Showing papers on "Tartrate-resistant acid phosphatase published in 2003"
TL;DR: The change in the levels of these key regulators of osteoclast differentiation may play a major role in the bone loss seen in periodontitis.
Abstract: Objectives and background: This study investigated the expression of key mediators that regulate differentiation of osteoclasts, receptor activator of nuclear factor κB ligand (RANKL), and its natural inhibitor, osteoprotegerin (OPG), in periodontitis. We aimed to compare the levels of the RANKL and OPG in the granulomatous tissue adjacent to areas of alveolar bone loss from patients with periodontitis to that present in tissue from patients without periodontitis. In addition, we aimed to determine the types of cells expressing these factors in these tissues and to demonstrate the expression of the osteoclastic markers, RANK and tartrate-resistant acid phosphatase (TRAP), in periodontitis.
Materials and methods: Frozen biopsy specimens were analysed using specific monoclonal antibodies and were evaluated by semiquantitative analysis and digital image analysis to compare levels of RANKL and OPG protein expression. Double labelling of frozen sections with antibodies to different cell lineage specific markers was used to determine the types of cells expressing these proteins. In situ hybridization was used to detect cells expressing RANK mRNA.
Results: Semiquantitative image analysis demonstrated that significantly higher levels of RANKL protein (P < 0.05) were expressed in the periodontitis tissue. Conversely, OPG protein was significantly lower (P < 0.05) in the periodontitis tissues. RANKL protein was associated with lymphocytes and macrophages. OPG protein was associated with endothelial cells in both tissues. Many leukocytes expressing RANK mRNA and TRAP were observed in periodontitis tissues.
Conclusion: The change in the levels of these key regulators of osteoclast differentiation may play a major role in the bone loss seen in periodontitis.
285 citations
TL;DR: In both in vitro and in vivo studies, RANKL and OPG are suggested to be important regulators of not only the terminal differentiation of OC but also their resorptive function.
Abstract: The differentiation and functions of osteoclasts (OC) are regulated by osteoblast-derived factors such as receptor activator of NFKB ligand (RANKL) that stimulates OC formation, and a novel secreted member of the TNF receptor superfamily, osteoprotegerin (OPG), that negatively regulates osteoclastogenesis. In examination of the preosteoclast (pOC) culture, pOCs formed without any additives expressed tartrate-resistant acid phosphatase (TRAP), but showed little resorptive activity. pOC treated with RANKL became TRAP-positive OC, which expressed intense vacuolar-type H(+)-ATPase and exhibited prominent resorptive activity. Such effects of RANKL on pOC were completely inhibited by addition of OPG. OPG inhibited ruffled border formation in mature OC and reduced their resorptive activity, and also induced apoptosis of some OC. Although OPG administration significantly reduced trabecular bone loss in the femurs of ovariectomized (OVX) mice, the number of TRAP-positive OC in OPG-administered OVX mice was not significantly decreased. Rather, OPG administration caused the disappearance of ruffled borders and decreased H(+)-ATPase expression in most OC. OPG deficiency causes severe osteoporosis. We also examined RANKL localization and OC induction in periodontal ligament (PDL) during experimental movement of incisors in OPG-deficient mice. Compared to wild-type OPG (+/+) littermates, after force application, TRAP-positive OC were markedly increased in the PDL and alveolar bone was severely destroyed in OPG-deficient mice. In both wild-type and OPG-deficient mice, RANKL expression in osteoblasts and fibroblasts became stronger by force application. These in vitro and in vivo studies suggest that RANKL and OPG are important regulators of not only the terminal differentiation of OC but also their resorptive function. To determine resorptive functions of OC, we further examined the effects of specific inhibitors of H(+)-ATPase, bafilomycin A1, and lysosomal cysteine proteinases (cathepsins), E-64, on the ultrastructure, expression of these enzymes and resorptive functions of cultured OC. In bafilomycin A1-treated cultures, OC lacked ruffled borders, and H(+)-ATPase expression and resorptive activity were significantly diminished. E-64 treatment did not affect the ultrastructure and the expression of enzyme molecules in OC, but significantly reduced resorption lacuna formation, by inhibition of cathepsin activity. Lastly, we examined the expression of H(+)-ATPase, cathepsin K, and matrix metalloproteinase-9 in odontoclasts (OdC) during physiological root resorption in human deciduous teeth, and found that there were no differences in the expression of these molecules between OC and OdC. RANKL was also detected in stromal cells located on resorbing dentine surfaces. This suggests that there is a common mechanism in cellular resorption of mineralized tissues such as bone and teeth.
132 citations
TL;DR: It is suggested that TRACP‐5b is increased in MM, reflects the extent of myeloma bone disease and may have a predictive value, and has also proved to be very useful for monitoring antimyeloma treatment, which had no effect on OPG levels.
