TRACP-5b Levels Research Articles

Role of interleukin-6 on RANKL-RANK/osteoprotegerin system in hypothyroid ovariectomized mice.

Postmenopausal women frequently develop hypothyroidism. Estrogen depletion is accompanied by an increase of IL-6, accelerating bone turnover. The influence of hypothyroidism on bone metabolism in postmenopausal women is poorly understood. The aim of the study was an attempt to clarify the role of interleukin-6 on RANKL-RANK/osteoprotegerin system in hypothyroid ovariectomized mice. The study was performed on 56, 12-13 weeks old, female mice: C57BL/6J (wild-type; WT) and C57BL/6JIL6-/-Kopf (IL-6 knock-out; IL6KO). The mice were randomly divided into 8 groups with 7 mice in each one: 1/ WT controls, 2/ IL6KO controls, 3/ WT hypothyroid mice, 4/ IL6KO hypothyroid mice, 5/ WT ovariectomized, 6/ IL6KO ovariectomized, 7/ WT ovariectomized hypothyroid mice and 8/ IL6KO ovariectomized hypothyroid mice. Experimental model of menopause was produced by bilateral ovariectomy carried out in 8-9 weeks old mice. Experimental model of hypothyroidism was induced by propylthiouracyl administration in driking water. The serum levels of TRACP 5b, osteocalcin, OPG and RANKL were determined by ELISA. Serum RANKL concentrations were elevated significantly in all groups of ovariectomized mice as compared to respective controls, but in a minor degree in IL6KO hypothyroid mice as compared to wild-type animals. Moreover sRANKL values were significantly lower in IL6KO as compared to WT controls and IL6KO PTU injected mice. Osteoprotegerin serum levels were decreased in all IL-6 deficient mice and in a highest degree in sham-operated hypothyroid mice. To sum up, the results of the present study suggest that estrogens deficit is a strong stimulus for RANKL-RANK/OPG pathway that breaks an inhibitory influence of hypothyroidism even in IL-6 deficient mice.

Estrogens Modulate RANKL-RANK/Osteoprotegerin Mediated Interleukin-6 Effect on Thyrotoxicosis-related Bone Turnover in Mice

Interleukin-6 has been shown to cause imbalance between bone resorption and formation in thyrotoxicosis. The aim of the present study was an attempt to estimate the influence of estrogens on thyrotoxicosis-related disturbances in bone turnover in relation to RANKL-RANK/osteoprotegerin system in IL-6 deficient mice. The study was performed on 56, 12-13 weeks old, female mice: C57BL/6J (wild-type; WT) and C57BL/6J (IL6-/-Kopf) (IL-6 knock-out; IL6KO). The mice were randomly divided into 8 groups with 7 mice in each one: 1. WT controls, 2. IL6KO controls, 3. WT mice with thyrotoxicosis, 4. IL6KO mice with thyrotoxicosis, 5. WT ovariectomized, 6. IL6KO ovariectomized, 7. WT ovariectomized mice with thyrotoxicosis, and 8. IL6KO ovariectomized mice with thyrotoxicosis. Experimental model of menopause was evoked by bilateral ovariectomy carried out in 8-9 weeks old mice. Thyrotoxicosis was induced by intraperitoneal injection of levothyroxine at a dose of 1 μg/g daily over 21 days. The serum levels of TRACP5b, osteocalcin, OPG, and RANKL were determined by ELISA. RANKL serum concentrations were elevated significantly in all groups of ovariectomized mice as compared to respective controls, however, in a minor degree in IL6KO thyrotoxic mice as compared to wild-type animals. Osteoprotegerin serum levels were significantly increased in all thyrotoxic groups of mice except ovariectomized IL6KO animals. To sum up, the results of the present study suggest that IL-6 plays a key role in stimulation of RANKL-RANK/OPG system and this effect is strongly enhanced in conditions of accelerated bone turnover such as thyrotoxicosis and/or estrogen depletion.

TRACP 5b and CTX as osteological markers of delayed fracture healing

Radiological studies are the standard method to monitor fracture healing but they do not allow a timely assessment of bone healing. Biochemical markers react rapidly to changes in bone metabolism during fracture healing and could be an additional tool to monitor this process.The goal of this study was to observe changes in serum biomarkers and evaluate the possible differences in the serum levels of tartrate-resistant acid phosphatase 5b (TRACP 5b), total N-terminal propeptide of type I collagen (PINP), bone-specific alkaline phosphatase (BAP), and C-terminal cross-linking telopeptide of type I collagen (CTX) in patients with normal and delayed fracture healing.Several serum samples were collected for one year after the surgical treatment of long bone fractures in 248 patients. From this large pool, 15 patients with atrophic nonunion were matched to 15 patients with normal bone healing. Post-operative changes in osteological markers were monitored during the 1st, 2nd, 4th, 8th, 12th and 52nd weeks. The patients were followed both clinically and radiologically for the entire one-year duration of the study.In the first week, the absolute values of CTX decreased significantly (p=0.0164) in cases of delayed fracture healing. The relative values of TRACP 5b were significantly decreased at weeks 4 (p=0.0066) and 8 (p=0.0043). BAP and PINP levels decreased in the first week followed by an increase, but there were no significant differences in the absolute or relative values during the healing process in both patient groups.For the first time, we have demonstrated changes in serum concentrations of TRACP 5b, PINP, BAP, and CTX during normal and delayed fracture healing. Characteristic changes in systemic TRACP 5b and CTX levels could reflect the initial process of successful fracture healing and may be used in clinical practice to monitor the healing process. Furthermore, it could be very important for determining the beneficial effects of additional treatments such as ultrasound or BMPs in clinical trials.

