Showing papers on "Tartrate-resistant acid phosphatase published in 2005"
TL;DR: New evidence is provided that skeletal sites rich in marrow cellularity under active turnover offer a more congenial microenvironment to facilitate cancer localization in the skeleton to facilitate metastatic prostate cancer in bone.
Abstract: Bone metastasis is a common untreatable complication associated with prostate cancer Metastatic cells seed in skeletal sites under active turnover containing dense marrow cellularity We hypothesized that differences in these skeletal-specific processes are among the critical factors that facilitate the preferential localization of metastatic prostate cancer in bone To test this, athymic mice were administered PTH to induce bone turnover and increase marrow cellularity daily 1 wk before and after intracardiac inoculation of luciferase-tagged PC-3 cells Tumor localization was monitored by bioluminescence imaging weekly for 5 wk At the time of tumor inoculation, PTH-treated mice demonstrated significant increases in serum levels of bone turnover markers such as osteocalcin and tartrate-resistant acid phosphatase 5b and in the number of tartrate-resistant acid phosphatase-positive osteoclasts per millimeter of bone when compared with the other groups Likewise, PTH treatment stimulated a qualitative increase in marrow cellular proliferation as determined by 5-bromo-2'-deoxyuridine immunostaining Skeletal metastases formed in the hind limb and craniofacial regions of young mice with no difference between groups In adult mice, however, bioluminescent signals in the hind limb and craniofacial regions were 3-fold higher in PTH-treated mice vs controls Fluorochrome labeling revealed increased bone formation activity in trabecular bone adjacent to tumors When zoledronic acid, a nitrogen-containing bisphosphonate that inhibits osteoclast-mediated bone resorption, was administered concurrently with PTH, a significant reduction in the incidence of bone tumors was observed Overall, these studies provide new evidence that skeletal sites rich in marrow cellularity under active turnover offer a more congenial microenvironment to facilitate cancer localization in the skeleton
196 citations
TL;DR: Findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.
Abstract: Background: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). Methods: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). Results: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. Conclusions: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.
173 citations
TL;DR: TRPV5 deficiency leads to an increase in osteoclast size and number, in which Ca(2+) resorption is nonfunctional, and the significance of transcellular Ca( 2+) transport in osteoclastic function is demonstrated.
Abstract: Bone remodeling involves the interplay of bone resorption and formation and is accurately controlled to maintain bone mass. Both processes require transcellular Ca(2+) transport, but the molecular mechanisms engaged remain largely elusive. The epithelial Ca(2+) channel TRPV5 is one of the most Ca(2+)-selective transient receptor potential (TRP) channels. In this study, the functional role of TRPV5 in bone was investigated. TRPV5 mRNA was expressed in human and murine bone samples and in osteoclasts along with other genes involved in transcellular Ca(2+) transport, including calbindin-D(9K) and calbindin-D(28K), Na(+)/Ca(2+) exchanger 1, and plasma membrane Ca(2+)-ATPase 1b. TRPV5 expression in murine osteoclasts was confirmed by immunostaining and showed predominant localization to the ruffled border membrane. However, TRPV5 was absent in osteoblasts. Analyses of femoral bone sections from TRPV5 knockout (TRPV5(-/-)) mice revealed increased osteoclast numbers and osteoclast area, whereas the urinary bone resorption marker deoxypyridinoline was reduced compared with WT (TRPV5(+/+)) mice. In an in vitro bone marrow culture system, the amount of osteoclasts and number of nuclei per osteoclast were significantly elevated in TRPV5(-/-) compared with TRPV5(+/+) mice. However, using a functional resorption pit assay, we found that bone resorption was nearly absent in osteoclast cultures from TRPV5(-/-) mice, supporting the impaired resorption observed in vivo. In conclusion, TRPV5 deficiency leads to an increase in osteoclast size and number, in which Ca(2+) resorption is nonfunctional. This report identifies TRPV5 as an epithelial Ca(2+) channel that is essential for osteoclastic bone resorption and demonstrates the significance of transcellular Ca(2+) transport in osteoclastic function.
170 citations
TL;DR: It is shown that aging is accompanied by increased RANKL and M‐CSF expression, increased stromal/osteoblastic cell‐induced osteoclastogenesis, and expansion of the osteOClast precursor pool.
