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Hyaluronate degradation as an alternative mechanism for proteoglycan release from cartilage during interleukin-1beta-stimulated catabolism.

ABSTRACT: Data presented previously suggest that release of components of the cartilage matrix, in response to catabolic agents, cannot be accounted for by proteolytic mechanisms alone. In the present study, the release of glycosaminoglycan-containing components from bovine nasal cartilage cultured in the presence of interleukin-1beta, and from bovine nasal, fetal bovine epiphyseal and adult human articular cartilage cultured in the presence of retinoic acid, was accompanied by the loss of link protein and hyaluronate into the culture medium. Chromatographic analysis of the released hyaluronate showed it to be markedly reduced in size relative to that extracted from the corresponding tissue. It is proposed that, under stimulation by catabolic agents, two independent, but concurrent, mechanisms act to promote the release of aggrecan from the cartilage matrix. First, proteolytic cleavage of the aggrecan core protein results in the production of glycosaminoglycan-containing fragments that are free to diffuse from the tissue. Secondly, cleavage of hyaluronate renders portions of the proteoglycan aggregate small enough so that complexes of aggrecan (or fragments containing its G1 domain) and link protein are released from the tissue. It is likely that both mechanisms contribute to cartilage metabolism in normal physiology and pathology.

SUBMITTER: Sztrolovics R

PROVIDER: S-EPMC1222409 | biostudies-other | 2002 Mar

REPOSITORIES: biostudies-other

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Lysosomal cysteine endopeptidases mediate interleukin 1-stimulated cartilage proteoglycan degradation.

Project description:The peptidyl diazomethane inactivator of cysteine endopeptidases, benzyloxycarbonyl-Tyr-Ala-CHN2, was tested as an inhibitor of interleukin 1 alpha-stimulated release of proteoglycan from bovine nasal septum cartilage explants. Like the previously tested epoxidyl peptide proinhibitor trans-epoxysuccinyl-leucylamido-(3-methyl)butane ethyl ester, it proved to be an effective inhibitor of proteoglycan release from cartilage, with significant inhibition at a concentration of 1 microM. The inhibition did not seem to be due to a general toxic effect. The rates of inactivation of the bovine cysteine endopeptidases by the peptidyl diazomethane, the epoxidyl peptide proinhibitor and its active form were determined. Benzyloxycarbonyl-Tyr-Ala-CHN2 proved to be a rapid inactivator of cathepsins L, S and B, but reacted much more slowly with cathepsin H and calpain. Thus it would appear that the latter two enzymes are not implicated in proteoglycan release in our test system. The peptidyl diazomethane and epoxidyl peptide proinhibitor (above) were also tested for their effects on three other interleukin 1-mediated cellular events, namely epidermal growth factor receptor transmodulation, and interleukin 6 and prostaglandin E2 production. In all cases the inactivators did not interfere with the response to interleukin 1 in human gingival fibroblasts. We conclude that one or more of the lysosomal cysteine endopeptidases cathepsins B, L and S mediate interleukin 1-stimulated cartilage proteoglycan degradation without affecting signal transduction.

| S-EPMC1133216 | biostudies-other

Inhibition of interleukin 1-stimulated cartilage proteoglycan degradation by a lipophilic inactivator of cysteine endopeptidases.

Project description:Inactivators of cysteine endopeptidases were tested as inhibitors of the cytokine-stimulated release of proteoglycan from cartilage. The test system consisted of bovine nasal septum cartilage maintained in organ culture, and the stimulus was provided by recombinant human interleukin 1 alpha. L-3-Carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E64) and L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(3-methyl)b utane (Ep475) showed no inhibition at concentrations up to 100 microM. In contrast, trans-epoxysuccinyl-leucylamido-(3-methyl)butane ethyl ester (Ep453), a 'prodrug' of Ep475, was an effective inhibitor. The LL-, LD- and DL-isomers gave significant inhibition at 10 microM, and the DD-isomer was inhibitory at 100 microM. None of the isomers had any detectable effect on protein synthesis or glycolysis, and their inhibitory effects were reversible. Iodoacetate inhibited proteoglycan release by a general toxic effect. Our results suggest that cysteine endopeptidase(s) play a part in cytokine-stimulated cartilage breakdown, but that effective inhibitors must pass through membranes.

| S-EPMC1130657 | biostudies-other

Articular cartilage cultured with interleukin 1. Increased release of link protein, hyaluronate-binding region and other proteoglycan fragments.