Changes in bone microstructure and serum Tracp-5b level in male rats exposed to cadmium

Changes in bone microstructure and serum Tracp-5b level in male rats exposed to cadmium

Abstract 2255: In vivo study of serum cancer biomarkers and pain-induced bone metastasis of human prostate tumor in Nude rats

Abstract Bone is one of the most frequent sites of spread for many cancers such as breast, prostate, lung or kidney. Management of metastatic bone disease and associated pain can control the symptoms and prevent further complications such as the decrease of quality of life, pathological fracture or compression of the spinal cord. Until recently, investigation of novel drugs in the treatment of cancer-induced bone pain was hampered by a lack of suitable models. The aim of this study was to characterize biochemical markers and behavioral pain responses in an experimental animal model of bone metastases induced by intracardiac injection of PC-3 human prostate cancer cells in Nude rats. Firstly, potential differences of nociceptive responses between healthy Nude rats (classically used in cancer studies) and healthy SD rats (routinely used in pain studies) were assessed in 2 mechanical tests (electronic Von Frey and paw pressure tests) and 2 thermal tests (plantar and acetone tests). Pharmacological response to morphine was also assessed in both strains using the paw pressure test. Secondly, Nude rats were intracardiacally inoculated with PC3 cancer cells or RPMI medium and nociceptive responses were measured between tumor-bearing and non tumor-bearing Nude rats. Behavioral tests were carried out once a week during 4 consecutive weeks. In a satellite group, 15 days after tumor cell injection, 5 Nude rats received 4 intravenous injections of paclitaxel (5 mg/kg every 4 days). Development of bone metastases was monitored by ELISA quantification of serum biomarkers such as CTX-1 and TRACP 5b (bone resorption) and PINP (bone formation). Nociceptive tests in healthy SD and Nude rats did not demonstrate any major difference of hypersensitivity between both strains. Morphine (3 mg/kg, s.c.) induced a similar and classically observed analgesic effect in both strains. Any significant difference between Nude rats inoculated or not with cancer cells was observed neither in mechanical nor in thermal hyperalgesia assays. CTX-1 and TRACP-5b levels were significantly higher in tumor-bearing Nude rats than in non tumor-bearing ones. PINP level was lower in Nude rats injected with PC-3 cells than in non injected-rats. Paclitaxel significantly increased the life span of tumor-bearing Nude rats (ILS > 120%) and affected the kinetic of the 3 serum biomarkers. At sacrifice, CTX-1 and TRACP-5b serum levels were decreased by 51% and 95% respectively, and PINP level was increased by 325% compared to the non-treated rats. These results indicated that intracardiac injection of PC-3 human prostate cancer cells in Nude rats associated with bone formation/resorption serum biomarkers monitoring is a useful experimental in vivo model for bone metastases study and anti-cancer drug evaluation. Surprisingly, pain induced by experimental bone metastases was not demonstrated in this model with classical painful assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2255.

Potential role of tartrate‐resistant acid phosphatase 5b (TRACP 5b) as a surrogate marker of late loosening in patients with total hip arthroplasty: A cohort study

In a cohort study, the role of the active tartrate-resistant acid phosphatase (TRACP 5b), a marker of bone-resorbing osteoclasts, for the assessment of loosening after total hip arthroplasty (THA), was analyzed, as well as its correlation with osteolysis and multinucleated cell appearance in the retrievals. Eighty THA patients, who went consecutively to the orthopedic department, were asked to participate, and 54 accepted and were enrolled in the study. Finally, 46 subjects were analyzed, clinical-radiographic evaluation was considered the gold standard, serum TRACP 5b was blindly measured, and a cut-off was obtained, by performing a ROC Curve. Based on the gold standard, patients were split by 19 stable and 27 loosened subjects, and results were matched. TRACP 5b was significantly higher in loosened patients than in stable ones (p < 0.001). A good specificity (89.5%), positive predictive value (90.0%), and likelihood ratio (6.33) were calculated, that provided strong evidence of loosening with TRACP 5b levels higher than the cut-off. Moreover, TRACP 5b and osteolysis (Fisher's exact test, p = 0.03) were found significantly correlated. TRACP 5b has been proven a reliable marker, specifically related to resorbing-multinucleated cells, to ascertain late loosening in THA, and could support standard procedures, if confirmed by performing prospective studies.

Diffuse MRI Marrow Pattern Correlates with Increased Angiogenesis, Advanced Disease Features and Poor Prognosis in Newly-Diagnosed Patients with Multiple Myeloma Treated with Novel Agents.

