Showing papers on "Tartrate-resistant acid phosphatase published in 2009"
TL;DR: The findings show that androgens act through the AR in mineralizing osteoblasts to maintain bone by regulating bone resorption and the coordination of bone matrix synthesis and mineralization, and that this action is most important during times of bone accrual and high rates of bone remodeling.
Abstract: Androgens play a key role in skeletal growth and bone maintenance; however, their mechanism of action remains unclear. To address this, we selectively deleted the androgen receptor (AR) in terminally differentiated, mineralizing osteoblasts using the Cre/loxP system in mice (osteocalcin-Cre AR knockouts [mOBL-ARKOs]). Male mOBL-ARKOs had decreased femoral trabecular bone volume compared with littermate controls because of a reduction in trabecular number at 6, 12, and 24 wk of age, indicative of increased bone resorption. The effects of AR inactivation in mineralizing osteoblasts was most marked in the young mutant mice at 6 wk of age when rates of bone turnover are high, with a 35% reduction in trabecular bone volume, decreased cortical thickness, and abnormalities in the mineralization of bone matrix, characterized by increased unmineralized bone matrix and a decrease in the amount of mineralizing surface. This impairment in bone architecture in the mOBL-ARKOs persisted throughout adulthood despite an unexpected compensatory increase in osteoblast activity. Our findings show that androgens act through the AR in mineralizing osteoblasts to maintain bone by regulating bone resorption and the coordination of bone matrix synthesis and mineralization, and that this action is most important during times of bone accrual and high rates of bone remodeling.
105 citations
TL;DR: In this paper, the effect of neutralizing antisera to CSF-1 on basal and parathyroid hormone (PTH)-induced bone resorption using two organ culture assays designed to examine the recruitment of osteoclast precursors and the activation of mature osteoclasts.
Abstract: Although colony stimulating factor-1 (CSF-1) plays a key role in osteoclast recruitment, studies examining the effect of CSF-1 on mature osteoclasts indicate that it may directly inhibit bone resorption by isolated rat osteoclasts. To define further CSF-1's role in bone remodeling, we examined the effect of neutralizing antisera to CSF-1 on basal and parathyroid hormone (PTH)-induced bone resorption using two organ culture assays designed to examine the recruitment of osteoclast precursors and the activation of mature osteoclasts, respectively. We first assessed whether PTH increases CSF-1 production from bone in organ culture by examining conditioned medium from 19-day-old fetal rat long bones in a mitogenesis assay employing a CSF-1-responsive cell line, CRX-1. Conditioned medium from untreated bones induced a titratable increase in CRX-1 cell proliferation, and treatment of bones with PTH for 72 h caused a significant increase in mitogenic activity. CSF-1 antiserum caused a significant decrease in mitogenic activity in conditioned medium, indicating that bone in organ culture produces CSF-1 constitutively and in response to PTH. To examine bone-derived CSF-1's role in bone resorption, we examined the effect of neutralizing antisera to CSF-1 on basal and PTH-induced bone resorption in the fetal rat long bone assay, which reflects activation of mature osteoclasts. Anti-CSF-1 caused a significant increase in unstimulated and PTH-induced bone resorption compared with control. By contrast, in the fetal mouse metacarpal assay, which examines proliferation and recruitment of osteoclast progenitors and precursors, anti-CSF-1 caused significant inhibition of PTH related protein (PTHrP)-induced bone resorption after 3 and 6 days of incubation. Consistent with these findings, histological examination of cultured 17-day-old fetal metacarpals demonstrated that anti-CSF-1 inhibits the formation of tartrate-resistant acid phosphatase-positive osteoclasts in PTHrP-treated explants, whereas it has no effect on unstimulated bones. We conclude that bone-derived CSF-1 may have a dual role in PTH/PTHrP-induced bone resorption by enhancing the appearance of osteoclast precursors while restraining the resorptive function of mature osteoclasts.
89 citations
TL;DR: The results suggest that RANKL may stimulate switching between Nox homologues during osteoclast differentiation, and Nox-derived ROS may be crucial for RankL-induced osteocline differentiation.
