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1.
Osteoporos Int ; 22(11): 2887-93, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21104229

ABSTRACT

UNLABELLED: Sagopilone, a fully synthetic epothilone and very potent anti-tumor agent, has proved to be efficient in inhibiting bone destruction and tumor burden in a mouse model of breast cancer bone metastasis. In addition to its antiproliferative effects, this study shows direct effects of sagopilone on bone resorption and osteoclast activity. INTRODUCTION: Sagopilone, a novel fully synthetic third-generation epothilone, has proved to be efficient in inhibiting bone destruction and tumor burden in a mouse model of breast cancer bone metastasis. The aim of this study was to investigate whether the effect was primarily due to sagopilone's antiproliferative effect and consequent inhibition of tumor cell growth, or if sagopilone exerts direct effects on bone resorption and osteoclast activity. METHODS: Sagopilone was studied and compared to paclitaxel in vitro in human osteoclast differentiation and activity cultures. For studying the potential of sagopilone for inhibiting bone resorption in vivo, a mouse model of ovariectomy (ovx)-induced osteoporosis was utilized. RESULTS: Sagopilone inhibited osteoclast differentiation and activity more efficiently than paclitaxel and showed less cytotoxicity. Whereas sagopilone showed inhibitory effects on human osteoclast differentiation and activity already at 5 and 15 nM, respectively, paclitaxel started to show effects only at 20 and 100 nM concentrations, respectively. Sagopilone treatment increased BMD In the mouse ovx model even though a non-optimized dose was used which is effective in tumor-bearing mice. CONCLUSION: This is the first study to evaluate sagopilone's effects on bone resorption in non-cancerous situation. The evidence that sagopilone is beneficial for bone will strengthen the status of sagopilone as an anti-cancer compound compared to other microtubule stabilizing agents.


Subject(s)
Benzothiazoles/pharmacology , Bone Resorption/drug therapy , Epothilones/pharmacology , Osteoclasts/drug effects , Osteoporosis/drug therapy , Paclitaxel/pharmacology , Tubulin Modulators/pharmacology , Animals , Bone Density , Bone Resorption/etiology , Disease Models, Animal , Female , Humans , Mice , Osteoporosis/etiology , Ovariectomy/adverse effects
2.
Anticancer Res ; 26(4B): 3127-32, 2006.
Article in English | MEDLINE | ID: mdl-16886645

ABSTRACT

BACKGROUND: The purpose of this cross-sectional study was to evaluate the value of serum tartrate-resistant acid phosphatase 5b (TRACP 5b) and carboxyterminal telopeptide of type I collagen (ICTP) separately and in combination as markers of bone metastases compared to total alkaline phosphatase (tALP) in breast cancer. MATERIALS AND METHODS: Two groups of patients were studied, one with verfied bone metastases (N=46) and one without bone metastases (N=141). Bone marker levels were correlated with the presence or absence of bone metastases. RESULTS: Serum TRACP 5b concentrations exhibited the largest area under the receiver-operating characteristics (ROC) curve (AUC=0.845), followed by ICTP (0.818) and tALP (0.814) when all patients were included in the analysis. With the combination of TRACP 5b and ICTP, the AUC increased to 0.881. In multivariate regression analysis, all three markers were significant predictors of bone metastases. CONCLUSION: Serum TRACP 5b, ICTP and tALP exhibited equal performances in the detection of bone metastases. The combination of TRACP with ICTP did not significantly improve the detection of bone metastases over tALP.


Subject(s)
Acid Phosphatase/blood , Biomarkers, Tumor/blood , Bone Neoplasms/blood , Bone Neoplasms/secondary , Breast Neoplasms/blood , Isoenzymes/blood , Peptide Fragments/blood , Procollagen/blood , Adult , Aged , Aged, 80 and over , Alkaline Phosphatase/blood , Bone Neoplasms/enzymology , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Collagen Type I , Cross-Sectional Studies , Female , Humans , Middle Aged , Peptides , Sensitivity and Specificity , Tartrate-Resistant Acid Phosphatase
3.
Acta Oncol ; 44(7): 742-7, 2005.
Article in English | MEDLINE | ID: mdl-16227166