Abstract 829The aim of this study was to evaluate the prognostic value of bone marrow magnetic resonance imaging (MRI) in newly-diagnosed myeloma (MM) patients who received novel agent-based therapy and to explore possible correlations of MRI pattern with clinical and laboratory data. Eighty-two consecutive MM patients (43M/39F, median age: 65 years) at diagnosis were studied. MRI of the spine was performed within 1-2 weeks following diagnosis. T1-weighted, STIR and contrast-enhanced T1-weighted sagittal MR images of the thoracic and lumbar spine were obtained with a 1.5 T unit (Phillips, Eindhoven, the Netherlands). Marrow involvement was characterized as normal, focal, diffuse or variegated. Microvessel density (MVD) was evaluated in marrow trephine biopsies at the time of diagnosis. The following serum indices of bone metabolism and angiogenesis were measured on the day of MRI: i) osteoclast stimulators [sRANKL, osteoprotegerin (OPG), osteopontin (OPN) and macrophage inflammatory protein-1alpha (MIP-1alpha)], ii) dickkopf-1 (Dkk-1), iii) bone resorption markers [C- and N-terminal cross-linking telopeptide of collagen type-I (CTX and NTX, respectively) and TRACP-5b], iv) bone formation markers [bone alkaline phosphatase (bALP) and osteocalcin (OC)], and v) angiogenic cytokines [VEGF, VEGF-A, angiogenin (ANG), angiopoietin-1 (ANGP-1), angiopoietin-2 (ANGP-2) and bFGF]. MRI evaluation of the spine revealed that 34 (41.5%) patients had focal, 26 (31.7%) diffuse, 18 (22%) normal, and 4 (4.9%) had a variegated pattern of marrow involvement. Low-grade angiogenesis, as assessed by MVD, was present in 32 (39%) patients, while 33 (40%) had intermediate-grade and 17 (20%) patients had high-grade angiogenesis. Twenty-one of 26 (80%) patients with a diffuse MR pattern had intermediate or high-grade angiogenesis in contrast to 14/34 (41%) patients with focal involvement (p=0.01) and 12/18 (66%) with normal MR pattern (p=0.037). Patients had increased values of VEGF (p<0.001), VEGF-A (p<0.001), ANGP-2 (p<0.001) and bFGF (p=0.001), and reduced values of ANGP1/ANGP2 ratio (p<0.01) compared to healthy controls (n=36). Patients with a diffuse pattern were the only patients who had increased ANG compared to controls (p=0.004). Patients with diffuse pattern had higher levels of serum VEGF and VEGF-A compared to patients with normal (p=0.028 and p=0.006, respectively) and focal pattern (p=0.009 and p<0.01, respectively) and borderline elevated ANG compared to normal pattern (p=0.06). Myeloma patients had increased values of sRANKL (p<0.001), sRANKL/OPG ratio (p=0.004), NTX (p<0.001), CTX (p<0.001), TRACP-5b (p<0.001), Dkk-1 (p=0.01), and OPN (p<0.001) and borderline reduced values of bALP (p=0.09) compared to controls. Patients with normal MRI pattern showed no difference in terms of Dkk-1 and bone formation markers compared to controls. Patients with diffuse pattern of marrow involvement had reduced values of bone formation markers (p=0.01), while patients with focal pattern had only a borderline reduction of OC (p=0.09). Both diffuse and focal MRI patterns had increased Dkk-1 levels. All abnormal MRI patterns had increased levels of NTX, CTX and TRACP-5b and increased levels of osteoclast stimulators sRANKL/OPG, MIP-1alpha and OPN. Diffuse and variegated patterns of infiltration correlated with greater bone marrow plasma cell infiltration (p=0.043) and advanced ISS stage (p=0.018). All patients received a combination regimen that contained at least one novel anti-myeloma agent (IMiD and/or bortezomib) at some point during the course of their disease. The median follow-up of the patients was 25 months. Patients with diffuse or variegated pattern had a median survival of 29 months, patients with focal pattern had a median survival of 62 months, while the median survival of patients with normal MRI pattern has not been reached at the time of the analysis (p=0.009). Multivariate analysis showed that only MRI pattern and ISS stage were independent prognostic factors for survival. In conclusion, our analysis provides strong evidence that diffuse MRI pattern of marrow involvement correlates with increased angiogenesis, adverse disease features and inferior survival in MM patients who were treated with novel agents. Such patients may be considered for upfront treatment with combinations of novel agents. Our results highlight the important prognostic value of MRI in MM and support its routine use in MM patients. Disclosures:No relevant conflicts of interest to declare.

IL-12 inhibits TNF-α induced osteoclastogenesis via a T cell-independent mechanism in vivo

