Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage.

The effect of oral glucose tolerance test on serum osteocalcin and bone turnover markers in young adults.

Osteocalcin (OC) is an osteoblast-derived protein implicated in the regulation of glucose tolerance and energy metabolism. This endocrine function has been suggested to be exerted via its undercarboxylated form, which has been shown to induce expression of adiponectin, insulin, and islet cell proliferation in mice. Furthermore, insulin has recently been shown to regulate the biological activity of OC in bone. Our aim was to explore the association between glucose and bone metabolism by evaluating the effect of a standard 75 g oral glucose tolerance test (OGTT) on serum OC, carboxylated OC (cOC) and bone-turnover markers (BTMs) C terminal telopeptide (ßCTX-I) and N terminal propeptide (PINP) of type I collagen and tartrate-resistant acid phosphatase 5b (TRACP5b). Serum samples collected at 0 and at 120 min were analyzed in a cohort of normoglycemic young adults (n = 23, mean age 23.6 years). During OGTT a significant decrease was observed in all BTMs (P < 0.001 for all variables). The median decreases from 0 to 120 min for OC, cOC, ßCTX-I, PINP, and TRACP5b were -32.1% (-37.9 to -19.6), -34.4% (-39.8 to -22.2), -61.4% (-68.5 to -53.0), -26.8% (-33.2 to -19.2), and -44.5% (-48.3 to -40.2), respectively. A strong association between the changes in OC and cOC was observed (r = 0.83, P < 0.001). The decrease in PINP was associated with changes in OC, whereas the changes in ßCTX-I and TRACP5b were not associated with decreases in OC or cOC. The observed OGTT-induced changes in bone-derived proteins were partially independent of each other and potentially mediated by different mechanisms.

Osteocyte-derived HB-GAM (pleiotrophin) is associated with bone formation and mechanical loading.

HB-GAM (also known as pleiotrophin) is a cell matrix-associated protein that is highly expressed in bone. It affects osteoblast function, and might therefore play a role in bone development and remodeling. We aimed to investigate the role of HB-GAM in bone in vivo and in vitro. The bones of HB-GAM deficient mice with an inbred mouse background were studied by histological, histomorphometrical, radiological, biomechanical and mu-CT analyses and the effect of immobilization was evaluated. HB-GAM localization in vivo was studied. MLO-Y4 osteocytes were subjected to fluid shear stress in vitro, and gene and protein expression were studied by subtractive hybridization, quantitative PCR and Western blot. Human osteoclasts were cultured in the presence of rhHB-GAM and their formation and resorption activities were assayed. In agreement with previous reports, the skeletal structure of the HB-GAM knockout mice developed normally. However, a growth retardation of the weight-bearing bones was observed by 2 months of age, suggesting a link to physical activity. Adult HB-GAM deficient mice were characterized by low bone formation and osteopenia, as well as resistance to immobilization-dependent bone remodeling. HB-GAM was localized around osteocytes and their processes in vivo and furthermore, osteocytic HB-GAM expression was upregulated by mechanical loading in vitro. HB-GAM did not affect on human osteoclast formation or resorption in vitro. Taken together, our results suggest that HB-GAM is an osteocyte-derived factor that could participate in mediating the osteogenic effects of mechanical loading on bone.

Effect of impact exercise on bone metabolism.

SUMMARY: Regular impact exercise in premenopausal women caused positive osteogenic effects associated to low basal serum parathormone (PTH) but had no effects on bone turnover markers PINP or TRACP5b. The low serum basal PTH levels during impact exercise may be a sign of increased incorporation of calcium to bone. INTRODUCTION: This study aimed to determine the long-term effects of high-impact exercise on bone turnover and calciotropic hormones. METHODS: We performed a 12-month population-based, randomized, controlled exercise trial in 120 women (age 35-40 years) randomly assigned to an exercise group (EG; n = 60) or a control group (CG; n = 60). The exercise regimen consisted of supervised high-impact exercises three times per week. Daily impact loading was assessed by using an accelerometer. Bone turnover markers and calciotropic hormones were analyzed at 0, 6, and 12 months. RESULTS: Twelve months of impact exercise did not reveal any treatment effects in bone turnover markers PINP or TRAPC5b, whereas serum basal PTH decreased significantly more in the EG than in the CG (-11.2 vs. -2.2 pg/mL; p = 0.03). The change in PTH was dose dependent and most clearly seen in subjects with 96 to 130 daily impacts at 2.5 to 5.3 g (e.g., running or jumping). CONCLUSIONS: Regular impact exercise does not cause persistent alterations in bone turnover emphasizing necessity of continuous training to achieve bone benefits. Impact exercise training lowers the serum basal PTH levels and possibly enables greater difference between the basal PTH and transient exercise-induced PTH peaks leading to osteogenic effects.