ABSTRACT

Skeletal metastases are a significant problem in prostate cancer (PC). The patients are also exposed to treatment-related skeletal changes. This cross-sectional study evaluated a marker of bone resorption, TRACP 5b in relation to the standard analyte total alkaline phosphatase (tALP) as a marker of skeletal changes. Serum levels of TRACP 5b, tALP and PSA were measured in 130 prostate cancer patients. Comparison was made between patients with (BM+, n = 25) and without (BM-, n = 105) skeletal metastases, and between those treated with (n = 64) or without (n = 66) androgen deprivation (AD). Sensitivities and specificities were calculated for each marker and diagnostic accuracy was evaluated by ROC curve analysis. ROC curves indicated the superior accuracy of tALP, whereas TRACP 5b and PSA were comparable. With tALP the best combination of sensitivity (96%) and specificity of (91%) was reached at a cut-off point 224 U/L, the corresponding values were for TRACP 5b sensitivity (76%), specificity (89%) with a cut-off point 4.89 U/L, and for PSA sensitivity (65%), specificity (81%) at 23 ng/L for skeletal metastases. Patients treated with AD showed with increasing duration an increase in TRACP 5b values. TRACP 5b was less specific than tALP as a marker of skeletal metastases. TRACP 5b may have a role in the diagnostics of skeletal changes in PC with a focus on treatment-related skeletal changes.


Subject(s)
Acid Phosphatase/blood , Biomarkers, Tumor/blood , Bone Neoplasms/enzymology , Isoenzymes/blood , Prostatic Neoplasms/enzymology , Aged , Aged, 80 and over , Alkaline Phosphatase/blood , Androgen Antagonists/therapeutic use , Bone Neoplasms/drug therapy , Bone Neoplasms/secondary , Bone Resorption , Cross-Sectional Studies , Humans , Male , Middle Aged , Prostate-Specific Antigen/blood , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , ROC Curve , Sensitivity and Specificity , Tartrate-Resistant Acid Phosphatase
4.
Bone ; 36(6): 1065-77, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15878315

ABSTRACT

Tartrate-resistant acid phosphatase (TRACP) is produced by macrophages and other cells of the monohistiocytic lineage. In particular, osteoclasts are characterized for a high expression of this enzyme. Yet, several data suggest that other bone cell types, such as osteocytes and osteoblasts, may also express activity of this enzyme. This is particularly obvious at sites were osteoclasts resorb bone, suggesting that osteoclasts (or their precursors) somehow induce TRACP activity in osteoblasts. In the present study, we investigated this by culturing human osteoblast-like cells with and without conditioned medium (MCM) from human blood monocytes (as a source of osteoclast precursors). High levels of TRACP activity were found in osteoblast-like cells cultured with MCM. Depletion of TRACP from this medium resulted in the absence of its activity in osteoblast-like cells, thus suggesting that the TRACP activity in these cells was the result of endocytosed TRACP that was released by the monocytes in the MCM. Osteoblast-like cells cultured in control (non-conditioned) medium contained very low levels of TRACP-like activity. However, the cells expressed TRACP mRNA and incubation of extracts of these cells with active cathepsin B did induce activity of a TRACP-like enzyme. Inhibition of the activity of cysteine proteinases in general and of cathepsin B in particular, completely blocked TRACP activity of the osteoblast-like cells. This TRACP-like enzyme but not the alleged endocytosed fraction of TRACP was inhibited by fluoride, suggesting that the fractions may be different isoenzymes. Our data seem to indicate that osteoblast-like cells may contain two different fractions of TRACP, one that is released by monocytes and subsequently endocytosed by osteoblast-like cells and a second endogenous fraction that is present in an inactive proform. We hypothesize that the capacity of osteoblast-like cells to endocytose TRACP is important for the removal of this enzyme during or following the bone resorptive activity of the osteoclast.