Abstract: Stromal/osteoblastic cell expression of RANKL and M-CSF regulates osteoclastogenesis. We show that aging is accompanied by increased RANKL and M-CSF expression, increased stromal/osteoblastic cell-induced osteoclastogenesis, and expansion of the osteoclast precursor pool. These changes correlate with age-related alterations in the relationship between osteoblasts and osteoclasts in cancellous bone.
Introduction: Bone mass is maintained through a balance between osteoblast and osteoclast activity. Osteoblasts regulate the number and activity of osteoclasts through expression of RANKL, osteoprotegerin (OPG), and macrophage-colony stimulation factor (M-CSF). To determine whether age-related changes in stromal/osteoblastic cell expression of RANKL, OPG, and M-CSF are associated with stimulation of osteoclastogenesis and whether the osteoclast precursor pool changes with age, we studied cultures of stromal/osteoblastic cells and osteoclast precursor cells from animals of different ages and examined how aging influences bone cell populations in vivo.
Materials and Methods: Osteoclast precursors from male C57BL/6 mice of 6 weeks (young), 6 months (adult), and 24 months (old) of age were either co-cultured with stromal/osteoblastic cells from young, adult, or old mice or treated with M-CSF, RANKL, and/or OPG. Osteoclast precursor pool size was determined by fluorescence-activated cell sorting (FACS), and osteoclast formation was assessed by measuring the number of multinucleated TRACP+ cells and pit formation. The levels of mRNA for RANKL, M-CSF, and OPG were determined by quantitative RT-PCR, and transcription was measured by PCR-based run-on assays. Osteoblast and osteoclast numbers in bone were measured by histomorphometry.
Results: Osteoclast formation increased dramatically when stromal/osteoblastic cells from old compared with young donors were used to induce osteoclastogenesis. Regardless of the origin of the stromal/osteoblastic cells, the number of osteoclasts formed from the nonadherent population of cells increased with increasing age. Stromal/osteoblastic cell expression of RANKL and M-CSF increased, whereas OPG decreased with aging. Exogenously administered RANKL and M-CSF increased, dose-dependently, osteoclast formation from all donors, but the response was greater in cells from old donors. Osteoclast formation in vitro positively, and the ratio of osteoblasts to osteoclasts in vivo negatively, correlated with the ratio of RANKL to OPG expression in stromal/osteoblastic cells for all ages. The effects of RANKL-induced osteoclastogenesis in vitro were blocked by OPG, suggesting a causal relationship between RANKL expression and osteoclast-inducing potential. The osteoclast precursor pool and expression of RANK and c-fms increased with age.
Conclusions: Our results show that aging significantly increases stromal/osteoblastic cell-induced osteoclastogenesis, promotes expansion of the osteoclast precursor pool and alters the relationship between osteoblasts and osteoclasts in cancellous bone.
168 citations
TL;DR: It is suggested that acidification is essential for normal bone remodeling and that attenuated acidification leads to uncoupling with decreased bone resorption and unaffected bone formation.
Abstract: Patients with defective osteoclastic acidification have increased numbers of osteoclasts, with decreased resorption, but bone formation that remains unchanged. We demonstrate that osteoclast survival is increased when acidification is impaired, and that impairment of acidification results in inhibition of bone resorption without inhibition of bone formation. We investigated the role of acidification in human osteoclastic resorption and life span in vitro using inhibitors of chloride channels (NS5818/NS3696), the proton pump (bafilomycin) and cathepsin K. We found that bafilomycin and NS5818 dose dependently inhibited acidification of the osteoclastic resorption compartment and bone resorption. Inhibition of bone resorption by inhibition of acidification, but not cathepsin K inhibition, augmented osteoclast survival, which resulted in a 150 to 300% increase in osteoclasts compared to controls. We investigated the effect of inhibition of osteoclastic acidification in vivo by using the rat ovariectomy model with twice daily oral dosing of NS3696 at 50 mg/kg for 6 weeks. We observed a 60% decrease in resorption (DPYR), increased tartrate-resistant acid phosphatase levels, and no effect on bone formation evaluated by osteocalcin. We speculate that attenuated acidification inhibits dissolution of the inorganic phase of bone and results in an increased number of nonresorbing osteoclasts that are responsible for the coupling to normal bone formation. Thus, we suggest that acidification is essential for normal bone remodeling and that attenuated acidification leads to uncoupling with decreased bone resorption and unaffected bone formation.
151 citations
Journal Article•
TL;DR: The determination of biochemical markers of bone turnover offers many advantages in clinical practice, since they are non-invasive, can be repeated often, and major changes occur in a short time.
