TRACP Activity Research Articles

Osteogenic Differentiation of Human Gingival Fibroblasts Inhibits Osteoclast Formation.

Gingival fibroblasts (GFs) can differentiate into osteoblast-like cells and induce osteoclast precursors to differentiate into osteoclasts. As it is unclear whether these two processes influence each other, we investigated how osteogenic differentiation of GFs affects their osteoclast-inducing capacity. To establish step-wise mineralization, GFs were cultured in four groups for 3 weeks, without or with osteogenic medium for the final 1, 2, or all 3 weeks. The mineralization was assessed by ALP activity, calcium concentration, scanning electron microscopy (SEM), Alizarin Red staining, and quantitative PCR (qPCR). To induce osteoclast differentiation, these cultures were then co-cultured for a further 3 weeks with peripheral blood mononuclear cells (PBMCs) containing osteoclast precursors. Osteoclast formation was assessed at different timepoints with qPCR, enzyme-linked immunosorbent assay (ELISA), TRAcP activity, and staining. ALP activity and calcium concentration increased significantly over time. As confirmed with the Alizarin Red staining, SEM images showed that the mineralization process occurred over time. Osteoclast numbers decreased in the GF cultures that had undergone osteogenesis. TNF-α secretion, a costimulatory molecule for osteoclast differentiation, was highest in the control group. GFs can differentiate into osteoblast-like cells and their degree of differentiation reduces their osteoclast-inducing capacity, indicating that, with appropriate stimulation, GFs could be used in regenerative periodontal treatments.

Effect of photoperiod on eggshell quality and quality characteristics of tibia, femur, and ulna in laying ducks

We investigated the effect of photoperiod on eggshell quality, bone quality characteristics and bone metabolism related enzymes and factors in laying ducks. After adaption, 300 Jinding laying ducks (252-day-old) were randomly divided into 5 treatments, receiving 12L (hours of light):12D (hours of darkness), 14L:10D, 16L:8D, 18L:6D or 20L:4D, respectively. Each group had 6 replicates of 10 birds each. The feeding experiment lasted 8 wk. Compared with 12L:12D, the higher values of eggshell breaking strength occurred in ≥18 h photoperiods at the end of 6 wk, and in ≥16 h photoperiods at the end of 4 wk, with the common highest values in 18 h photoperiod (P ˂ 0.05). Besides, 18L:6D had higher values of ultimate load Fu and cortical cross-sectional area A in tibia, femur, and ulna (P ˂ 0.05), compared with 12L:12D. The higher values of proximal bone mineral content (BMC; tibia), distal BMC (ulna), total Ca (tibia), and cortical volumetric bone mineral density (vBMC; tibia and ulna) were observed in 16L:8D and 18L:6D treatments (P ˂ 0.05). Meanwhile, 18 h photoperiod group had the higher proximal BMC (femur) and total Ca in ulna (P ˂ 0.05). In serum, compared with 12L:12D group, the higher ALP activity occurred in ≥16 h photoperiods (0:00 and 18:00), with the highest values in 18L:6D treatment (P ˂ 0.05); the higher values of TGF-β (6:00) and OC (6:00 and 18:00) were simultaneously observed in 18 h photoperiod (P ˂ 0.05). Moreover, values of trACP activity, TNF-α and IL-6 contents decreased in ≥18 h photoperiods at 0:00 (P ˂ 0.05), compared with 12L:12D group. To sum up, an appropriate photoperiod could improve eggshell quality, bone strength and mineral content through increasing osteogenesis during the light time and decreasing resorption activity during the dark, and 18 h is an adequate photoperiod for the eggshell and bone quality of laying ducks.

Dynamics of biochemical bone and endothelial parameters during the replacement of bone defects in dogs with hydroxyapatite ceramic doped with silicon

