A high throughput enzymatic assay for stromelysin-1.
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Acetylation↗
Biomedical subjects
E C Arner
Publications and source records attributed to E C Arner.
A fluorescent enzyme-linked immunosorbent assay (FELISA) for stromelysin using a polyclonal antisera to human stromelysin with broad species cross-reactivity.
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Animals↗
A high-throughput assay for stromelysin using a casein-resorufin substrate.
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Body Fluids↗
Isothiazolones interfere with normal matrix metalloproteinase activation and inhibit cartilage proteoglycan degradation.
A series of isothiazolones that inhibit pro-(matrix metallo-proteinase) (proMMP) activation but do not inhibit the active enzyme are effective as cartilage protectants in bovine nasal cartilage organ culture, preventing interleukin-1 (IL-1)-induced proteoglycan (aggrecan) degradation without affecting its synthesis. These compounds were found to bind to prostromelysin (proMMP-3) in a non-dialysable and stoichiometric manner. Preincubation with cartilage-protectant isothiazolones prevented the binding of [14C]iodoacetamide to Cys75 of the MMP-3 propeptide, suggesting that the activity of these compounds involves their binding to the Cys75 of the MMP zymogen. Studies following chymotrypsin activation of proMMP-3 by SDS/PAGE indicated that altered processing of the 57 kDa zymogen to the active form occurred in the presence of compound. The 53 kDa intermediate seen on normal activation was not formed; instead a different intermediate appeared with a molecular mass of approx. 46 kDa. N-terminal sequence analysis indicated that this intermediate was formed by cleavage at the putative 4-aminophenylmercuric acid cleavage site. Importantly the 45 kDa active MMP-3 species formed in the presence of compound was one amino acid residue shorter than the native MMP-3. These results suggest that the inhibition of cartilage proteoglycan degradation by isothiazolones might be due to their ability to bind to the Cys75 in the propeptide region of the MMP zymogen and interfere with its normal activation process.
Amino Acid Sequence↗
2,5-Diarylisothiazolone: novel inhibitors of cytokine-induced cartilage destruction.
A series of 2,5-diarylisothiazolones is reported that inhibit the IL-1 beta-induced breakdown of bovine nasal septum cartilage in an organ culture assay. The synthesis and preliminary SAR of these compounds are described. These compounds represent a novel, nonpeptide lead series approach to the mediation of the chronic cartilage breakdown associated with arthritic disease. These compounds are relatively resistant to reductive metabolism by liver microsomal preparations and appear to inhibit cartilage breakdown by interfering with the proteolytic activation of matrix metalloproteinases.
Animals↗
Signal transduction through chondrocyte integrin receptors induces matrix metalloproteinase synthesis and synergizes with interleukin-1.
OBJECTIVE: To study the role of signal transduction via integrin receptors in the production of metalloproteinase by rabbit articular chondrocytes. METHODS: Confluent, primary rabbit articular chondrocytes (RAC) were incubated for 72 hours in the presence of interleukin-1 (IL-1), Arg-Gly-Asp (RGD) peptide, or a combination of IL-1 and RGD peptide. Media were analyzed for stromelysin enzymatic activity using a 3H-labeled transferrin substrate, and for stromelysin and collagenase protein by Western analysis. Gelatinase activity was analyzed by gelatin zymography. IL-1 receptor antagonist (IL-1Ra) protein was used to determine the involvement of IL-1 in mediating the effects of RGD peptide, and fluorescence-activated cell sorter analysis (FACS) was used to examine the effect of IL-1 on chondrocyte integrin subunit expression. RESULTS: RGD peptides induced chondrocyte synthesis of stromelysin, collagenase, and 92-kd gelatinase B, and increased synthesis of the constitutively expressed 72-kd gelatinase A. Further studies focusing on stromelysin demonstrated that this up-regulation was concentration dependent and that RGD peptides synergized with IL-1 in inducing stromelysin synthesis. RGD-induced stromelysin production was inhibited by the IL-1Ra in a concentration-dependent manner, indicating that induction by RGD requires binding of IL-1 to its receptor. FACS analysis of RAC showed that IL-1 stimulation increased the expression of beta 1 and alpha v integrin subunits on the chondrocyte surface. CONCLUSION: Our data demonstrate that signal transduction through chondrocyte integrin receptors up-regulates metalloproteinase expression and that this is likely mediated through induction of IL-1. They also suggest that the binding of adhesion molecules to their chondrocyte integrin receptors reduces the amount of IL-1 required to induce stromelysin synthesis. Up-regulation of chondrocyte integrin expression by IL-1 may play a role in the synergistic effects seen with a combination of IL-1 and RGD peptides. Since elevated levels of both IL-1 and adhesion molecules are present in rheumatoid arthritis and osteoarthritis synovial fluid, our data suggest that this interaction may be important in mediating the cartilage destruction accompanying these diseases.