Abstract: Bone remodelling is a process by which bone grows and turns over. This process involves a series of highly regulated steps that depend on the interaction of two cell lineages, the osteoclasts and the osteoblasts. Information on metabolic activity of bone tissue are achieved with the determination, in blood and in urine, of biochemical products derived from the activity of this cells. The ability to determine bone turnover with biochemical markers has been enhanced considerably in recent years with the development of new assays for more sensitive and specific markers. These new markers can now replace the outdated and non-specific markers of bone remodeling such as serum total alkaline phosphatase (ALP) and urinary hydroxyproline (Hyp) determination. Biochemical markers of bone turnover can be classified according to the process that underlie in markers of bone formation, products of the osteoblast activity [bone ALP, osteocalcin (OC), procollagene I C- and N-terminal propeptides] and markers of bone resorption, products of the osteocalst activity [pyridinuim crosslinks, collagen I C- and N-terminal telopeptides (CTX-I and NTX-I), tartrate resistent acid phosphatase (TRACP) isoform 5b]. The interpretation of laboratory results should always include the consideration of potential sources of variability. Variation in the results of biochemical markers of bone metabolism can compromise their ability to characterize disorders of bone metabolism. Variation can be categorized into pre-analytical, analytical and biological sources. However, the determination of biochemical markers of bone turnover offers many advantages in clinical practice, since they are non-invasive, can be repeated often, and major changes occur in a short time.
127 citations
TL;DR: It is demonstrated that PEMF with different intensities could regulate osteoclastogenesis, bone resorption, OPG, RANKL, and M‐CSF concentrations in marrow culture system.
93 citations
TL;DR: In vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis.
Abstract: In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock-out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild-type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild-type and CD44 k.o. mice. Bone marrow cells from wild-type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild-type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild-type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild-type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild-type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment.
93 citations
TL;DR: Findings suggest that the osteoclastogenesis by P. gingivalis, T. denticola, and T. socranskii is mediated by a RANKL-dependent pathway and that PGE(2) is a main factor in the pathway by the enhancing of RANKl expression and the depression of osteoprotegerin, a RankL inhibitor.
Abstract: Background: Periodontitis is an inflammatory disease that often leads to destruction of alveolar bone; a number of bacteria in subgingival plaque are associated with bone destruction in periodontitis. To understand the mechanism of how periodontopathogens induce osteoclastogenesis, we determined which mediators are involved in the osteoclastogenesis. Methods: We investigated effects of sonicates from three periodontopathic bacteria, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, on osteoclast formation in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. The osteoclast formation was determined by tartrate resistant acid phosphatase (TRAP) staining. The expression of the receptor activator of nuclear factor-κ B ligand (RANKL), prostaglandin E2 (PGE2) and osteoprotegerin (OPG) in mouse calvaria-derived osteoblasts was determined by immunoassay. Results: Each bacterial sonicate induced the osteoclast formation in the co-culture system. These bacterial ...
73 citations
TL;DR: Interestingly, the magnitude and mode of inhibition by fluoride depends not only on the proteolytic state but also pH, and the combined kinetic data imply a role of the loop residue D158 in catalysis in the cleaved enzyme.
71 citations
TL;DR: Compared free radical production in macrophages of TRACP overexpressing (TRACP+) and wild-type (WT) mice, results suggest that TRACP has an important biological function in immune defense system.
TL;DR: Data show that the highly phosphorylated human OPN‐1 can stimulate trophoblastic cell migration and provides evidence for the involvement of the PI3K/mTOR/p70 S6 kinase pathway in the JAR cells response.
