Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present report, we show that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/ml) by an increased DNA binding activity of NF-kappaB and AP-1 transcription factors. Incubation of the cells with 10(-5) M rhein for 24 h was found to reduce this activity, particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to 1-h treatment with IL-1beta. This effect was greater in hypoxia (3% O(2)) than in normoxia (21% O(2)). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extracellular matrix synthesis. IL-1-induced collagenase (MMP1) expression was significantly decreased by rhein in the same conditions. In conclusion, rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several pro-inflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMP1 synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the anti-osteoarthritic properties of rhein and its disease-modifying effects on OA cartilage, in spite of absence of inhibition at prostaglandin level.
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PMID:Articular chondrocytes cultured in hypoxia: their response to interleukin-1beta and rhein, the active metabolite of diacerhein. 1529 86

Chondrocytes were released from articular cartilage fragments of 6-week-old Wistar rats by a 2-hour treatment with bacterial collagenase. The cells from one animal were seeded in a 25-cm2 culture flask at a density of 10(5) cells/cm2. After 1 h, the flask was gently shaken and the medium, containing nonadherent cells, was transferred to a new flask. The attached cells were incubated with 5 ml of fresh medium. This procedure was repeated after 3, 24, 48 and 96 h. Resulting cell populations were then analyzed. The earlier cells attached, the more rapidly they proliferated, and the less collagen and proteoglycan (PG) they produced. The cells that attached after 24 h grew much more slowly, piled up in many areas, exhibited strong alkaline phosphatase activity and calcified extracellular matrix (ECM). Differences in deoxyribonucleic acid (DNA) and protein/PG synthesis were also observed when these cell populations were challenged with growth factors and 12-myristate 13-acetate (PMA). Pretreatment of cells for 2 h with PMA strongly enhanced DNA and PG synthesis only in cultures containing insulin-like growth factor-1. Nonselected rat articular chondrocytes (AC) subcultured at least four times as monolayers still expressed mRNA specific for aggrecan and type II collagen, suggesting conservation of the chondrogenic phenotype. In conclusion, AC of young individuals seem to be heterogeneous with respect to their capacity to proliferate and synthesize ECM. By selecting and expanding in vitro the appropriate cell population, this method could be potentially useful for studies aimed at repairing damaged cartilage.
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PMID:Method for selecting populations of rat articular chondrocytes that exhibit distinct growth and metabolic characteristics, and their responses to growth factors, PMA and vitamin D3. 1545 76

Hyaluronic-acid-based biomaterials used for cartilage repair allow the expression of specific extracellular matrix molecules by human chondrocytes grown onto them. We investigated whether these biomaterials could also create an environment in which the cells downregulate the expression of some catabolic factors. Chondrocytes were isolated from human articular cartilage obtained from the knees of patients with a history of trauma. First, the cells were expanded in monolayers and then they were seeded on a hyaluronic-acid derivative scaffold. Constructs and surnatants were collected and analysed at 1, 3, 7, 14 and 21 days after seeding. Immunohistochemical analysis for CD44 and caspase was carried out on paraffin-embedded sections. The Tunel method was used to identify chondrocyte apoptosis status. Secretion of MMP-1 and MMP-13 in the surnatants of the cells grown onto the biomaterial was measured by enzyme-linked immunosorbent assay. Nitric oxide (NO) production was evaluated by estimating the stable NO metabolite nitrite by the Griess method. A real-time RT-PCR analysis was performed on the constructs to evaluate the expression of type I and II collagens, aggrecan, Sox-9, MMP-1 and MMP-13 mRNAs at the different experimental times evaluated. Decreased levels of metalloproteinases and nitric oxide were observed in the surnatants of chondrocytes grown onto the hyaluronan-based scaffold. This was also confirmed by real-time PCR analysis which showed that the cells expressed the specific differentiated phenotype downregulating the expression of some catabolic molecules. Cells apoptosis decreased during the culture period, which further supported the biochemical data. The ability of the hyaluronan scaffold to reduce the expression and production of molecules involved in cartilage degenerative diseases indicates its use to treat early lesions of osteoarthritic patients.
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PMID:Down regulation of degenerative cartilage molecules in chondrocytes grown on a hyaluronan-based scaffold. 1587 72