Abstract: Reactive oxygen species (ROS) derived from NADPH oxidase (Nox) homologues have been suggested to regulate osteoclast differentiation. However, no bone abnormalities have been documented in Nox1 deficient, Nox2 deficient, or Nox3 mutant mice. During receptor activator of nuclear factor-kappaB ligand (RANKL)-stimulated differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts, mRNA levels of Nox enzymes (Nox1-4) and their adaptor proteins were monitored by real-time reverse transcriptase PCR. RAW264.7 cells constitutively expressed abundant Nox2 mRNA and small amounts of Nox1 and Nox3 transcripts. RANKL markedly attenuated Nox2 mRNA expression in association with reciprocal up-regulation of Nox1 and Nox3 transcripts. Introduction of small interference RNA targeting p67(phox) or p22(phox) into RAW264.7 cells effectively down-regulated ROS generation and significantly suppressed the RANKL-stimulated differentiation, which was assessed by appearance of tartrate resistant acid phosphatase (TRAP)-positive, multinucleated cells having an ability to form resorption pits on calcium phosphate thin film-coated disks, and by expression of osteoclast marker genes (TRAP, cathepsin K, Atp6i, ClC-7, and NFATc1). Our results suggest that RANKL may stimulate switching between Nox homologues during osteoclast differentiation, and Nox-derived ROS may be crucial for RANKL-induced osteoclast differentiation.
87 citations
TL;DR: The polyclonal antibody was used to develop a competitive fluorescence immunoassay for measuring serum TRAP concentrations, and according to the assay, children have higher serum TR AP concentrations than adults, and postmenopausal women have higher concentrations than pre menopausal women.
Abstract: Tartrate-resistant acid phosphatase (TRAP) was purified 20,000-fold to apparent homogeneity from human bone. The purified enzyme consisted of one 32 kd subunit, which was cleaved by β-mercaptoethanol into two subunits of 15 kd and 20 kd, as shown by sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The purified enzyme was identified by N-terminal amino acid sequencing, and it was shown to be homologous with previously purified TRAPs from other sources. We developed a polyclonal antiserum against the purified enzyme in mice. In immunohistochemistry, the antiserum recognized osteoclasts from human bone and alveolar macrophages from human lung tissue, but no cells from human spleen tissue. It also stained osteoclasts from rat bone cells cultured on bovine bone slices. Purified TRAP could be inhibited by vanadate and molybdate, but not by tartrate, and it was activated 2-fold by β-mercaptoethanol. The glycoprotein structure of human bone TRAP was analyzed, and it was shown to contain only high-mannose type carbohydrates. We used the polyclonal antibody to develop a competitive fluorescence immunoassay for measuring serum TRAP concentrations. According to the assay, children have higher serum TRAP concentrations than adults, and postmenopausal women have higher concentrations than premenopausal women. Postmenopausal women also have higher serum TRAP concentrations than postmenopausal women on estrogen replacement therapy.
80 citations
TL;DR: The use of Type 5 tartrate-resistant acid phosphatase (TRAP) as a biomarker for osteoclast number and bone resorption has been studied for over 50 years as mentioned in this paper.
Abstract: Type 5 tartrate-resistant acid phosphatase (TRAP) has been a clinically relevant biomarker for about 50 years. It has always been a reliable and specific cytochemical marker for hairy cell leukemia and for differentiated cells of monocytic lineage. Only recently has the test for serum TRAP activity been accepted as sensitive and specific enough for clinical use as a marker of osteoclasts and bone resorption. This has come about through steady advances in knowledge about TRAP enzymology, structure, function, and molecular regulation and a consequent appreciation that TRAP isoforms 5a and 5b have very different clinical significance. As a measure of osteoclast number and bone resorption, TRAP 5b has diagnostic and prognostic applications in osteoporosis, cancers with bone metastasis, chronic renal failure, and perhaps other metabolic and pathologic bone diseases. Serum TRAP 5a, on the other hand, has no relationship to bone metabolism but seems instead to be a measure of activated macrophages and chronic inflammation. Exploration of the real clinical usefulness of serum TRAP 5a for diagnosis and disease management in a wide variety of chronic inflammatory diseases is only now beginning. This perspective traces the important basic scientific developments that have led up to the refinement of serum TRAP isoform immunoassays and their validation as biomarkers of disease. Many unanswered questions remain, providing a wealth of opportunity for continued research of this multifaceted enzyme.
79 citations
TL;DR: Results indicated that phenolic compounds are antiosteoporotic chemical constituents from Curculigo orchioides, and their activities are related with chemical structures.
73 citations
TL;DR: An enhancement of osteoblast differentiation in the 3D mineralized environment that in turn promoted earlier osteoclast differentiation was observed and led to a deposition of extracellular matrix that faithfully reflected the morphology of bone tissue.