Abstract: This study examined the role of osteopontin (OPN), a phosphorylated secreted glycoprotein, in the promotion of trophoblastic cell migration, an early event in the embryo implantation process. Three human choriocarcinoma cell lines, namely JAR, BeWo, and JEG-3, were treated with variants of OPN differing in the extent of phosphorylation following sequential dephosphorylation with tartrate-resistant acid phosphatase (TRAP), and their migratory response was measured. The highly phosphorylated human milk form of OPN (OPN-1) strongly triggered migration in all three cell lines, whereas the less phosphorylated variants, OPN-2a and OPN-2b, failed to stimulate migration. JAR cell migration in response to OPN-1 was accompanied by a rapid rearrangement of actin filaments to the cellular membrane. Using broad spectrum protein kinase profiling, we identified p70 S6 kinase as a major signal transduction pathway activated by OPN-1 during the migratory response in JAR cells. Activation was blocked completely by rapamycin and LY294002, thus demonstrating that OPN-1-stimulated migration occurs through mTOR and PI3K pathways, respectively. Conversely, PD98059 did not affect the activation of p70 S6 kinase by OPN-1, therefore, this response does not involve the Ras/ MAPK signaling cascade. Together, these data show that the highly phosphorylated human OPN-1 can stimulate trophoblastic cell migration and provides evidence for the involvement of the PI3K/mTOR/p70 S6 kinase pathway in the JAR cells response. Because both OPN and TRAP are expressed in the uterus during early pregnancy, it is conceivable that extracellular phosphatases such as TRAP may modify OPN charge state and thus modulate cell migration.
TL;DR: The results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.
TL;DR: It is shown that mice lacking β3 integrin are protected from OVX‐induced bone loss and that β3S752, but not β3Y747/Y759, is important for osteoclastic bone resorption in vivo.
Abstract: We show that mice lacking β3 integrin are protected from OVX-induced bone loss. Using a lentiviral-based strategy to express β3 mutants in β3−/− mice, we also show that β3S752, but not β3Y747/Y759, is important for osteoclastic bone resorption in vivo.
Introduction: Mice lacking the β3 integrin have dysfunctional osteoclasts and therefore accumulate bone mass with age. Thus, the αvβ3 integrin is a potential anti-osteoporosis target. Identifying components of the β3 integrin that determine its function in vivo is essential for therapeutically exploiting the antiresorptive properties of αvβ3.
Materials and Methods: We used DXA and histomorphometry to assess bone loss after ovariectomy in wildtype and β3 integrin null mice. We used lentiviral vectors carrying various human β3 (hβ3) integrin constructs to transduce β3−/− bone marrow and reconstituted lethally irradiated β3−/− mice with the transduced marrow. The expressed constructs include the intact integrin and two mutants, namely hβ3Y747F/Y759F and hβ3S752P, each of which induces the bleeding dyscrasia, Glanzmann's thrombasthenia, in humans. Two months after transplantation, the expression of hβ3 was measured by flow cytometry of marrow-derived macrophages. Osteoclast differentiation and function were assessed ex vivo by TRACP and actin-ring staining, respectively. Reconstituted mice were ovariectomized, and bone loss was assessed by DXA, histomorphometry, and serum TRACP5b assay.
Results: β3−/− mice are protected from ovariectomy-induced bone loss, showing no difference in BMD compared with sham-operated controls. We successfully expressed hβ3 integrins in β3−/− hosts using lentiviral transduction of bone marrow. Two months after transplantation, 25–35% of marrow-derived macrophages expressed the hβ3 constructs. Similar to its effect in vitro, hβ3WT completely rescued the osteoclast and platelet phenotype of β3−/− mice. Whereas platelet function remained deranged in β3−/− mice overexpressing hβ3Y747F/Y759F, osteoclast function was fully restored. In contrast, β3−/− mice expressing hβ3S752P continued to exhibit prolonged bleeding times and dysfunctional osteoclasts in vitro and ex vivo. Most importantly, hβ3WT and hβ3Y747F/Y759F transplanted mice underwent equivalent ovariectomy-induced bone loss, whereas, like those bearing the control vector, hβ3S752P transplanted mice were protected.
Conclusions: Functional β3 integrin is required for ovariectomy-induced bone loss. β3S752, but not β3Y747/Y759, is critical for osteoclast function in vivo.
TL;DR: The co-localization of PAP/TRAP and the cysteine protease cathepsin L could suggest a role for cathePSin L in the in vivo proteolytic processing of P AP/ TRAP in monocytic cells.
Abstract: Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase (TRAP), uteroferrin or type 5 acid phosphatase (Acp5) is synthesized as an N-glycosylated monomeric latent precursor, which can be processed by limited proteolysis to a disulfide-linked two-subunit form with increased enzyme activity. In this study, we disclosed that the proteolytically processed two-subunit form constitutes the major PAP/TRAP variant in monocytic cells in spleen, thymus, liver and colon. In addition significant expression of the monomeric PAP/TRAP, indicating a non-enzymatic function, was detected in epithelial cells of colon, lung and kidney. Interestingly, proteolytic processing alone did not activate the enzyme but rendered the enzyme more susceptible to activation by reductants. Thus, beside limited proteolysis, the subcellular redox state could also be a determinant of enzyme action in vivo. The co-localization of PAP/TRAP and the cysteine protease cathepsin L could suggest a role for cathepsin L in the in vivo proteolytic processing of PAP/TRAP in monocytic cells.