Animals↗
Interleukin-1 receptor antagonist inhibits proteoglycan breakdown in antigen induced but not polycation induced arthritis in the rabbit.
OBJECTIVE: To compare the alterations in proteoglycan metabolism in antigen induced arthritis and polycation induced arthritis and to determine the involvement of interleukin-1 (IL-1) in the cartilage degradation that occurs in these models of rheumatoid arthritis (RA). METHODS: The time course for loss of proteoglycan into the synovial fluid (SF) and inhibition of proteoglycan synthesis, as well as depletion of articular cartilage proteoglycan content, was compared in rabbit antigen arthritis and polycation arthritis. The ability of recombinant IL-1 receptor antagonist IL-1ra to block the acute cartilage loss at 24 h in these models was investigated, compared to its ability to block the cartilage breakdown induced by direct administration of IL-1 in rabbits. RESULTS: Initial loss of cartilage proteoglycan was accompanied by release of high levels of glycosaminoglycan (GAG) into the SF and decrease in proteoglycan synthetic rates in both antigen and polycation induced arthritis SF GAG rapidly returned to control levels, while proteoglycan synthesis and cartilage proteoglycan content remained depressed, suggesting that the inhibition in proteoglycan synthesis prevented recovery to normal levels. GAG loss from the cartilage into the SF in response to IL-1 injection, as well as other effects of IL-1 challenge, was blocked in a dose dependent manner by IL-1ra administered either intraarticularly (ED50 = 160 ng) or intravenously (iv) (ED50 = 0.09mg/kg). In the antigen induced arthritis model, IL-1ra (20 mg/kg, iv -2h) inhibited GAG release by 40%, whereas in polycation induced arthritis no inhibition was observed even with repeated administration of high doses of inhibitor. CONCLUSION: These studies suggest that sustained depression of proteoglycan synthesis may be responsible for the chronic depletion of articular cartilage proteoglycan in the antigen and the polycation model of RA. However, while IL-1 may play a role in the initial breakdown of articular cartilage in antigen induced arthritis, it does not appear to be involved in polycation induced arthritis in the rabbit.
Animals↗
Effect of animal age and chronicity of interleukin-1 exposure on cartilage proteoglycan depletion in vivo.
Rheumatoid arthritis and osteoarthritis are characterized by an early depletion of cartilage proteoglycans, which leads to a decrease in cartilage compressibility and, eventually, to a loss of joint function. Interleukin-1, which is thought to have a role in mediating this loss of proteoglycans in arthritis, induces an acute depletion of proteoglycans from articular cartilage following intra-articular injection in rabbits. As the structure and metabolism of proteoglycans are known to change with age, my laboratory investigated the effect of age on depletion and recovery of proteoglycans in response to interleukin-1 in the rabbit. Loss of cartilage proteoglycans induced by interleukin-1 was less severe in immature animals, increased until the age of sexual maturity, and then remained constant. The rate of recovery and compensatory overshoot in the rate of proteoglycan synthesis following challenge with interleukin-1 was more rapid in immature animals and may have been responsible for the quicker return of the cartilage proteoglycan content to control levels in younger animals. With multiple exposures to interleukin-1 at time intervals too short for recovery to occur, smaller amounts of interleukin-1 induced loss of proteoglycans, and the proteoglycan content and the rate of synthesis remained depressed longer after treatment had stopped. The decreased ability of mature cartilage to replace proteoglycans rapidly after exposure to cytokines would increase the probability of subsequent inflammatory episodes before recovery is complete; this may result in increased susceptibility of adult cartilage to proteoglycan depletion.
Aging↗
Interleukin-1 differentially modulates chondrocyte expression of cyclooxygenase-2 and phospholipase A2.
Interleukin-1 beta (IL-1) increases the synthesis of prostaglandins as well as the expression of synovial fluid phospholipase A2 (PLA2) mRNA and activity by chondrocytes. In order to examine the potential involvement of cyclooxygenase in the induction of prostaglandins by IL-1, the effect of IL-1 on rabbit articular chondrocyte expression of cyclooxygenase enzymes was investigated. By Northern analyses, mRNA for cyclooxygenase-2 (COX-2) was found to be constitutively expressed, and its expression was increased in cultures treated for 24 h with IL-1 (100 ng/ml). Cyclooxygenase-1 mRNA was not detected in control or IL-1-treated cultures. IL-1 caused a concentration-dependent increase in steady-state levels of COX-2 message as assessed by slot-blot analyses. Half-maximal induction of the COX-2 message levels was estimated to require 1.2 ng/ml IL-1. In duplicate slot blots probed with a cDNA for synovial fluid PLA2, half-maximal induction of PLA2 mRNA was estimated to require 0.15 ng/ml IL-1, approximately 10-fold less IL-1 than required for COX-2. In addition, maximal increase in COX-2 message levels was only 3-fold as compared with a 13-fold induction of PLA2. No change in message levels for the intracellular protein, actin, was found between control and experimental cultures. Cycloheximide enhanced COX-2 transcript levels, but did not modulate IL-1 induction of COX-2. Actinomycin D did not inhibit IL-1 augmentation of COX-2 mRNA levels; whereas, induction of PLA2 mRNA levels was completely inhibited, implicating transcriptional mechanisms in the induction of PLA2 but not COX-2 mRNA. These data demonstrate that chondrocyte expression of COX-2 and synovial fluid PLA2 are differentially modulated by IL-1.
