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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gel-coated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000-40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (approximately 1000 cells per head). The purity of TRAP+ MNCs was 5.6 +/- 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30-40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 +/- 0.7 x 10(-10) M and 16.5 +/- 1.5 x 10(6) binding sites per cell. These results were similar to the Kd value (3.5 x 10(-10) M) and the number of binding sites (3.3 x 10(6) per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: slmon CT at 10(-10) M, human CT at 10(-8) M; ED50s: salmon CT, 2.2 x 10(-11) M, human CT, 1.3 x 10(-9) M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.
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PMID:Preparation and characterization of a mouse osteoclast-like multinucleated cell population. 128 5

Recent investigations have indicated that cytokines such as tumor necrosis factor-alpha (TNF-alpha) play a potential role in the bone resorption associated with inflammatory diseases. In this immunoperoxidase study, TNF-alpha was localized in mononuclear cells, macrophages, fibroblasts, osteoblasts, and osteoclasts adjacent to bone resorption areas in both human and experimental middle ear cholesteatomas. In vitro, TNF-alpha stimulated monocytes to form multinucleated cells that demonstrate tartrate-resistant acid phosphatase activity, a marker enzyme for osteoclasts. These multinucleated osteoclast-like cells induce resorption of devitalized bone. The extent of bone resorption was increased by the co-cultures of osteoblasts and osteoclasts in the presence of TNF-alpha, suggesting that cell to cell interaction plays a significant role in bone resorption. Moreover, TNF-alpha was capable of stimulating macrophages to produce acid phosphatase and collagenase, and osteoblasts to produce prostaglandin E2 and collagenase. These chemical mediators have been known to lead to bone resorption. Our findings suggest that TNF-alpha may play an important clinical role in the destructive process of cholesteatoma.
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PMID:The role of tumor necrosis factor-alpha in bone resorption of cholesteatoma. 165 Jan 49

Biochemical and molecular studies of osteoclasts generally require cells in a reasonable degree of purity. The chicken has been extremely useful in this regard, as abundant avian osteoclasts can be generated in vitro entirely from pure populations of marrow macrophage precursors. Propagation of murine osteoclasts is, in contrast, far less efficient, demanding the presence of stromal cells. The aims of this study were to develop a method by which murine osteoclasts generated in culture, can be effectively enriched while maintaining viability and, to explore the mechanisms by which stromal cells promote murine osteoclast generation and survival. We find that 10(6) fractionated murine marrow cells enriched, for marrow-residing colony-forming units (CFU-cs), yield 3000-4000 tartrate-resistant acid phosphatase (TRAP)-expressing multinucleated giant cells when cultured for 12 days with ST-2 stromal cells. These cells are osteoclasts as evidenced by their ability to "pit" bone slices, resorb radiolabeled bone particles, and generate cyclic AMP in response to calcitonin. Treatment of these generated osteoclast cultures with bacterial collagenase for 2 hours at 37 degrees selectively removes virtually all ST-2 cells, yielding a > 60% pure population of TRAP and calcitonin receptor-expressing cells, 90% of which are viable. These cells continue to respond to calcitonin and survive for 24 hours in the absence of ST-2 cells. We also found that murine osteoclast generation depends upon contact of osteoclast precursors with viable ST-2 cells. Furthermore, the stromal cells secrete macrophage colony-stimulating factor (CSF-1), and the anti-CSF-1 antibody 5A1 inhibits murine osteoclastogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enrichment of generated murine osteoclasts. 753 41

The odontoclast, which is morphologically similar to osteoclast, is thought to play a major role in root resorption of deciduous teeth. High collagenolytic activity has been detected in the root resorbing tissue. In order to identify collagenase-producing cells and the role of collagenase in root resorption of deciduous tooth, in situ hybridization of collagenase mRNA in bovine root-resorbing tissue sections was performed using a digoxigenin-labeled, nonradioactive RNA probe. Collagenase mRNA expression was clearly observed in odontoclasts in addition to the macrophages, fibroblasts, odontoblasts, and cementoblasts. Multinuclear odontoclasts showed intense tartrate-resistant acid phosphatase (TRAP) activity. We also examined interleukin-1 (IL-1) mRNA expression in the root-resorbing tissue by in situ hybridization. IL-1 transcripts were found to be expressed in odontoclasts, fibroblasts, and macrophages suggesting that IL-1 might be an important factor for promoting root resorption. These results suggest that the collagenase produced by odontoclasts may play a key role in dentin collagen degradation in root resorption.
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PMID:Detection of collagenase mRNA in odontoclasts of bovine root-resorbing tissue by in situ hybridization. 846 13