TL;DR: It is demonstrated that cathepsin K secretion was coupled to ruffled border formation in clasts irrespective of whether the number of polarized clasts was low (in rickets) or high (in healing), suggesting a role for secreted TRAP as a modulator of resorptive activity.
Abstract: The activation sequence of clasts (the designation clast was used because ultrastructurally in this tissue, it is not always possible to differentiate between chondroclasts sitting on cartilage and osteoclasts sitting on bone matrix) was studied in vivo using the healing of low-phosphate, vitamin D-deficiency rickets as a model system. Thus, the bones of 7-week-old rachitic animals were analyzed with a combination of morphological, biochemical, and molecular biological methods at 48 and 72 h, respectively, after change to normal food. A quantitative ultrastructural analysis showed that the number of clast profiles exhibiting the characteristic polarized features of actively resorbing cells, i.e., ruffled borders and clear zones, had reached normal levels after 48 h. By combining the data with quantitative analyses by the immunogold technique, we demonstrated that cathepsin K secretion was coupled to ruffled border formation in clasts irrespective of whether the number of polarized clasts was low (in rickets) or high (in healing). In contrast, the levels of tartrate-resistant acid phosphatase (TRAP) both between ruffles and in the outside matrix adjoining the ruffled border were low in polarized clasts both in rickets and at the early (48 h) healing time-point, but were increased at the latest (72 h) healing time-point. Interestingly, expression of TRAP and the cathepsin K at the mRNA level, as well as protein expression and the activity of TRAP, were not different during the healing sequence. Although the two enzymes are confined to the same clast populations, their secretion during the resorption process is apparently differentially regulated: cathepsin K secretion is coupled to ruffled border formation in clasts, whereas TRAP is secreted at a later stage during the resorption sequence, suggesting a role for secreted TRAP as a modulator of resorptive activity.
TL;DR: The capacity of osteoblast-like cells to endocytose TRACP is important for the removal of this enzyme during or following the bone resorptive activity of the osteoclast, as well as two different fractions that may be different isoenzymes.
TL;DR: In this paper, studies with this natural indolic pigment at a concentration of 10.0 μmol L− 1 demonstrated a 20% activation of total protein phosphatase extracted from human lymphocytes.
Abstract: Given the importance of protein phosphorylation in the context of cellular functions, abnormal protein phosphatase activity has been implicated in several diseases, including cancer. These critical roles of protein phosphatases qualify them as potential targets for the development of medicinal compounds that possess distinct modes of action such as violacein. In this work, studies with this natural indolic pigment at a concentration of 10.0 μmol L− 1 demonstrated a 20% activation of total protein phosphatase extracted from human lymphocytes. Although no alteration was observed on protein tyrosine phosphatase (CD45), 30% of inhibition was achieved in cytoplasmatic protein phosphatase activity after incubation with 10.0 μmol L− 1 violacein. Additionally, 5.0 μmol L− 1 of violacein inhibited by 50% the serum tartrate-resistant acid phosphatase activity. Violacein presented toxic effect on lymphocytes with IC50 values of 3 and 10 μmol L− 1 for protein content and protein phosphatase activity, respectively. Th...
TL;DR: It is demonstrated that AA inhibits RANKL-induced differentiation of OCL precursor cells into mature OCL and reduces the formation of bone resorption pits in vitro.
Abstract: Ascorbic acid (AA) plays a key role in the regulation of differentiation and activation of osteoclast (OCL). It was reported that AA might induce the formation of OCL in cocultures of mouse bone marrow cells and ST2 cells, but it is not clear whether AA has a direct impact on the OCL precursors. The purpose of this study was to examine the effect of AA on the differentiation of OCL precursor RAW264.7 cells, cultured with receptor-activated nuclear factor kappaB ligand (RANKL). The results showed that AA remarkably inhibited the cell proliferation at a higher concentration and RANKL alone is sufficient for osteoclastogenesis. The expression of carbonic anhydrase (CAII) mRNA and protein, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), and the percentage area of resorption lacunae induced by RANKL were decreased when AA was added to the cultures. The results demonstrate that AA inhibits RANKL-induced differentiation of OCL precursor cells into mature OCL and reduces the formation of bone resorption pits in vitro.