It has been reported that TNF-α plays an important role in bone resorption in pathological conditions. IL-12, which is a T cell mediator, is also an important inflammatory cytokine. We previously reported that IL-12 induces apoptosis in bone marrow cells treated with TNF-α in vitro via an interaction between TNF-α-induced Fas and IL-12-induced Fas ligand (FasL), and that, as a result, osteoclastogenesis is inhibited. The purpose of this study was to investigate the effects of IL-12 on TNF-α-mediated osteoclastogenesis in vivo. We administered TNF-α with and without IL-12 into the supracalvaria in mice. The numbers of osteoclasts in the sutures in the calvaria were higher in mice administered TNF-α than in control mice not administered TNF-α. The numbers of osteoclasts in mice administered both TNF-α and IL-12 were lower than those in mice administered only TNF-α. Next, we determined the levels of mRNAs for cathepsin K and tartrate-resistant acid phosphatase (TRAP). mRNA levels were increased in mice administered TNF-α compared with control mice, but not in mice administered both TNF-α and IL-12. We also evaluated the amounts of tartrate-resistant acid phosphatase 5b (TRACP 5b) in mouse sera. The levels of TRACP 5b in mice administered TNF-α were higher than those in control mice. On the other hand, in mice administered both TNF-α and IL-12, the levels were lower than those in mice administered TNF-α alone. Fas and FasL expression levels were analyzed by real-time RT-PCR. The levels of Fas mRNA were increased in the calvaria of mice administered TNF-α compared with control mice, while those of FasL mRNAs were increased in the calvaria of mice administered IL-12. In TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assays, many apoptotic cells were found in the sutures in the calvaria of mice administered both TNF-α and IL-12. IL-12 also inhibited TNF-α-induced osteoclastogenesis in mice whose T cells were blocked by anti-CD4 and anti-CD8 antibodies. These results suggest that IL-12 inhibits TNF-α-mediated osteoclastogenesis and induces apoptotic changes through an interaction between TNF-α-induced Fas and IL-12-induced FasL, in vivo, via a T cell-independent mechanism.

Epimedium-derived flavonoids promote osteoblastogenesis and suppress adipogenesis in bone marrow stromal cells while exerting an anabolic effect on osteoporotic bone

Background Epimedium-derived flavonoids (EFs) have been reported to prevent bone loss in ovariectomized (OVX) rats and late postmenopausal women but the underlying mechanism of the anabolic effect is unknown. Objective This study aimed to investigate the effect of EFs on osteoporotic bone using histomorphometry and on osteoblastogenesis/adipogenesis of bone marrow stromal cells (BMSCs). Methods 11-month-old female Wistar rats were divided into Sham, OVX, Sham + soluble vehicle (Sham + SV), OVX + SV and OVX + EFs (10 mg/kg/day) groups. 3 months after surgery, rats from the first two groups were euthanized to verify the establishment of OVX-induced osteoporosis. Other groups were orally treated with either daily SV or EFs for 4 months. At sacrifice, serum was analyzed for the levels of osteocalcin and TRACP 5b, BMD in the proximal femur was measured by pQCT. Static and dynamic bone histomorphometry was performed in proximal tibiae with microCT and undecalcified sections, respectively. The effect of EF treatment on differentiation of rat BMSCs was assessed by colony formation assays and gene expression analysis, respectively. Gene expression, ALP activity and adipocyte numbers were determined in differentiating human BMSCs after exposure to conditioned serum from SV- or EFs-treated OVX rats. Results The serum level of osteocalcin was higher and TRACP 5b was lower in EFs versus SV-treated OVX rats. BMD, BV/TV, Tb.N and Conn.D in EFs-treated OVX rats were significantly greater than those of SV-treated OVX rats. Bone histomorphometric parameters OS/BS, MAR, and BFR/BS were significantly higher in EFs versus SV-treated OVX rats. EFs significantly increased osteogenesis and decreased adipogenesis of BMSCs, as evidenced by CFU-ALP and CFU-Adipo assays, respectively. The mRNA level of Runx2 and bone sailoprotein was significantly higher while PPARγ2 was significantly lower in BMSCs from EFs-treated versus SV-treated OVX rats. ALP activity and Runx2 mRNA was significantly higher while adipocyte number and PPARγ2 mRNA was significantly lower in human BMSCs after exposure to conditioned serum from EFs versus SV-treated OVX rats. Conclusion EFs exerted anabolic effect on osteoporotic bone by concomitantly promoting osteogenic and suppressing adipogenic differentiation of BMSCs.

Bone loss in survival motor neuron (Smn−/−SMN2) genetic mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is characterized by degenerating lower motor neurons and an increased incidence of congenital bone fractures. Survival motor neuron (SMN) levels are significantly reduced due to deletions/mutations in the telomeric SMN1 gene in these patients. We utilized the Smn(-/-) SMN2 mouse model of SMA to determine the functional role for SMN in bone remodelling. microCT analysis of lumber vertebrae, tibia and femur bones from SMA mice revealed an osteoporotic bone phenotype. Histological analysis demonstrated a thin porous cortex of cortical bone and thin trabeculae at the proximal end of the growth plate in the vertebrae of SMA mice compared to wild-type mice. Histochemical staining of the vertebrae showed the presence of abundant activated osteoclasts on the sparse trabeculae and on the endosteal surface of the thin cortex in SMA mice. Histomorphometric analysis of vertebrae from SMA mice showed an increased number of osteoclasts. Serum TRAcP5b and urinary NTx levels were elevated, consistent with increased bone resorption in these mice. SMA mice showed a significant decrease in the levels of osteoblast differentiation markers, osteocalcin, osteopontin and osterix mRNA expression; however, there were no change in the levels of alkaline phosphatase expression compared to WT mice. SMA mouse bone marrow cultures revealed an increased rate of osteoclast formation (54%) and bone resorption capacity (46%) compared to WT mice. Pre-osteoclast cells from SMA mice showed constitutive up-regulation of RANK receptor signalling molecules critical for osteoclast differentiation. Our results implicate SMN function in bone remodelling and skeletal pathogenesis in SMA. Understanding basic mechanisms of SMN action in bone remodelling may uncover new therapeutic targets for preventing bone loss/fracture risk in SMA.