Can elevated serum TRACP-5b levels predict stress fractures? A cohort study.

BACKGROUND AND AIMS: Stress fracture is a common overuse injury in athletes and military conscripts. The reliable diagnosis of stress fractures is often difficult, however, because it is usually based solely on radiographic findings. Biochemical markers of bone resorption reflect bone degradation and may also reflect the rate of bone loss. The aim of the study was to examine whether elevated serum tartrate-resistant acid phosphatase isoform 5b (TRACP-5b) levels reflect enhanced bone remodeling and predict the occurrence of stress fractures in military conscripts. MATERIAL AND METHODS: Randomly selected military conscripts [mean age, 19.8 (range 18-28) years; n = 820] were followed for 3 months. Baseline blood samples were drawn upon arrival to the service. Four subsequent samples were obtained from subjects that developed stress fractures and one sample each was obtained from two asymptomatic control subjects for each fracture case. RESULTS: Plain radiography was used to diagnose stress fractures in 20 of the 820 conscripts (2.4%). Follow-up data were available for 14 subjects with 21 stress fractures and 28 control subjects. Subjects with proportionally increasing serum TRACP-5b levels had an 8-fold greater probability of stress fracture than controls. No statistically significant difference was detected. CONCLUSIONS: Although assessing serum TRACP-5b levels appears to be a promising method to predict bone stress injuries, the present study failed to give a conclusive statement of its usefulness as a diagnostic tool.

Differential effects of selective oestrogen receptor modulators (SERMs) tamoxifen, ospemifene and raloxifene on human osteoclasts in vitro.

BACKGROUND AND PURPOSE: Several selective oestrogen receptor modulators (SERMs) with oestrogen agonist effects in bone cells and without increased risk of breast and endometrial cancer have been developed. Here, we have investigated the effects of different types of SERMs on osteoclast differentiation, bone resorption and apoptosis in vitro. EXPERIMENTAL APPROACH: Human peripheral blood-derived CD14+ monocytes were cultured on bovine bone slices in the presence of RANKL, M-CSF, TNF-alpha and dexamethasone for seven days. Also, CD14+ monocytes were co-cultured either with human SaOS-2 or MG-63 osteosarcoma cells, in the presence of parathyroid hormone. Osteoclast cultures were treated with different SERMs. TRACP+ multinucleated cells and C-terminal telopeptide of type I collagen were used as markers for osteoclast formation and bone resorption, respectively. KEY RESULTS: In CD14+ monocyte cultures, tamoxifen directly inhibited human osteoclast formation and bone resorption, while raloxifene and ospemifene had no inhibitory effect. In the co-cultures either with SaOS-2 or MG-63 cells, ospemifene and raloxifene as well as tamoxifen inhibited osteoclast formation in a concentration-dependent manner. The inhibitory effect was associated with an increased production of osteoprotegerin. The anti-oestrogen ICI 182 780 completely reversed the effects of these SERMs. CONCLUSION AND IMPLICATIONS: Tamoxifen had an oestrogen receptor dependent, direct, inhibitory effect on human osteoclast differentiation and bone resorption, whereas ospemifene and raloxifene required osteoblastic cells to achieve a similar inhibition. The effects of ospemifene and raloxifene were mediated by oestrogen receptors by a mechanism involving paracrine induction of osteoprotegerin in cultures with osteoblast derived osteosarcoma cells.

Serum TRACP 5b and ICTP as markers of bone metastases in breast cancer.

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.

Serum tartrate-resistant acid phosphatase 5b (TRACP 5b) as a marker of skeletal changes in prostate cancer.

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.

Estrogen protects primary osteocytes against glucocorticoid-induced apoptosis.