TL;DR: The differences in substrate affinity and catalytic activity were abolished and partially restored, respectively, by proteolytic cleavage in the loop domain, indicating that the extent of N-glycosylation influences the interaction of the repressive loop domain with catalytically important residues.
TL;DR: A differentiation-dependent effect of growth factors on osteoclasts is indicated, which is important during bone formation and fracture healing.
Abstract: Previous in vivo studies were able to demonstrate the efficacy of locally released growth factors IGF-I, TGF-beta1, and BMP-2 from a poly(D,L-lactide) (PDLLA) implant coating on fracture healing. In vitro studies using human osteoblast-like cells showed an enhanced collagen-1 production due to growth factor application without an effect of the PDLLA on the investigated parameter. Both bone-forming osteoblasts and bone-resorbing osteoclasts are important during bone formation and fracture healing. The aim of this study was to investigate the influence of different growth factors and the polylactide coating into which they were incorporated on isolated osteoclasts. In vitro studies using human osteoclast-like cells derived from peripheral blood mononuclear cells (PBMNCs) were performed. Titanium K-wires coated with the lactide loaded with IGF-I and TGF-β1 (alone and in combination) or BMP-2 were added to the culture in a non-contact manner and the fusion, resorption activity (pit formation assay), and TRAP 5b synthesis of the cells were analyzed. Differences in the effect of the growth factors were seen depending on the differentiation state of the cells. The fusion of the monocytes to multinuclear osteoclasts was significantly enhanced by the application of TGF-β1 both alone and in combination with IGF-I. No effect was seen after application of IGF-I alone or BMP-2. The resorption activity of the osteoclasts analyzed on dentine chips was significantly enhanced after application of TGF-β1 or BMP-2. These results indicate a differentiation-dependent effect of growth factors on osteoclasts. TGF-β1 affects both the osteoclastogenesis and the activity of osteoclasts, whereas BMP-2 had an effect only on the activity of mature osteoclasts but not on the fusion of the PBMNCs.
TL;DR: In this paper, a mixture of a GGT composite, that is, a novel biodegradable composite containing genipin crosslinked gelatin and tricalcium phosphate, and traditional Chinese medicine (TCM) was prepared as a gGT-TCM composite.
Abstract: This study investigates the biological effects of traditional Chinese medicines on the activities of bone cells using rat bone cells. Then, a mixture of a GGT composite, that is, a novel biodegradable composite containing genipin crosslinked gelatin and tricalcium phosphate, and traditional Chinese medicine (TCM) was prepared as a GGT-TCM composite. A cultured neonatal rat calvarias organ was used to measure the potential of GGT-TCM composite for use in promoting the regeneration of defective bone tissue. The mitochondria activity of the bone cells following exposure to various concentrations of crude extracts of five herbal Chinese medicines was measured by colorimetric assay. Biochemical markers, such as alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) titers were analyzed to evaluate the activities of bone cells. Finally, we examined the organ culture units, which were maintained in cultured medium for 5 weeks. Morphology of tissue was observed, and the quantitative evaluation of the regenerated bone was determined. In a bone cells culture experiment, adding Cuscuta chinensis Lam. (TCM-5) to the bone cells culture clearly promoted the proliferation and differentiation of the osteoblasts from their precursor cells; but the reduced amount of TRAP indicated that the medicine significantly inhibited the osteoclasts activities. Opposite bone cell responses were observed when Loranthus parasiticus Merr. (TCM-3) and Achyranthes bidentata Bl. (TCM-4) were added to the bone cells culture. Eucommia ulmoides Oliv. (TCM-1) and Dipsacus asper Wall. (TCM-2) potentially influence the proliferation and differentiation of the osteoblasts from their precursor cells, but they did not affect the osteoclasts activities. The finding from the organ culture indicated that Chinese medicine effectively increased the rate of tissue regeneration of damaged bones.
TL;DR: Small interfering RNA molecules were used to knock down V‐ATPase subunit a3 in rat osteoclast cultures and found that Expression of a1 did not respond to decreased a3 levels, suggesting that a1 does not compensate for a3 for bone resorption in osteOClast cultures.
TL;DR: The results indicate that E MD induces the formation of osteoclasts through RANKL expressed by osteoblastic cells, and suggest that EMD may regulate both bone formation and bone resorption during periodontal tissue regeneration.