Abstract: Tartrate-resistant acid phosphatase isoform-5b (TRACP-5b), a new marker reflecting osteoclast activity, and osteoprotegerin (OPG) were measured in 121 patients with multiple myeloma (MM) at diagnosis, and in 63 of them during pamidronate administration, to define their correlation with the extent of bone disease and disease activity in MM. Radiographic evaluation of the skeleton, measurement of other markers of bone remodelling, including N-terminal cross-linking telopeptide of type-I collagen (NTX), bone alkaline phosphatase and osteocalcin and of markers of disease activity (beta2-microglobulin, paraprotein, interleukin-6 (IL-6), were also performed. Levels of TRACP-5b were increased (p <.0001), while OPG was decreased in MM patients compared to controls (p <.01). TRACP-5b levels were associated with the radiographically assessed severity of bone disease (p <.0001) as well as with levels of NTX, IL-6 and beta2-microglobulin (p <.001, for each biochemical parameter, respectively). The combination of pamidronate with VAD-chemotherapy produced a reduction in TRACP-5b, NTX, IL-6, paraprotein and beta2-microglobulin levels from the 2nd month of treatment, with no effect on bone formation and OPG. A strong correlation was observed between changes in TRACP-5b and changes in NTX, IL-6 and beta2-microglobulin, while TRACP-5b predicted the disease progression in 5 patients. These findings suggest that TRACP-5b is increased in MM, reflects the extent of myeloma bone disease and may have a predictive value. TRACP-5b has also proved to be very useful for monitoring antimyeloma treatment, which had no effect on OPG levels.
95 citations
TL;DR: Serum TRACP levels correlate well with histological indices of osteoclasts and may serve as a specific marker for osteoclastic activity in patients with renal bone disease, suggesting a link between histomorphometric parameters of bone resorption and TRACP.
94 citations
TL;DR: It is hypothesized that binding to TRAP may be one mechanism by which the full osteoblast phenotype is expressed during the process of bone remodeling.
91 citations
TL;DR: This study is the first to demonstrate that bisphenol A functions to suppress directly osteoblasts and osteoclasts among vertebrates, which strongly suggests that this scale in an in vitro assay system can be utilized for the evaluation of the effects of endocrine disrupters on bone cells.
82 citations
TL;DR: It is proposed that TRACP may be an important regulator of osteopontin/η‐1 activity common to both the immune system and skeleton and possibly also in endochondral ossification.
Abstract: TRACP is a lysosomal enzyme found in diverse tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages. To investigate the function of TRACP in vivo, we have generated mice in which the gene-encoding TRACP has been selectively disrupted by targeted homologous recombination in murine embryonic stem cells. Homozygous TRACP "knockout" mice have progressive foreshortening and deformity of the long bones and axial skeleton suggesting a role for TRACP in endochondral ossification. There is increased mineralization reflecting a mild osteopetrosis caused by reduced osteoclast modeling activity. These knockout mice also display an impairment of macrophage function with abnormal immunomodulatory cytokine responses. Superoxide formation and nitrite production were enhanced in stimulated macrophages lacking TRACP as was the secretion of the proinflammatory cytokines TNF-alpha, interleukin (IL)-1beta, and IL-12. TRACP knockout mice showed delayed clearance of the microbial pathogen Staphylococcus aureus after sublethal intraperitoneal inoculation. The macrophages lacking TRACP showed an increase in tartrate-sensitive lysosomal acid phosphatase activity (LAP). The TRACP knockout mice were bred with mice lacking LAP. Mice lacking both TRACP and LAP had even shorter bones than the TRACP single knockouts. Osteopontin, identical to the T-cell cytokine eta-1, accumulated adjacent to actively resorbing osteoclasts suggesting that both phosphatases are important for processing this protein. We propose that TRACP may be an important regulator of osteopontin/eta-1 activity common to both the immune system and skeleton.
82 citations
TL;DR: It is proposed that cathepsin K acts as a physiological activator of TRACP in osteoclasts, whereas cat hepsin L might fulfill a similar role in different types of macrophages, as well as a more general function for TRACP as a regulator of OPN phosphorylation and bioactivity.
Abstract: TRACP is synthesized as a latent proenzyme requiring proteolytic processing to attain maximal phosphatase activity. Excision of an exposed loop domain abolishes the interaction between the loop residue Asp146 and a ligand to the redox-sensitive iron of the active site, most likely Asn91, providing a mechanism for the enzyme repression. Both cathepsin K and L efficiently cleave in the loop domain and activate the latent enzyme, and we propose that cathepsin K acts as a physiological activator of TRACP in osteoclasts, whereas cathepsin L might fulfill a similar role in different types of macrophages. Considering the rather broad substrate specificity of TRACP, a tight regulation of its activity in the cell appears warranted. Besides proteolytic cleavage, the enzyme should need a specific local environment with a slightly acidic pH and reducing equivalents to keep the enzyme fully active. Cellular subcompartments where these required conditions prevail are potential subcellular site(s) of TRACP action. Of bone phosphoproteins shown to be substrates for TRACP, both osteopontin and bone sialoprotein are colocalized with TRACP in the resorption lacuna of the osteoclasts, and dephosphorylation of OPN impair its ability to promote adhesion as well as migration of osteoclasts in vitro. A role for TRACP as an osteopontin phosphatase in bone is therefore suggested. The expression of TRACP as well as OPN in other tissues with possible interactions between the two could suggest a more general function for TRACP as a regulator of OPN phosphorylation and bioactivity.
