Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the alphavbeta3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase. The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human alphavbeta3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype. The cryopreserved cells can be cultured on bovine cortical bone for 24-48 h and resorption can be measured by either confocal microscopy or biochemical assays. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds. In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay. Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.
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PMID:Development and characterization of a human in vitro resorption assay: demonstration of utility using novel antiresorptive agents. 1046 85

Brefeldin-A (BFA) is a specific and potent inhibitor of the intracellular transport of clathlin-uncoated transitional vesicles from the cisterns of rough-surfaced endoplasmic reticulum (RER) to the Golgi lamellae. This study was designed to clarify the effects of BFA on ultrastructure, subcellular localization of vacuolar-type H+-ATPase and a lysosomal cysteine proteinase, cathepsin K, in cultured osteoclasts and their resorptive function. H+-ATPase and cathepsin K are the most important enzymes for decalcification of apatite crystals and degradation of type-I collagen, respectively. In control cultures without BFA, osteoclasts were structurally characterized by the development of broad ruffled borders and clear zones, and formed many resorption lacunae in cocultured dentine slices. In BFA-treated cultures, osteoclasts lacked ruffled borders, and the cytoplasm was filled with regular-size and extremely large pale vacuoles over 2 microm in diameter, which were produced by fusion of adjacent vacuoles. BFA did not, however, inhibit clear zone formation and adhesion of osteoclasts to dentine slices. Resorption lacuna formation was markedly diminished by BFA treatment. Although H+-ATPase and cathepsin K were strongly expressed in osteoclast ruffled borders in control cultures, BFA treatment altered the subcellular localization and decreased the expression of these molecules. In BFA-treated cultures, H+-ATPase immunoreaction in osteoclasts was observed along the limiting membranes of some, but not all, regular-size pale vacuoles, but neither in extremely large vacuoles nor along the smooth plasma membranes facing the dentine slices. Similarly, cathepsin K was localized within lysosomes and some regular-size pale vacuoles, but its secretion toward the dentine slices through the ruffled borders was strongly inhibited by BFA treatment. These results suggest that 1.) formation of the osteoclast ruffled borders and their resorptive function are closely associated with the intracellular transport of these molecules from the RER cisterns and the Golgi lamellae to the ruffled borders, and 2.) both H+-ATPase and cathepsin K are selectively transported to the ruffled border membranes by pale vacuoles.
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PMID:Effects of brefeldin-A: potent inhibitor of intracellular protein transport on ultrastructure and resorptive function of cultured osteoclasts. 1136 Feb 30

Osteoclasts differentiate from hematopoietic precursors of the monocyte/macrophage lineage to mononuclear preosteoclasts and multinuclear mature osteoclasts. In the present study, we report on the establishment of macrophage like cell lines from mouse bone marrow by coculturing bone marrow cells with mouse chondrocytes. Isolated clones (MLC-6 and MLC-7 cells) expressed fully differentiated osteoclast markers, such as calcitonin receptors, vitronectin receptors, tartrate-resistant acid phosphatase and vacuolar H+ -ATPase, in the absence of osteoclast differentiation factor/osteoprotegerin ligand/RANKL/TRANCE, which was essential for osteoclast differentiation. Most clones also maintained expression of a macrophage-associated protein, namely non-specific esterase. Both MLC-6 and MLC-7 cells released cathepsin K into the culture medium. Both clones resolved dentine slices when cocultured with the osteoblast cell line ST2. The cloned cell lines are considered to be useful tools in the study of osteoclast differentiation.
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PMID:Establishment and characterization of macrophage-like cell lines expressing osteoclast-specific markers. 1144 14

To investigate the cellular mechanisms of physiological root resorption in human deciduous teeth, the authors examined the immunocytochemical localization of vacuolar-type H+-ATPase, a lysosomal cysteine proteinase, cathepsin K, matrix metalloproteinase-9 (MMP-9), and receptor activator of NFKB ligand (RANKL) in odontoclasts. H+-ATPase, cathepsin K, and MMP-9 are the most important enzymes for decalcification of apatite crystals and degradation of type-I collagen. In addition, RANKL is one of the key regulatory molecules in osteoclast formation and functions. Odontoclasts developed extensive ruffled borders and clear zones apposed to the resorbing root dentine surfaces. On immunoelectron microscopy, the expression of vacuolar-type H+-ATPase was detected along the limiting membranes of pale vacuoles and the ruffled border membranes of odontoclasts. Cathepsin K in odontoclasts was localized within pale vacuoles, lysosomes, the extracellular canals of ruffled borders, and the underlying resorbing dentine surfaces. MMP-9 localization in odontoclasts was similar to those of cathepsin K. RANKL was detected in both mononuclear stromal cells and odontoclasts located on resorbing dentine surfaces. These results suggest that (1) odontoclasts are directly involved in decalcification of apatite crystals by active extrusion of proton ions mediated by H+-ATPase and (2) extracellular degradation of dentine type-I collagen by both cathepsin K and MMP-9, and (3) odontoclast differentiation and activity are regulated, at least in part, by RANKL, possibly produced by mononuclear stromal cells and odontoclasts themselves in the resorbing tissues. Thus, the cellular mechanisms of physiological root resorption appear to be quite similar to those of osteoclastic bone resorption.
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PMID:Immunolocalization of vacuolar-type H+-ATPase, cathepsin K, matrix metalloproteinase-9, and receptor activator of NFkappaB ligand in odontoclasts during physiological root resorption of human deciduous teeth. 1159 12