Abstract: There is increasing interest in developing new in vitro tissue models using typical tissue engineering approaches. This study was designed to (1) develop a novel three-dimensional (3D) in vitro model of bone by seeding murine primary osteoblasts and osteoclast precursors on a resorbable porous ceramic scaffold based on silicon-stabilized tricalcium phosphate (Skelite), and (2) investigate bone cell interactions in a 3D environment mimicking an in vivo condition and compare it to traditional two-dimensional (2D) cultures. Murine primary osteoblasts from C57Bl6/J mice and osteoclast precursors from C57Bl/6-Tg(ACTB-EGFP)1Osb/J mice were co-cultured on 3D Skelite scaffolds and on standard plastic culture dishes. The differentiation of these cells in both culture conditions was compared by histology (hematoxylin-eosin staining and polarized light analysis), immunohistochemistry (collagen type I), and gene expression analysis by real-time PCR for Runt-related transcription factor 2, osterix, osteocalcin, cathepsin K, and tartrate resistant acid phosphatase. To analyze and compare bone turnover in 3D and 2D co-cultures, we evaluated the modulation of RANKL and OPG mRNA expression. We observed an enhancement of osteoblast differentiation in the 3D mineralized environment that in turn promoted earlier osteoclast differentiation. In this paper, we also report that the increased osteoblast differentiation in the 3D model led to a deposition of extracellular matrix that faithfully reflected the morphology of bone tissue.
73 citations
TL;DR: In this paper, the effect of cell-cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co-culture.
Abstract: The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell-cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell-cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co-culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule-1 (ICAM-1) and of osteoclastogenesis-related genes (RANKL, RANK, TNF-alpha, and IL-1beta) was highly up-regulated in the co-cultures compared to mono-cultures and the 5-10-fold up-regulation reflected a synergistic increase due to direct cell-cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)(2)VitD(3) and dexamethasone. In case of indirect cell-cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast-like cells that were formed in co-culture with periodontal ligament fibroblasts was significantly augmented compared to mono-cultures. Our data indicate that cell-cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts.
72 citations
TL;DR: A technique to investigate osteocyte gene expression in vivo, using the reverse transcriptase linked polymerase chain reaction (PCR), has shown that they express mRNA for β‐actin (β‐ACT), osteocalcin (OC), connexin‐43 (Cx43), insulin‐like growth factor I (IGF‐I), c‐fos, and c‐jun, but not tumor necrosis factor alpha (TNF‐α) or tartrate‐resistant acid phosph
Abstract: Osteocytes have been proposed to be the cells primarily responsible for sensing the effects of mechanical loading in bone. Osteocytes respond to loading in vivo, and have been shown to express osteotropic agents and their receptors, and cell/matrix adhesion molecules in vitro, but the functional significance of such findings is not clear. One obstacle to increased understanding of the role of osteocytes in the regulation of bone mass is that the cells are not easily accessible for study. In situ studies are difficult, and although it is possible to extract and culture osteocytes from neonatal bones, the responses of such cells might be very different from those in older bones in situ. We have developed a technique to investigate osteocyte gene expression in vivo, using the reverse transcriptase linked polymerase chain reaction (PCR), and have shown that they express mRNA for beta-actin (beta-ACT), osteocalcin (OC), connexin-43 (Cx43), insulin-like growth factor I (IGF-I), c-fos and c-jun, but not tumor necrosis factor alpha (TNF-alpha) or tartrate-resistant acid phosphatase (TRAP). The principle behind the method is that after removal of the periosteum, tangential cryostat sections of a tubular bone contain RNA only from osteocytes and a very small number of endothelial cells as long as the marrow cavity is not broached. Using this method, we have investigated gene expression in cells from rat ulnar cortical bone under forming and resorbing bone surfaces. In addition, we have investigated the effect on gene expression of mechanical loading which, if repeated daily, initiates new bone formation on quiescent or resorbing surfaces. Although the expression of the genes we have studied in osteocytes is different from those expressed by the periosteal surfaces overlying the cortex, we have not detected loading-related changes in osteocyte gene expression in any cortical bones. This may be because of the extreme sensitivity of the PCR technique which can only resolve large differences in expression. The use of quantitative methods in the future may allow demonstration of regulated gene expression in osteocytes.
68 citations
TL;DR: Results suggested that curcumin reduced diabetes-stimulated bone resorptive activity and the number of osteoclasts through the prevention of osteoclastogenesis associated with inhibition of the expression of c-fos and c-jun in the diabetic rats.
68 citations
TL;DR: Human bone TRAP hydrolyzes aryl phosphates, nucleoside di‐ and triphosphates, pyrophosphate, and phosphoproteins and is activated by mild reducing agents but inhibited by molybdate, fluoride, arsenate, phosphate, and dithionite.