Mechanical stimulation by intermittent compressive force (ICF) stimulates bone formation and inhibits bone resorption in cultured fetal mouse bone. Fetal bone tissue can produce autocrine factors that stimulate bone cell replication and matrix formation, and paracrine factors that increase the formation of osteoclast precursor-like cells from bone marrow. In the present study, we have tested whether ICF affects the production of such local factors in fetal mouse calvariae. Calvariae were cultured for 4 days in the presence and absence of ICF (130 mbar, 0.3 Hz). Conditioned medium was collected daily and pooled. We found that conditioned medium from ICF-exposed cultures stimulated [3H]-TdR incorporation into DNA, and [3H]-proline incorporation into collagenase digestible protein but not into non-collagen protein in fresh calvarial cultures. Treatment with conditioned medium from ICF-exposed cultures had earlier effects on [3H]-TdR and [3H]-proline incorporation than direct treatment with ICF. Conditioned medium from ICF-exposed cultures decreased the number of osteoclast precursor-like cells in bone marrow cultures stained for tartrate-resistant acid phosphatase. We conclude that ICF stimulates the release (activity) of an autocrine growth-factor from bone. In addition, ICF can stimulate the release (activity) of a paracrine factor, inhibiting the growth and/or differentiation of osteoclast precursor-like cells. These data suggest that mechanical forces may modulate skeletal (re)modeling by affecting the production of local growth factors.
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PMID:Effect of mechanical stimulation on the production of soluble bone factors in cultured fetal mouse calvariae. 847 8

We have reported that short calcitonin (CT) treatment of mature mouse osteoclast-like cells (OCLs) in culture induced prolonged down-regulation of the CT receptor (CTR) and desensitization to CT rechallenge, at the level of adenylate cyclase activity. In this study, we have extended those studies to examine the bone resorbing activity of OCLs pretreated with CT. OCLs, which formed on gelled type I collagen, were pretreated with salmon CT (sCT)(10(-9)M, 1 h) and 24 h later were replated onto plastic dishes or dentine slices after removal from the gel by collagenase digestion. The number and population of either mononuclear or multinuclear OCLs that adhere to either surface was not affected by sCT pretreatment. It was found that OCLs pretreated with sCT regained reduced but significant bone resorbing capacity, which was quantitated as the surface area resorbed by OCLs on dentine slices. However, compared with control, the number of resorption pits produced by sCT- pretreated OCLs was slightly reduced, and the total pit area was decreased by approximately 40-50%. The distribution of individual pit sizes was altered by sCT-pretreatment so that the number of larger pits was predominantly reduced, suggesting that short sCT treatment may produce a long lasting decrease in osteoclast mobility. sCT was able to inhibit bone resorption activity of CT-pretreated OCLs (ED50:10(-13)-10(-12)M). Importantly, the ED50 of sCT inhibition of bone resorption in sCT-pretreated OCLs was approximately 100-fold greater than for control, indicating resistance of the OCLs to CT rechallenge. Consistent with these results, treatment of OCLs with sCT greatly decreased the expression of CTR messenger RNA, whereas no significant effect was observed on the tartrate-resistant acid phosphatase messenger RNA expression, a marker of resorptive capacity of osteoclasts. These results indicate, therefore, that an important component of escape of osteoclastic resorption from CT inhibition is CT resistance of mature osteoclasts, which regain bone resorbing function.
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PMID:Calcitonin receptor down-regulation relates to calcitonin resistance in mature mouse osteoclasts. 860 72