We report the effects of specific and potent inhibitors of vacular-type H(+)-ATPase and lysosomal cysteine proteinases, cathepsins, on the ultrastructure, expression of these enzymes, and resorptive functions of cultured osteoclasts. Osteoclasts were formed by co-culture of marrow cells and calvarial primary osteoblasts of ddY mice. Formed osteoclasts were cultured on dentine slices for 6-48 hr with either an H(+)-ATPase inhibitor, bafilomycin A1, or a cysteine proteinase inhibitor, E-64. In control cultures with no additive, osteoclasts were structurally characterized by the development of ruffled borders and clear zones, and formed many resorption lacunae on dentine slices. Both H(+)-ATPase and cathepsin K were strongly expressed in the ruffled borders of these osteoclasts. In bafilomycin A1-treated cultures, osteoclasts lacked ruffled borders, and resorption lacuna formation was markedly diminished. This effect of bafilomycin A1 on osteoclast structure was reversible by removal of the compound. Bafilomycin A1 treatment altered the subcellular localization and decreased the expression of H(+)-ATPase molecules. H(+)-ATPase expression was observed throughout the cytoplasm, but not along the plasma membranes facing dentine slices. On the other hand, E-64 treatment did not affect the ultrastructure of osteoclasts and the expression of enzyme molecules. Although E-64 showed no effect on demineralization of dentine slices, it dose-dependently reduced resorption lacuna formation. Our results suggest that 1) bafilomycin A1 dose-dependently inhibits resorption lacuna formation via inhibition of ruffled border formation, 2) H(+)-ATPase expression is closely associated with the cytoskeleton of osteoclasts, and 3) E-64 treatment decreases the depth of resorption lacunae, by inhibition of secreted cathepsin K activity, but does not impair ruffled border formation and the associated expression of H(+)-ATPase and cathepsin K in osteoclasts.
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PMID:Specific biological functions of vacuolar-type H(+)-ATPase and lysosomal cysteine proteinase, cathepsin K, in osteoclasts. 1252 90

Subosteoclastic bone resorption is a result of HCl and proteinase secretion through a late endosome-like bone facing membrane domain called ruffled border. As bone matrix is degraded, it enters osteoclasts' transcytotic vesicles for further processing and is then finally exocytosed to the intercellular space. The present study clarifies the spatial relationship between these vesicle fusion and matrix uptake processes at the ruffled border. Our results show the presence of vacuolar H+-ATPase, small GTPase rab7 as well as dense aggregates of F-actin at the peripheral ruffled border, where basolaterally endocytosed transferrin and cathepsin K are delivered. On the contrary, rhodamine-labeled bone matrix enters transcytotic vesicles at the central ruffled border, where the vesicle budding proteins such as clathrin, AP-2 and dynamin II are also localized. We present a model for the mechanism of ruffled border turnover and suggest that, due to its late endosomal characteristics, the ruffled border serves as a valuable model for studying the dynamic organization of other endosomal compartments as well.
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PMID:Osteoclast ruffled border has distinct subdomains for secretion and degraded matrix uptake. 1255 37

Over the past decade, advances in molecular tools, stem cell differentiation, osteoclast and osteoblast signaling mechanisms, and genetically manipulated mice models have resulted in major breakthroughs in understanding osteoclast biology. This review focuses on key advances in our understanding of molecular mechanisms underlying the formation, function, and survival of osteoclasts. These include key signals mediating osteoclast differentiation, including PU.1, RANK, CSF-1/c-fms, and src, and key specializations of the osteoclast including HCl secretion driven by H+-ATPase and the secretion of collagenolytic enzymes including cathepsin K and matrix metalloproteinases (MMPs). These pathways and highly expressed proteins provide targets for specific therapies to modify bone degradation. The main outstanding issues, basic and translational, will be considered in relation to the osteoclast as a target for antiresorptive therapies.
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PMID:Osteoclastogenesis, bone resorption, and osteoclast-based therapeutics. 1267 20

