Tartrate-resistant Acid Phosphatase Enzyme Activity Research Articles

Effect of Chitosan Degradation Products, Glucosamine and Chitosan Oligosaccharide, on Osteoclastic Differentiation.

Chitosan, a natural cationic polysaccharide derived from crustaceans and shellfish shells, is known for its advantageous biological properties, including biodegradability, biocompatibility, and antibacterial activity. Chitosan and its composite materials are studied for their potential for bone tissue repair. However, the effects of chitosan degradation products, glucosamine (GlcN) and chitosan oligosaccharide (COS), on osteoclasts remain unclear. If these chitosan degradation products promote osteoclastic differentiation, careful consideration is required for the use of chitosan and related materials in bone repair applications. Here, we assessed the effects of high (500 μg/mL) and low (0.5 μg/mL) concentrations of GlcN and COS on osteoclastic differentiation in human peripheral blood mononuclear cells (PBMCs) and murine macrophage-like RAW264 cells. A tartrate-resistant acid phosphatase (TRAP) enzyme activity assay, TRAP staining, and actin staining were used to assess osteoclastic differentiation. High concentrations of GlcN and COS, but not low concentrations, suppressed macrophage colony-stimulating factor (M-CSF)- and RANKL-dependent increases in TRAP enzyme activity, TRAP-positive multinuclear osteoclast formation, and actin ring formation in PBMCs without cytotoxicity. Similar effects were observed in the RANKL-dependent osteoclastic differentiation of RAW264 cells. In conclusion, chitosan degradation products do not possess osteoclast-inducing properties, suggesting that chitosan and its composite materials can be safely used for bone tissue repair.

Mineral trioxide aggregate (MTA) inhibits osteoclastogenesis and osteoclast activation through calcium and aluminum activities.

To evaluate the effect(s) of mineral trioxide aggregate (MTA) on in vitro RANKL-mediated osteoclast-dependent bone resorption events and the influence of Ca2+ and Al3+ on the osteoclastogenesis inhibition by MTA. Two types of osteoclast precursors, RAW 264.7 (RAW) cell line or bone marrow cells (obtained from BALB/c mice and stimulated with recombinant (r)macrophage colony stimulation factor (M-CSF), were stimulated with or withoutrecombinant (r) activator of nuclear kappa B ligand (RANKL), in the presence or absence ofMTA for 6 to 8days. White Angelus MTA and Bios MTA (Angelus, Londrina, Paraná, Brazil) were prepared and inserted into capillary tubes (direct contact surface = 0.50mm2 and 0.01mm2). Influence of MTA on these types of osteoclast precursors was measured by the number of differentiated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (RAW and bone marrow cells), TRAP enzyme activity (RAW cells), cathepsin K gene expression (RAW cells), and resorptive pit formation (RAW cells) by mature osteoclasts. Besides, RAW cells were also stimulated with Ca2+ and Al3+ to evaluate the influence of theseions on MTA anti-osteoclastogenic potential. In bone marrow and RAW cells, the number of TRAP-positive mature osteoclast cells induced by rRANKL was significantly inhibited by the presence of MTA compared with control rRANKL stimulation without MTA (p < 0.05), along with the reduction of TRAP enzyme activity (p < 0.05) and the low expression of cathepsin K gene (p < 0.05). In contrast, to control mature osteoclasts, the resorption area on dentin was significantly decreased for mature osteoclasts incubated with MTA (p < 0.05). rRANKL-stimulated RAW cells treated with Ca2+ and Al3+ decreased the number of osteoclasts cells. Besides, the aluminum oxide was the dominant suppressor of the osteoclastogenesis process. MTA significantly suppressed RANKL-mediated osteoclastogenesis and osteoclast activity and, therefore, appears able to suppress bone resorption events in periapical lesions. This process might be related to Ca2+ and Al3+ activities. MTA is an important worldwidely acknowleged biomaterial. The knowledge about its molecular activities on osteoclasts might contribute to improving the understanding of itsclinical efficacy.