Project description:Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.

| S-EPMC1147171 | biostudies-other

Resistance of small leucine-rich repeat proteoglycans to proteolytic degradation during interleukin-1-stimulated cartilage catabolism.

Project description:A bovine nasal-cartilage culture system has been utilized to analyse the catabolic events occurring in response to interleukin-1beta over a 14-day period. An early event following the start of interleukin-1 treatment was the release of glycosaminoglycan into the culture medium. This release was accompanied by the appearance in the tissue, and shortly thereafter also in the culture media, of a globular domain (G1)-containing aggrecan degradation product generated by the action of aggrecanase. Link protein was also released from the cartilage with a similar timeframe to that of the G1 fragment, although there was no evidence of its proteolytic degradation. By comparison with aggrecan, the small leucine-rich repeat proteoglycans decorin, biglycan and lumican showed a resistance to both proteolytic cleavage and release throughout the culture period. In contrast, fibromodulin exhibited a marked decrease in size after day 4, presumably due to proteolytic modification, but the major degradation product was retained throughout the culture period. Also in contrast with the early changes in the components of the proteoglycan aggregate, type II collagen did not display signs of extensive degradation until much later in the culture period. Collagen degradation products compatible with collagenase action first appeared in the medium by day 10 and increased thereafter. These data demonstrate that the leucine-rich repeat proteoglycans are resistant to proteolytic action during interleukin-1-stimulated cartilage catabolism, compared with aggrecan. This resistance and continued interaction with the surface of the collagen fibrils may help to stabilize the collagen fibrillar network and protect it from extensive proteolytic attack during the early phases of cartilage degeneration.

| S-EPMC1220192 | biostudies-other

Dual effects of 17beta-oestradiol on interleukin 1beta-induced proteoglycan degradation in chondrocytes.

Project description:OBJECTIVE: To determine whether 17beta-oestradiol (E2) modulates interleukin (IL) 1beta-induced proteoglycan degradation in chondrocytes, and to analyse the part played by metalloproteinases (MMPs) in this process. METHODS: Primary cultured rabbit articular chondrocytes were prepared and treated with 10 ng/ml IL1beta combined or not with 0.1-10 nM E2. Neosynthesised proteoglycans (PGs) were evaluated after incorporation of [(35)SO(4)]sulphate and further analysed after chromatography on a Sepharose 2B column. Chondrocyte mRNA levels of aggrecan, MMP-1, -3, -13, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were studied by northern blot. MMP-1 activity was measured by zymography. MMP-1 gene transcription was studied by transient transfection of chondrocytes with an MMP-1-luciferase construct. RESULTS: E2 modulated the IL1beta-induced total sulphated PGs in rabbit articular chondrocytes, which decreased as the E2 concentration was increased. At a low concentration (0.1 nmol/l) E2 counteracts the IL1beta-induced decrease in sulphated PG, while at high concentration (10 nmol/l) E2 enhances the IL1beta effects. A biphasic E2 effect was also observed on IL1beta-induced disaggregation of PG, 53-58 kDa gelatinolytic activity, and MMP-1, -3, and -13 mRNA levels. In contrast, E2 did not modify the level of aggrecan mRNA and had no effect on TIMP-1 mRNA expression. Finally, simultaneous addition of IL1beta and E2 (0.1-10 nmol/l) did not modify IL1beta-induced MMP-1-luciferase activity, suggesting that E2 effects probably occur at the post-transcriptional level of MMP gene expression. CONCLUSION: Oestrogen concentration may have an inverse effect on IL1beta stimulated proteoglycan degradation and MMP production by chondrocytes.

| S-EPMC1754890 | biostudies-other

Involvement of ADAMTS5 and hyaluronidase in aggrecan degradation and release from OSM-stimulated cartilage.