TL;DR: The results suggest that the development of specific P2X7 receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts.
Abstract: The P2X7 nucleotide receptor is an ATP-gated ion channel that plays an important role in bone cell function. Here, we investigated the effects of L: -tyrosine derivatives 1-3 as potent P2X7 antagonists on human primary osteoclasts. We found that the level of expression of P2X7 receptor increased after treatment with the derivatives 1-3, together with the induction of high levels of apoptosis. This effect is associated with activation of caspase-3 and inhibition of expression of IL-6. Interestingly, no pro-apoptotic effect of compounds 1-3 was found on human osteoblasts. Our results suggest that the development of specific P2X7 receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts. Since bone loss due to osteoclast-mediated resorption represents one of the major unsolved problem in osteopenic disorders, the identification of molecules able to induce apoptosis of osteoclasts is of great interest for the development of novel therapeutic strategies.
TL;DR: These studies show that confocal microscopy of neonatal calvaria in culture can be used to assess the cytological relationships between osteoclast and osteoblastic cells in response to agents that regulate bone remodeling in situ, avoiding systemic effects that can compromise in vivo studies and artifacts associated with studies of isolated osteoclasts.
Abstract: Because the development and activity of osteoclasts in bone remodeling is critically dependent on cell-cell and cell-matrix interactions, we used laser confocal microscopy to study the response of osteoclasts to lipopolysaccharide (LPS; 10 μg/ml), parathyroid hormone (PTH; 10−8 M), and bisphosphonates (BPs; 1–25 μM clodronate or 0.1–2.5 μM risedronate) in cultured neonatal calvaria. Following treatment with LPS or PTH (<48 hr), osteopontin (OPN) and the αvβ3 integrin were found colocalized with the actin ring in the sealing zone of actively resorbing osteoclasts. In contrast, non-resorbing osteoclasts in BP-treated cultures showed morphological abnormalities, including retraction of pseudopods and vacuolization of cytoplasm. In the combined presence of LPS and BP, bone-resorbing osteoclasts were smaller and the sealing zone diffuse, reflecting reduced actin, OPN, and β3 integrin staining. Depth analyses of calvaria showed that the area of resorbed bone was filled with proliferating osteoblastic cells that...
TL;DR: Results demonstrate that a basal constitutive neuronal NOS activity positively affects the RANKL/M-CSF-related osteoclastic differentiation.
Abstract: It has been clearly established that osteoclasts, which play a crucial role in bone resorption, differentiate from hematopoietic cells belonging to the monocyte/macrophage lineage in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). We have here investigated the M-CSF- and RANKL-induced osteoclastic differentiation of two distinct clones of the murine monocytic/macrophagic RAW 264.7 cell line, known as TIB-71 and CRL-2278, the latter cell clone being defective for the expression of the inducible nitric oxide synthase isoform in response to interferon-gamma or lipopolysaccharide. CRL-2278 cells demonstrated a more rapid osteoclastic differentiation than TIB-71 cells, as documented by morphology, tartrate-resistant acid phosphatase positivity, and bone resorption activity. The enhanced osteoclastic differentiation of CRL-2278 was accompanied by a higher rate of cells in the S/G2-M phases of cell cycle as compared to TIB-71. The analysis of nitric oxide synthase (NOS) isoforms clearly demonstrated that only neuronal NOS was detectable at high levels in CRL-2278 but not in TIB cells under all tested conditions. Moreover, the broad inhibitor of NOS activity L-NAME significantly inhibited osteoclastic differentiation of CRL-2278 cells. Altogether, these results demonstrate that a basal constitutive neuronal NOS activity positively affects the RANKL/M-CSF-related osteoclastic differentiation.
TL;DR: Serum PINP and TRACP5b are compared as markers for distinguishing between responders and nonresponders to bisphosphonate treatment with alendronate or risedronate.
Abstract: Bone markers to monitor the efficacy of antiresorptive therapy of osteoporosis are of great value to clinicians. Considerable decreases in markers can be seen within 3 to 6 months after the start of an efficient treatment, with considerable increases in bone mineral density (BMD) being observed in 1 to 2 years (1)(2)(3). Decreases in marker concentrations reflect the patient’s compliance to treatment and, particularly for bisphosphonate therapy, the intestinal absorption of the drug, which may be poor. By indicating that the treatment is efficacious, biomarkers may also encourage the patient to continue therapy (4). Economic restrictions and the many bone turnover markers available make it challenging to choose the one that best serves these purposes and most reliably predicts fracture prevention. The most important consideration in practice may be to choose a marker that enables the clinician to make a clear distinction between responders and nonresponders to treatment.