Subject(s)
Acid Phosphatase/metabolism , Endocytosis/physiology , Gene Expression/genetics , Isoenzymes/metabolism , Osteoblasts/enzymology , Acid Phosphatase/drug effects , Acid Phosphatase/genetics , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Bone Resorption/metabolism , Cathepsin B/antagonists & inhibitors , Cathepsin B/metabolism , Cathepsin B/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Coculture Techniques , Culture Media/pharmacology , Culture Media, Conditioned/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Cytoplasmic Vesicles/chemistry , Dipeptides/pharmacology , Enzyme Activation , Humans , Isoenzymes/drug effects , Isoenzymes/genetics , Models, Biological , Monocytes/cytology , Monocytes/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Peptide Hydrolases/metabolism , Protease Inhibitors/pharmacology , Sodium Fluoride/pharmacology , Tartrate-Resistant Acid Phosphatase
5.
Bone ; 33(1): 132-43, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12919708

ABSTRACT

In this randomized, double-blind, placebo-controlled 12-month trial we evaluated effects of weight- bearing jumping exercise and oral alendronate, alone or in combination, on the mass and structure of bone, risk factors for falling (muscle strength and power, postural sway, and dynamic balance), and cardiorespiratory fitness in postmenopausal women. A total of 164 healthy, sedentary, early postmenopausal women were randomly assigned to one of four experimental groups: (1) 5 mg of alendronate daily plus progressive jumping exercise, (2) 5 mg alendronate, (3) placebo plus progressive jumping exercise, or (4) placebo. The primary endpoint was 12-month change in bone mass and geometry (measured with dual-energy X-ray absorptiometry and peripheral computed tomography at several axial and limb sites) and physical performance; the secondary endpoint was change in biochemical markers of bone turnover. The jumping exercise was conducted an average 1.6 +/- 0.9 (mean +/- SD) times a week. Alendronate daily was effective in increasing bone mass at the lumbar spine (alendronate vs placebo 3.5%; 95% CI, 2.2-4.9%) and femoral neck (1.3%; 95% CI, 0.2-2.4%) but did not affect other bone sites. Exercise alone had no effect on bone mass at the lumbar spine or femoral neck; it had neither an additive nor an interactive effect with alendronate at these bone sites. However, at the distal tibia the mean increase of 3.6% (0.3-7.1%) in the section modulus (that is, bone strength) and 3.7% (0.1-7.3%) increase in the ratio of cortical bone to total bone area were statistically significant in the exercise group compared to the nonexercise group, indicating exercise-induced thickening of the bone cortex. Bone turnover was reduced in alendronate groups only. Alendronate had no effect on physical performance while the jumping exercise improved leg extensor power, dynamic balance, and cardiorespiratory fitness. As conclusion Alendronate is effective in increasing bone mass at the lumbar spine and femoral neck, while exercise is effective in increasing the mechanical properties of bone at some of the most loaded bone sites, as well as improving the participants' muscular performance and dynamic balance. Together alendronate and exercise may effectively decrease the risk of osteoporotic fractures.


Subject(s)
Alendronate/pharmacology , Bone Density/drug effects , Exercise/physiology , Postmenopause/drug effects , Bone Density/physiology , Bone Remodeling/drug effects , Bone Remodeling/physiology , Confidence Intervals , Double-Blind Method , Female , Femur Neck/drug effects , Femur Neck/physiology , Humans , Lumbar Vertebrae/drug effects , Lumbar Vertebrae/physiology , Middle Aged , Postmenopause/physiology , Risk Factors
6.
Calcif Tissue Int ; 71(1): 20-5, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12073156

ABSTRACT

Human serum contains two isoforms of tartrate-resistant acid phosphatase (TRACP) known as TRACP 5a and TRACP 5b with pH optima of 5.0 and 5.8, respectively. Preliminary data suggest that serum TRACP 5b is derived from osteoclasts and serum TRACP 5a from some other cells. It has been reported that heparin inhibits TRACP 5a but has no effect on the activity of TRACP 5b. Here we show that heparin has no effect on serum TRACP activity, as determined using our previously published immunoassay, suggesting that the immunoassay does not detect TRACP 5a. The change of serum TRACP 5b activity after 6 months HRT, determined by this immunoassay, correlated significantly with the changes of all markers of bone turnover determined, including serum N- and C-terminal propeptides of type I collagen and urinary-free deoxypyridinoline. Serum TRACP 5b activity was significantly elevated in patients with osteoporosis and had a significant negative correlation with bone mineral density (BMD). Serum TRACP 5a activity, determined by an immunoassay, showed no correlation with serum TRACP 5b activity, with BMD, or with any of the markers of bone turnover. These results show that serum TRACP 5b, but not 5a, reflects the bone resorption rate, and that our TRACP 5b immunoassay may be a specific method for the determination of the bone resorption rate from serum samples.