Glucosamine Suppresses Osteoclast Differentiation through the Modulation of Glycosylation Including O-GlcNAcylation.

Osteoclasts represent the only bone resorbing cells in an organism. In this study, we investigated the effect of glucosamine (GlcN), a nutrient used to prevent joint pain and bone loss, on the osteoclastogenesis of murine macrophage-like RAW264 cells. GlcN supplementation suppressed the upregulation of osteoclast-specific genes (tartrate-resistant acid phosphatase (TRAP), cathepsin K, matrix metallopeptidase 9, and nuclear factor of activated T cell c1 (NFATc1)), receptor activator of nuclear factor-κB ligand (RANKL)-dependent upregulation of TRAP enzyme activity, and the formation of TRAP-positive multinuclear cells more effectively than N-acetylglucosamine (GlcNAc), which we have previously shown to inhibit osteoclast differentiation. To clarify the mechanism by which GlcN suppresses osteoclastogenesis, we further investigated the effect of GlcN on O-GlcNAcylation by Western blotting and on other types of glycosylation by lectin blotting. We found that, upon addition of GlcN, the O-GlcNAcylation of cellular proteins was increased whereas α2,6-linked sialic acid modification was decreased. Therefore, these glycan modifications in cellular proteins may contribute to the suppression of osteoclastogenesis.

Abstract 3791: TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents

Abstract Background: Multiple myeloma and its associated bone disease are generally incurable. Hence, better therapies are needed, ideally targeting biomolecules implicated in the aberrant biology of the disease. Overexpression of transient receptor potential cation channel TRPV6, a highly selective calcium channel has been observed in breast, colon, thyroid, ovary and prostate cancer tissues, and in several tumour cell lines. TRPV6 expression has also been seen in osteoclasts, however its role in bone metabolism remains unclear. The reciprocal interaction between osteoclasts and myeloma cells is pivotal to the generation of bone lesions that characterize myeloma. While myeloma cells secrete factors promoting osteoclast activity, osteoclasts in turn are known to induce myeloma cell growth and survival. The TRPV6 peptide antagonist SOR-C13 is currently in phase I clinical trials as an anti-cancer therapy for advanced cancers. We investigated the expression and potential therapeutic significance of TRPV6 in human osteoclasts and myeloma cells using shrew venom derived peptide antagonists. Methods: High affinity TRPV6 peptide antagonists SOR-C13 and SOR-C27 derived from the C-terminus of venom from the short-tailed shrew, Blarina brevicauda were used. Human primary osteoclasts were generated in vitro from human bone marrow (BM) aspirates; characterized by Hoechst-phalloidin staining, Tartrate resistant acid phosphatase (TRAP) staining, TRAP enzyme activity and Cathepsin K expression. CD138 positive myeloma cells were isolated from patient bone marrow specimens by EasySep immunomagnetic separation, or examined in tissue microarrays of patient BM core biopsies. TRPV6 expression in primary human osteoclasts, myeloma cell lines and myeloma patient BM microarray was checked by qPCR, immunohistochemistry or immunoblotting. Anti-resorptive potential of SOR peptides using human osteoclasts was evaluated in Osteoassay plates that mimic bone, and myeloma cell growth inhibition was determined by prestoblue cell viability assays. Results: We found strong expression of TRPV6 protein in human myeloma cells and osteoclasts by immunohistochemical staining on myeloma patient bone marrow sections. Human osteoclasts generated in vitro and CD138 positive myeloma patient bone marrow plasma cells were found to express TRPV6. We saw dose-dependent inhibition of osteoclast activity in vitro by SOR-C13 and SOR-C27, including markedly reduced osteoclast formation, decreased TRAP activity and reduced osteoassay surface resorption. TRPV6 peptide antagonists were also found to inhibit the growth of human myeloma cell lines U266 and KMM-1. Conclusion: Anti-myeloma and anti-osteoclast activity of human TRPV6 antagonist peptides was seen in the current study. Taken together, our findings suggest a novel therapeutic approach for multiple myeloma involving TRPV6 inhibition to target both myeloma cells and osteoclasts. Citation Format: Alli Murugesan, Philippe Tremblay, Ming Han, Bithika Ray, Tyler Lutes, Tony Reiman. TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3791.

Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

Osteoclasts (OCs) are bone-resorptive cells critical for maintaining skeletal integrity through coupled bone turnover. OC differentiation and activation requires receptor activator of NF-kB ligand (RANKL) signaling through the p38 MAPK pathway. However the role of the p38 MAPK substrate, MAPK-activated protein kinase 2 (MK2), is not clearly delineated. Within the bone marrow exists a specific subpopulation of defined osteoclast progenitor cells (dOCPs) with surface expression of B220-Gr1-CD11blo/-CD115+ (dOCPlo/-). In this study, we isolated dOCPs from male and female mice to determine sex-specific effects of MK2 signaling in osteoclastogenesis (OCgen). Male Mk2-/- mice display an increase in the dOCPlo cell population when compared to Mk2+/+ mice, while female Mk2-/- and Mk2+/+ mice exhibit no difference. Defined OCPs from male and female Mk2+/+ and Mk2-/- bone marrow were treated with macrophage colony stimulation factor (M-CSF) and RANKL cytokines to promote OCgen. RANKL treatment of dOCPlo cells stimulated p38 and MK2 phosphorylation. Tartrate-resistant acid phosphatase (TRAP) assays were used to quantify OC number, size, and TRAP enzyme activity post-RANKL stimulation. MK2 signaling was critical for male dOCPlo OCgen, yet MK2 signaling regulated OCgen from female dOCP- and CD11bhi subpopulations as well. The functional gene, Ctsk, was attenuated in both male and female Mk2-/- dOCPlo-derived OCs. Conversely, MK2 signaling was only critical for gene expression of pre-OC fusion genes, Oc-stamp andTm7sf4, in male OCgen. Therefore, these data suggest there is a sexual dimorphism in MK2 signaling of OCP subpopulations.

Increased dietary phosphorous prevents vertebral deformities in triploid Atlantic salmon (Salmo salarL.)

Triplicate groups of triploid and diploid Atlantic salmon were fed diets with a low (LP, total P: 7.1 g kg−1), medium (MP, total P: 9.4 g kg−1) or high (HP, total P: 16.3 g kg−1) phosphorous (P) level from first feeding (0.18 g) to transfer to sea water (~50 g, duration: 203 days) and subsequently fed a commercial diet in sea water for 426 days (~3 kg). This study examined the short- and long-term effects of dietary P on freshwater performance (mortality, growth), vertebral deformities (radiology), bone cell activity (ALP and TRACP enzyme activity in vertebrae and scales, and fgf23, bgp and igf-I relative gene expression in vertebrae), bone mineralization (ash content) and some parameters related to fish condition (heart and liver size). Irrespective of ploidy, at seawater transfer, fish fed the MP diet had significantly highest length and weight and those fed the LP diet significantly lowest length and weight, while those fed the HP diet had intermediate lengths and weights. Increased dietary phosphorus reduced deformities in both ploidies at seawater transfer; however, triploids fed the LP and MP diets had more deformities than diploids fed the respective diets, while there was no ploidy effect observed for fish fed the HP diet. The vertebral bone ash content at seawater transfer was significantly higher in diploids than in triploids when fed the MP diet only. Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRACP) enzyme activities and relative gene expression of bone hormones involved in metabolism of plasma phosphate (fgf23) and bone growth (bgp) were not affected by ploidy at seawater transfer, but by dietary P level; LP increased ALP activity and reduced TRACP activity and fgf23 and bgp expression levels in vertebral bone. In scales, LP increased both ALP and TRACP activity. At the termination of the seawater period, the group-wise pattern in occurrence of vertebral deformities was the same as at seawater transfer. The present results on mortality, growth, bone mineralization and development of skeletal deformities all demonstrate that triploids have a higher P requirement than diploids in fresh water. This study shows that an optimalization of P nutrition for triploid Atlantic salmon can improve health and welfare and reduce down-grading of triploid salmon.