β-Arrestin2 Regulates RANKL and Ephrins Gene Expression in Response to Bone Remodeling in Mice

PTH-stimulated intracellular signaling is regulated by the cytoplasmic adaptor molecule beta-arrestin. We reported that the response of cancellous bone to intermittent PTH is reduced in beta-arrestin2(-/-) mice and suggested that beta-arrestins could influence the bone mineral balance by controlling RANKL and osteoprotegerin (OPG) gene expression. Here, we study the role of beta-arrestin2 on the in vitro development and activity of bone marrow (BM) osteoclasts (OCs) and Ephrins ligand (Efn), and receptor (Eph) mRNA levels in bone in response to PTH and the changes of bone microarchitecture in wildtype (WT) and beta-arrestin2(-/-) mice in models of bone remodeling: a low calcium diet (LoCa) and ovariectomy (OVX). The number of PTH-stimulated OCs was higher in BM cultures from beta-arrestin2(-/-) compared with WT, because of a higher RANKL/OPG mRNA and protein ratio, without directly influencing osteoclast activity. In vivo, high PTH levels induced by LoCa led to greater changes in TRACP5b levels in beta-arrestin2(-/-) compared with WT. LoCa caused a loss of BMD and bone microarchitecture, which was most prominent in beta-arrestin2(-/-). PTH downregulated Efn and Eph genes in beta-arrestin2(-/-), but not WT. After OVX, vertebral trabecular bone volume fraction and trabecular number were lower in beta-arrestin2(-/-) compared with WT. Histomorphometry showed that OC number was higher in OVX-beta-arrestin2(-/-) compared with WT. These results indicate that beta-arrestin2 inhibits osteoclastogenesis in vitro, which resulted in decreased bone resorption in vivo by regulating RANKL/OPG production and ephrins mRNAs. As such, beta-arrestins should be considered an important mechanism for the control of bone remodeling in response to PTH and estrogen deprivation.

Single-Dose, Randomized, Double-Blind, Placebo-Controlled Study of ACE-011 (ActRIIA-IgG1) in Postmenopausal Women

The effects of ACE-011 on safety, pharmacokinetics, and bone biomarkers were evaluated in healthy, postmenopausal women. Our data indicate that ACE-011 results in a sustained increase in biomarkers of bone formation and reduction in markers of bone resorption. The activin type IIA receptor (ActRIIA) is the high-affinity receptor for activin. ACE-011 is a dimeric fusion protein consisting of the extracellular domain of the human ActRIIA linked to the Fc portion of human IgG1. ACE-011 binds to activin, preventing activin from binding endogenous receptors. A randomized, double-blind, placebo-controlled study was conducted to evaluate the safety and tolerability of ACE-011. Forty-eight healthy, postmenopausal women were randomized to receive either a single dose of ACE-011 or placebo and were followed for 4 mo. Dose levels ranged from 0.01 to 3.0 mg/kg intravenously and from 0.03 to 0.1 mg/kg subcutaneously. Safety and pharmacokinetic (PK) analyses and the biological activity of ACE-011, as assessed by markers of bone turnover, and follicle stimulating hormone (FSH) levels were measured. No serious adverse events (AEs) were reported. AEs were generally mild and transient. The PK of ACE-011 was linear over the dose range studied, with a mean half-life of 24-32 days. The absorption after subcutaneous dosing was essentially complete. ACE-011 caused a rapid and sustained dose-dependent increase in serum levels of bone-specific alkaline phosphatase (BSALP) and a dose-dependent decrease in C-terminal type 1 collagen telopeptide (CTX) and TRACP-5b levels. There was also a dose-dependent decrease in serum FSH levels consistent with inhibition of activin. ACE-011 is a novel agent with biological evidence of both an increase in bone formation and a decrease in bone resorption. ACE-011 may be an effective therapy in a variety of diseases involving bone loss.

Overexpression of Bone Sialoprotein Leads to an Uncoupling of Bone Formation and Bone Resorption in Mice

The purpose of this study was to determine the effects of bone sialoprotein (BSP) overexpression in bone metabolism in vivo by using a homozygous transgenic mouse line that constitutively overexpresses mouse BSP cDNA driven by the cytomegalovirus (CMV) promoter. CMV-BSP transgenic (TG) mice and wildtype mice were weighed, and their length, BMD, and trabecular bone volume were measured. Serum levels of RANKL, osteocalcin, osteoprotegerin (OPG), TRACP5b, and PTH were determined. Bone histomorphometry, von Kossa staining, RT-PCR analysis, Western blot, MTS assay, in vitro mineralization assay, and TRACP staining were also performed to delineate phenotypes of this transgenic mouse line. Compared with wildtype mice, adult TG mice exhibit mild dwarfism, lower values of BMD, and lower trabecular bone volume. TG mice serum contained increased calcium levels and decreased PTH levels, whereas the levels of phosphorus and magnesium were within normal limits. TG mice serum also exhibited lower levels of osteoblast differentiation markers and higher levels of markers, indicating osteoclastic activity and bone resorption. H&E staining, TRACP staining, and bone histomorphometry showed that adult TG bones were thinner and the number of giant osteoclasts in TG mice was higher, whereas there were no significant alterations in osteoblast numbers between TG mice and WT mice. Furthermore, the vertical length of the hypertrophic zone in TG mice was slightly enlarged. Moreover, ex vivo experiments indicated that overexpression of BSP decreased osteoblast population and increased osteoclastic activity. Partly because of its effects in enhancing osteoclastic activity and decreasing osteoblast population, BSP overexpression leads to an uncoupling of bone formation and resorption, which in turn results in osteopenia and mild dwarfism in mice. These findings are expected to help the development of therapies to metabolic bone diseases characterized by high serum level of BSP.