Abstract: Tartrate-resistant acid phosphatase (TRAP) is a histochemical marker for osteoclasts, the multinucleated bone resorbing cell. This type 5 acid phosphatase has been purified 500-fold from human bone by three chromatographic steps: cation exchange, gel filtration, and HPLC cation exchange. Like most other TRAPs isolated, it is a basic glycoprotein of a molecular weight about 33,000. Its pH optimum Km, and Vmax for p-nitrophenyl phosphate are 5.7, 0.8 mM, and 12 units/mg, respectively. Human bone TRAP hydrolyzes aryl phosphates, nucleoside di- and triphosphates, pyrophosphate, and phosphoproteins. It is activated by mild reducing agents but inhibited by molybdate, fluoride, arsenate, phosphate, and dithionite. Its activity is not inhibited by tartrate, a feature that distinguishes it from other acid phosphatases. Sodium etridonate, the bisphosphonate used clinically to reduce bone resorption, is a relatively poor inhibitor of bone TRAP. Human bone TRAP is immunologically related to the porcine uterine secretory TRAP, uteroferrin. Monospecific rabbit antibodies to the bone TRAP have been immunopurified by using affinity chromatography with uteroferrin immobilized on Sepharose and can be used to detect low amounts of the enzyme in a simple dot-blot assay.
TL;DR: Results indicated that significantly greater bone loss occurred with more inflammatory infiltrate and a significant increase in osteoclastogenesis compared with Mkp-1−/− control sites or either wild-type group, indicating that MKP-1 plays a key role in the regulation of inflammatory bone loss.
Abstract: The mitogen-activated protein (MAP) kinase phosphatase (MKP) family plays an important function in regulating the pro-inflammatory cytokines by deactivating MAP kinases. MKP-1 is essential for the dephosphorylation of p38 MAP kinase that regulates expression of IL-6, TNF-alpha, and IL-1 beta. We hypothesized that MKP-1 regulates inflammatory bone loss in experimental periodontitis. Wild-type and Mkp-1(-/-) mice received A. actinomycetemcomitans LPS injection in the palatal region or PBS control 3 times/wk for 30 days. Mice were killed, and maxillae were assessed by microcomputed tomography, histological analysis, and TRAP staining for measurement of bone loss, extent of inflammation, and degree of osteoclastogenesis. Results indicated that, in LPS-injected Mkp-1(-/-) mice, significantly greater bone loss occurred with more inflammatory infiltrate and a significant increase in osteoclastogenesis compared with Mkp-1(-/-) control sites or either wild-type group. Analysis of these data indicates that MKP-1 plays a key role in the regulation of inflammatory bone loss.
TL;DR: Data support an osteoclast-independent role for N-BP therapy in bone metastasis and the impact of treatment with ZA on B16 melanoma bone tumor burden in irradiated mice transplanted with splenic cells from src(-/-) mice, which have non-functioning OCs.
TL;DR: A defined model for studying osteoclast differentiation and activity in the absence of serum is described, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.
Abstract: Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete α-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1α,25-dihydroxyvitamin D3 (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.
TL;DR: Although there was a temperature-dependent delay in bone formation after heat stress, the 48 degrees C heat stress did not obstruct bone formation eventually and was probably caused by slow periosteal membrane regeneration.
Abstract: Objectives: It is important to know the etiology of implant failure. It has been reported that heat stress during drilling was one of the causes for failure and the threshold was 47°C. However, clinically, we encounter cases in which overheating does not seem to affect osseointegration eventually. The purpose of this study was to assess histologically the spatio-temporal effect of heat stress on bone formation after overheating the bone matrix.
Material and methods: Rat calvarial bone was heated to 37°C, 43°C, 45°C and 48°C for 15 min by a temperature stimulator. Paraffin sections were prepared 1, 3 and 5 weeks after heating and investigated histologically under light microscopy. Hematoxylin and eosin staining, alkaline phosphatase (ALP), osteopontin (OPN), heat shock protein 27 (Hsp27) and heat shock protein 70 (Hsp70) immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry were carried out. The area of dead osteocytes was calculated and statistically analyzed. Apoptotic osteocytes were detected by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) method.
Results: Along with the temperature increase, the area of dead osteocytes increased and regeneration of the periosteal membrane was delayed. Hsps- and TUNEL-positive cells were only seen in the 48°C group. Spatio-temporal changes of TRAP- and ALP-positive cell numbers were observed, while OPN expression was mostly absent. Even after 48°C stimulation, bone formation on the calvarial surface was observed after 5 weeks.