Project description:The relative contribution of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4 and ADAMTS5 to aggrecan degradation under oncostatin M (OSM) stimulation, the role of the ancillary domains of the aggrecanases on their ability to cleave within the chondroitin sulfate (CS)-2 region, the role of hyaluronidases (HYAL) in stimulating aggrecan release in the absence of proteolysis, and the identity of the hyaluronidase involved in OSM-mediated cartilage breakdown were investigated. Bovine articular cartilage explants were cultured in the presence of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha) and/or OSM, or treated with trypsin and/or hyaluronidase. Aggrecan was digested with various domain-truncated isoforms of ADAMTS4 and ADAMTS5. Aggrecan and link protein degradation and release were analyzed by immunoblotting. Aggrecanase and HYAL gene expression were determined. ADAMTS4 was the most inducible aggrecanase upon cytokine stimulation, whereas ADAMTS5 was the most abundant aggrecanase. ADAMTS5 was the most active aggrecanase and was responsible for the generation of an OSM-specific degradation pattern in the CS-2 region. Its ability to cleave at the OSM-specific site adjacent to the aggrecan G3 region was enhanced by truncation of the C-terminal thrombospondin domain, but reduced by further truncation of both the spacer and cysteine-rich domains of the enzyme. OSM has the ability to mediate proteoglycan release through hyaluronan degradation, under conditions where HYAL-2 is the predominant hyaluronidase being expressed. Compared to other catabolic cytokines, OSM exhibits a unique potential at degrading the proteoglycan aggregate, by promoting early robust aggrecanolysis, primarily through the action of ADAMTS5, and hyaluronan degradation.

| S-EPMC3249595 | biostudies-literature

Curcumin reduces prostaglandin E2, matrix metalloproteinase-3 and proteoglycan release in the secretome of interleukin 1β-treated articular cartilage.

Project description:ObjectiveCurcumin (diferuloylmethane) is a phytochemical with potent anti-inflammatory and anti-oxidant properties, and has therapeutic potential for the treatment of a range of inflammatory diseases, including osteoarthritis (OA). The aim of this study was to determine whether non-toxic concentrations of curcumin can reduce interleukin-1beta (IL-1β)-stimulated inflammation and catabolism in an explant model of cartilage inflammation.MethodsArticular cartilage explants and primary chondrocytes were obtained from equine metacarpophalangeal joints. Curcumin was added to monolayer cultured primary chondrocytes and cartilage explants in concentrations ranging from 3μM-100μM. Prostaglandin E 2 (PGE 2) and matrix metalloproteinase (MMP)-3 release into the secretome of IL-1β-stimulated explants was measured using a competitive ELISA and western blotting respectively. Proteoglycan (PG) release in the secretome was measured using the 1,9-dimethylmethylene blue (DMMB) assay. Cytotoxicity was assessed with a live/dead assay in monolayer cultures after 24 hours, 48 hours and five days, and in explants after five days.ResultsCurcumin induced chondrocyte death in primary cultures (50μM p<0.001 and 100μM p<0.001) after 24 hours. After 48 hours and five days, curcumin (≥25μM) significantly increased cell death ( p<0.001 both time points). In explants, curcumin toxicity was not observed at concentrations up to and including 25μM after five days. Curcumin (≥3μM) significantly reduced IL-1β-stimulated PG ( p<0.05) and PGE 2 release ( p<0.001) from explants, whilst curcumin (≥12μM) significantly reduced MMP-3 release ( p<0.01).ConclusionNon-cytotoxic concentrations of curcumin exert anti-catabolic and anti-inflammatory effects in cartilage explants.

| S-EPMC3901452 | biostudies-literature

Early response genes induced in chondrocytes stimulated with the inflammatory cytokine interleukin-1beta.