Metal structures for osteosynthesis available in veterinary orthopedics are not able to compensate for the lost elements of bone tissue in complex splinter fractures. It is prompt the use of hydroxyappatite materials that replaced bone defects for maintenance of osteoconductive function, and ideally would combine osteointegration and osteoinductive properties. However, their influence on the biological processes of fracture consolidation which go through a number of successive stages and end with the formation of bone tissue in the fracture zone identical to the maternal, is insufficiently substantiated according to the criteria of the molecular biological phase of reparative osteogenesis. The aim of the study was to investigate the dynamics of biochemical osteotropic parameters and the level of NO using silicon-doped ceramics for fractures heeling in dogs. Materials and methods. The animals suffering of fractures that were admitted to the faculty clinic were divided into control (n=7) and experimental (n=7) groups. In both groups, extracortical osteosynthesis was performed with a support plate from an unalloyed titanium alloy. In the control group, bone defects were left to heal under spontaneous blood clot, and in the experimental group, they were replaced with ceramic based on hydroxyapatite with β-tricalciumphosphate doped with silicon (HA/β-TCP/l-Si–3).Blood samples were taken after the injury no later than the 48th day, and on the 3th, 12th, 21th, 42th and 60th days after osteosynthesis. To increase the objectivity of the biochemical analysis, we additionally formed a group of clinically healthy dogs that were admitted to the clinic for routine vaccination (n=10). It included the spectrophotometric determination of the content of NO, BALP, TRACP, Ca, P, Mg, total protein in blood serum, and fibrinogen in blood plasma. Research results. A clinical study showed that in the case of using HA/β-TCP/l-Si–3for splinter fractures, the stages of reparative osteogenesis are more optimized in time, and their consolidation occurs on average 19 days earlier than in the control group. The results of the biochemical study showed that when using HA/β-TCP/l-Si–3, it is accompanied by a peak NO value already on the third day, which is significantly higher than in the control group and indicates early angiogenesis in the research group. In terms of TRACP, the period of osteoresorption in the control group was permanent with little expressed peaks of activity. However, in the research group, the peak of TRACP activity is limited to 12 and 21 days, which is evidence of an optimized inflammatoryresorptive phase. In parallel with this, the activity of BALP increases, which indicates the consistency of the stages of reparative osteogenesis and provides an optimized and accelerated consolidation of fractures in the research group. Conclusion. The dynamics of NO, BALP and TRACP pathochemically substantiates the optimized reparative osteogenesis when using HA/β-TCP/l-Si–3 for bone defects replacement in cases of splinter fractures of tubular bones. Key words: bone markers, bone isoenzyme of alkaline phosphatase, NO, tartrate-resistant acid phosphatase, fibrinogen, calcium, phosphorus.

SUN-347 Glucagon-like Peptide 1 (GLP-1) Acts Directly On Human Osteoclasts To Increase Differentiation And Bone Resorptive Activity

Glucagon-like peptide 1 (GLP-1) is an intestinal hormone released in response to nutrient intake that promotes glucose-dependent insulin secretion by acting upon the pancreatic GLP-1 receptor (GLP-1R). GLP-1R agonists (GLP-1RAs) are widely used in treatment of type 2 diabetes. Preclinical data indicate that GLP-1RAs could be repurposed to treat low bone mass as GLP-1R-depleted mice have higher bone resorption and thinner cortical bones, while insulinopenic and insulin resistant rats have improved bone formation and reduced bone mass deterioration when treated with GLP-1 or GLP-1RAs. However, the effect of GLP-1 and GLP-1RAs on human bone cells remains undetermined. We aimed to elucidate the effect of GLP-1 on primary human osteoclast (OC) and osteoblast (OB) cultures. OCs were differentiated over 10 days from human blood-derived CD14+ monocytes and OBs over 4–6 weeks from human bone. Cells were seeded on bovine bone slices and studies performed using fetal bovine serum, MCSF and RANKL (OC monocultures) or MCSF only (OB monocultures and OB+OC co-cultures). We first investigated the effect of GLP-1 on bone resorptive activity of mature OCs on bovine bone slices. GLP-1 increased the eroded bone surface percentage compared to vehicle in both OC monocultures (1nM P=0.002; 10nM P=0.023; n=8 donors) and OC+OB co-cultures (1nM P=0.013; 10nM P=0.012; n=8 donors). We then tested the effects of GLP-1 on osteoblast activity in OC+OB co-cultures by measuring alkaline phosphatase (ALP). We found that GLP-1 increased ALP in OC+OB cultures (1nM, P=0.049; 10nM, P=0.019) and these effects were reversed by the GLP-1R antagonist exendin 9–39 (1nM, P=0.93, 10nM, P=0.64). However, in OB monocultures GLP-1 had no effects on ALP (1nM P=0.93, 10nM P=0.64) indicating a GLP-1-driven increase in osteoblast activity through osteoclast-osteoblast coupling. We then assessed the effect of GLP-1 on OC differentiation by assessing TRAcP activity. Although there was a trend towards increased TRAcP activity upon stimulation with GLP-1 on day 10 of osteoclastogenesis, this was not statistically significant (1nM P=0.12; n=8 donors; 10nM P=0.29, n=4 donors). Our studies indicated GLP-1 may have a direct effect on osteoclasts, and we therefore sought to characterise GLP-1-mediated signalling in these cells. We assessed the effect of GLP-1 on cAMP signalling using LANCE assays and assessed phosphorylation of ERK proteins by Western blot analysis in human OC cultures. OCs treated with 10nM GLP-1 for 30 minutes had increased cAMP signaling (P=0.004, n=12 bone slices from 2 donors) when compared to vehicle. Furthermore, 10nM GLP-1 induced rapid increases in phosphorylated ERK (P=0.03 following 2 minutes exposure, n=4 blots). In conclusion, our studies reveal that GLP-1 increases activity in primary mature human OCs, and OBs, via OCs. Our signaling studies in OCs indicate this is mediated by direct action of GLP-1 on human bone cells.