In vivo impact of a 4 bp deletion mutation in the DLX3 gene on bone development

Distal-less 3 (DLX3) gene mutations are etiologic for Tricho-Dento-Osseous syndrome. To investigate the in vivo impact of mutant DLX3 on bone development, we established transgenic (TG) mice expressing the c.571_574delGGGG DLX-3 gene mutation (MT-DLX3) driven by a mouse 2.3 Col1A1 promoter. Microcomputed tomographic analyses demonstrated markedly increased trabecular bone volume and bone mineral density in femora from TG mice. In ex vivo experiments, TG mice showed enhanced differentiation of bone marrow stromal cells to osteoblasts and increased expression levels of bone formation markers. However, TG mice did not show enhanced dynamic bone formation rates in in vivo fluorochrome double labeling experiments. Osteoclastic differentiation capacities of bone marrow monocytes were reduced in TG mice in the presence of osteoclastogenic factors and the numbers of TRAP(+) osteoclasts on distal metaphyseal trabecular bone surfaces were significantly decreased. TRACP 5b and CTX serum levels were significantly decreased in TG mice, while IFN-γ levels were significantly increased. These data demonstrate that increased levels of IFN-γ decrease osteoclast bone resorption activities, contributing to the enhanced trabecular bone volume and mineral density in these TG mice. These data suggest a novel role for this DLX-3 mutation in osteoclast differentiation and bone resorption.

Trabecular Bone Deterioration in col9a1+/− Mice Associated With Enlarged Osteoclasts Adhered to Collagen IX–Deficient Bone

Short collagen IX, the exclusive isoform expressed by osteoblasts, is synthesized through alternative transcription of the col9a1 gene. The function of short collagen IX in bone was characterized in col9a1-null mutant mice. Trabecular bone morphometry of lumbar bones and tibias was evaluated by muCT and nondecalcified histology. Osteoblastic and osteoclastic activities were evaluated by PCR- and microarray-based gene expression assays and TRACP-5b and C-terminal telopeptide (CTX) assays, as well as in vitro using bone marrow stromal cells and splenocytes. The effect of col9a1(+/-) mutation on osteoclast morphology was evaluated using RAW264.7-derived osteoclastic cells cultured on the mutant or wildtype calvarial bone substrates. Col9a1 knockout mutation caused little effects on the skeletal development; however, young adult female col9a1(-/-) and col9a1(+/-) mice exhibited significant loss of trabecular bone. The trabecular bone architecture was progressively deteriorated in both male and female heterozygous col9a1(+/-) mice while aging. The aged mutant mice also exhibited signs of thoracic kyphosis and weight loss, resembling the clinical signs of osteoporosis. The col9a1(+/-) osteoblasts synthesized short col9a1 transcripts at decreased rates. Whereas bone formation activities in vitro and in vivo were not affected, the mutant osteoblast expressed the elevated ratio of RANKL/osteoprotegerin. Increased serum TRACP-5b and CTX levels were found in col9a1(+/-) mice, whose bone surface was associated with osteoclastic cells that were abnormally flattened and enlarged. The mutant and wildtype splenocytes underwent similar osteoclastogenesis in vitro; however, RAW264.7-derived osteoclastic cells, when cultured on the col9a1(+/-) calvaria, widely spread over the bone surface and formed large resorption pits. The surface of col9a1(+/-) calvaria was found to lack the typical nanotopography. The mineralized bone matrix deficient of short collagen IX may become susceptible to osteoclastic bone resorption, possibly through a novel non-cell-autonomous mechanism. The data suggest the involvement of bone collagen IX in the pathogenesis of osteoporosis.

Pharmacological Chaperone Therapy for the Treatment of Gaucher Disease: Results of Phase 1 Clinical Trials and a Clinical Ex Vivo Response Study with a Survey of Blood Markers for 53 Gaucher Patients.

Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme β-glucocerebrosidase (GCase). Deficient GCase activity leads to symptoms such as anemia, thrombocytopenia, hepatosplenomegaly, bone necrosis, infarcts and osteoporosis, and in some cases, neuropathic disease. The pharmacological chaperone AT2101 (isofagamine tartrate) selectively binds and stabilizes GCase in the ER and increases trafficking of the enzyme to the lysosome. In single- and repeat-dose Phase 1 clinical trials involving 72 healthy volunteers, AT2101 was well tolerated with no serious adverse events. In the repeat-dose study, a dose-dependent increase in GCase levels in white blood cells (up to 3.5-fold) was observed during the 7 day treatment period, and enzyme levels remained elevated for more than a week after removal of the drug. To evaluate the effects of AT2101 on a range of different GCase variants, we conducted an ex vivo response study using macrophages and EBV-transformed lymphoblasts derived from GD patients. The study was conducted on samples from 53 patients enrolled at 5 sites in the United States. The study included 26 males and 26 females with type I GD, and one male with type III GD. Patients ranged in age from 7 to 83 years; 50 of 53 patients were receiving imiglucerase and blood was drawn prior to enzyme infusion. Incubation of cells with AT2101 (5 days) increased GCase levels in macrophages or lymphoblasts derived from 52 of 53 patients representing 18 different genotypes (mean: 2.6-fold, range: 1.4- to 8.6-fold). Plasma was also screened for potential biomarkers associated with inflammation, bone metabolism, multiple myeloma and neurodegeneration. Analysis of 40 markers showed elevated levels of chitotriosidase activity, TRACP 5b, PARC, IL-8, IL-17, VEGF, MIP-1α and α-synuclein and reduced bone-specific alkaline phosphatase levels in some patients. These results show that an imbalance between osteoclast and osteoblast activities may remain even though treatment with imiglucerase (Wenstrup et al. 2007. Journal of Bone and Mineral Research , 22: 119–26) and bisphosphonates (Wenstrup et al. 2004. Blood , 104: 1253–7) have been shown to increase bone mass in GD patients. Interestingly, increases in IL-8, IL-17, VEGF, MIP-1α, impaired osteoblast activity and increased osteoclast activity have also been implicated in the pathogenesis of multiple myeloma, and it has been reported that GD patients have an increased risk of developing multiple myeloma (Rosenbloom et al. 2005. Blood , 105: 4569–72). To determine if these biomarkers respond to treatment with AT2101, they are being monitored in an ongoing 6-month Phase 2 clinical trial with AT2101 in GD patients. Additionally, a 4-week Phase 2 clinical trial with AT2101 is being conducted in GD patients and preliminary results are expected by the end of 2007.

A Crucial Role of Interleukin-6 in the Pathogenesis of Thyrotoxicosis-related Disturbances of Bone Turnover in Mice

Interleukin-6 (IL-6) has been shown to be involved in the pathogenesis of several bone diseases characterized by a negative balance between bone resorption and formation. The aim of the study was to estimate serum markers of bone turnover: osteoclast-derived tartrate-resistant acid phosphatase form 5a (TRACP 5b) reflecting resorption, and osteocalcin as a marker of bone formation in IL-6 knock-out mice to assess the role of IL-6 in the pathogenesis of thyrotoxicosis-related disturbances of bone metabolism. The study was performed on forty, 14-15 weeks old, female mice: C57BL/6J (wild-type; WT) and C57BL/6J (IL6-/-Kopf) (IL-6 knock-out; IL6KO). The mice were randomly divided into 4 groups with 10 mice in each one: 1. WT mice in thyrotoxicosis (WT-thx), 2. WT controls (WT-ctrl), 3. IL6KO mice with thyrotoxicosis (IL6KO-thx), and 4. IL6KO controls. Experimental model of hyperthyroidism was induced by intraperitoneal injection of levothyroxine at a dose of 1 microg/g, daily over 21 days. The serum levels of TRACP 5b and osteocalcin were determined by ELISA. Serum concentration of TRACP 5b (median and interquartile ranges) were significantly increased in both groups of mice with thyrotoxicosis: WT [28.2(18.8-41.6) U/l] and IL6KO [26.4(23.0-31.2) U/l] as compared to the respective controls. Osteocalcin serum levels in IL6KO-thx mice [111.9(103.1-175.6) ng/ml] were significantly elevated in comparison to WT-thx animals [46.1(32.5-58.9) ng/ml]. In summary, the results of the present study suggest that IL-6 plays a crucial role in thyrotoxicosis-related disturbances of bone turnover in mice, determining the imbalance between bone resorption and bone formation caused by excess of thyroid hormones predominantly by inhibition of bone formation.

Effects of Calcitriol on Type 5b Tartrate-Resistant Acid Phosphatase and Interleukin-6 in Secondary Hyperparathyroidism