Subject(s)
Acid Phosphatase/blood , Bone Density/physiology , Bone and Bones/metabolism , Isoenzymes/blood , Acid Phosphatase/antagonists & inhibitors , Biomarkers/blood , Female , Heparin/pharmacology , Humans , Hydrogen-Ion Concentration , Isoenzymes/antagonists & inhibitors , Kinetics , Middle Aged , Postmenopause , Regression Analysis , Tartrate-Resistant Acid Phosphatase
7.
Calcif Tissue Int ; 70(1): 40-7, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11907706

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.


Subject(s)
Alendronate/pharmacology , Bone Resorption/chemically induced , Bone and Bones/drug effects , Osteoclasts/drug effects , Transport Vesicles/drug effects , Acid Phosphatase/metabolism , Alendronate/administration & dosage , Animals , Animals, Newborn , Apoptosis/drug effects , Biological Transport/drug effects , Bone Resorption/pathology , Bone and Bones/cytology , Cattle , In Vitro Techniques , Isoenzymes/metabolism , Liposomes , Osteoclasts/ultrastructure , Rats , Rats, Sprague-Dawley , Tartrate-Resistant Acid Phosphatase , Transport Vesicles/metabolism , Transport Vesicles/ultrastructure
8.
J Endocrinol ; 171(3): 425-33, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11739008

ABSTRACT

Estrogens are important for the male skeleton. Osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), interleukin-6 (IL-6), IL-1 and tumor necrosis factor alpha (TNFalpha) have been suggested to be involved in the skeletal effects of estrogen. We treated orchidectomized mice with estradiol for 2 weeks and observed a 143% increase in the trabecular bone mineral density of the distal metaphysis of femur that was associated with a decreased OPG/RANKL mRNA ratio in vertebral bone. A similar decreased OPG/RANKL ratio was also seen after estrogen treatment of ovariectomized female mice. The effect of estrogen receptor (ER) inactivation on the OPG/RANKL ratio was dissected by using intact male mice lacking ER alpha (ERKO), ER beta (BERKO) or both receptors (DERKO). The expression of OPG was increased in ERKO and DERKO but not in BERKO male mice, resulting in an increased OPG/RANKL ratio. Furthermore, serum levels of IL-6 and tartrate-resistant acid phosphatase 5b (TRAP 5b) were decreased in ERKO and DERKO, but not in BERKO male mice. These results demonstrate that ER alpha, but not ER beta, is involved in the regulation of the vertebral OPG/RANKL ratio, serum levels of IL-6 and TRAP 5b in male mice.


Subject(s)
Carrier Proteins , Glycoproteins/metabolism , Interleukin-6/blood , Membrane Glycoproteins , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Estrogen/physiology , Receptors, Tumor Necrosis Factor/metabolism , Acid Phosphatase/blood , Animals , Biomarkers/blood , Bone Density/drug effects , Estradiol/pharmacology , Estrogen Receptor alpha , Estrogen Receptor beta , Female , Isoenzymes/blood , Ligands , Male , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Orchiectomy , Osteoprotegerin , Ovariectomy , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Tartrate-Resistant Acid Phosphatase
9.
J Endocrinol ; 171(2): 229-36, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11691642