Abeliophyllum distichum Nakai alleviates postmenopausal osteoporosis in ovariectomized rats and prevents RANKL-induced osteoclastogenesis in vitro

Ethnopharmacological relevanceAbeliophyllum distichum Nakai (AD), called Miseon, is one of Korea's monotypic endemic species. As a folk remedy, the AD has been used to treat inflammatory disease, stomachaches, diarrhea, and gynecologic disease in Korea. Some researchers have reported that the AD has anti-cancer, anti-inflammatory, and anti-oxidant effects. But the protective effect of AD leaf for osteoporosis has not been reported yet. Aim of the studyThis study aimed to analyze the effects and mechanism of AD-ethyl acetate fraction (EA) extract on the osteoporosis, one of the gynecologic disease. Materials and methodsThe RAW 264.7 cells were used as a model for RANKL-induced osteoclastogenesis. We measured the TRAcP activity, expressions of NFATc1, c-fos, and MAPK to investigate the effect of AD-EA. OVX-induced osteoporosis rat model was used as menopausal osteoporosis. After both ovaries were removed through a surgical procedure, and AD-EA or 17b-estradiol was orally administered for 8 weeks. BMD of femurs was measured as well as the bone morphometric parameter, such as BV/TV, trabecular thickness, number and surface using a micro CT. ResultsAD-EA significantly inhibited TRAcP activity, actin ring formation, pit formation and the expressions of osteoclast-related genes in a dose-dependent manner through the inhibition of the MAPK and c-fos/NFATc1 pathway. In addition, low dose administration of AD-EA improved the deterioration of trabecular bone microarchitecture caused by OVX through the inhibition of bone resorption by TRAcP and CTK. ConclusionsThese results suggest that AD-EA may contribute to the therapy of osteoporosis caused by menopause in women.

Combined low-dose LiCl and LY294002 for the treatment of osteoporosis in ovariectomized rats

BackgroundTo provide a low-toxicity and high-efficacy clinical treatment for osteoporosis via a novel combination of LiCl and LY294002.MethodsThe protein levels of p-AKT, AKT, p-GSK3β, GSK3β, β-catenin, p-β-catenin, and NFATC1 were measured in osteoblasts and osteoclasts by Western blot. ALP activity and TRACP activity were measured using the corresponding kit. The levels of BALP, PINP, CTX, and TRACP-5b were determined in accordance with the requirements of the ELISA kits. Microstructural analysis was performed on the left distal femur using microcomputed tomography.ResultsTreatment with the combination of LiCl and LY294002 led to a markedly increased osteoblast activity but significantly decreased osteoclast differentiation and bone absorption capacity compared with the treatment with LiCl or LY294002 alone (P < 0.01). In serum, the low-dose combination of LiCl and LY294002 significantly enhanced BALP levels (P < 0.01) and significantly decreased PINP, TRACP-5b, and CTX levels (P < 0.01) compared with the application of either drug alone.ConclusionsThis study indicates that drug combinations directed at multiple targets could be used for osteoporosis treatment by promoting osteoblast proliferation and inhibiting differentiation with high efficiency.

Osteoclastogenesis Behavior of Zirconia for Dental Implant.

Zirconia is worth studying as an alternative to dental titanium implants to overcome the disadvantages of titanium. This study investigated the surface characteristics of the zirconia implant material and osteoclastogenesis responses on the surface compared with titanium. Yttrium oxide-stabilized 5% tetragonal zirconia polycrystalline specimens were manufactured, and osteoclast-precursor cells were cultured and differentiated into osteoclasts on the specimens. Surface shape, roughness, and chemical composition were evaluated. After culturing, cell morphologies and differentiation capacity were analyzed using tartrate-resistant acid phosphatase activity (TRACP). mRNA of two critical transcription factors, nuclear factor of activated T-cells 1 (NFATc1) and c-Fos were measured, and protein levels of NFATc1 and c-Fos were investigated. The zirconia specimens had rhomboid-like shapes with smooth surfaces and exhibited no difference in surface roughness compared to the titanium specimens. Morphologies of differentiated osteoclasts on both materials were similar. TRACP activity on the zirconia showed comparable results to that on the titanium. The mRNA value of NFATc1 on the zirconia was higher than that on the titanium at day four. The protein level of c-Fos was expressed thicker on the zirconia when compared to the titanium at day two. The results of this study suggest that zirconia material provides adequate osteoclastogenesis behaviors for dental implant use.