Animals↗
Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) is induced in rabbit articular chondrocytes by cotreatment with interleukin 1 beta and a protein kinase C activator.
The synthesis of an 88-kDa gelatinolytic enzyme, identified as a zymogen of matrix metalloproteinase (proMMP)-9, was induced in the primary culture of rabbit articular chondrocytes by cotreatment with recombinant interleukin 1 beta (rIL-1 beta) and the protein kinase C (PKC) agonists, phorbol 12,13-dibutyrate (PDBu) or mezerein. Negligible 88-kDa gelatinolytic activity was produced by unstimulated cells or cells treated with a PKC activator alone at concentrations up to 100 ng/ml, and only a modest induction occurred with rIL-1 beta alone at concentrations of 1-100 ng/ml. However, when these cells were treated with a PKC activator in the presence of IL-1 beta (1 ng/ml), induction was striking, with enzymic activity detectable at a concentration as low as 1 ng/ml of mezerein or 10 ng/ml of PDBu. Rabbit chondrocytes in culture constitutively produced the zymogen of MMP-2 (proMMP-2) and its production was not altered by treatment with IL-1 beta or PKC agonists alone or in combination. Recombinant tumor necrosis factor alpha (rTNF alpha) did not substitute for IL-1 beta in inducing proMMP-9 in the presence of PKC activators, nor was the combination of IL-1 beta or TNF alpha alone effective. These data indicate that rabbit articular chondrocytes have a potential to synthesize and secrete proMMP-9 under certain biological and pathological conditions but that the expression of proMMP-9 is differently regulated from that of other MMPs.
Animals↗
Modulation of interleukin-1-induced alterations in cartilage proteoglycan metabolism by activation of protein kinase C.
Interleukin-1 (IL-1) stimulates proteoglycan degradation and prostaglandin E2 (PGE2) release and inhibits proteoglycan synthesis by cartilage in organ culture. Addition of the protein kinase C (PKC) activator, mezerein, resulted in the concentration-dependent inhibition of IL-1 activity on proteoglycan metabolism. Similar effects were seen with other compounds which stimulated PKC, such as teleocidin B4 and phorbol dibutyrate (PDBu), but not with a phorbol analog that is inactive in stimulating PKC. Simultaneous addition of the PKC antagonist, staurosporine, blocked the mezerein-induced inhibition of IL-1 activity on both proteoglycan degradation and synthesis in a concentration-related manner. In contrast to its inhibition of the effect of IL-1 on proteoglycan metabolism, mezerein did not block the release of PGE2 by cartilage in response to IL-1 but caused a synergistic stimulation of PGE2 release. Importantly, in cultures made deficient in PKC by prolonged incubation with PDBu, the effects of this PKC agonist on proteoglycan breakdown and PGE2 were blocked, while stimulation by IL-1 persisted. These data indicate that the effects of IL-1 on proteoglycan metabolism and prostaglandin production are mediated by an intracellular signal distinct from PKC and suggest that activation of PKC in chondrocytes may play a role in modulating the action of IL-1 on proteoglycan metabolism.
Alkaloids↗
Effects of IL-1 muteins on cartilage degradation and as inducers of acute inflammation.
IL-1 peptides with N-terminal amino acid mutations were cloned and expressed to help characterize structural requirements for activity. Addition of Thr-Asn to the N-terminus (DP516) or substitution of the first two residues (Ala-Pro) of mature, native IL-1 beta with Thr-Met (DuP118) had no effect on the potency of the muteins in stimulating proteoglycan breakdown and inhibiting proteoglycan synthesis in vitro, or inducing mouse paw swelling in vivo. When Arg in position 4 of DuP118 was replaced by Glu (Glu-4), proteoglycan-synthesis-inhibitory activity was reduced to 20% and proteoglycan-degrading activity to 2% of that induced by native IL-1 beta. Glu-4 was much less active in inducing mouse paw swelling and gave maximal swelling about 40% that of native IL-1 beta. The data suggest that the presence of the positively charged side chain of Arg in position 4 may be important for the activity of IL-1 and may be useful in designing specific IL-1 receptor agonists/antagonists.
Amino Acid Sequence↗
The effects of interleukin-1 on the expression of thrombospondin and fibronectin by rabbit articular chondrocytes.