73 citations
Journal Article•
TL;DR: Serum TRACP 5b activity is normal in breast cancer patients without bone metastases, and elevated in approximately 80% of breast cancers patients with bone metastased patients, suggesting that serum TRACP5b activity may be a useful marker for the early detection of the spreading of breast cancer cells to bone.
Abstract: Bone resorbing osteoclasts contain high amounts of tartrate-resistant acid phosphatase (TRACP) 5b and secrete it into the blood circulation. Circulating TRACP 5b activity is derived exclusively from osteoclasts. We have developed a TRACP 5b-specific-immunoassay using a monoclonal antibody O1A that was developed using TRACP 5b purified from human osteoclasts as antigen. Serum TRACP 5b activity has a low diurnal variability, and it does not accumulate in the circulation in renal or hepatic failure. It is elevated in 80% of patients with osteoporosis, while decreased 40-50% after antiresorptive therapy with estrogen and the bisphosphonate alendronate. Preliminary results show that serum TRACP 5b activity is normal in breast cancer patients without bone metastases, and elevated in approximately 80% of breast cancer patients with bone metastases. These results suggest that serum TRACP 5b activity may be a useful marker for the early detection of the spreading of breast cancer cells to bone.
72 citations
TL;DR: It is suggested that ELF‐PEMF can both enhance (approximately 50%) and suppress (approximately 27%) the formation of osteoclast‐like cells in bone marrow culture, depending on the induced electric field intensity.
Abstract: Over the past 30 years, the beneficial therapeutic effects of selected low energy, time varying electromagnetic fields (EMF) have been documented with increasing frequency to treat therapeutically resistant problems of the musculoskeletal system. However, the underlying mechanisms at a cellular level are still not completely understood. In this study, the effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMF) on osteoclastogenesis, cultured from murine bone marrow cells and stimulated by 1,25(OH)(2)D(3), were examined. Primary bone marrow cells were cultured from mature Wistar rats and exposed to ELF-PEMF stimulation daily for 7 days with different intensities of induced electric field (4.8, 8.7, and 12.2 micro V/cm rms) and stimulation times (0.5, 2, and 8 h/day). Recruitment and authentication of osteoclast-like cells were evaluated, respectively, by determining multinuclear, tartrate resistant acid phosphatase (TRAP) positive cells on day 8 of culture and by the pit formation assay. During the experiments, cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and prostaglandin-E(2) (PGE(2)) were assayed using the enzyme linked immunosorbent assay (ELISA). These findings suggest that ELF-PEMF can both enhance (approximately 50%) and suppress (approximately 27%) the formation of osteoclast-like cells in bone marrow culture, depending on the induced electric field intensity. In addition, consistent correlations were observed between TNF-alpha, IL-1beta, and osteoclast-like cell number after exposure to different induced electric field intensities of ELF-PEMF. This in vitro study could be considered as groundwork for in vivo ELF-PEMF clinical applications on some osteoclast-associated bone diseases.
69 citations
TL;DR: The results showed that several TRAP-positive osteoclasts exhibited large vacuoles containing TUNEL positive apoptotic structures, probably derived from osteoblasts/lining cells and/or osteocytes, which support the idea that alveolar bone osteoclast are able to internalize dying apoptotic bone cells.
Abstract: Although it is generally accepted that osteoclasts breakdown and resorb bone matrix, the possibility that they may also be able to engulf apoptotic osteoblasts/lining cells and/or osteocytes remains controversial. Apoptosis of osteoblasts/ lining cells and/or osteocytes and interactions between these cells and osteoclasts are extremely rapid events that are difficult to observe in vivo. A suitable in vivo model for studying these events is the alveolar bone of young rats because it is continuously undergoing intense resorption/remodeling. Thus, sections of aldehyde fixed alveolar bone of young rats were stained by the combined terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and the tartrate-resistant acid phosphatase (TRAP) method for the simultaneous visualization of apoptotic cells and osteoclasts in the same section. The combined TUNEL and TRAP reactions, in the same section, greatly facilitated visualization of relationship between osteoclasts and apoptotic bone cells during alveolar bone remodeling. Our results showed that several TRAP-positive osteoclasts exhibited large vacuoles containing TUNEL positive apoptotic structures, probably derived from osteoblasts/lining cells and/or osteocytes. These results support the idea that alveolar bone osteoclasts are able to internalize dying apoptotic bone cells.
TL;DR: RANKL was shown to exert in vitro pro-resorptive effects by increasing osteoclast marker expressions, M MP-9 expression, and pro-MMP-9 activity and by diminishing TIMP-1 expression, leading to an up-regulation of the MMP- 9/TIMp-1 ratio.
TL;DR: It is concluded that PTH stimulates OCL formation and bone resorption in mice in part by endogenous FGF-2 synthesis by osteoblasts, and RANKL- and interleukin-11-induced OCL formations were reduced in bone marrow cultures from Fgf2-/mice.