IL1β and TNFα promote RANKL-dependent adseverin expression and osteoclastogenesis.

Adseverin is an actin-binding protein involved in osteoclastogenesis, but its role in inflammation-induced bone loss is not well-defined. Here, we examined whether IL1β and TNFα regulate adseverin expression to control osteoclastogenesis in mouse primary monocytes and RAW264.7 cells. Adseverin was colocalized with subcortical actin filaments and was enriched in the fusopods of fusing cells. In precursor cells, adseverin overexpression boosted the formation of RANKL-induced multinucleated cells. Both IL1β and TNFα enhanced RANKL-dependent TRAcP activity by 1.6-fold and multinucleated cell formation (cells with ≥3 nuclei) by 2.6- and 3.3-fold, respectively. However, IL1β and TNFα did not enhance osteoclast formation in adseverin-knockdown cells. RANKL-dependent adseverin expression in bone marrow cells was increased by both IL1β (5.4-fold) and TNFα (3.3-fold). Luciferase assays demonstrated that this expression involved transcriptional regulation of the adseverin promoter. Activation of the promoter was restricted to a 1118 bp sequence containing an NF-κB binding site, upstream of the transcription start site. TNFα also promoted RANKL-induced osteoclast precursor cell migration. We conclude that IL1β and TNFα enhance RANKL-dependent expression of adseverin, which contributes to fusion processes in osteoclastogenesis.

Osteoclast Fusion: Time-Lapse Reveals Involvement of CD47 and Syncytin-1 at Different Stages of Nuclearity.

Investigations addressing the molecular keys of osteoclast fusion are primarily based on end‐point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multi‐nucleated cells, the number of nuclei per multinucleated cell or TRAcP activity. But end‐point analyses do not show how the fusion came about. This would not be a problem if fusion of osteoclasts was a random process and occurred by the same molecular mechanism from beginning to end. However, we and others have in the recent period published data suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore, we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed individual fusion events using time‐lapse and antagonists of CD47 and syncytin‐1. All time‐lapse recordings have been studied by two independent observers. A total of 1808 fusion events were analyzed. The present study shows that CD47 and syncytin‐1 have different roles in osteoclast fusion depending on the nuclearity of fusion partners. While CD47 promotes cell fusions involving mono‐nucleated pre‐osteoclasts, syncytin‐1 promotes fusion of two multi‐nucleated osteoclasts, but also reduces the number of fusions between mono‐nucleated pre‐osteoclasts. Furthermore, CD47 seems to mediate fusion mostly through broad contact surfaces between the partners’ cell membrane while syncytin‐1 mediate fusion through phagocytic‐cup like structure. J. Cell. Physiol. 232: 1396–1403, 2017. © 2016 Wiley Periodicals, Inc.

Regulation of osteoclast function by poly(ethylene sodium phosphate)