Project description:Recent work has established that IL-1beta plays a central role in the inflammation and connective tissue destruction observed in both rheumatoid arthritis and osteoarthritis. These processes result from the ability of this inflammatory cytokine to activate expression of genes for neutral proteases, such as the matrix metalloproteinases. While IL-1beta activates matrix metalloproteinase genes within several hours, it also activates immediate early genes, which are required for the later expression of matrix metalloproteinases and other arthritis-perpetuating genes, are also activated. To identify putative immediate early genes involved in IL-1beta-mediated arthritic disease, a chondrocytic cell line (SW1353) was stimulated with this cytokine for 2 hours, total RNA was isolated, and expressed genes were identified by microarray analysis. This analysis identified alterations in the expression of multiple transcription factors, cytokines, growth factors and their receptors, adhesion molecules, proteases, and signaling intermediates that may contribute to inflammation and cartilage destruction in arthritis. Interestingly, confirmation of the expression of activating protein-1 family members by reverse transcriptase polymerase chain reaction revealed a preferential increase in junB, a known transcriptional antagonist of c-jun. The failure to observe induction of early growth response gene-1, which was detected by reverse transcriptase polymerase chain reaction to be substantially and transiently induced by 1 hour of IL-1 treatment, may be explained by the known instability of the message after early induction. However, this analysis has identified numerous IL-1beta-responsive genes that warrant further investigation as mediators of disease in arthritis.

| S-EPMC64850 | biostudies-literature

IFN-gamma impairs release of IL-8 by IL-1beta-stimulated A549 lung carcinoma cells.

Project description:BACKGROUND: Production of interferon (IFN)-gamma is key to efficient anti-tumor immunity. The present study was set out to investigate effects of IFNgamma on the release of the potent pro-angiogenic mediator IL-8 by human A549 lung carcinoma cells. METHODS: A549 cells were cultured and stimulated with interleukin (IL)-1beta alone or in combination with IFNgamma. IL-8 production by these cells was analyzed with enzyme linked immuno sorbent assay (ELISA). mRNA-expression was analyzed by real-time PCR and RNase protection assay (RPA), respectively. Expression of inhibitor-kappa Balpha, cellular IL-8, and cyclooxygenase-2 was analyzed by Western blot analysis. RESULTS: Here we demonstrate that IFNgamma efficiently reduced IL-8 secretion under the influence of IL-1beta. Surprisingly, real-time PCR analysis and RPA revealed that the inhibitory effect of IFNgamma on IL-8 was not associated with significant changes in mRNA levels. These observations concurred with lack of a modulatory activity of IFNgamma on IL-1beta-induced NF-kappaB activation as assessed by cellular IkappaB levels. Moreover, analysis of intracellular IL-8 suggests that IFNgamma modulated IL-8 secretion by action on the posttranslational level. In contrast to IL-8, IL-1beta-induced cyclooxygenase-2 expression and release of IL-6 were not affected by IFNgamma indicating that modulation of IL-1beta action by this cytokine displays specificity. CONCLUSION: Data presented herein agree with an angiostatic role of IFNgamma as seen in rodent models of solid tumors and suggest that increasing T helper type 1 (Th1)-like functions in lung cancer patients e.g. by local delivery of IFNgamma may mediate therapeutic benefit via mechanisms that potentially include modulation of pro-angiogenic IL-8.

| S-EPMC2556346 | biostudies-literature

A comparative study of the binding of cartilage link protein and the hyaluronate-binding region of the cartilage proteoglycan to hyaluronate-substituted Sepharose gel.

Project description:The hyaluronate-binding proteins from bovine nasal cartilage, i.e. the hyaluronate-binding region of the proteoglycan and the link protein, were labelled with 125I and separated from each other by gel chromatography. The proteins were characterized by molecular-weight determinations and their purity was established by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and immunodiffusion. The binding properties of the two proteins by hyaluronate-substituted Sepharose gel were compared. It was found that both proteins behaved similarly. They bound with the same efficiency to the gel, they showed the same time course of binding, had slightly different pH optima for binding and both proteins had a decreasing affinity for the gel with increasing ionic strength. The binding to the gel could be inhibited by soluble hyaluronate, and the minimum size of a hyaluronate oligosaccharide required for inhibition was in both cases a decasaccharide (only even-numbered oligosaccharides were tested). The proteins did not show any co-operative binding in the system tested, which could be explained by the large number of binding sites in the hyaluronate-substituted gel. Binding constants for the protein-hyaluronate interaction were estimated. A value of 1.3 x 10(7) M-1 was obtained for the hyaluronate-binding region of the proteoglycan, in agreement with literature data. The corresponding value for the link protein was 0.7 x 10(7) M-1.

| S-EPMC1163372 | biostudies-other

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