TL;DR: It is suggested that TRAP positive mononuclear and multinucleated cells located in the synovium at the cartilage-synovial interface produce MMP-2 and MMP -9, and may have an important role in articular cartilage destruction in patients with rheumatoid arthritis.
Abstract: Objective: To examine the role of tartrate resistant acid phosphatase (TRAP) positive mononuclear and multinucleated cells in the destruction of articular cartilage in patients with rheumatoid arthritis (RA). Methods: The presence of TRAP positive cells in the synovial tissue of patients with RA was examined by enzyme histochemistry and immunohistochemistry. Expression of mRNAs for matrix metalloproteinases (MMPs) was assessed by the reverse transcriptase-polymerase chain reaction (RT-PCR) and northern blot analysis. Production of MMPs by mononuclear and multinucleated TRAP positive cells was examined by immunocytochemistry, enzyme linked immunosorbent assay (ELISA) of conditioned medium, and immunohistochemistry of human RA synovial tissue. In addition, a cartilage degradation assay was performed by incubation of 35 S prelabelled cartilage discs with TRAP positive cells. Results: TRAP positive mononuclear cells and multinucleated cells were found in proliferating synovial tissue adjacent to the bone-cartilage interface in patients with RA. Expression of MMP-2 (gelatinase A), MMP-9 (gelatinase B), MMP-12 (macrophage metalloelastase), and MMP-14 (MT1-MMP) mRNA was detected in TRAP positive mononuclear and multinucleated cells by both RT-PCR and northern blot analysis. Immunocytochemistry for these MMPs showed that MMP-2 and MMP-9 were produced by both TRAP positive mononuclear and multinucleated cells, whereas MMP-12 and MMP-14 were produced by TRAP positive multinucleated cells. MMP-2 and MMP-9 were detected in the conditioned medium of TRAP positive mononuclear cells. TRAP positive mononuclear cells also induced the release of 35 S from prelabelled cartilage discs. Conclusion: This study suggests that TRAP positive mononuclear and multinucleated cells located in the synovium at the cartilage-synovial interface produce MMP-2 and MMP-9, and may have an important role in articular cartilage destruction in patients with RA.
Journal Article•
TL;DR: Serum levels of TRAP 5b were significantly higher in patients with breast cancer and clinical signs of bone metastases before therapy than in healthy women, indicating a reduction of bone alteration under bisphosphonate therapy.
Abstract: Background: The osteoclast-specific active TRAP 5b isoform is detectable in serum and claimed to be a specific marker of bone resorption. The present study was undertaken to evaluate the usefulness of TRAP 5b as a serum marker of bone resorption in breast cancer patients with bone metastases. Materials and Methods: TRAP 5b serum levels were measured in 192 samples from patients with breast cancer with and without bone metastases and in 53 healthy pre- and postmenopausal women using the enzyme immunoassay Bone-TRAP®. Results: Serum levels of TRAP 5b were significantly higher in patients with breast cancer and clinical signs of bone metastases before therapy than in healthy women. There was also a significant difference between patients with bone metastases before and during bisphosphonate therapy, indicating a reduction of bone alteration under this treatment. The subgroup with progression of bone metastases under bisphosphonate therapy showed the highest difference in TRAP 5b concentrations compared to patients with stable disease. Conclusion: Serum TRAP 5b levels are elevated in patients with bone metastases and breast cancer. The TRAP 5b levels decline under bisphosphonate therapy when no progression is detectable. When progress of the bone metastases occurs, TRAP 5b levels rise again. Therefore, active TRAP 5b seems to be a useful serum marker for bone metastases in breast cancer patients, especially to detect progressive disease under bisphosphonate treatment. Further studies with larger numbers of patients are required to confirm these data.
TL;DR: A method for evaluating bone resorption by quantifying area percentages of positive TRAP localization using image analysis and demonstrating a strong correlation between the conventional histomorphometric osteoclast index and positive TR AP localization is demonstrated, validating the latter as an alternative method to assess bone Resorption.
Abstract: Tartrate resistant acid phosphatase (TRAP) activity of bone is a suitable biochemical marker for osteoclastic bone resorption. Qualitatively, the histochemical distribution of TRAP has been used to identify osteoclasts responsible for bone resorption; however, there have been few attempts to quantify TRAP localization. We describe a method for evaluating bone resorption by quantifying area percentages of positive TRAP localization using image analysis. Mouse tibiae were paraffin embedded following demineralization in disodium ethylenediamine tetraacetic acid. Longitudinal sections of tibia were cut from 15 levels in the left and the right limbs of six mice (180 sections total) and stained for TRAP distribution. Positive TRAP localization was quantified by pixel area count and reported as a percentage of the total tissue area specified. The 1.85 mm2 region of interest was placed at the midpoint of the epiphyseal growth plate containing the provisional calcification layer and the primary spongiosa, while excluding cortical bone of each mouse tibia. The percentage of TRAP localization ranged from 0.95 to 1.31% and was not significantly different from level to level or limb to limb in each mouse (p > 0.100). Within the same region of interest, an osteoclast count along the bone perimeter also was performed. We demonstrated a strong correlation (r2 = 0.903) between the conventional histomorphometric osteoclast index and positive TRAP localization, validating the latter as an alternative method to assess bone resorption. Quantitative analysis of TRAP is significant because it allows statistical comparisons between treatment groups, promotes precise pathological diagnoses and facilitates a reference data base that may aid the study of bone related diseases involving increased bone resorption.