Conclusions: Although there was a temperature-dependent delay in bone formation after heat stress, the 48°C heat stress did not obstruct bone formation eventually. This delay was probably caused by slow periosteal membrane regeneration.
TL;DR: IL-12 inhibited TNF-alpha-induced osteoclastogenesis in mice whose T cells were blocked by anti-CD4 and anti- CD8 antibodies and induces apoptotic changes through a T cell-independent mechanism.
TL;DR: It is suggested that TLR3 promotes osteoclastogenesis in the RA synovium both directly and indirectly and induces RANKL expression indirectly in RA-FLS, and the expression of RankL promotes the differentiation of osteoclasts in theRAsynovium.
TL;DR: A combination of MTX and ZA prevented both bone erosion and systemic bone loss in a rat model of arthritis, providing support for the use of a combination of these two drugs to improve the prevention of structural joint damage in RA.
Abstract: Osteoclasts play a key role in the pathogenesis of bone erosion and systemic bone mass loss during rheumatoid arthritis (RA). In this study, we aimed to determine the effect of methotrexate (MTX) and zoledronic acid (ZA), used alone or in combination, on osteoclast-mediated bone erosions and systemic bone mass loss in a rat model of collagen induced arthritis (CIA). We hypothesized that MTX and ZA could have an additive effect to prevent both bone erosion and systemic bone loss. Arthritis was induced in 64 female Sprague-Dawley rats. After the clinical onset of CIA, rats were assigned to treatment with MTX (1 mg/kg/week), ZA (100 μg/kg twice weekly), both treatments at the same regimens, or vehicle. Arthritis score and paw thickness were recorded twice weekly. The rats were sacrificed on D28 and hind paws were removed for radiographic, histological and immunohistochemical analysis. The effects of treatments on osteoclastogenesis were determined by Tartrate resistant acid phosphatase (TRAP) staining. Micro-CT of the tibia was carried out for histomorphometric analysis. Bone mass density was evaluated by densitometry. MTX significantly decreased the severity of CIA, whereas ZA slightly exacerbated it. When these two drugs were used in combination, MTX prevented the pro-inflammatory effect of ZA. The combination of ZA with MTX was more effective than MTX alone for reducing structural joint damage with a dramatic decrease of osteoclasts' number in the eroded joints. However, MTX alone also significantly reduced the number of osteoclasts and the number of CD68+ mononuclear cells. ZA alone, or ZA with MTX, significantly increased the systemic bone mass density measured by densitometry and bone volume on histomorphometric analysis. A combination of MTX and ZA prevented both bone erosion and systemic bone loss in a rat model of arthritis. Both treatments independently decreased the number of osteoclasts in the eroded joint. However, while MTX probably acts mainly through a decrease of inflammation, ZA has a direct effect on osteoclasts, allowing a dramatic down-regulation of these cells in inflamed joints. These two different mechanisms of action provide support for the use of a combination of these two drugs to improve the prevention of structural joint damage in RA.
TL;DR: A transient increase in markers of osteoclast activity observed in six heavily pretreated patients with multiple myeloma suggests that HD‐Sim may be harmful rather than beneficial for MM patients.
Abstract: 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.
TL;DR: In vitro sAC mediates the inhibition of osteoclast function by acting as a bicarbonate sensor, and a 1‐week mouse calvaria culture system where osteoclasts were shown to be viable was developed.
Abstract: High [HCO(3)(-)] inhibits and low [HCO(3)(-)] stimulates bone resorption, which mediates part of the effect of chronic acidosis or acid feeding on bone. Soluble adenylyl cyclase (sAC) is a bicarbonate sensor that can potentially mediate the effect of bicarbonate on osteoclasts. Osteoclasts were incubated in 0, 12, and 24 mM HCO(3)(-) at pH 7.4 for 7-8 days and assayed for tartrate-resistant acid phosphatase (TRAP) and vacuolar-ATPase expression, and H+ accumulation. Total number and area of TRAP (+) multinucleated osteoclasts was decreased by HCO(3)(-) in a dose-dependent manner. V-ATPase expression and H+ accumulation normalized to cell cross-sectional area or protein were not significantly changed. The HCO(3)(-) -induced inhibition of osteoclast growth and differentiation was blocked by either 2-hydroxyestradiol, an inhibitor of sAC or sAC knockdown by sAC specific siRNA. The model of HCO(3)(-) inhibiting osteoclast via sAC was further supported by the fact that the HCO(3)(-) dose-response on osteoclasts is flat when cells were saturated with 8-bromo-cAMP, a permeant cAMP analog downstream from sAC thus simulating sAC activation. To confirm our in vitro findings in intact bone, we developed a 1-week mouse calvaria culture system where osteoclasts were shown to be viable. Bone volume density (BV/TV) determined by micro-computed tomography (microCT), was higher in 24 mM HCO(3)(-) compared to 12 mM HCO(3)(-) treated calvaria. This HCO(3)(-) effect on BV/TV was blocked by 2-hydroxyestradiol. In summary, sAC mediates the inhibition of osteoclast function by HCO(3)(-), by acting as a HCO(3)(-) sensor.