Cartilage destruction in the arthritides is thought to be mediated by two main enzyme families: the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, and enzymes from the ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family mediate cartilage aggrecan loss. Many genes subject to transcriptional control are regulated, at least in part, by modifications to chromatin, including acetylation of histones. The aim of this study was to examine the impact of histone deacetylase (HDAC) inhibitors on the expression of metalloproteinase genes in chondrocytes and to explore the potential of these inhibitors as chondroprotective agents. The effects of HDAC inhibitors on cartilage degradation were assessed using a bovine nasal cartilage explant assay. The expression and activity of metalloproteinases was measured using real-time RT-PCR, western blot, gelatin zymography, and collagenase activity assays using both SW1353 chondrosarcoma cells and primary human chondrocytes. The HDAC inhibitors trichostatin A and sodium butyrate potently inhibit cartilage degradation in an explant assay. These compounds decrease the level of collagenolytic enzymes in explant-conditioned culture medium and also the activation of these enzymes. In cell culture, these effects are explained by the ability of HDAC inhibitors to block the induction of key MMPs (e.g. MMP-1 and MMP-13) by proinflammatory cytokines at both the mRNA and protein levels. The induction of aggrecan-degrading enzymes (e.g. ADAMTS4, ADAMTS5, and ADAMTS9) is also inhibited at the mRNA level. HDAC inhibitors may therefore be novel chondroprotective therapeutic agents in arthritis by virtue of their ability to inhibit the expression of destructive metalloproteinases by chondrocytes.
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PMID:Histone deacetylase inhibitors modulate metalloproteinase gene expression in chondrocytes and block cartilage resorption. 1598 98

The irreversible destruction of the cartilage, tendon, and bone that comprise synovial joints is the hallmark of both rheumatoid arthritis (RA) and osteoarthritis (OA). While cartilage is made up of proteoglycans and type II collagen, tendon and bone are composed primarily of type I collagen. RA is an autoimmune disease afflicting numerous joints throughout the body; in contrast, OA develops in a small number of joints, usually resulting from chronic overuse or injury. In both diseases, inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) stimulate the production of matrix metalloproteinases (MMPs), enzymes that can degrade all components of the extracellular matrix. The collagenases, MMP-1 and MMP-13, have predominant roles in RA and OA because they are rate limiting in the process of collagen degradation. MMP-1 is produced primarily by the synovial cells that line the joints, and MMP-13 is a product of the chondrocytes that reside in the cartilage. In addition to collagen, MMP-13 also degrades the proteoglycan molecule, aggrecan, giving it a dual role in matrix destruction. Expression of other MMPs such as MMP-2, MMP-3 and MMP-9, is also elevated in arthritis and these enzymes degrade non-collagen matrix components of the joints. Significant effort has been expended in attempts to design effective inhibitors of MMP activity and/or synthesis with the goal of curbing connective tissues destruction within the joints. To date, however, no effective clinical inhibitors exist. Increasing our knowledge of the crystal structures of these enzymes and of the signal transduction pathways and molecular mechanisms that control MMP gene expression may provide new opportunities for the development of therapeutics to prevent the joint destruction seen in arthritis.
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PMID:Matrix metalloproteinases: role in arthritis. 1614 51