TL;DR: A human homolog of OCIL and its gene is identified, its regulation in human osteoblast cell lines is determined, and it is established that it can inhibit murine and human osteoclast formation and resorption.
Abstract: Osteoclast inhibitory lectin (OCIL) is a newly recognized inhibitor of osteoclast formation. We identified a human homolog of OCIL and its gene, determined its regulation in human osteoblast cell lines, and established that it can inhibit murine and human osteoclast formation and resorption. OCIL shows promise as a new antiresorptive.
Introduction: Murine and rat osteoclast inhibitory lectins (mOCIL and rOCIL, respectively) are type II membrane C-type lectins expressed by osteoblasts and other extraskeletal tissues, with the extracellular domain of each, expressed as a recombinant protein, able to inhibit in vitro osteoclast formation.
Materials and Methods: We isolated the human homolog of OCIL (hOCIL) from a human fetal cDNA library that predicts a 191 amino acid type II membrane protein, with the 112 amino acid C-type lectin region in the extracellular domain having 53% identity with the C-type lectin sequences of rOCIL and mOCIL. The extracellular domain of hOCIL was expressed as a soluble recombinant protein in E. coli, and its biological effects were determined.
Results and Conclusions: The hOCIL gene is 25 kb in length, comprised of five exons, and is a member of a superfamily of natural killer (NK) cell receptors encoded by the NK gene complex located on chromosome 12. Human OCIL mRNA expression is upregulated by interleukin (IL)-1α and prostaglandin E2 (PGE2) in a time-dependent manner in human osteogenic sarcoma MG63 cells, but not by dexamethasone or 1,25 dihydroxyvitamin D3. Soluble recombinant hOCIL had biological effects comparable with recombinant mOCIL on human and murine osteoclastogenesis. In addition to its capacity to limit osteoclast formation, OCIL was also able to inhibit bone resorption by mature, giant-cell tumor-derived osteoclasts. Thus, a human homolog of OCIL exists that is highly conserved with mOCIL in its primary amino acid sequence (C-lectin domain), genomic structure, and activity to inhibit osteoclastogenesis.
TL;DR: It is found that myoblastic C2C12 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells in the presence of 10−7 M 1α, 25-dihydroxyvitamin D3 [1α,25(OH)2D3] and that BMP-2 might play an important role in the differentiationof cells that support osteoclastogenesis.
Abstract: The interaction of osteoclast precursors with osteoblasts and/or stromal cells is essential for the formation of mature osteoclasts and the resorption of bone We found that myoblastic C2C12 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells in the presence of 10(-7) M 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and that C2C12 cells that had been treated with bone morphogenetic protein-2 (BMP-2) dose-dependently stimulated the formation of osteoclasts The newly developed TRAP-positive multinucleated cells were capable of resorbing mineralized tissues Treatment of C2C12 cells with BMP-2 for 24 h enhanced the subsequent expression in C2C12 cells of mRNA for the receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of 1alpha,25(OH)2D3 Since the formation of osteoclasts was inhibited dose-dependently by exogenous OPG, the expression of RANKL in response to BMP-2 appeared to be critical for the formation of osteoclasts Our findings suggest that BMP-2 might play an important role in the differentiation of cells that support osteoclastogenesis
TL;DR: It is proposed that the osteogenic action of fluoride is mediated, at least in part, by the fluoride‐mediated inhibition of osteoblastic type 5 TRACP/protein tyrosine phosphorylation, which leads to a stimulation of osteoblast proliferation and differentiation, and subsequently, an increase in bone formation.
Abstract: Although type 5 TRACP is recognized as a histochemical and biochemical marker of osteoclasts, there is evidence that bone forming cells, osteoblasts, and osteocytes also express a type 5 TRACP. Accordingly, an osteoblastic type 5 TRACP has been purified from human osteoblasts and from bovine cortical bone matrices. Comparison of biochemical properties of osteoblastic type 5 TRACP with those of osteoclastic type 5 TRACP suggests that osteoblastic type 5 TRACP is a different isoenzyme from osteoclastic type 5 TRACP. Two properties of osteoblastic type 5 TRACP may be relevant to its physiological functions: (1) it acts as a protein-tyrosine phosphatase (protein tyrosine phosphorylation) under physiologically relevant conditions, and (2) it is sensitive to inhibition by clinically relevant concentrations of fluoride. Because fluoride is a stimulator of osteoblastic proliferation and differentiation and a potent osteogenic agent and because protein tyrosine phosphorylation plays an important regulatory role in cell proliferation and differentiation, these unique properties and other evidence summarized in this review led to the proposal that the osteogenic action of fluoride is mediated, at least in part, by the fluoride-mediated inhibition of osteoblastic type 5 TRACP/protein tyrosine phosphorylation, which leads to a stimulation of osteoblast proliferation and differentiation, and subsequently, an increase in bone formation.