ABSTRACT

There are two known estrogen receptors, estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta), which may mediate the actions of estrogen. The aim of the present study was to compare fat content, skeletal growth and adult bone metabolism in female mice lacking ER alpha (ERKO), ER beta (BERKO) or both ERs (DERKO). We demonstrate that endogenous estrogens decrease the fat content in female mice via ER alpha and not ER beta. Interestingly, the longitudinal bone growth was decreased in ERKO, increased in BERKO, but was intermediate in DERKO females, demonstrating that ER alpha and ER beta exert opposing effects in the regulation of longitudinal bone growth. The effects on longitudinal bone growth were correlated with similar effects on serum levels of IGF-I. A complex regulation of the trabecular bone mineral density (BMD), probably caused by a disturbed feedback regulation of estrogen and testosterone, was observed in female ER-inactivated mice. Nevertheless, a partial functional redundancy for ER alpha and ER beta in the maintenance of the trabecular BMD was observed in the female mice at 60 days of age. Thus, ER alpha and ER beta may have separate effects (regulation of fat), opposing effects (longitudinal bone growth) or partial redundant effects (trabecular BMD at 60 days of age), depending on which parameter is studied.


Subject(s)
Femur/metabolism , Receptors, Estrogen/metabolism , Absorptiometry, Photon/methods , Animals , Body Constitution , Bone Density/physiology , Estrogen Receptor alpha , Estrogen Receptor beta , Female , Femur/diagnostic imaging , Femur/growth & development , Insulin-Like Growth Factor I/metabolism , Linear Models , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Estrogen/genetics , Tomography, X-Ray Computed
10.
Endocrinology ; 142(12): 5371-8, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11713237

ABSTRACT

Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-terminal fragment, the C-terminal cross-linked peptide of type I collagen. E2 significantly inhibited the release of the C-terminal cross-linked peptide of type I collagen into the culture medium without affecting the release of cross-linked carboxyl-terminal telopeptide of type I collagen in osteoclast cultures. These results suggest that organic matrix degradation is initiated by MMPs and continued by cysteine proteases; the latter event is regulated by E2.


Subject(s)
Bone Matrix/metabolism , Bone Resorption/pathology , Estradiol/pharmacology , Osteoclasts/drug effects , Osteoclasts/physiology , Animals , Biomarkers , Bone and Bones/metabolism , Cathepsin K , Cathepsins/pharmacology , Cell Count , Cells, Cultured , Cellular Senescence/physiology , Collagen/metabolism , In Vitro Techniques , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase 9/pharmacology , Microscopy, Electron, Scanning , Osteoclasts/ultrastructure , Protease Inhibitors/pharmacology , Rats , Recombinant Proteins
11.
Biochem Biophys Res Commun ; 288(1): 142-50, 2001 Oct 19.
Article in English | MEDLINE | ID: mdl-11594765

ABSTRACT

Tartrate-resistant acid phosphatase (TRAP) is an enzyme expressed specifically in osteoclasts and activated macrophages, two phagocytosing cell types originating from the same hematopoietic stem cells. TRAP contains a binuclear iron centre which has been shown to generate reactive oxygen species (ROS). In this study murine macrophage like cell line RAW-264 overexpressing TRAP was shown to produce elevated levels of hydroxyl radicals compared to parental cells. TRAP transfected cells also had reduced growth rate indicating harmful effects of excessive intracellular ROS levels. Using TRAP specific antibody TRAP protein was shown in alveolar macrophages partially colocalize with late endosomal/lysosomal markers Rab7, Lamp 1 and MHC II molecules that bind antigenic peptides. TRAP also colocalized into compartments where Staphylococcus aureus were phagocytosed. These results suggest that TRAP may have an important biological function in the defence mechanism of macrophages by generating intracellular ROS which would be targeted to destroy phagocytosed foreign material.


