF alpha, 41.7 +/- 3.7%; anti-IL-1 alpha + anti-TNF alpha, 40.5 +/- 1.3%; and control, 100%), and the ratio of mononuclear to multinuclear cells had changed (anti-IL-1 alpha, 90:10; anti-TNF alpha, 75:25; anti-IL-1 alpha+ anti-TNF alpha, 88:12; and control, 60:40). The total pit areas per dentin slice also decreased with the addition of antibodies (anti-IL-1 alpha, 28,828; anti-TNF alpha, 49,249; anti-IL-1 alpha + anti-TNF alpha, 30,685; and control, 303,139 mm2). These results suggest that local production of IL-1 alpha and TNF alpha by osteoclasts is an important mechanism for regulating the osteoclast differentiation and bone resorptive process.

Two-Site Immunoassays for Osteoclastic Tartrate-Resistant Acid Phosphatase Based on Characterization of Six Monoclonal Antibodies

Abstract: Tartrate-resistant acid phosphatase (TRAP), an enzyme expressed in bone-resorbing osteoclasts, is secreted into the circulation during bone resorption. We used six monoclonal antibodies (MAbs) to optimize direct two-site fluoroimmunoassays for determining serum TRAP concentrations. Four of the MABs, 1F1, 2H1, 4E6, and 5C1, were raised against recombinant human TRAP, and the other two, O1A and J1B, against human bone TRAP. 2H1, J1B, and O1A appeared to be highly specific for TRAP. 1F1 and 4E6 were poor in recognizing bone TRAP and were not useful in the assay. 5C1, while having a good affinity for the bone enzyme, was not specific. Serum TRAP is relatively stable, because 7 days of storage of serum samples at 4 degreesC and -20 degreesC or five thawing-freezing cycles, did not change the TRAP concentration detected using the two-site assays. All studied assays detected an increase in serum TRAP concentrations of postmenopausal women compared with premenopausal women, the difference being highest with MAB pairs 2H1-5C1 and O1A-J1B. These results suggest that serum TRAP may be a useful bone resorption marker, and the MAB pairs 2H1-5C1 and O1A-J1B may be useful in determining the bone resorption rate.

T cell numbers relate to bone involvement in Gaucher disease.

Abstract: The major elements of bone pathology in Gaucher disease are a failure of osteoclast and osteoblast function, resulting in osteopenia and also osteonecrosis. T lymphocytes have recently been found to be involved in the regulation of osteoblast/osteoclast activity in vitro. In the present report the peripheral blood T major lymphocyte subsets were investigated in a group of genotyped type 1 Gaucher disease patients. A total of 31 patients were studied: 21 non-splenectomized (5 N370S homozygotes) and 10 splenectomized (of whom 1 was a N370S homozygote). The results show that non-splenectomized patients present a decrease in absolute numbers of peripheral blood T lymphocytes, specially the CD4+ T subset. However, when patients were analyzed with respect to the presence of bone disease, the number of CD8+ T lymphocytes was found to be statistically significantly lower in patients presenting bone involvement. Furthermore, lower numbers of CD8+ T lymphocytes were significantly correlated with higher levels of plasma tartrate resistant acid phosphatase (TRAP) activity, a putative marker of osteoclast cell activity. These in vivo findings are in agreement with the results reached in vitro by others. They provide an additional marker of disease severity in Gaucher disease. In the group of genotyped Gaucher disease patients, the majority of the N370S homozygous patients presented a clinically milder phenotype, including the absence of bone involvement, confirming earlier reports predicting that a number of these patients may remain undiagnosed. Collectively the homozygosity for the N370S mutation and normal T cell numbers may provide additional markers for the clinical heterogeneity of Gaucher disease.

Mechanisms of osteoclast dysfunction in human osteopetrosis: abnormal osteoclastogenesis and lack of osteoclast-specific adhesion structures.

Abstract: Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast-like cells.

The characterization of macrophages and osteoclasts in tissues harvested from revised total hip prostheses.

Abstract: The differentiation and maturation of macrophages and osteoclasts at the prosthetic interface in cases of implant loosening are poorly understood. Using histochemical and immunohistochemical staining methods, we compare macrophage differentiation in tissues from revised hip replacements in patients with specific clinical-radiological appearances. Periprosthetic tissues were harvested from 12 cemented acetabular and 12 cemented femoral components in 24 patients undergoing revision hip replacement. The prostheses were all radiographically and clinically loose. Six acetabular and six femoral components demonstrated radiographic ballooning osteolysis. Serial 6 microm frozen sections of the periprosthetic tissues were processed with hematoxylin and eosin for general tissue morphology, and analyzed for the presence of tartrate resistant acid phosphatase (TRAP, an osteoclast marker). Immunoperoxidase staining using monoclonal antibodies to CD68 (macrophages and osteoclasts) and CD51 (the alpha chain of the vitronectin receptor, an osteoclast marker) was also performed. Approximately 8-30% of the total cells in the tissues were positive for TRAP and the vitronectin receptor, and comprised a subset of the CD68 positive macrophages and macrophage polykaryons. However, there were no statistically significant differences between specific groups (femoral vs. acetabular, osteolysis vs. no osteolysis) for the numbers or percentages of macrophages or osteoclast-like cells. Once prosthetic loosening has occurred, few differences in the macrophage-osteoclast profile of tissues from different periprosthetic locations, with and without osteolysis, are noted. This suggests a final common biologic pathway for periprosthetic bone resorption, once implant loosening has transpired.