TL;DR: The data show that USF1 and USF2 play a functional role in RANKL-dependent TRAP expression during osteoclast differentiation, and mutation of the USF-binding site partially blocks RankL-induced TRAP transcription in RAW264.7 cells.
TL;DR: The tissue distribution of Runx2/Cbaf1/Pebp2αA in ossifying bones of the human fetus is similar to that in neonatal rodent tissues, and positive immunostaining of osteoclasts for typical osteogenic/chondrogenic markers has to be interpreted with caution due to the phagocytosing capacity of these cells.
Abstract: Runx2/Cbfa1 is a transcription factor, essential for the osteogenic/chondrogenic and odontogenic lineage. Three isoforms of Cbfa1 have been identified, type I (Pebp2alphaA isoform), type II (til-1 isoform), and type III (Osf2 isoform). Here we examined the expression of the Runx2/Cbfa1 during intramembranous and enchondral bone formation in the craniofacial tissues of neonatal rodents (hamster, rat, mouse) and the human fetus. We used a monoclonal antibody raised against the Pebp2alphaA portion and thus potentially recognizing all three isoforms of Runx2/Cbaf1. We report Cbfa1 at the mRNA and protein level in periosteum, preosteoblasts, osteoblasts, young osteocytes, perichondrium, resting and hypertrophic chondrocytes. During active bone remodeling, almost one third of tartrate resistant acid phosphatase (TRAP) positive multinuclear cells identified as osteoclasts were also stained with anti-Pebp2alphaA antibodies. Osteoclasts, however, did not express mRNA transcripts of the Pebp2alphaA gene. Some of the immunopositive structures within these osteoclasts resembled (ingested) cells. TRAP-positive mononuclear cells not attached to bone surfaces did not stain with anti-Pebp2alphaA antibodies. We concluded that the tissue distribution of Runx2/Cbaf1/Pebp2alphaA in ossifying bones of the human fetus is similar to that in neonatal rodent tissues. Osteoclasts do not transcribe the Runx2/Cbfa1 gene but become immunostained by phagocytosing and digesting osteocytes/hypertrophic chondrocytes. The substantial number of osteoclasts involved in phagocytosis of Runx2/Cbfa1 immunopositive cells suggests that phagocytosis is a major way of removing osteocytes/hypertrophic chondrocytes during resorption of bone and cartilage. Finally, the data indicate that positive immunostaining of osteoclasts for typical osteogenic/chondrogenic markers has to be interpreted with caution due to the phagocytosing capacity of these cells.
TL;DR: The results suggest that PVS has a hyperplastic property consisting of the CD68-positive monocytic cell lineage with differentiation of osteoclastic giant cells from monocyte and probably controlled against proliferation by wild-type p53, p21, and p16.
Journal Article•
TL;DR: The CWR22 human prostate cell line used in an intra-tibial nude rat model provides a useful system to study mechanisms involved in osteoblastic and osteolytic bony metastases and may provide a critical tool for drug development efforts focused on developing integrated systemic therapy targeting the tumor in its specific primary or/and metastatic microenvironments.
Abstract: We investigated the utility of CWR22 human prostate cancer cells for modeling human metastatic prostate cancer, specifically their ability to induce bone formation following intra-tibial injections in the nude rat. Prostate cancer is unique in regard to its tropism for bone and ability to induce new bone formation. In contrast to humans, other mammalian species rarely develop prostatic cancer spontaneously upon aging and do not have the propensity for bone metastasis that is the hallmark of cancer malignancy in men. We chose human prostate cancer cell line CWR22 based on its properties, which closely resemble all of the features that characterize the early stages of prostatic cancer in human patients including slow growth rate, hormone dependence/independence and secretion of prostate-specific antigen. When CWR22 cells were injected directly into the proximal tibia of immunodeficient male rats, both osteoblastic and osteolytic features became evident after 4 to 6 weeks, with elevated levels of serum prostate-specific antigen. However, osteosclerosis dominates the skeletal response to tumor burden. Radiological and histological evidence revealed osteosclerotic lesions with trabeculae of newly formed bone lined by active osteoblasts and surrounded by tumor cells. Toward the end of the 7-week study, osteolytic bone lesions become more evident on X-rays. Paraffin and immunohistochemical evaluations revealed mature bone matrix resorption as evidenced by the presence of many tartrate resistant acid phosphatase positive multinucleated osteoclasts. We conclude that the CWR22 human prostate cell line used in an intra-tibial nude rat model provides a useful system to study mechanisms involved in osteoblastic and osteolytic bony metastases. This type of in vivo model that closely mimics all major features of metastatic disease in humans may provide a critical tool for drug development efforts focused on developing integrated systemic therapy targeting the tumor in its specific primary or/and metastatic microenvironments. In addition to targeting bone marrow stroma, this strategy will help to overcome classical drug resistance seen at the sites of prostate cancer metastasis to bones.