TL;DR: Data suggest that the TRAP promoter is complex and contains multiple regulatory elements, which may permit production of transgenic mice, which can be used to develop previously unavailable osteoclast cell lines.
Abstract: Little information is available on the molecular mechanisms controlling osteoclastic bone resorption. We used tartrate-resistant acid phosphatase (TRAP) to begin to investigate the regulation of bone resorption at the molecular level. TRAP is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. Therefore, we isolated the murine TRAP gene from a mouse spleen genomic library and characterized its promoter. A restriction map was generated for the 17 kb TRAP insert. A 2 kb SmaI fragment, containing the 5'-flanking region, was subcloned and the nucleotide sequence determined. Sequence analysis of the SmaI fragment revealed the presence of numerous candidate transcription factor binding sequences, including those for AP1 and H-APF-1. The H-APF-1 site matches the consensus sequence for the IL-6-regulated transcription factor. An intron was identified at -1 to -393 bp relative to the ATG. The presence of an intron was confirmed by PCR analysis of RNA isolated from murine osteoclasts. Primer extension analysis indicated the presence of a transcription initiation site at -552 bp from the ATG. The region from -1846 to 2bp relative to the ATG initiation codon drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. PMA treatment of HRE H9 cells enhanced luciferase transcription approximately threefold. These data suggest that the TRAP promoter is complex and contains multiple regulatory elements. The availability of the TRAP promoter may also permit production of transgenic mice, which can be used to develop previously unavailable osteoclast cell lines.
TL;DR: The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption, and suggests that the classical NF-κB pathway plays a pivotal role in osteOClast bone-resorbing activity.
Abstract: The classical NF-κB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-κB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-κB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-κB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p < 0.001) and depths (p < 0.05) of pits. The reduced resorption by the peptide was due to reduced osteoclast bone-resorbing activity, but not reduced differentiation as the number of osteoclasts was similar in all groups. The peptide inhibited bone resorption by reducing TRAP activity, disrupting actin rings and preventing osteoclast migration. Gene expressions of a panel of bone resorption markers were significantly reduced. The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption. Therefore, these data suggest that the classical NF-κB pathway plays a pivotal role in osteoclast bone-resorbing activity.
TL;DR: The results demonstrate that saurolactam potentially inhibits osteoclast differentiation by preventing the activation of MAP kinases and transcription factors that consequently affect the regulation of genes required for osteooclastogenesis, and the bone resorptive activity of mature osteoclasts by inhibiting osteOClast survival‐related signaling pathways and triggering the apoptotic signaling cascade.
Abstract: The receptor activator of nuclear factor-kappaB ligand (RANKL) plays a critical role in the differentiation and bone resorptive activity of osteoclasts. Recently, the development of anti-resorptive agents from natural substances has become a subject of interest. Therefore, we evaluated the effects of 222 natural compounds on the RANKL-induced tartrate-resistance acid phosphatase (TRAP; a marker for osteoclast differentiation) activity and multinucleated osteoclast formation in RAW264.7 murine macrophage cells. We found that saurolactam was one of the compounds inhibiting the RANKL-induced osteoclastogenesis; it significantly inhibited the RANKL-induced TRAP activity and formation of multinucleated osteoclasts without any cytotoxicity. Interestingly, saurolactam prevented RANKL-induced activation of MAP kinases and NF-kappaB, and mRNA expression of osteoclast-related genes and transcription factors (c-Fos, Fra-2, and NFATc1). We also observed the inhibitory effect of saurolactam on the differentiation of mouse bone marrow-derived macrophages into osteoclasts. Furthermore, saurolactam inhibited the bone resorptive activity of mature osteoclasts with the induction of apoptotic signaling cascade and the inhibition of survival signaling pathways such as c-Src/PI3K/Akt, Ras/ERK, and JNK/c-Jun. In conclusion, although further studies are needed to determine the precise mechanism and biological efficacy of saurolactam in osteoclast-mediated bone disorders, our results demonstrate that saurolactam potentially inhibits osteoclast differentiation by preventing the activation of MAP kinases and transcription factors that consequently affect the regulation of genes required for osteoclastogenesis, and the bone resorptive activity of mature osteoclasts by inhibiting osteoclast survival-related signaling pathways and triggering the apoptotic signaling cascade.