Studies to evaluate the effects of recombinant interleukin-1 beta (IL-1) on the expression of matrix proteins by rabbit articular chondrocytes were conducted. Chondrocytes expressed high levels of message for thrombospondin (Tsp) and fibronectin (Fn). RNA slot-blot analysis demonstrated that treatment of the cultures with IL-1 (100 ng/ml) for 24 h caused a 70% suppression of their steady-state Tsp mRNA levels whereas those of Fn were not affected. Steady-state mRNA levels for the intracellular protein, actin, were not modulated by treatment with IL-1. The suppression of Tsp mRNA levels by IL-1 (100 ng/ml) was maximal by 4 h and was concentration dependent; half-maximal suppression was estimated to require 0.12 ng/ml IL-1. Cycloheximide treatment enhanced Tsp mRNA levels, but did not modulate IL-1 suppression of Tsp mRNA. Using pulse-labeling and immunoprecipitation techniques, we found that IL-1 suppression of Tsp mRNA levels was reflected in a coordinate inhibition of Tsp protein synthesis. Chondrocyte synthesis of Fn was not affected by IL-1. These data suggest that IL-1 specifically regulates chondrocyte expression of Tsp at least in part by decreasing the amount of Tsp mRNA available for translation.
Actins↗
Eicosanoid production and cell accumulation induced by intrapleural injection of sodium arachidonate in the rat. Characterization of the model.
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Animals↗
Interleukin-1 beta stimulates phospholipase A2 mRNA synthesis in rabbit articular chondrocytes.
A cDNA that codes for human rheumatoid synovial fluid phospholipase A2 (PLA2) hybridized with a RNA of the same size (900 base pairs) isolated from rabbit articular chondrocytes (RAC). Treatment of RAC with 100 ng/ml recombinant human interleukin-1 beta (IL-1) for 24 hours caused a 13-fold increase in mRNA for this PLA2. Timecourse studies demonstrated that maximal induction of PLA2 mRNA occurred by 16 hours post addition of IL-1 (100 ng/ml). Augmentation of RAC PLA2 mRNA levels was dose-dependent; half-maximal induction was estimated to require 0.15 ng/ml IL-1. Actinomycin D inhibited IL-1 effects on PLA2 mRNA levels. Coordinate effects of IL-1 on RAC PLA2 activity were observed with respect to time and dose dependence as well as actinomycin D sensitivity.
Animals↗
Effect of interleukin-1 on the size distribution of cartilage proteoglycans as determined by sedimentation field flow fractionation.
The effect of interleukin-1 (IL-1) on the size distribution of cartilage proteoglycans was studied using sedimentation field flow fractionation (SdFFF), a rapid, high-resolution technique for the separation of proteoglycan monomers and aggregates. During incubation of cartilage in control media, 35S-prelabeled proteoglycan was lost primarily from proteoglycan present in the monomer form; aggregates were conserved. In the presence of IL-1, both 35S-proteoglycan monomers and aggregates were lost, suggesting that IL-1 increases the susceptibility of aggregates to loss from the cartilage matrix. Evaluation of uronic acid as a measure of net change in proteoglycan content indicated that IL-1 causes a net decrease in both monomers and aggregates. Kinetic studies suggested that aggregates are degraded to monomers which then diffuse out of the matrix. Incorporation of [35S]sulfate into cartilage proteoglycans following exposure to IL-1 showed that synthesis of monomers and aggregates is inhibited similarly. SdFFF is a valuable technique for studying proteoglycan metabolism. With its use, changes in proteoglycan monomer and aggregate populations can be detected in response to cytokines such as IL-1.
Animals↗
Independent effects of interleukin-1 on proteoglycan breakdown, proteoglycan synthesis, and prostaglandin E2 release from cartilage in organ culture.
Exposure of bovine nasal cartilage in culture to interleukin-1 (IL-1) leads to a time- and concentration-dependent stimulation of proteoglycan breakdown and prostaglandin E2 (PGE2) release, and to inhibition of proteoglycan synthesis. The threshold levels of IL-1 required for initiating these effects were different, and IL-1 was 10 times more potent in inhibiting synthesis than in stimulating breakdown of proteoglycan. Kinetic studies indicated that the effects on proteoglycan metabolism occurred earlier (16-24 hours) than those for PGE2 release (48 hours). Selective effects were observed with inhibitors. Nonsteroidal antiinflammatory drugs blocked PGE2 production in response to IL-1, but had no effect on proteoglycan metabolism, and the antiarthritic drugs that blocked IL-1-stimulated breakdown augmented the inhibition of proteoglycan synthesis. We suggest that the effects of IL-1 on proteoglycan breakdown, proteoglycan synthesis, and PGE2 release are mediated by independent post-receptor mechanisms.
Animals↗