Event Abstract Back to Event Regulation of osteoclast function by poly(ethylene sodium phosphate) Yasuhiko Iwasaki1*, Sujit Kootala2* and Jöns Hilborn2* 1 Kansai University, Department of Chemistry and Materials Engineering, Japan 2 Uppsala University, Department of Chemistry, Sweden Introduction: An imbalance in bone resorption relative to bone formation is the origin of many metabolic bone diseases, including osteoporosis [1]. In the current field of clinical osteology, bisphosphonates (BPs), which reduce bone resorption, comprise a major class of drugs used in the treatment of osteoporosis. Although BPs are reliable molecules for bone treatment, medication-related osteonecrosis of the jaw has emerged as a serious complication in patients treated with high BP doses. Therefore, new therapeutic agents showing BP-like activity could be beneficial. Recently, we have clarified that polyphosphoester ionomers exhibited clearly superior physicochemical properties for mineralization and biocompatibility when compared with pamidronate [2]. Our hypothesis is that polyphosphoesters, in similarity to bisphosphonates, inhibit osteoclast growth without affecting osteoblasts. Therefore, we studied the effects of poly(ethylene sodium phosphate) (PEP·Na) treatment on bone slice-cultured osteoclasts. Materials and Method: Poly(ethylene sodium phosphate) (PEP·Na) was synthesized as previously reported [3]. Peripheral blood mononuclear cells (PBMCs) were seeded on one side of each bovine cortical bone slice placed in a 24-well cell culture insert; each culture contained a poly(ethylene terephthalate) (PET) trans well membrane and was maintained for 3 weeks with a change of medium every 3 days to induce monocyte differentiation into mature osteoclasts. Cultures of mature human osteoclasts on bone slices were exposed for 24 h to six different concentrations of PEP·Na to assess the toxicity. As a measure of cell viability the numbers of adherent cells were determined from microscopic images using Image J software. TRAcP activity was assessed with a leukocyte acid phosphatase kit. TRAcP-positive multinucleated cells were defined as cells with 3 or more nuclei. Results and Discussion: Optical microscopic data of osteoclasts incubated with different concentrations of PEP·Na are shown in Figure 1. The cells were stained for TRAcP and included giant cells. In the control, which was not treated with PEP·Na, many cells adhered to the bone slice and large, well-spread cells were observed. When a low concentration of PEP·Na was added to the cultivation medium, the number of mature osteoclasts that remained adhered increased, although many smaller adherent cells were also observed. By increasing the concentration of PEP·Na, the number of adherent cells decreased significantly, and their sizes were also reduced. Kanatani and colleagues reported that inorganic phosphate inhibited osteoclast differentiation both by upregulating osteoprotegrin (OPG) expression and through direct action on osteoclast precursor cells [4]. Polyphosphoester degradation is likely to increase the concentration of inorganic phosphate in the cultivation medium and upregulate both the intracellular osteoclast and extracellular inorganic phosphate concentrations. Conclusion: In the present study, the possibility of using polyphosphoesters for selective inhibition of human osteoclast functions was validated. Polyphosphoesters then have a potential to be used as a prodrug for treatment of osteoporosis. JSPS KAKENHI Grant Number 26107719

Zebrafish (Danio rerio) in calcium-poor water mobilise calcium and phosphorus from scales

Summary Calcium is of vital importance in vertebrates and plasma levels are tightly regulated. Terrestrial vertebrates depend for their calcium uptake exclusively on the diet, while fish have an essentially infinite directly accessible source of calcium in the surrounding water. The need to store calcium seems thus less evident in fish. It was the goal of the present study to investigate the possible role of the scale compartment of zebrafish (Danio rerio) as an accessible pool for calcium and phosphorus. We restricted the calcium availability to zebrafish both in water and diet. Following 12 days restriction, we found that scalar contents of calcium and phosphorus had declined, while magnesium was unaffected, indicating resorption of calcium hydroxyapatite. Osteoclast activity on scales of calcium-restricted fish increased, as we conclude from enlarged demineralized areas on scales, increased TRAcP activity staining, and higher ctsk gene expression. Osteoblasts respond to calcium limitation by increasing expression of col1a2 and alpl. In the presence of water and dietary calcium, removal of scales from one flank of the fish does not affect scalar mineral contents, scale demineralization and osteoclastic TRAcP activity; expression of sp7, alpl and ctsk are up-regulated. Apparently, a high demand for calcium in zebrafish is preferentially complemented with exogenous calcium, while calcium is recruited from the scales when environmental calcium is limited. We conclude that the scales represent an important deposit of available calcium and phosphorus from which these minerals can be recruited in periods of high demand. This finding contributes to a niche for scales as model in bone research.

Magnesium deficiency results in an increased formation of osteoclasts

Magnesium (Mg2+) deficiency is a frequently occurring disorder that leads to loss of bone mass, abnormal bone growth and skeletal weakness. It is not clear whether Mg2+ deficiency affects the formation and/or activity of osteoclasts. We evaluated the effect of Mg2+ restriction on these parameters. Bone marrow cells from long bone and jaw of mice were seeded on plastic and on bone in medium containing different concentrations of Mg2+ (0.8 mM which is 100% of the normal value, 0.4, 0.08 and 0 mM). The effect of Mg2+ deficiency was evaluated on osteoclast precursors for their viability after 3 days and proliferation rate after 3 and 6 days, as was mRNA expression of osteoclastogenesis-related genes and Mg2+-related genes. After 6 days of incubation, the number of tartrate resistant acid phosphatase-positive (TRACP+) multinucleated cells was determined, and the TRACP activity of the medium was measured. Osteoclastic activity was assessed at 8 days by resorption pit analysis. Mg2+ deficiency resulted in increased numbers of osteoclast-like cells, a phenomenon found for both types of marrow. Mg2+ deficiency had no effect on cell viability and proliferation. Increased osteoclastogenesis due to Mg2+ deficiency was reflected in higher expression of osteoclast-related genes. However, resorption per osteoclast and TRACP activity were lower in the absence of Mg2+. In conclusion, Mg2+ deficiency augmented osteoclastogenesis but appeared to inhibit the activity of these cells. Together, our in vitro data suggest that altered osteoclast numbers and activity may contribute to the skeletal phenotype as seen in Mg2+ deficient patients.