Subject(s)
Acid Phosphatase/physiology , Hydroxyl Radical/metabolism , Isoenzymes/physiology , Macrophages, Alveolar/enzymology , Macrophages, Alveolar/microbiology , Phagocytosis , Acid Phosphatase/genetics , Acid Phosphatase/immunology , Animals , Antibodies/immunology , Cell Division , Cell Line , Cells, Cultured , Humans , Isoenzymes/genetics , Isoenzymes/immunology , Kinetics , Macrophages, Alveolar/cytology , Mice , Phagosomes/chemistry , RNA, Messenger/biosynthesis , Staphylococcus aureus , Tartrate-Resistant Acid Phosphatase , Transfection
14.
Calcif Tissue Int ; 69(5): 274-80, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11768197

ABSTRACT

Osteopenia develops in experimental animals following surgical removal of the ovaries (ovariectomy. Ovx) or the stomach (gastrectomy, Gx). Though the effect of Ovx has been ascribed to estrogen deficiency, the mechanism behind the Gx-evoked osteopenia remains unknown. In order to compare Gx- and Ovx-evoked osteopenia, young female rats were subjected to Ovx, gx, the combination of ovx and Gx, or sham operation (SHAM). Serum osteoclast-derived tartrate-resistant acid phosphatase 5b was measured as an index of bone resorption, and serum osteocalcin as an index of bone formation/turnover. Bone resorption predominated over bone formation during the first 4 days after Ovx but not later. Bone resorption predominated over bone formation throughout the first 4-week period after Gx. the changes were not additive in the ovx+Gx group. Transillumination and histomorphometry of the calvariae revealed extensive osteopenia in the Gx and the Ovx+Gx groups but not in the Ovx group. Peripheral quantitative computerized tomography of the femur metaphysis showed a decrease in the trabecular bone mineral density (BMD) in all three groups although Ovx+Gx seemed to induce greater trabecular bone loss than Gx alone. However, dual energy X-ray absorptiometry (DXA) of the intact femurs revealed reduced bone mineral content (BMC) in the Gx and Ovx+Gx groups but not in the Ovx group. Indeed, cortical bone was impaired by Gx and Ovx+Gx but not by Ovx. Hence, it seems clear that the Gx-evoked osteopenia differs from that induced by Ovx but that the osteopenia induced by Ovx+Gx is only marginally greater than that induced by Gx alone.


Subject(s)
Bone Diseases, Metabolic/etiology , Gastrectomy/adverse effects , Ovariectomy/adverse effects , Animals , Body Weight , Bone Density , Bone Diseases, Metabolic/blood , Bone Diseases, Metabolic/pathology , Female , Osteocalcin/blood , Radiography , Rats , Skull/diagnostic imaging , Skull/ultrastructure
15.
Clin Chem ; 46(11): 1751-4, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11067809

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<0.0001; n = 120). 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.


Subject(s)
Acid Phosphatase/metabolism , Isoenzymes/metabolism , Osteoclasts/metabolism , Acid Phosphatase/immunology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/enzymology , Bone Marrow Cells/metabolism , Cell Differentiation , Cells, Cultured , Humans , Immune Sera , Immunoassay , Isoenzymes/immunology , Mice , Osteoclasts/cytology , Osteoclasts/enzymology , Rabbits , Tartrate-Resistant Acid Phosphatase
16.
J Bone Miner Res ; 15(7): 1337-45, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10893682

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.


Subject(s)
Acid Phosphatase/blood , Bone Resorption/diagnosis , Estrogen Replacement Therapy , Isoenzymes/blood , Antibodies, Monoclonal , Biomarkers/blood , Bone Resorption/blood , Bone Resorption/enzymology , Double-Blind Method , Enzyme Stability , Estradiol/therapeutic use , Female , Humans , Middle Aged , Neuraminidase , Norethindrone/therapeutic use , Placebos , Postmenopause , Reference Values , Tartrate-Resistant Acid Phosphatase
17.
J Cell Sci ; 113 ( Pt 3): 377-81, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10639325

ABSTRACT

Osteoclasts are multinucleated cells responsible for bone resorption. They have developed an efficient machinery for dissolving crystalline hydroxyapatite and degrading organic bone matrix rich in collagen fibers. When initiating bone resorption, osteoclasts become polarized, and three distinct membrane domains appear: a ruffled border, a sealing zone and a functional secretory domain. Simultaneously, the cytoskeleton undergoes extensive re-organisation. During this process, the actin cytoskeleton forms an attachment ring at the sealing zone, the membrane domain that anchors the resorbing cell to bone matrix. The ruffled border appears inside the sealing zone, and has several characteristics of late endosomal membrane. Extensive vesicle transport to the ruffled border delivers hydrochloric acid and proteases to an area between the ruffled border and the bone surface called the resorption lacuna. In this extracellular compartment, crystalline hydroxyapatite is dissolved by acid, and a mixture of proteases degrades the organic matrix. The degradation products of collagen and other matrix components are endocytosed, transported through the cell and exocytosed through a functional secretory domain. This transcytotic route allows osteoclasts to remove large amounts of matrix-degradation products without losing their tight attachment to underlying bone. It also facilitates further processing of the degradation products intracellularly during the passage through the cell.