The goal of studies of autoimmune disease biomarkers is to identity markers that fluctuate with disease development and severity, but then normalize following successful therapy. The perfect marker could thus serve as a diagnostic tool, as well as a monitoring device for therapeutic drug efficacy. Current biomarker discovery efforts are focused on three groups of proteins reflective of the autoimmune disease process: (1) degradation products arising from destruction of affected tissues, (2) enzymes that play a role in tissue degradation and (3) cytokines and other proteins associated with immune activation. Potential biomarkers for two autoimmune diseases, rheumatoid arthritis and multiple sclerosis, have been described in recent publications. For rheumatoid arthritis, these markers (by group) include (1) aggrecan fragments, C-propeptide of type II collagen and cartilage oligomeric matrix protein, (2) matrix metalloprotease (MMP)-1, MMP-3 and MMP-1/inhibitor complexes and (3) thioredoxin, IL-16 and tumour necrosis factor (TNF)-alpha. For multiple sclerosis, they include (1) neurofilament light protein and glial fibrillary acidic protein, (2) MMP-2 and MMP-9 and (3) TNF-alpha and soluble vascular adhesion molecule-1. The utility of most of these markers is limited by their restriction to relatively inaccessible anatomic sites (synovial or cerebrospinal fluid). Thus, from a practical standpoint, the most useful autoimmune biomarkers will be those measurable in serum or plasma.
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PMID:Biomarkers for diagnosing and monitoring autoimmune diseases. 1629 11

Articular cartilage degeneration in osteoarthritis (OA) involves type II collagen degradation and chondrocyte differentiation (hypertrophy). Because these changes resemble growth plate remodeling, we hypothesized that collagen degradation may be inhibitable by growth factors known to suppress growth plate hypertrophy, namely transforming growth factor (TGF)-beta2, fibroblast growth factor (FGF)-2, and insulin. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with TGF-beta2, FGF-2, and insulin in combination (growth factors) or individually. In cultured explants from five OA patients, collagenase-mediated type II collagen cleavage was significantly down-regulated by combined growth factors as measured by enzyme-linked immunosorbent assay. Individually, FGF-2 and insulin failed to inhibit collagen cleavage in some OA explants whereas TGF-beta2 reduced collagen cleavage in these 5 explants and in 19 additional explants. Moreover, TGF-beta2 effectively suppressed cleavage at low concentrations. Together or individually these growth factors did not inhibit glycosaminoglycan (primarily aggrecan) degradation while TGF-beta2 occasionally did. Semiquantitative reverse transcriptase-polymerase chain reaction of articular cartilage from six OA patients revealed that TGF-beta2 suppressed expression of matrix metalloproteinase-13 and matrix metalloproteinase-9, early (PTHrP) and late (COL10A1) differentiation-related genes, and proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha). In contrast, TGF-beta2 up-regulated PGES-1 expression and prostaglandin E(2) release. These observations show that TGF-beta2 can suppress collagen resorption and chondrocyte differentiation in OA cartilage and that this may be mediated by prostaglandin E(2). Therefore TGF-beta2 could provide therapeutic control of type II collagen degeneration in OA.
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PMID:Transforming growth factor-beta2 suppresses collagen cleavage in cultured human osteoarthritic cartilage, reduces expression of genes associated with chondrocyte hypertrophy and degradation, and increases prostaglandin E(2) production. 1640 16

The objective of the study was to improve the biological understanding of degenerative disc disease using a rabbit model in which different stages of disc degeneration are induced by variation of the duration of loading with an external compression-device applying 2.4 MPa. Gene expression and protein distribution were analyzed in controls and after 1, 28, and 56 days of hyperphysiologic loading. To evaluate extracellular matrix genes, quantitative real-time reverse-transcriptase polymerase chain reaction was applied for collagen I, collagen II, biglycan, decorin, fibromodulin, fibronectin, aggrecan, and osteonectin. As representatives of catabolic, anticatabolic, and anabolic factors, matrix metalloproteinase-13 (MMP-13), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and bone morphogenetic protein-2 (BMP-2) were chosen. To evaluate protein distribution, immunohistochemistry was performed for collagen I, collagen II, and BMP-2/4. Matrix gene expression was characterized by two major developments: collagen I and II, biglycan, and decorin showed early elevation followed by later downregulation to control levels, whereas fibromodulin, fibronectin, aggrecan, and osteonectin showed continuous upregulation or remained at similar levels. Induction of MMP-13 gene expression was found in degenerated discs. TIMP-1 and BMP-2 were elevated immediately after hyperphysiologic loading and presented highest levels in the 56-day group. Immunohistochemistry showed less collagen II and BMP-2/4 positive cells after compression. In conclusion, elevated matrix gene expression represents an early cellular response to hyperphysiologic loading. As degeneration progresses, some matrix genes increase upregulation, whereas others start downregulation. Continuous upregulation of catabolic, anticatabolic, and anabolic factors indicates their important role in the degeneration process.
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PMID:Changes in gene expression and protein distribution at different stages of mechanically induced disc degeneration--an in vivo study on the New Zealand white rabbit. 1647 72