A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts

Osteoclasts are known to be important for the coupling process between bone resorption and formation.The aim of this study was to address when osteoclasts are anabolically active.Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional or non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine with or without diphyllin, E64 or GM6001. Osteoclasts numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2T3 was treated with 50% of CM or non-CM for 12days. Bone formation was assessed by Alizarin Red extraction.CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed little resorption, but still an induction of bone formation by osteoblasts. Mimicking the reduction in bone resorption using the V-ATPase inhibitor Diphyllin, the cysteine proteinase inhibitor E64 and the MMP-inhibitor GM6001 showed that CM from diphyllin and E64 treated osteoclasts showed reduced ability to induce bone formation compared to CM from vehicle treated osteoclasts, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities.In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.

P-0264 Carboxy-Terminal Telopeptide of Type I Collagene and Tartrate-Resistant Acid Posphatase Measurement in Patients with Colorectal Cancer and Bone Metastases

ABSTRACT Introduction Despite significant advances in detection and treatment, colorectal cancer (CRC) still remains one of the most prevalent cancers, and one of the leading causes of mortality due to malignancy. In patients with stage I-II CRC surgical resection usually represents the sole treatment, while in those with metastasized tumor (stage IV) any therapy is rarely successful. Unfortunately, up to 60% of patients with CRC undergoing primary surgery with curative intention die from metastatic disease. Thus, occult tumor cells, not detected preoperatively, likely colonize and remain vital in different tissues of these patients, such as lymph nodes, blood, and bone marrow. Usually, CRC metastasizes to the liver and lungs more frequently than to bone. Isolated bone metastasis (BM) is considered truly rare, but it has been observed that the improved survival for patients with metastatic CRC following expanded treatment options is associate with an increased incidence of BMs. Because metastases in uncommon sites often indicates the terminal phase of CRC, clinicians should be more vigilant about possible BMs. For this purpose, several serum biomarkers have been tested for early detection of BMs, such as carboxy-terminal telopeptide of type I collagen (ICTP), a cross-link product of collagen I degradation, and tartrate-resistant acid phosphatase 5b (TRACP), specifically derived from osteoclasts. Recent studies showed that TRACP activity and ICTP were increased in up to 90% of patients with breast cancer and BM. The aim of this study was to evaluate the usefulness of TRACP, ICTP, and bone alkaline phosphatase (BAP) measurements in patients with CRC and BM. Methods Fourteen patients (9 men, 5 women, mean age 56.1±4.8, range 49-63 years) with CRC and confirmed BMs (cases), and a group of 15 age- and gender matched (10 men, 4 women, age 57.1±4.9 years, p=0.08) patients (controls) without BM (negative F-18 FDG PET/CT) underwent serum TRACP, ICTP, and BAP measurements. Written informed consent was obtained from all the participants. TRACP and BAP were measured by two-site enzyme-linked immunosorbent assay (ELISA), while ICTP was measured by commercially available radioimmunoassay. The sensitivity and specificity of serum TRACP, ICTP, and BAP as a marker for BM were estimated by receiver operator characteristic (ROC) curves. Results The mean levels of TRACP, ICTP, and BAP (cases vs. controls) were: 5.9±1.6 vs. 4.8±1.3 U/L (95% CI 0.11-2.11, p=0.08), 6.9±1.4 vs. 5.9±1.3 U/L (95% CI 0.94-3.04, p=0.0003), and 82.6±18.2 vs. 79.3±16.2 (95% CI 9.81-16.39, p=0.59). ROC analysis established a cutoff value for ICTP of 4.51 U/mL to identify patients with extensive BM with a specificity of 97% and a sensitivity of 92% (area under the curve=0.97; 95% CI=0.95-0.98). A strong relationship was found only between TRACP and ICTP (R=0.95, p Conclusion Serum ICTP is a useful diagnostic marker in the detection of BMs in patients with CRC, more accurate than TRACP and BAP.

Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion

Generation of osteoclasts through fusion of mono-nucleated precursors is a key event of bone physiology and bone resorption is inefficient without osteoclast fusion. Several factors playing a critical role in the fusion process have already been recognized, but the factors involved in the actual fusion of the lipid bilayers of their cell membranes are still unknown. Syncytin-1 is a protein encoded by a human endogenous retroviral gene which was stably integrated into the human ancestor genome more than 24 million years ago. Upon activation, syncytin-1 is able to destabilize the lipid bilayer of the target cell and to force the merging of plasma membranes. This protein is a key player in the fusion of cytotrophoblasts. In the present study, syncytin-1 as well as its putative receptor ASCT2 was found to be expressed in differentiating osteoclasts in vitro, both on mRNA and protein level. This was documented through Q-PCR, Western blot and immunofluorescence analyses. These in vitro findings were confirmed by immunohistochemical stainings in human iliac crest biopsies. A syncytin-1 inhibitory peptide reduced the number of nuclei per osteoclast by 30%, as well as TRACP activity. From a mechanistic point of view, it is interesting that the distribution of syncytin-1 immunoreactivity on the cell surface parallels that of actin, another important player in cell fusion, and that cell–cell proximity induces particular patterns of distribution of syncytin-1 and actin in the respective cells. These complementary observations support a critical role of syncytin-1 in osteoclast fusion, which is of special interest in view of its well-known ability to force the merging of plasma membranes.

Alterations in osteoclast function and phenotype induced by different inhibitors of bone resorption - implications for osteoclast quality

BackgroundNormal osteoclasts resorb bone by secretion of acid and proteases. Recent studies of patients with loss of function mutations affecting either of these processes have indicated a divergence in osteoclastic phenotypes. These difference in osteoclast phenotypes may directly or indirectly have secondary effects on bone remodeling, a process which is of importance for the pathogenesis of both osteoporosis and osteoarthritis. We treated human osteoclasts with different inhibitors and characterized their resulting function.MethodsHuman CD14 + monocytes were differentiated into mature osteoclasts using RANKL and M-CSF. The osteoclasts were cultured on bone in the presence or absence of various inhibitors: Inhibitors of acidification (bafilomycin A1, diphyllin, ethoxyzolamide), inhibitors of proteolysis (E64, GM6001), or a bisphosphonate (ibandronate). Osteoclast numbers and bone resorption were monitored by measurements of TRACP activity, the release of calcium, CTX-I and ICTP, as well as by counting resorption pits.ResultsAll inhibitors of acidification were equally potent with respect to inhibition of both organic and inorganic resorption. In contrast, inhibition of proteolysis by E64 potently reduced organic resorption, but only modestly suppressed inorganic resorption. GM6001 alone did not greatly affect bone resorption. However, when GM6001 and E64 were combined, a complete abrogation of organic bone resorption was observed, without a great effect on inorganic resorption. Ibandronate abrogated both organic and inorganic resorption at all concentrations tested [0.3-100 μM], however, this treatment dramatically reduced TRACP activity.ConclusionsWe present evidence highlighting important differences with respect to osteoclast function, when comparing the different types of osteoclast inhibitors. Each class of osteoclast inhibitors will lead to different alterations in osteoclast quality, which secondarily may lead to different bone qualities.

A phase II clinical trial does not show that high dose simvastatin has beneficial effect on markers of bone turnover in multiple myeloma

Several studies have evaluated the impact of low dose statin (20-80 mg/day) on bone metabolism with inconclusive results despite promising data of preclinical studies. In this study, we investigated the effect of high dose simvastatin (HD-Sim) on biochemical markers of bone turnover and disease activity in six heavily pretreated patients with multiple myeloma (MM). These patients were treated with simvastatin (15 mg/kg/day) for 7 days followed by a rest period of 21 days in two 4-week cycles. Endpoints were changes in the level of biochemical markers of (i) osteoclast activity (tartrate resistant acid phosphatase, TRACP); (ii) bone resorption (collagen fragments CTX and NTX); (iii) bone formation (osteocalcin and aminoterminal propeptide of type I collagen PINP); (iv) cholesterol; (v) regulators of bone metabolism [osteoprotegerin (OPG) and Dickkopf-1 (DKK-1)] and (vi) disease activity (monoclonal proteins or free light chains in serum). TRACP activity in serum and levels of collagen fragments (NTX) in urine increased for all patients temporarily during the 7 days of treatment with HD-Sim indicating that osteoclasts may have been stimulated rather than inhibited. The other markers of bone metabolism showed no change. None of the patients showed any reduction in free monoclonal light chains or monoclonal proteins in serum during treatment with HD-Sim. In spite of the fact that bone turn over effects of HD-Sim may have been blunted by concomitant treatment of patients with other drugs we observed a transient increase in markers of osteoclast activity. This sign of a transient stimulation of osteoclast activity suggests that HD-Sim may be harmful rather than beneficial for MM patients. For this reason and because of gastro-intestinal side effects the study was stopped prematurely.