The amino-terminal propeptide of type I procollagen (PINP) is liberated into the circulation during type I collagen formation, and its serum concentration reflects bone formation rate (5). Because of the coupling between bone resorption and formation, PINP shows promise as a sensitive indicator of the efficacy of antiresorptive therapy (6). Tartrate-resistant acid phosphatase (TRACP), an iron-containing 35-kDa enzyme, is produced in osteoclasts, beginning early in their development. TRACP has several known functions in the osteoclasts from which it is liberated into the circulation in active form (7)(8)(9). In addition to osteoclastic isoform TRACP5b, human serum contains another, differently glycosylated isoform, TRACP5a. Secreted TRACP5b activity, which can be measured specifically with a novel immunoextraction method (10), is believed to reflect bone resorption.
We compared serum PINP and TRACP5b as markers for distinguishing between responders and nonresponders to bisphosphonate treatment with alendronate or risedronate. The response …
TL;DR: In this paper, immunohistochemical staining for CD117 (c-Kit), MITF, TRAP, and HAM-56 was studied in 35 GCTBs, 15 GCTTSs, and 5 foreign-body GC controls.
Abstract: Context.—Osteoclast-like giant cells (GCs) in giant cell tumors (GCTs) are thought to derive from a monocyte-macrophage lineage. Microphthalmia transcription factor (MITF) is necessary for osteoclast gene expression and tartrate-resistant acid phosphatase (TRAP) activation; c-Kit plays a role in regulation of MITF. Objective.—To gain insight into the differentiation of GCTs of bone (GCTBs) and GCTs tendon sheath (GCTTSs) by investigating immunohistochemical staining for c-Kit, MITF, TRAP, and HAM-56 in the GCs and stroma. Design.—Immunoreactivity for CD117 (c-Kit), MITF, TRAP, and HAM-56 was studied in 35 GCTBs, 15 GCTTSs, and 5 foreign-body GC controls. Results.—Across tumors, MITF and TRAP but not c-Kit were generally expressed in GCs; TRAP was variably expressed in stromal cells. The MITF was expressed more consistently in stromal cells of GCTTSs than GCTBs (P < .001). The GCTBs showed more intense MITF stromal (P < .001) and TRAP GC staining (P = .04) than GCTTSs. HAM-56 staining by stromal c...
Journal Article•
TL;DR: In patients without further osseous progression, TRACP 5b is able to monitor the effectiveness of local radiotherapy and demonstrates that the ability to discriminate between those patients with or without osseus progression increases with time.
Abstract: Background: The aim of our study was to evaluate if the determination of the active isoform 5b of tartrate-resistant acid phosphatase (TRACP 5b) provides the possibility to monitor the effect of local radiotherapy in bone metastases and if TRACP 5b will predict further osseous progression. Materials and Methods: In 48 breast cancer patients with bone metastases, patients' characteristics, diagnostic imaging and laboratory investigation, tumor- and therapy-related parameters were registered at the beginning and the end of radiotherapy, as well as 6 and 12 weeks afterwards. TRACP 5b activity was measured using a solid phase immunofixed enzyme activity assay with the monoclonal antibody O1A. Results: During follow-up, progression in another part of the skeleton was diagnosed in 31 patients (65%). There was a significant decrease of TRACP 5b in patients without progression in non-irradiated regions, whereas in progressive disease, TRACP 5b levels remained stable with a slightly increasing tendency (p 3 metastases (p=0.01). Conclusion: In patients without further osseous progression, TRACP 5b is able to monitor the effectiveness of local radiotherapy. The estimation of sensitivity and specificity based on each TRACP 5b value demonstrates that the ability to discriminate between those patients with or without osseous progression increases with time. In addition to routine clinical investigation, evaluation of pain scores and the use of X-ray imaging and/or bone scintigraphy (1), the evaluation and monitoring of local radiotherapy in the treatment of bone metastases, as well as the disease activity, may be improved by biochemical markers of bone formation and/or bone resorption. However, neither in diagnosis nor in monitoring systemic or local therapies of osseous metastases, are bone markers - with the exception of the alkaline phosphatase (AP) with its