Glucocorticoid-induced osteoporosis may be at least in part due to the increased apoptosis of osteocytes. To study the role of osteocyte apoptosis in glucocorticoid-induced osteoporosis, we isolated primary osteocytes from murine calvaria for the analysis of the effects of dexamethasone in in vitro culture. The cells were identified by morphology, cytochemical staining, immunocytochemical staining and mRNA expression of phosphate-regulating gene with homology to endopeptidases on the X chromosome (PHEX) and sclerosteosis/van Buchem disease gene (SOST). We found that dexamethasone induced osteocyte apoptosis in a dose-dependent manner. A glucocorticoid receptor antagonist, mifepristone (RU486), suppressed dexamethasone-induced osteocyte apoptosis, suggesting that it was mediated by glucocorticoid receptor. Immunocytochemical stainings showed that glucocorticoid receptors are present in primary osteocytes, and they were translocated to nuclei after the exposure to dexamethasone. Addition of estrogen prevented glucocorticoid receptor translocation into nuclei. Corresponding antiapoptotic effects in primary osteocytes were also seen after the pretreatment of primary osteocytes with a picomolar concentration of estrogen. The pure antiestrogen ICI 182,780 inhibited estrogen effect on apoptosis induced by dexamethasone. These data suggest that glucocorticoid receptors play an important role in glucocorticoid-induced osteocyte apoptosis. Most importantly, estrogen has a protective effect against osteocyte apoptosis. To conclude, the mechanism of glucocorticoid-induced osteoporosis may be due to the apoptosis of osteocytes, which can be opposed by estrogen.

Characterization of circulating human osteoclast progenitors: development of in vitro resorption assay.

Several cell surface markers were used to isolate monocytes as osteoclast progenitors with an immunomagnetic cell separation system. Use of this system with specific monocyte antibodies produced 99% pure monocytes. When purified monocytes were cultured on bovine bone slices in the presence of receptor activator of nuclear factor-kappaB (RANKL), macrophage-colony stimulating factor (M-CSF), tumor necrosis factor alpha (TNF-alpha), and dexamethasone for 14 days, CD14(+) CD11b(+), and CD61(+) monocytes had approximately 90-, 30- and 20-fold higher osteoclast formation capacities/plated cells compared to the control culture. CD15(+) monocytes generated few tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP+ MNC), and CD169(+) monocytes generated no TRACP+ MNC. This suggests, that there are various subsets of monocytes in the blood circulation and that they have different capacities in osteoclast formation. These results show that circulating human osteoclast progenitors can be efficiently purified by immunomagnetic cell separation system using anti-CD14, -CD11b, and -CD61 antibodies. These purified monocyte fractions had different ability to give rise to osteoclasts. CD169 was not found to be suitable for osteoclast progenitor isolation. Optimal concentration of dexamethasone for osteoclast formation and bone resorption was 10 nM. To develop a human resorption assay, osteoclasts were first induced for 7 days, whole media were replaced, cultures were continued for additional 3 days and C-terminal telopeptide of type I collagen was determined from culture media. This assay was shown to be functional, since two well-known resorption inhibitors, bafilomycin A(1) and calcitonin, dose-dependently inhibited the resorption activity of osteoclasts.

Osteoblast-like cells complete osteoclastic bone resorption and form new mineralized bone matrix in vitro.

Bone remodeling involves old bone resorption by osteoclasts and new bone formation by osteoblasts. However, the precise cellular mechanisms underlying these consecutive events remain obscure. To address this question in vitro, we have established a cell culture model in which the resorption lacunae are first created by osteoclasts and osteoblast-like cells accomplish the subsequent bone formation. We isolated osteoclasts from rat bone marrow and cultured them on bovine bone slices for 48 hours to create resorption lacunae. After removing osteoclasts, confluent differentiated primary osteoblast cultures were trypsinized and the cells were replaced on the resorbed bone slices for up to 14 days. The cultures were then examined by confocal microscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). Our data suggest that after osteoclastic bone resorption, osteoblast-like cells, not macrophages, remove the remaining organic matrix in the lacuna. After cleaning the lacuna, osteoblast-like cells deposit new collagen fibrils at the bottom of the lacuna and calcify the newly formed matrix only, as visualized by labeled tetracycline accumulation merely in the lacuna during the osteoblast culture. Furthermore, an electron-dense layer rich in osteopontin separates the old and new matrices suggesting formation of the cement line. Since the morphology of the newly formed matrix is similar to the natural bone with respect to the cement line and osteoid formation as well as matrix mineralization, the present method provides for the first time a powerful in vitro method to study the cellular mechanisms leading to bone remodeling also in vivo.