TL;DR: Results suggested that a deficiency of Asc stimulated bone resorption at an early stage, followed by a decrease in bone formation in mature ODS rats which already had a well-developed collagen matrix and fully differentiated osteoblasts.
Abstract: The effect of ascorbic acid deficiency on bone metabolism was evaluated using the ascorbate-requiring Osteogenic Disorder Shionogi (ODS) rat model. Ascorbic acid (Asc)-deficient rats gained body weight in a manner similar to Asc-supplemented rats (control) during 3 weeks , but began to lose weight during the 4th week of Asc deficiency. The tartrate-resistant acid phosphatase (TRAP) activity in serum increased to about 2-fold the control value in the rats fed the Asc-free diet for 2, 3, and 4 weeks (AscD2, AscD3, and AscD4), while a decrease in the alkaline phosphatase (ALP) activity was observed only in AscD4 rats. The serum pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) level significantly increased to 1.3-, 1.4-, and 1.9-fold of that in the controls in AscD2, D3, and D4, respectively. The ALP activity in the distal femur was unchanged in AscD1, D2, and D3, but decreased to 50% of the control level in AscD4 rats. The TRAP activity in the distal femur increased to about 2-fold of that in the controls in the AscD2 and D3 and decreased to the control level in the AscD4 rats. The amount of hydroxyproline in the distal femur significantly decreased to about 80%, 70%, and 60% of the control in AscD2, D3, and D4 rats, respectively. These decreases were associated with a similar reduction in the calcium content of the distal femur. Histochemical analysis of the distal femur showed an increase in TRAP-positive cells in AscD2 and AscD3 rats and a decrease in the trabecular bone in AscD2, D3, and D4 rats. These results suggested that a deficiency of Asc stimulated bone resorption at an early stage, followed by a decrease in bone formation in mature ODS rats which already had a well-developed collagen matrix and fully differentiated osteoblasts.
TL;DR: It is suggested that osteoclast-mediated bone resorption was inhibited by incadronate administration, and that immature bones including the BMP pellets remained for a long time, indicating that the process of bone maturation was blocked.
Abstract: To determine the effects of a bisphosphonate on the quality of bone morphogenetic protein-(BMP-) induced bone, incadronate was administered to rats in which subcutaneous ectopic bones were induced by recombinant human BMP-2. Incadronate (1 μg/kg/day) was administered to rats carrying the BMP-induced bones three times per week, from the 3rd to 7th week after BMP implantation (incadronate group). Aliquots of phosphate-buffered saline were administered in the same protocol without incadronate to the control group. During the 3rd, 4th, 7th, or 10th week, the BMP-induced bones were removed and observed by contact microradiography (CMR), H&E staining, enzyme histochemistry for tartrate-resistant acid phosphatase (TRAP), and immunohistochemistry for cathepsin K. By 3 weeks when administration of incadronate began, woven bones formed in the periphery of the BMP pellets and osteoclasts were attached to these bones. At 4 weeks, in both the incadronate and control groups, bone formation advanced inward. However, in the incadronate group, poorly calcified areas, corresponding to the remaining BMP pellets, were found in the middle areas of bone formation, whereas osteoclasts decreased when compared with those of the control group. During the 10 weeks, bone marrow was formed and the characteristics of lamellar bones, in which the lacunae of small osteocytes were regularly arranged, were noted in the control group. In contrast, the poorly calcified areas were still present up to 10 weeks in incadronate group in which the osteoclasts were also scarcer than in the control group. These findings suggested that osteoclast-mediated bone resorption was inhibited by incadronate administration, and that immature bones including the BMP pellets remained for a long time, indicating that the process of bone maturation was blocked. The possibility of using incadronate for the purpose of inhibiting osteoclastic bone resorption was confirmed, but further study is needed before clinical application.
TL;DR: Serial sections of rat metaphyses prepared from paraffin embedded tissue blocks provided direct evidence of PTH/PTHrP receptors in osteoclasts, which was greater for the older animals than for the young animals.
Abstract: Serial sections of rat metaphyses were prepared from paraffin embedded tissue blocks and analyzed in sets of three. The central section was stained for tartrate resistant acid phosphatase (TRAP) in order to identify osteoclasts, one adjacent section was immunostained with an affinity purified antibody to a 15 amino acid sequence unique to rat PTH/ PTHrP receptor, and the other adjacent section in the set served as an immunostaining control. This allowed each of the 110 osteoclasts examined to be identified by TRAP and to be tested for the presence or absence of PTH/PTHrP receptor. All antibody solutions and rinses contained 1% donkey serum and 0.5% Tween 20 to ensure antibody integrity and good rinsing procedure. Confocal microscopy was used to evaluate fluorescence intensity of the immunostained osteoclasts. Pixel intensities of 58 osteoclasts from young (4 month) rats and 52 osteoclasts from old (15 month) rats were obtained. Pixel intensities were similar (P = 0.89) for both young and old animals. However, the number of PTH/PTHrP receptor deficient osteoclasts was greater for the older animals (14.29% vs. 7.24%). This provides direct evidence of PTH/PTHrP receptors in osteoclasts.