Increased Tartrate-Resistant Acid Phosphatase Expression in Osteoblasts and Osteocytes in Experimental Osteoporosis in Rats

Tartrate-resistant acid phosphatase (TRAP) is known as an osteoclast marker, but osteoblasts and osteocytes in the vicinity of bone remodeling sites also express TRAP. Cell culture studies suggest that osteoblasts endocytose osteoclastic TRAP for inactivation. To evaluate whether changes in osteoclast activity could alter TRAP expression in osteoblasts and/or osteocytes in vivo, we studied the ovariectomized and vitamin D-deficient rat (Ovx-D) and rats healing from rickets. Bone sections were analyzed for TRAP gene expression by in situ hybridization, TRAP protein by immunogold labeling, and TRAP enzyme activity using the fluorescent substrate ELF97. Osteoblasts and osteocytes close to intracortical remodeling sites and bone surfaces demonstrated TRAP, most prominently in cancellous bone and osteocytes. Intracellular TRAP was located to electron-dense vesicles with similar morphology in both cell types. Ovx-D increased osteoclast activity (p < 0.001) and ELF97+ osteocytes (p < 0.05) in cancellous bone, but no corresponding increase was observed in the osteocyte lacunar area. The level of TRAP+ vesicles in cortical osteoblasts (p < 0.01) in Ovx-D rats was also increased. Enhanced osteoclast activity was noted in healing rickets after 72 h (p < 0.05), but no differences in TRAP expression were detected in osteoblasts or osteocytes. Thus, increased osteoclast activity does not affect TRAP expression in osteoblasts and osteocytes, favoring the notion that increased TRAP in these cells is rather due to increased synthesis. Although the role of TRAP in osteoblasts and osteocytes remains elusive, we speculate that the function is related to the capability of the enzyme to regulate the phosphorylation of proteins known to be expressed by these cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00223-013-9834-3) contains supplementary material, which is available to authorized users.

Eccentric Localization of Osteocytes Expressing Enzymatic Activities, Protein, and mRNA Signals for Type 5 Tartrate-resistant Acid Phosphatase (TRAP)

Enzymatic activity of type 5 tartrate-resistant acid phosphatase (TRAP) has been regarded as one of the reliable markers for osteoclasts and their precursors. The presence of TRAP activity in osteocytes near the bone resorbing surface has also been pointed out in some reports. However, the significance of TRAP reactions in osteocytes remains controversial and, in fact, there is no agreement as to whether the histochemical enzyme reactions in osteocytes represent the TRAP enzyme generated by the respective osteocytes or is a mere diffusion artifact of the reaction products derived from the nearby osteoclasts. Current histochemical, immunohistochemical, and in situ hybridization studies of rat and canine bones confirmed TRAP enzyme activity, TRAP immunoreactivity, and the expression of Trap mRNA signals in osteocytes located close to the bone-resorbing surface. TRAP/Trap- positive osteocytes thus identified were confined to the areas no further than 200 microm from the bone-resorbing surface and showed apparent upregulation of TRAP/Trap expression toward the active osteoclasts. Spatial and temporal patterns of TRAP/Trap expression in the osteocytes should serve as a valuable parameter for further analyses of biological interactions between the osteocytes and the osteoclasts associated with bone remodeling.

The effects of colony-stimulating factor-1 (CSF-1) on the development of osteoclasts and their expression of tartrate-resistant acid phosphatase (TRAP) in toothless (tl-osteopetrotic) rats.