Subject(s)
Bone Resorption/pathology , Osteoclasts/cytology , Animals , Biological Transport , Bone Matrix/metabolism , Cell Movement , Cell Polarity , Cells, Cultured , Collagen/metabolism , Cytoskeleton/ultrastructure , Durapatite/metabolism , Endopeptidases/metabolism , Humans , Hydrochloric Acid/metabolism , Mice , Mice, Knockout , Organelles/physiology , Osteoclasts/physiology , Proton Pumps/metabolism , Reactive Oxygen Species
18.
Clin Chem ; 45(12): 2150-7, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10585347

ABSTRACT

BACKGROUND: Tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) 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) microg/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/microg) was similar to that in healthy subjects (0.091 U/microg). By contrast, the specific activity of TRAP in the 31 of 99 rheumatic sera with increased TRAP protein (0.035 U/microg) 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.


Subject(s)
Acid Phosphatase/analysis , Immunoassay/methods , Isoenzymes/analysis , Acid Phosphatase/blood , Acid Phosphatase/immunology , Adult , Antibody Specificity , Bone Resorption/blood , Female , Horseradish Peroxidase , Humans , Isoenzymes/blood , Isoenzymes/immunology , Kidney Failure, Chronic/blood , Male , Renal Dialysis , Rheumatic Diseases/blood , Sensitivity and Specificity , Tartrate-Resistant Acid Phosphatase
19.
J Biol Chem ; 274(33): 22907-10, 1999 Aug 13.
Article in English | MEDLINE | ID: mdl-10438453

ABSTRACT

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in bone-resorbing osteoclasts and activated macrophages. It has been suggested that a redox-active iron in the binuclear iron center of TRAP could have the capacity to react with hydrogen peroxide to produce highly destructive reactive oxygen species (ROS). Here we show that TRAP can generate ROS in vitro and that cells over-expressing TRAP produce higher amounts of intracellular ROS than their parent cells. We further demonstrate that these ROS can be targeted to destroy collagen and other proteins. In resorbing osteoclasts, TRAP was found in transcytotic vesicles transporting matrix degradation products through the cell, suggesting that TRAP-facilitated fragmentation of endocytosed material takes place in a specific cellular compartment. These results suggest that bone matrix degradation occurs not only extracellularly in the resorption lacunae but also intracellularly in the transcytotic vesicles. We propose that proteins containing redox-active iron could represent a novel mechanism of physiological fragmentation of organic molecules. This mechanism could be important in tissue remodeling and as a defense mechanism of phagocytosing cells.


Subject(s)
Acid Phosphatase/metabolism , Bone Resorption , Isoenzymes/metabolism , Reactive Oxygen Species/metabolism , Animals , Bone and Bones/cytology , Bone and Bones/metabolism , Cells, Cultured , Collagen/metabolism , Rats , Tartrate-Resistant Acid Phosphatase
20.
J Bone Miner Res ; 14(3): 464-9, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10027912

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.


Subject(s)
Acid Phosphatase/analysis , Acid Phosphatase/immunology , Antibodies, Monoclonal , Immunoassay/methods , Isoenzymes/analysis , Isoenzymes/immunology , Osteoclasts/enzymology , Acid Phosphatase/blood , Adult , Animals , Bone Resorption/enzymology , Enzyme Stability , Epitope Mapping , Female , Humans , Immunohistochemistry , Isoenzymes/blood , Menopause/metabolism , Mice , Middle Aged , Tartrate-Resistant Acid Phosphatase
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