A New Heterozygous Mutation (R714C) of the Osteopetrosis Gene, Pleckstrin Homolog Domain Containing Family M (With Run Domain) Member 1 (PLEKHM1), Impairs Vesicular Acidification and Increases TRACP Secretion in Osteoclasts

We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast-osteoblast cross-talk. The gene PLEKHM1 encodes a nonsecretory adaptor protein that localizes to endosomal vesicles. A highly truncated Plekhm1 protein was previously found in a patient with intermediate autosomal recessive osteopetrosis. We describe a new heterozygous mutation in the PLEKHM1 gene in a patient presenting with low vertebral and femoral T-scores and areas of focal sclerosis. Clinical evaluation, mutational analysis, assessment of in vitro osteoclast morphology and activity, transfection studies, and evaluation of osteoclast-osteoblast cross-talk were carried out. Direct DNA sequencing showed a heterozygous C to T substitution on cDNA position 2140 of the PLEKHM1 gene, predicted to lead to an R714C mutant protein. The mutation was not found in 104 control chromosomes. In vitro, patient's osteoclasts showed normal formation rate, morphology, number of nuclei, and actin rings but lower TRACP activity and higher endosomal pH than control osteoclasts. The patient had high serum PTH and TRACP, despite low TRACP activity in osteoclasts in vitro. HEK293 cells overexpressing either wildtype or Plekhm1-R714C showed no difference in localization of the variants, and co-transfection with a TRACP vector confirmed low TRACP activity in cells carrying the R714C mutation. RAW 264.7 osteoclast-like cells expressing the Plekhm1-R714C variant also showed low TRACP activity and reduced ability to acidify endosomal compartments compared with cells expressing the wildtype protein. Reduced intracellular TRACP was caused by increased protein secretion rather than reduced expression. TRACP-containing conditioned medium was able to increase osteoblast alkaline phosphatase, suggesting the focal osteosclerosis is a result of increased osteoclast-osteoblast coupling. We provide further evidence for a role of Plekhm-1 in osteoclasts by showing that a novel mutation in PLEKHM1 is associated with a complex bone phenotype of generalized osteopenia combined with "focal osteosclerosis." Our data suggest that the mutation affects endosomal acidification/maturation and TRACP exocytosis, with implications for osteoclast-osteoblast cross-talk.

Cannabinoid receptor 2 selective agonists stimulate osteoclast formation in vitro but act as anabolic agents in vivo by stimulating bone formation

Glucocorticoids are used as powerful drugs in the treatment of various diseases. One of the most severe side effects of this treatment is bone loss. Glucocorticoids are known to strongly inhibit and kill osteoblasts, whereas glucocorticoid-treated osteoclasts remain active and possibly show even increased activity. This imbalance causes a rapid and pronounced bone loss. However, in many cases there are no alternatives to glucocorticoid therapy. Thus, it would be optimal to find a treatment that minimizes the adverse effects of glucocorticoids while still keeping its beneficial effects. From investigations on other tissues it is known that high doses of glucocorticoids stimulate degradation of essential factors through the proteasome. We have therefore combined high dose glucocorticoid treatment with bortezomib, a proteasome inhibitor used for treating multiple myeloma, and investigated the effect of this combination on osteoblasts and osteoclasts in vitro. We found that culturing osteoblast-like cells (MC3T3) with therapeutic doses of prednisolone (1.6 microM) had strong toxic effects as expected, but that this was partly overcome by pre-treating the cells with bortezomib. Furthermore, the expression of key osteoblast genes was significantly up-regulated in the bortezomib pretreated osteoblasts as compared to prednisolone alone. This was found to be true for the vitamin D3 receptor, collagen type 1, osteocalcin, CD56 and osteopontin genes. In addition, we found that osteoclast survival was unaffected by prednisolone (0.1 and 1.6 microM) or bortezomib alone, whereas release of TRACP activity by the osteoclasts was strongly reduced by either drug alone. However, when the osteoclasts were pre-treated with bortezomib followed by prednisolone, survival was compromised and TRACP release was severely affected. Thus, bortezomib reduces the anti-osteoblastic activity of glucocorticoids and turns them into strong anti-osteoclastic drugs. The molecular mechanism explaining how proteasome inhibition counteracts the adverse effects of glucocorticoids on bone cells remains to be elucidated.

Diphyllin, a Novel and Naturally Potent V-ATPase Inhibitor, Abrogates Acidification of the Osteoclastic Resorption Lacunae and Bone Resorption

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1. In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.