A synthetic peptide corresponding to the N-terminal amino acid residues of stanniocalcin (STC1-20) and including a region that is known to be an active site in teleosts was prepared and tested for its effects on the metabolism of mammalian bone in vitro. STC1-20 (10(-10)-10(-12) M) inhibited increases in the number of tartrate-resistant acid phosphatase-positive, multinucleated cells promoted by an N-terminal fragment of human parathyroid hormone (hPTH1-34) in cultures of murine hemopoietic cells. STC1-20 also slightly decreased the rate of loss of radioactivity from calvariae of fetal rats that had been prelabeled with 45Ca, both with and without stimulation by hPTH1-34. The accumulation of cAMP induced by hPTH1-34 in ROS 17/2.8-5 cells was suppressed by STC1-20 (10(-10)-10(-12) M). Treatment with STC1-20 (10(-11)-10(-13) M) caused increases of the rate of incorporation of [3H]proline into the collagenase-digestible protein of calvariae in newborn mice. From these results, it appears that STC1-20 has diverse effects on the metabolism of mammalian bone, causing a biphasic response. Such effects have not been observed with intact stanniocalcin or with materials from the corpuscles of Stannius and they are also different from the effects of hPTH1-34.
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PMID:Effects of a synthetic N-terminal fragment of stanniocalcin on the metabolism of mammalian bone in vitro. 866 40

We describe a method for the isolation and culture of osteoclast-like cells from cancellous bone chips of iliac crests from patients undergoing reconstructive maxillofacial surgery. Under aseptic conditions, bone chips were cut into small pieces, incubated briefly with collagenase, and the isolated bone cells were separated from the bone chips by filtration using a nylon mesh. Bone cells were then cultured on a variety of surfaces for up to 10 days. Cell motility and fusion, together with the development of tartrate-resistant acid phosphatase activity, were seen in many cells soon after culture. The large osteoclast-like cells adhered to human cortical bone slices and produced resorption pits. These morphological and functional characteristics suggest that the cells we isolated and cultured were human osteoclasts and their precursors. Thus this method may provide a reliable means of obtaining human osteoclasts from normal tissue for short-term studies of their metabolism or from various skeletal diseases to study pathological aberrations and mechanisms.
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PMID:Human osteoclast-like cells in primary culture. 883 79

The identification and purification of human osteoclast precursors is essential to further our understanding of the mechanisms that control human osteoclast differentiation. Osteoclastoma tissue potentially provides a rich source of human osteoclast precursors, and in previous studies we have demonstrated the existence of a population of mononuclear cells within this tissue that is reactive with osteoclast-selective vitronectin receptor monoclonal antibodies. In this study, mononuclear cells expressing the vitronectin receptor, as defined by their ability to react with a murine monoclonal antibody to the beta 3 chain of the vitronectin receptor (87MEM1), were isolated from collagenase digests of osteoclastoma tissue using a fluorescence activated cell sorter. Based on their fluorescence signal and size, approximately 2-3% of the viable cells (typically 2 x 10(5)) were obtained and prepared for further phenotyping. The isolated cells demonstrated a number of phenotypic characteristics of osteoclasts: positive tartrate-resistant acid phosphatase (TRAP) activity, reactivity with human osteoclast-selective antibodies, expression of calcitonin receptors, cathepsin K (a novel osteoclast-selective cysteine proteinase) mRNA, and osteopontin mRNA and protein. These phenotypic characteristics were also detected in mononuclear cells within cryostat sections of the native osteoclastoma tissue as well as in resorption lacunae of sections of human bone. In contrast, isolated peripheral blood monocytes were negative for TRAP activity and osteopontin expression and, unlike the osteoclastoma-derived cells, demonstrated strong nonspecific esterase activity. Significantly, when the osteoclastoma-derived 87MEM1 positive cells were cocultured on whale dentine for 1-3 weeks with stromal cells, extensive resorption of the dentine surface was observed. This is the first demonstration of the purification of human osteoclast precursors. These cells provide an homogeneous cell population for studying cellular events that occur during human osteoclast differentiation.
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PMID:Purification and characterization of fully functional human osteoclast precursors. 891 68

We have previously demonstrated that a tartrate-resistant acid phosphatase (TRAP)-positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKbeta-8, RANTES, and MIP-1alpha elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP-1beta, MCP-1, MCP-2, MCP-3, MCP-4, HCC-1, eotaxin-2, PARC, SLC, ELC) and 3 CXC chemokines (IL-8, GROalpha, SDF-1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG-63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross-desensitization studies demonstrate that RANTES and MIP-1alpha can partially inhibit the chemotactic response elicited by CKbeta-8. CKbeta-8, the most potent of the active CC chemokines (EC(max) 0.1-0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKbeta-8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKbeta-8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKbeta-8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption.
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PMID:CKbeta-8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues. 1073 95


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