Fluid shear stress increases interleukin-11 expression in human osteoblast-like cells: its role in osteoclast induction.

Abstract: It is unclear how mechanical stress influences bone cells. Mechanical stress causes fluid shear stress (FSS) in the bone. Osteoblast lineage cells are thought to sense FSS and regulate bone remodeling. We therefore investigated the effects of FSS on human osteoblast-like osteosarcoma cells: SaOS-2 cells in vitro. The conditioned medium of the SaOS-2 cells after 24 h of FSS (24 h-FSS CM) showed such osteoclastic phenotype inductions as significantly increasing the number of tartrate-resistant acid phosphatase (TRAP) positive multinuclear cells in rat bone marrow cells and TRAP-positive cells in human preosteoclastic cells: FLG 29.1 cells. An enzyme-linked immunosorbent assay showed interleukin-11 (IL-11) protein to increase 7-fold in the 24 h-FSS CM. A Northern analysis showed that IL-11 mRNA increased 4-fold in the SaOS-2 cells after 6 h-FSS; however, no IL-6 mRNA expression was detected. Furthermore, the anti-human IL-11 antibody significantly neutralized the osteoclastic phenotype induction of the 24 h-FSS CM. The IL-11 mRNA up-regulation in SaOS-2 cells by the 6 h-FSS was not inhibited by the anti-human transforming growth factor-beta1 antibody, but it was significantly inhibited by indomethacin. An enzymeimmunoassay showed prostaglandin E2 to increase 7-fold in the 1 h-FSS CM. These findings thus suggest that FSS induces osteoblasts to produce IL-11 (mediated by prostaglandins) and thus stimulates bone remodeling.

Tartrate-Resistant Bone Acid Phosphatase: Large-Scale Production and Purification of the Recombinant Enzyme, Characterization, and Crystallization

Abstract: Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed in bone-resorbing osteoclasts and certain tissue macrophages in human tissues. The functions of TRAP in biological systems are not known. Elucidation of the three-dimensional structure of the active site could yield important information about the physiological substrate(s) of the enzyme. We have produced recombinant rat bone TRAP using a baculovirus expression vector system. The production was scaled up to a 30-l bioreactor, and a method of purification in large scale was developed. The enzyme is composed of one 34 kDa polypeptide chain. Trypsin digestion resulted in a preparation where two subunits of approximately 23 kDa and approximately 16 kDa appeared after disulfide reduction. Trypsin digestion activated the enzyme. We generated monoclonal antibodies against recombinant TRAP. One of the selected antibodies detected the 23 kDa subunit in Western blotting. The reduced and oxidized forms of the enzyme could be separated by Mono-S cation-exchange chromatography. Crystals of TRAP have been obtained with ammonium sulfate/polyethylene glycol as precipitant. They belong to space group P212121 or P21212 with unit cell dimensions a = 57.2 A, b = 69.5 A, and c = 87.2 A and diffract to at least 2.2 A resolution. A packing density value of 2.55 A3/Da is consistent with one subunit in the asymmetric unit.

Tartrate-Resistant Acid Phosphatase Forms Complexes with α2-Macroglobulin in Serum

Abstract: Tartrate-resistant acid phosphatase (TRAP) is a standard histochemical marker of differentiated osteoclasts and has been proposed as a serum/plasma marker for osteoclast activity. Enzyme assays have been described that show elevated TRAP enzyme activity in the serum or plasma of patient groups known to have increased bone metabolism. However, the poor stability of the enzyme and potential contribution from nonosteoclastic sources make it problematic to measure in patient samples. Immunoassays developed to measure TRAP in serum and plasma have yielded widely varying TRAP levels in both normal and disease states. It is not clear if this variability is caused by differences in assay calibration, antibody specificity, and/or TRAP instability. In this paper, we report that purified TRAP spiked into serum forms high molecular weight complexes. Complex formation results in greatly decreased TRAP enzyme activity and immunoreactivity. The complexing protein in serum has been identified as alpha2-macroglobulin (alpha2M). Similar complexes are observed in stored patient samples. In vitro studies with purified components show that TRAP binds to alpha2M primarily through noncovalent ionic interactions. Our results demonstrate that one mechanism of TRAP instability in serum is complex formation with alpha2M and suggest further that current TRAP enzyme and immunoassays may not accurately measure the circulating level of TRAP.

Serum tartrate resistant acid phosphatase as a potential marker of bone metastasis from breast cancer.