The aim of the study was to investigate the relationship between invasion and proliferation in rheumatoid arthritis synovial fibroblasts (RASFs). In vitro, RASFs, normal synovial fibroblasts (NSFs), and RASFs transformed with SV40 T-antigen (RASF(SV40)) were analyzed for the expression of cell surface markers (Thy1, VCAM-1, ICAM-1, CD40, CD44) and their proliferation by flow cytometry. Furthermore, colony-forming unit assays were performed and the expression of matrix metalloproteinases (MMP)-14 and cathepsin K mRNA were determined by real-time polymerase chain reaction. In vivo, in the severe combined immunodeficiency (SCID) mouse co-implantation model, RASFs, NSFs, and RASF(SV40) were tested for cartilage invasion, cellular density, and for their expression of the cell cycle-associated protein Ki67. In the SCID mouse co-implantation model, RASFs invaded significantly stronger into the cartilage than NSFs and RASF(SV40). Of note, RASF(SV40) cells formed tumor-like tissues, and the cellular density adjacent to the cartilage was significantly higher than in RASFs or NSFs. In turn, the proliferation marker Ki67 was strongly expressed in the SV40-transformed synoviocytes in SCID mice, but not in RASFs, and specifically not at sites of cartilage invasion. Using the colony-forming unit assay, RASFs and NSFs did not form colonies, whereas RASF(SV40) lost contact inhibition. In vitro, the proliferative rate of RASFs was low (4.3% S phase) in contrast to RASF(SV40) (24.4%). Expression of VCAM-1 was significantly higher, whereas of ICAM-1 was significantly lower, in RASFs than in RASF(SV40). CD40 was significantly stronger expressed in RASF(SV40), whereas CD44 and AS02 were present at the same degree in almost all synoviocytes. Expression of cathepsin K and matrix metalloproteinase-14 mRNA was significantly higher in RASFs than in the RASF(SV40). Our data demonstrate clearly that invasion of cartilage is mediated by activated RASFs characterized by increased expression of adhesion molecules, matrix-degrading enzymes, but does not depend on cellular proliferation, suggesting the dissociation of invasion and proliferation in RASFs.
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PMID:Cartilage destruction mediated by synovial fibroblasts does not depend on proliferation in rheumatoid arthritis. 1270 22

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.
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PMID:Differentiation and functions of osteoclasts and odontoclasts in mineralized tissue resorption. 1287 16

The osteoclast is a bone-degrading polykaryon. Recent studies have clarified the differentiation of this cell and the biochemical mechanisms it uses to resorb bone. The osteoclast derives from a monocyte/macrophage precursor. Osteoclast formation requires permissive concentrations of M-CSF and is driven by contact with mesenchymal cells in bone that bear the TNF-family ligand RANKL. Osteoclast precursors express RANK, and the interaction between RANKL and RANK (which is inhibited by OPG) is the major determinant of osteoclast formation. Hormones, such as PTH/PTHrP, glucocorticoids and 1,25(OH)2D3, and humoral factors, including TNFalpha, interleukin-1, TGFss and prostaglandins, influence osteoclast formation by altering expression of these molecular factors. TNFalpha, IL-6 and IL-11 have also been shown to promote osteoclast formation by RANKL-independent processes. RANKL-dependent/independent osteoclast formation is likely to play an important role in conditions where there is pathological bone resorption such as inflammatory arthritis and malignant bone resorption. Osteoclast functional defects cause sclerotic bone disorders, many of which have recently been identified as specific genetic defects. Osteoclasts express specialized proteins including a vacuolar-type H+-ATPase that drives HCl secretion for dissolution of bone mineral. One v-ATPase component, the 116 kD V0 subunit, has several isoforms. Only one isoform, TCIRG1, is up-regulated in osteoclasts. Defects in TCIRG1 are common causes of osteopetrosis. HCl secretion is dependent on chloride channels; a chloride channel homologue, CLCN7, is another common defect in osteopetrosis. Humans who are deficient in carbonic anhydrase II or who have defects in phagocytosis also have variable defects in bone remodelling. Organic bone matrix is degraded by thiol proteinases, principally cathepsin K, and abnormalities in cathepsin K cause another sclerotic bone disorder, pycnodysostosis. Thus, bone turnover in normal subjects depends on relative expression of key cytokines, and defects in osteoclastic turnover usually reflect defects in specific ion transporters or enzymes that play essential roles in bone degradation.
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PMID:Recent advances in osteoclast biology and pathological bone resorption. 1470 87


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