The osteopetrotic mutation toothless (tl) in the rat is characterized by a limited number of osteoclasts with reduced amounts and/or activity of tartrate-resistant acid phosphatase (TRAP). Treatment of tl/tl mutants with the cytokine colony-stimulating factor (CSF)-1 increases both osteoclast number and enzyme activity, consistent with a loss-of-function mutation in the CSF-1 gene recently detected in this mutant. We have pursued these observations to demonstrate that there is a dose-dependent increase in osteoclast number, but not to normal levels. Osteoclasts in CSF-1-treated tl/tl mutants are large, have well-developed clear zones and ruffled borders, and secrete TRAP into resorption lacunae. The expression of TRAP mRNA, protein, and enzyme activity per bone appear normal after CSF-1 treatment. However, in contrast to the predominantly apical intracellular distribution in normal osteoclasts, an enrichment of TRAP enzyme activity in osteoclasts of CSF-1-treated tl/tl mutants is observed in the basal part of the cell. Our observations suggest that the CSF-1-treated mutant bones contain an abundance of mature osteoclasts, actively expressing markers for osteoclasts such as TRAP, cathepsin K, and matrix metalloproteinase (MMP)-9. Accumulation of TRAP at the end of the endocytic pathway in mature osteoclasts formed during CSF-1 treatment suggests that the TRAP enzyme has a rapid turnover in these highly active cells and uses a transcytotic pathway.

Tissue responses to nacreous implants in rat femur: an in situ hybridization and histochemical study

The interface of bone and aragonite nacre ( Margaritifera, fresh water pearl mussel) was studied by in situ hybridization and a tartrate-resistant acid phosphatase (TRAP) histochemical assay. Columnar implants were inserted into rat femora for 4, 7, 14, 28 and 56 days. In medullary region, a burst of transient bone formation was observed, which propagated from the periphery towards the nacre implant. A fused interface of bone and nacre was observed at 14 days. Later, the new medullary bone was resorbed and bone marrow was re-established while a thin layer of bone tissue remained covering the implant surface. Expressions of collagen α1( I), osteocalcin, osteopontin mRNAs and TRAP in the surrounding tissue were monitored. Correlated with the histology events, a strong transient induction of collagen α1( I) and osteocalcin mRNAs as well as TRAP expression, exhibiting a peak signal intensity on day 7 and subsequent down-regulation after day 14 was observed. Osteopontin mRNA, in contrast, was expressed continuously. The degrading nacre surface appeared in direct contact with macrophages and multinucleated giant cells at both days 14 and 28. These cells expressed osteopontin mRNA intensively and some TRAP enzyme activity occasionally.

Tartrate-resistant acid phosphatase forms complexes with alpha2-macroglobulin in serum.

Tartrate-resistant acid phosphatase (TRAP) is a standard histochemical marker of differentiated osteoclasts and has been proposed as a serum/plasma marker for osteoclast activity. Enzyme assays have been described that show elevated TRAP enzyme activity in the serum or plasma of patient groups known to have increased bone metabolism. However, the poor stability of the enzyme and potential contribution from nonosteoclastic sources make it problematic to measure in patient samples. Immunoassays developed to measure TRAP in serum and plasma have yielded widely varying TRAP levels in both normal and disease states. It is not clear if this variability is caused by differences in assay calibration, antibody specificity, and/or TRAP instability. In this paper, we report that purified TRAP spiked into serum forms high molecular weight complexes. Complex formation results in greatly decreased TRAP enzyme activity and immunoreactivity. The complexing protein in serum has been identified as alpha2-macroglobulin (alpha2M). Similar complexes are observed in stored patient samples. In vitro studies with purified components show that TRAP binds to alpha2M primarily through noncovalent ionic interactions. Our results demonstrate that one mechanism of TRAP instability in serum is complex formation with alpha2M and suggest further that current TRAP enzyme and immunoassays may not accurately measure the circulating level of TRAP.

Human giant cell tumors of the bone (osteoclastomas) are estrogen target cells.