Abstract: BACKGROUND Tartrate resistant acid phosphatase (TRACP) is exclusively localized in osteoclasts and has been suggested to be a unique marker of bone metastasis. PATIENTS AND METHODS TRACP activity using an improved spectrophotometric assay was measured in 56 healthy volunteers and 113 breast cancer patients, including 35 with bone metastases (BM). RESULTS TRACP activities (IU/l, mean +/- SD) of normal subjects in pre- and post-menopausal women were 5.7 +/- 1.3 and 6.6 +/- 1.2, respectively (p < 0.02). The specificity, sensitivity, and accuracy of TRACP in patients, were 91.0%, 65.7%, and 83.2%, respectively. TRACP was significantly higher in patients with BM than in patients without BM (p < 0.01). In patients with BM, TRACP increased in proportion to the number of BM. Regarding clinical assessment of treatment of BM, TRACP was significantly higher in patients with progressive disease. CONCLUSION TRACP is a useful marker of metastatic bone disease and response to treatment in breast cancer patients.

A putative role for c‐Fos in the pathophysiology of paget's disease

Abstract: The molecular mechanisms underlying Paget's disease and subsequent osteosarcoma formation are not well understood. In this study, we aim to delineate the function of the c-Fos oncogene in Paget's disease using transgenic mice, based on previous findings that c-Fos is highly expressed in Pagetic osteoclasts and that c-Fos is an essential gene for osteoclast differentiation and skeletal neoplasia. We have generated transgenic mice in which c-Fos is overexpressed specifically in osteoclasts using the tartrate-resistant acid phosphatase (TRAP) promoter, and five founder mice have been identified. All transgene-expressing animals developed severe bone remodeling lesions, some of which progressed to large bone tumors. Histopathologic analysis indicated that the lesions contained a marked increase in the number of osteoclasts that contained a large number of nuclei. Osteoclasts were identified by histochemical staining for TRAP and by in situ hybridization for matrix metalloproteinase-9 (MMP-9) expression. Moreover, transgenic osteoclasts, and in some cases, osteoblasts and chondrocytes, expressed high levels of c-Fos protein as judged by immunocytochemistry. This phenotype of increased osteoclast number and activity, together with an apparently high rate of bone turnover, resembles some characteristics of Paget's disease. These data therefore support an important function for c-Fos in the Pagetic phenotype, and further support the notion that this gene is important in osteoclastogenesis and in bone remodeling disorders.

Transgenic mice overexpressing tartrate-resistant acid phosphatase exhibit an increased rate of bone turnover

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.

Expression of CSF-1 receptor on TRAP-positive multinuclear cells around the erupting molars in rats.

Abstract: Bone resorption overlying a developing tooth is a necessary event in the creation of an eruption pathway. The formation and function of osteoclasts, which play a major role in bone resorption, are controlled by several factors. Although CSF-1 and its mRNA are expressed in dental follicle cells required for eruption, little is known about the contribution of CSF-1 to osteoclast formation on the bony crypt around the tooth germ. The receptor protein of the CSF-1 encoded proto-oncogene c-fms was identified on multinucleated cells adjacent to the dental follicle, in conjunction with TRAP staining as a marker enzyme for osteoclasts in rat. c-Fms was highly expressed in TRAP-positive multinuclear cells at 3 days postnatal and the number of c-Fms-expressing cells was reduced thereafter. Administration of IL-1alpha, which enhances formation and function of osteoclasts, caused an increase in the number of c-Fms and TRAP-positive cells in rat. On the contrary, injection of calcitonin, which depresses osteoclast formation, caused a decrease in the number. It is obvious that the receptor of CSF-1 is expressed on the surface of osteoclasts around the tooth germ, on the dental follicle. These findings suggested that CSF-1 directly enhances the influx of osteoclasts adjacent to the erupting tooth, resulting in the formation of an eruption pathway.

Biology of the calcium phosphate integration in human long bones

Abstract: Calcium phosphate ceramics implanted in bones are integrated following a sequence of biological events which has already been described. We had the opportunity to analyze histologically some HA-ceramic samples that had been implanted in humans for various periods of time. Eight biopsies were obtained from calcium phosphate ceramics implanted in different sites. All the samples were implanted for periods ranging from 4 to 15 months. The samples were fixed in ethanol and embedded in hydroxyethylmetacrylate or polymethyl methacrylate. 5 μm thick sections were prepared and stained with Giemsa or by Von Kossa method. Alkaline phosphatase and tartrate resistant acid phosphatase (TRAP) were revealed by histochemical methods. Different osteointegration stages were evidenced. In early stages, hematoma remnants were still visible. These implants were invaded by a loose connective tissue in which some cell condensations were apparent. They lead to the formation of bone trabeculae by direct ossification. Some osteoblasts differentiated at the ceramic surface and synthesized osteoid tissue on the material. The fibroblast-like cells of the loose connective tissue were TRAP+. Most of them had TRAP+ nucleus and contained no cytoplasm vesicles. Multinucleated TRAP+ osteoclasts showing TRAP- negative nuclei were found in Howship's lacunae at the bone surface. Certain implants were integrated within mature bone in a later stage.