Background/Aims: Secondary hyperparathyroidism (SHP) is characterized by high bone turnover and elevated serum bone remodeling markers. Elevation of serum interleukin-6 (IL-6) levels is also characteristic of end-stage renal disease. This study investigates the effects of intravenous calcitriol on serum bone resorptive markers, namely, type 5b tartrate-resistant acid phosphatase (TRACP5b) and IL-6 in patients with SHP. Methods: Intravenous calcitriol therapy was given for 16 weeks to 24 patients on maintenance hemodialysis with plasma intact parathyroid hormone (iPTH) levels >300 pg/ml. Blood was drawn at baseline and every 4 weeks for 16 weeks for determination of the levels of biochemical parameters, iPTH, IL-6 and bone remodeling markers, including bone-specific alkaline phosphatase (bAP) and TRACP5b. Results: Only 21 patients responded to the calcitriol therapy, with significant decrements in serum iPTH after 4 weeks of therapy and thereafter. After 16 weeks of calcitriol therapy, 21 patients had significant decrements in serum iPTH (707.9 ± 317.8 vs. 205.0 ± 63.1 pg/ml, p < 0.01). Prior to treatment, a significant correlation was found between increased levels of serum iPTH and IL-6 levels (r = 0.45, p < 0.05). After treatment, there was also a significant and parallel lowering of levels of serum iPTH, IL-6 (8.52 ± 3.59 vs. 7.24 ± 2.81 pg/ml, p < 0.01), bAP (54.68 ± 36.17 vs. 24.55 ± 13.84 U/l, p < 0.01) and TRACP5b (3.41 ± 1.89 vs. 1.80 ± 0.55 U/l, p < 0.01). Our results additionally showed significant positive correlationsbetween baseline levels of serum IL-6 and those of iPTH, bAP and TRACP5b. After 16 weeks of calcitriol treatment, the correlation between IL-6 and iPTH levels lost significance but levels of serum IL-6, bAP and TRACP5b remained significantly correlated. Conclusions: Elevated levels of serum IL-6 and bone remodeling markers, namely, bAP and TRACP5b which are common features of SHP, are effectively suppressed by calcitriol therapy. This indicates that hyperparathyroidism not only accelerates bone remodeling but may also aggravate inflammation in patients on maintenance hemodialysis.

Association of 12 serum biochemical markers of angiogenesis, tumour invasion and bone turnover with bone metastases from breast cancer: a crossectional and longitudinal evaluation

Complex biological pathways including angiogenesis, invasion, osteoclastic activation and bone matrix degradation are involved in the formation of bone metastasis (BM). The aim of our study was to investigate the cross-sectional and longitudinal associations of a panel of 12 serum biochemical markers reflecting biological pathways underlying BM development. In a cross-sectional study, we investigated 29 patients with primary breast carcinoma without BM (BC/BM−), 28 patients with breast carcinoma and BM (BC/BM+) and 15 healthy women. In longitudinal analyses, we investigated 34 patients for whom serum was obtained a two different time points: at the time of primary BC diagnosis and after a median time of 3 years. During this follow-up, 15 patients developed BM, whereas the other 19 remained free of BM. In patients who developed BM, the second samples were obtained before BM was documented by bone scan. The cross-sectional analyses have shown all biochemical markers to be significantly elevated in patients with BM, when compared to the patients without BM and healthy controls, except TGFβ1 that was significantly decreased. Multivariable analyses showed that only the bone resorption markers TRACP 5b, CTX and ICTP, and the marker of angiogenesis VEGF were independently associated with BM. Those markers correctly distinguished 85% of BC patients with or without BM from normal individuals. Longitudinal analyses showed that patients with primary BC who developed BM during follow-up had higher levels of TRACP5b (+95%, P=0.08) at the time of primary diagnosis, those patients had also a higher increases of ICTP (P=0.006), MMP-7 (P=0.004) and TIMP-1 (P=0.017) during follow-up than patients who did not progress toward bone metastasis. This study provides evidence of increase and interrelationship of circulating markers of angiogenesis, invasion and bone resorption in patients with BC with and without BM. Markers of bone resorption have the highest independent diagnostic value for detecting and potentially predicting BM in breast carcinoma patients.

Evaluation of the Activity of Tartrate-resistant Acid Phosphatase Isoform 5b in Normal Chinese Children - A Novel Marker for Bone Growth

Most parents are very concerned about the height of their children. Biochemical markers of bone formation and resorption may provide useful clinical predictors for bone growth. Tartrate-resistant acid phosphatase 5b (TRAcP 5b) has been advocated as a biomarker of osteoclast activity and bone resorption. However, the TRAcP 5b levels of children at different ages are still unknown. It is necessary to accumulate and analyze the data for healthy children at different ages. We use an improved immunoassay for bone TRAcP 5b to examine sera from children to see whether it is a significant marker of bone growth. Serum, including cord blood, was collected from 404 normal Chinese children (age range 0-17 years; 225 male, 179 female). The venous blood was withdrawn from the peripheral vein and stored at 4 degrees C before centrifugation for serum collection. All sera were stored at -70 degrees C and thawed at 37 degrees C immediately before TRAcP 5b levels were measured. Bone-specific alkaline phosphatase (BAP) was also used for comparison with TRAcP 5b levels at different ages. TRAcP 5b levels were extremely high in infants of both genders, gradually decreasing with age (p <0.001). A second peak in TRAcP 5b values occurred at 12-13 and 10-11 years in males and females, respectively (p <0.001). Age alone, as well as age-related changes between the male and female groups, were independent predictors of TRAcP 5b levels (p <0.001). There was no significant between-gender difference in serum TRAcP 5b levels for any age group (p = 0.682). BAP values did not show a significant second peak in females. Age and gender alone, as well as the age-related changes between male and female groups, were independent predictors of BAP values (p <0.001). Preliminary results were established for serum TRAcP 5b and BAP values of normal Chinese children of different ages. Elevated serum TRAcP 5b values were observed during infancy and puberty for both genders. The pattern of this age-related change in serum TRAcP 5b levels is similar to the shape of the standard height velocity curve for healthy children. Values of BAP were less specific than TRAcP 5b. These data may prove valuable as a normal reference in future research about bone markers.