FGF-2 is a regulator of chondrocyte proliferation in the developing growth plate and has been shown to bind to perlecan, a heparan sulfate proteoglycan. We evaluated the effect of perlecan isolated from the growth plate on the binding of FGF-2 to its low and high affinity receptors on resting and proliferating chondrocytes. Chondrocytes were isolated by pronase/collagenase digestion of 1 mm thick slices from the resting and proliferating zones of fetal bovine ribs and were plated in serum-free DMEM. Chondrocytes maintained their zone-specific level of DNA and matrix synthesis over a two-day culture period. The collagen, aggrecan, and perlecan components of the matrix produced were associated with the cell layer and were secreted into the medium. Most of the perlecan made by the chondrocytes was secreted into the medium. Western blots showed medium perlecan to contain two high molecular weight core proteins and overlay assays showed only the large core protein bound FGF-2. Cell layer perlecan contained only the smaller core protein. Immunoprecipitation assays of media showed that the medium perlecan bound (125)I-FGF-2, that the bound FGF-2 was eluted from perlecan by 2 M NaCl at pH 7.4, and that this binding was eliminated by prior digestion with heparatinase. This indicates that the perlecan secreted into the medium is a low affinity receptor for FGF-2. (125)I-FGF-2 also bound to the chondrocytes in cell culture. Competition studies showed exogenous FGF-2 reduced (125)I-FGF-2 binding to high affinity receptor but not the low affinity receptor in the cell layer. Exogenous perlecan, however, reduced (125)I-FGF-2 binding to both the low and the high affinity receptors in the cell layer by approximately 60%. The results suggest that perlecan made by growth plate chondrocytes is a low affinity receptor for FGF-2 and acts to sequester FGF-2 away from the high affinity receptor.
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PMID:Modulation of FGF-2 binding to chondrocytes from the developing growth plate by perlecan. 1648 Nov 52

Aims-To investigate the level of matrix metalloproteinase activity during the time-course of cartilage explant proteoglycan breakdown; to determine the effects of selective small-molecule inhibitors of matrix metalloproteinases on proteoglycan degradation.Methods-The levels of matrix metalloproteinase activity in cartilage explant cultures and conditioned media were monitored by use of a quenched fluorescent substrate. The constants for inhibition of certain matrix metalloproteinases by a series of synthetic inhibitors were determined. Bovine and human cartilage explant cultures were treated with interleukin-1, tumor necrosis factor or retinoic acid and the amount of proteoglycan released into the culture medium in the absence and presence of the inhibitors was quantified. Control experiments, examining the inhibition of other proteinases, and investigating possible toxic or non-specific effects of the inhibitors, were carried out.Results-The profile of inhibition of proteoglycan release suggested the involvement of interstitial collagenase-like, rather than gelatinase- or possibly stromelysin-like, proteinases. No evidence was found for toxic or non-specific mechanisms of inhibition. Very low levels of activity of the known matrix metalloproteinases were present during the time-course of aggrecan breakdown.Conclusions-A novel collagenase-like proteinase(s) may be involved in cartilage proteoglycan breakdown. Gelatinase-type matrix metalloproteinases do not seem to be involved in this process. Specific collagenase inhibitors may be therapeutically efficacious in the treatment of arthritis.
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PMID:Inhibitors of collagenase but not of gelatinase reduce cartilage explant proteoglycan breakdown despite only low levels of matrix metalloproteinase activity. 1669 99


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