TL;DR: GDF-15 inhibits expression of carbonic anhydrase II and cathepsin K, key osteoclast enzymes, and induces changes in SMAD and p38 signaling, and the lack of functional osteoclasts can contribute to accumulation of bone matrix by reduction of bone resorption.
TL;DR: Results demonstrate that OxLDL, by generation of an intracellular oxidative stress, prevents the differentiation of osteoclasts by inhibition of RANKL signaling pathway, related to the fact that atherosclerosis is accompanied by perturbations in bone and vascular remodeling, leading to osteoporosis and vascular calcification.
Abstract: The role of OxLDL in the generation and progression of atherosclerosis is well admitted. In addition, it is well known that atherosclerosis is often accompanied by perturbations in bone remodeling, resulting in osteoporosis. In the current studies, the effect of Cu(2+)-oxidized LDL (OxLDL) on RANKL-induced RAW264.7 mouse monocytes-macrophages differentiation to osteoclasts and on RANKL signaling pathway was investigated. OxLDL, within the range of 10-50 microg protein/ml, prevented RANKL-induced generation of multinucleated osteoclast-like cells and RANKL-induced tartrate resistant acid phosphatase (TRAP) activity. OxLDL also prevented the RANKL-induced phosphorylation of ERK, p38 and JNK kinases, together with the RANKL-induced DNA binding activities of NFkappaB and NFAT transcription factors. Concomitantly, OxLDL enhanced RANKL-induced generation of reactive oxygen species in a dose-dependent manner. The antioxidant glutathione (GSH) prevented whereas the prooxidant compound buthionine-sulfoximine (BSO) enhanced the effect of OxLDL on RANKL-induced oxidative stress and RANKL-induced differentiation. Finally, OxLDL also prevented RANKL-induced TRAP activity and RANKL-induced bone resorbing activity of human peripheral blood mononuclear cells. These results demonstrate that OxLDL, by generation of an intracellular oxidative stress, prevents the differentiation of osteoclasts by inhibition of RANKL signaling pathway. This might be related to the fact that atherosclerosis is accompanied by perturbations in bone and vascular remodeling, leading to osteoporosis and vascular calcification.
TL;DR: Results suggest that Ti(IV) ions released by biocorrosion from orthopedic implants induce differentiation of monocytes toward mature, functional osteoclasts, which may well contribute the pathomechanism of aseptic loosening.
Abstract: There is increasing evidence that titanium (Ti) ions are released from orthopedic implants, with concentrations in the range of 1 microM in tissue and blood, and may play a role in aseptic loosening of orthopedic implants. This study investigated whether Ti(IV) ions induce differentiation of monocytic osteoclast precursors into osteo-resorptive multinucleated cells and influence the activation and function of in vitro generated osteoclasts. Human monocytes and in vitro generated osteoclasts were exposed to 1 microM Ti(IV) ions for 10 days. Thereafter, osteoclast differentiation, activation, and function were evaluated. Transcription of specific osteoclastic genes was measured using quantitative reverse transcription polymerase chain reactions, which showed increased expression of tartrate-resistant acid phosphatase (TRAP) in approximately 20% of Ti(IV)-treated monocytes. Detection and quantification of intracellular TRAP activity using ELF97 as a fluorescent substrate revealed a significant increase of TRAP-positive cells in Ti(IV)-treated monocytes. Additionally, as demonstrated on dentin slide cultures, Ti(IV)-treated monocytes became functional bone resorbing cells, significantly increasing their osteo-resorptive activity to similar levels as osteoclasts in vitro. These results suggest that Ti(IV) ions released by biocorrosion from orthopedic implants induce differentiation of monocytes toward mature, functional osteoclasts, which may well contribute the pathomechanism of aseptic loosening.
TL;DR: H(2)S application caused a transient increase of osteoclast differentiation with up-regulation of RANKL expression in osteoblasts, which may also contribute to alveolar bone resorption through RankL expression.
TL;DR: It is concluded that CT has two actions on TRAP in isolated rat osteoclasts: the first to inhibit its release, the second to inhibitIts synthesis and/or increase its degradation.