Serum tartrate-resistant acid phosphatase 5b, but not 5a, correlates with other markers of bone turnover and bone mineral density.

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.

Bone morphogenetic protein 3b expressing reindeer antler.

A cDNA sequence of bone morphogenetic protein 3b (BMP-3b) of reindeer antler was produced with degenerative homology primers in polymerase chain reaction (PCR). An in situ hybridization study of BMP-3b mRNA in 1-month-old antler showed expression in most differentiated cells in the antler center. In addition, the bone-inductive capacity of the reindeer antler matrix was evaluated. Decalcified and powdered antler matrix of different stages of antler maturity was implanted in gelatin capsules under the rat dorsal muscle fascia for two implantation periods: 3 and 8 weeks. Allogenic matrix prepared from rat long bones was used as a positive control implant. Heterotopic ossification was evaluated histomorphometrically and densitometrically. Allogenic bone matrix induced rapid osteogenesis and mineral accumulation. Both endochondral and intramembranous ossification was evident, endochondral ossification being the dominant form. Mineral density in the induced ossicle was 115 +/- 48 mg/cm(3) as early as at 3 weeks and 350 +/- 69 mg/cm(3) at 8 weeks. The proportional areas of von Kossa-stained mineral were 3.67 +/- 2.1% and 11.6 +/- 0.07%, respectively. The antler preparations induced mineralization, but significantly less than the allogenic bone matrix. At 8 weeks, mineral density was significantly lower in the cast antler preparation than in the allogenic implants. The morphology of the mineralized areas of the antler preparations showed no ossification.

Estrogen reduces the depth of resorption pits by disturbing the organic bone matrix degradation activity of mature osteoclasts.

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.

Tartrate-resistant acid phosphatase facilitates hydroxyl radical formation and colocalizes with phagocytosed Staphylococcus aureus in alveolar macrophages.

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.

Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption.

During skeletal growth and remodeling the mineralized bone matrix is resorbed by osteoclasts through the constant secretion of protons and proteases to the bone surface. This relies on the formation of specialized plasma membrane domains, the sealing zone and the ruffled border, and vectorial transportation of intracellular vesicles in bone-resorbing osteoclasts. Here we show that Rab7, a small GTPase that is associated with late endosomes, is highly expressed and is predominantly localized at the ruffled border in bone-resorbing osteoclasts. The decreased expression of Rab7 in cultured osteoclasts by antisense oligodeoxynucleotides disrupted the polarization of the osteoclasts and the targeting of vesicles to the ruffled border. These impairments caused a significant inhibition of bone resorption in vitro. The results indicate that the late endocytotic pathway is involved in the osteoclast polarization and bone resorption and underscore the importance of Rab7 in osteoclast function.

Strong prediction of fractures among older adults by the ratio of carboxylated to total serum osteocalcin.

We examined serum total osteocalcin (TOC), carboxylated osteocalcin (COC), and their ratio (COC/TOC) by one-step two-site immunofluorescent assays in 87% (n = 792) of all home-dwelling persons of 70 years or older living in a defined area in northern Finland. Other baseline subject-related risk factors of fractures were assessed by postal questionnaires, interviews, clinical examinations, and tests. During a 5-year follow-up period, all falls and fractures (n = 106) were recorded by regular phone calls and by examining all the medical records yearly. Serum TOC and COC concentrations increased with advancing age and were higher in women than in men, but corresponding differences were not found in the case of COC/TOC. The adjusted relative risk of fracture was elevated in association with low (< or =-1 SD from the mean) COC; hazard ratio (HR, 95% CI) 2.00 (1.20-3.36) and low COC/TOC; HR 5.32 (3.26-8.68), the relative risk being highest in the population older than 80 years; and HR 7.02 (2.42-20.39). The predictive value of low COC/TOC lasted 3 years. The multivariable-adjusted relative risk of hip fracture (n = 26) in regard to low COC/TOC ratio was 3.49 (1.12-10.86), as compared with the persons who did not suffer hip fractures. Our results suggest that serum COC concentrations and, more strongly, COC/TOC, predict the occurrence of fractures in older community-dwelling adults. The risk of fracture associated with low COC/TOC equals the hip fracture risk previously verified for concomitant high serum undercarboxylated OC concentrations and low bone mineral density.

Rapid screening method for osteoclast differentiation in vitro that measures tartrate-resistant acid phosphatase 5b activity secreted into the culture medium.

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.

Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption.

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.