TL;DR: Results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.
Abstract: Alveolar bone resorption by osteoclasts is essential for tooth eruption. Osteoclast-deficient Csfm(op) homozygous (op/op) mice, which lack functional macrophage colony-stimulating factor (M-CSF), suffer from osteopetrosis and completely lack tooth eruption. Although osteoclasts appear, and osteopetrosis is cured with age in op/op mice, tooth eruption is never seen. This fact suggests that there is a critical period when osteoclasts are required for tooth eruption. In this study, to detect the critical period, we administered an antagonistic antibody directed against c-Fms, a receptor for M-CSF, to inbred C57BL/6 mice for various periods. Administration of this antibody decreased tartrate-resistant acid phosphatase-positive (TRAP) osteoclasts, and incisor eruption was completely inhibited by continual administration of this antibody from embryonic day 15.5 (E15.5) until postnatal day 12.5 (D12.5). A 1-day delay of this administration abolished the inhibition of incisor eruption. The number of TRAP-positive osteoclasts was significantly reduced between E16.5 and E18.5 in the mice treated with antibody from E15.5 compared with those treated from E16.5. These results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.
TL;DR: A lack of absolute specificity for bone has held back progress on the use of biochemical assay of TRACP activity for bone diseases, and a handful of special cytochemical reactions useful for the differentiation of various blood cell types are found.
Abstract: AS A YOUNG HEMATOLOGIST in the late 1960s at the New England Medical Center in Boston, one of us (LTY) was assigned to take charge of the Cytology Section of the Hematology Laboratory. It was the section’s responsibility to render a diagnosis from cytologic examination of bone marrow aspirations. In addition, research specimens from humans and animals were submitted for cytological interpretation. Although our diagnostic accuracy was very good, we soon recognized the need for more objective means of diagnosis through accurate cell identification. The section staff embarked on investigations to develop specific cell markers for cells in the hemopoietic system. We reasoned that the different lineages and cell types perform different biological functions; therefore, their biochemical constituents may reflect these specific functions. After examining and testing a wide array of special cytochemical reactions, we found a handful useful for the differentiation of various blood cell types. Among these were -naphthyl butyrate esterase, chloroacetate esterase, and acid phosphatase. At the time, serum acid phosphatase was recognized as a potential disease marker. Patients with prostatic cancer were known to have high levels of acid phosphatase activity in both blood and urine. Soon, however, it was realized that serum contained other acid phosphatases that were of nonprostatic origin. In a search to increase specificity of the biochemical tests for prostatic acid phosphatase, it was found that tartaric acid was a strong and effective inhibitor of prostatic acid phosphatase. Thus, to increase the specificity for prostate disease, acid phosphatase was measured as the tartrate-sensitive fraction. The tartrate-resistant fraction was believed to come primarily from red blood cells and was considered simply an interference. However, it was also noted that patients with bone diseases, particularly cancer patients with lytic bone lesions, also had increased acid phosphatase activity in the serum, and this acid phosphatase was resistant to inhibition by tartaric acid. Histochemically, TRACP activity was very strong in osteoclasts, and therefore, it was believed that the origin of the high TRACP in serum of patients with bone diseases may come from osteoclasts. However, because other acid phosphatase activities, particularly from red blood cells, were also resistant to tartrate, it was difficult to distinguish osteoclastic from non-osteoclastic TRACP activity by biochemical assay. This lack of absolute specificity for bone has held back progress on the use of biochemical assay of TRACP activity for bone diseases.
Journal Article•
TL;DR: The results have indicated that a predominance of bone resorption over bone formation is not involved in the pathogenesis of diabetes-associated osteopenia.
Abstract: The pathophysiological processes underlying the development of diabetic osteopenia has not hitherto been elucidated. Induction of streptozotocin diabetes leads in our experiments to decrease of bone density, ash, mineral content and to thinner cortical width compared to control male rats. In order to investigate the pathogenetic role of bone resorption by osteoclasts in streptozotocin-induced diabetes, we determined the circulating levels of tartrate-resistant acid phosphatase (TRAP), a biochemical marker for bone resorption. Plasma TRAP values in diabetic rats did not differ from their corresponding controls. Streptozotocin diabetes by itself did not have any effect on the weight of seminal vesicles which are highly testosterone-dependent. Low doses of nitric oxide cause bone resorption, but higher doses of NO inhibit bone resorbing activity. We examined the effect of L-NAME (inhibitor of nitric oxide production) after six weeks of administration to diabetic rats. There was no further significant loss of bone mineral density, ash and mineral content or tibia weight in diabetic rats treated with L-NAME. L-NAME itself did not decrease bone metabolism. In our study no evidence of an increased bone resorption was found. Our results have indicated that a predominance of bone resorption over bone formation is not involved in the pathogenesis of diabetes-associated osteopenia. Inhibition of NO neither increased osteoclastic activity (TRAP) nor induced osteopenia in L-NAME-treated rats. This suggests a possibility that NO is not involved in the pathogenesis of diabetic osteopenia.