The decrease in estrogen levels that follows the onset of menopause results in rapid bone loss and osteoporosis. The major effect of estrogen deficiency on bone metabolism is an increase in the rate of bone resorption, but the precise mechanism by which this occurs remains unresolved. A recently developed technique for the isolation of avian osteoclasts has been modified to obtain highly purified multinucleated cells from human giant cell tumors. These osteoclast-like cells have been examined for evidence of estrogen receptors (ERs) and responses to 17 beta-estradiol (17 beta-E2). Analysis of giant-cell RNA demonstrated expression of ER mRNA. Furthermore, immunoblot analysis revealed that the giant cells contained a 66-kDa protein that was recognized by a monoclonal antibody specific for the human ER. When isolated multinucleated cells were cultured on slices of bone, there was a dose-dependent decrease in resorption in response to treatment detectable at 10 pM 17 beta-E2. Treatment with 10 nM 17 alpha-estradiol or vehicle (control) did not inhibit resorption. Moreover, the multinucleated cells isolated from these tumors had decreased mRNA levels for cathepsin B, cathepsin D, and tartrate-resistant acid phosphatase (TRAP) as well as secreted cathepsin B and TRAP enzyme activity in response to treatment with 10 nM 17 beta-E2. In contrast to these data, no change in gene expression was detected in mononuclear cells from these tumors in response to 17 beta-E2 treatment. These data support the proposition that human osteoclasts are target cells for estrogen and that estrogen can inhibit bone resorption by human osteoclasts.

Transcriptional regulation of the tartrate-resistant acid phosphatase (TRAP) gene by iron

Tartrate-resistant acid phosphatase (TRAP) was first identified in cells from patients with hairy cell leukaemia. Subsequently, it has been found in other leukaemias, B-lymphoblastoid cell lines, osteoclasts and subsets of normal lymphocytes, macrophages, and granulocytes. Recent data indicate that TRAP and porcine uteroferrin, a placental iron-transport protein, represent a single gene product. However, the intracellular role of TRAP is unknown. We used a full-length human placental TRAP cDNA probe to examine TRAP expression in human peripheral mononuclear cells (PMCs). TRAP mRNA increased 50-75-fold after 24 h in unstimulated PMC cultures. Cell-fractionation experiments indicated that monocytes were the main cell population accounting for increased TRAP mRNA transcripts, and this was confirmed by histochemical staining for TRAP enzyme activity. Because expression of other iron-binding and -transport proteins is controlled by iron availability, we examined the role of iron in regulating TRAP expression. Increase of TRAP mRNA transcripts in PMCs was inhibited by 50 microM desferrioxamine, a potent iron chelator. The 5' flanking region of the TRAP gene was cloned from a mouse genomic library. In preliminary transient transfection experiments, it was determined that the 5'-flanking region of the TRAP gene contained iron-responsive elements. Therefore, a series of stably transfected HRE H9 cell lines was developed bearing genetic constructs containing various segments of the murine TRAP 5' promoter region driving a luciferase reporter gene. Treatment of transfectants with 100 micrograms/ml iron-saturated human transferrin (FeTF) was performed to assess iron responsiveness of the constructs. Constructs containing a full-length TRAP promoter (comprising base pairs -1846 to +2) responded to FeTF with a 4-5-fold increase of luciferase activity whereas constructs containing only base pairs -363 to +2 of the TRAP promoter did not respond. Constructs containing 1240 or 881 bp of the TRAP promoter gave only a 1.5- to 2-fold increase of luciferase activity with FeTF. In all cases, increase of luciferase activity was blocked by desferrioxamine. Cells transfected with another luciferase construct driven by a simian virus 40 promoter did not show any increase of luciferase activity with FeTF. These data indicate that expression of TRAP is regulated by iron and that this regulation is exerted at the level of gene transcription. The transfection experiments also suggest that the region of the TRAP 5'-flanking sequence between base pairs -1846 and -1240 contains an iron regulatory element.