Abstract: Tartrate-resistant acid phosphatase (TRAP) has been implicated as being involved in osteoclastic bone resorption, and calcitonin (CT) is known to inhibit the resorptive process. This study investigates the kinetics of CT action on TRAP activity in isolated rat osteoclasts using both biochemical and quantitative cytochemical methods. The latter technique has been developed to detect very small changes in intracellular TRAP activity at the single-cell level. The biochemical study showed that 10−9 M salmon CT (sCT) decreased TRAP activity in medium throughout the experimental period; TRAP activity in the cells was increased during the first 2 h but subsequently declined and was decreased to a significant level at 6 h. TRAP activity in sCT-treated osteoclasts measured by the cytochemical method showed significant increases within the first hour. This response was dose dependent between 1016 and 10−11 M sCT with EC50 at 8 × 10−14 M. After 1 h, the initial increase in intracellular TRAP activity in CT-treated osteoclasts was followed by a decline to below control levels, reaching statistical significance at 9 h. Treatment with forskolin (10−5 M) showed a similar trend, suggesting that this response is mediated by cyclic AMP-regulated phosphorylation events. From these results, we conclude that CT has two actions on TRAP in isolated rat osteoclasts: the first to inhibit its release, the second to inhibit its synthesis and/or increase its degradation.
TL;DR: It is suggested that the “escape” phenomenon may result from a prolonged CT‐induced loss of CT responsiveness due, at least in part, both to reduced synthesis of CTR, and to the appearance in bone of CTR‐deficient osteoclasts.
Abstract: Continuous treatment with calcitonin (CT) to inhibit osteoclastic bone resorption results in acquired resistance. The mechanisms of this escape phenomenon are not yet established. The aim of this study was to examine the effects of continuous treatment with CT on the generation of osteoclasts and calcitonin receptor (CTR) expression in mouse bone marrow cultures. This was done by daily CT treatment of mouse bone marrow cultures from day 0, when only undifferentiated mononuclear precursors of osteoclast-like cells were present, or commencing from day 6, when differentiated osteoclast-like cells were abundant. The response to CT treatment was determined by quantitation of cells positive for tartrate-resistant acid phosphatase (TRAP) and binding of 125 I-salmon CT. Calcitonin receptor and TRAP mRNA levels were determined using semi-quantitative reverse transcription/polymerase chain reaction. When cultures were treated with CT from day 0, TRAP-positive multinucleated cells appeared. These cells expressed only very low levels of CTR or CTR mRNA and were morphologically indistinguishable from osteoclast-like cells formed in control cultures. They also displayed the ability to resorb bone. Continuous CT treatment of cultures from day 6 rapidly reduced the CTR mRNA levels, with a t 1/2 of 6 to 12 h, and these levels remained low thereafter. 125 I-salmon CT binding capacity, as determined by autoradiography, was lost in parallel. These effects were specific for the CTR since there was no consistent effect on TRAP mRNA levels. Based on these data, we suggest that the escape phenomenon may result from a prolonged CT-induced loss of CT responsiveness due, at least in part, both to reduced synthesis of CTR, and to the appearance in bone of CTR-deficient osteoclasts.
TL;DR: It is demonstrated that hydroxyapatite substrate might be able to induce a self-production of RANKL cytokine that directly stimulates a different behaviour in terms of phenotype expression from monocyte/macrophage lineage to mature and functional osteoclasts without the addition of exogenous factors.
Abstract: Summary Background Hydroxyapatite surface coatings of dental implants have been introduced to obtain more rapid and complete osteointegration. A possible complication associated with hydroxyapatite implant surface is the release of particles. Those particles may be phagocytosed by monocytes, the first cells to colonize the inflammatory sites. The activated monocytes produce cytokines that could cause osteoclast activation. Methodology In order to establish the biological effect of particles released on monocyte differentiation to an osteoclast phenotype, we have used the murine monocyte/macrophage cell line, RAW 264.7 clone CRL-2278 cultured on a hydroxyapatite substrate. The direct action of hydroxyapatite on monocyte differentiation was examined using tartrate-resistant acid phosphatase (TRAP), immunohistochemistry and transmission electron microscopy (TEM) and Western Blot analysis. Results The present study demonstrated that hydroxyapatite substrate might be able to induce a self-production of RANKL cytokine that directly stimulates a different behaviour in terms of phenotype expression from monocyte/macrophage lineage to mature and functional osteoclasts without the addition of exogenous factors. Conclusions These studies were designed to test a model in which osteoclasts could be formed from HA-activated monocytes via positive feedback elicited by RANKL, allowing for identification of innovative targets for therapeutic approaches.