Differential Toll-like receptor-dependent collagenase expression in chondrocytes.

OBJECTIVES: To characterise the catabolic response of osteoarthritic chondrocytes to Toll-like receptor (TLR) ligands. METHODS: Induction of the collagenases, matrix metalloproteinase (MMP)1 and MMP13, by TLR ligands was assessed in chondrocytes by real-time reverse transcriptase (RT)-PCR. TLR signalling pathway activation and their involvement in collagenase induction were confirmed by immunoblotting and use of pathway inhibitors and siRNA. TLR expression was compared in the femoral head cartilage of normal controls and patients with osteoarthritis (OA) by real-time RT-PCR. RESULTS: Ligands for TLR6/2 and TLR3 showed the greatest upregulation of MMP1 and MMP13 respectively, although all TLR ligands upregulated these MMPs. MMP1 and MMP13 induction by TLR3 and TLR1/2 or TLR6/2 ligands were dependent on Trif and MyD88, respectively. These inductions were dependent upon the nuclear factor (NF)kappaB pathway, but were differentially inhibited by various mitogen-activated protein kinase inhibitors, with MMP13 induction most reliant on the extracellular signal-regulated kinase pathway. In addition, ligands for TLR1/2 and TLR6/2, but not TLR3, induced significant collagenolysis in a cartilage resorption assay. Finally, TLR2 was significantly downregulated and TLR3 upregulated in OA, compared to normal, cartilage. CONCLUSIONS: Activation of chondrocyte TLRs leads to differential collagenase gene activation. Treatment of chondrocytes with TLR1/2 or TLR6/2 ligands resulted in collagen resorption. The modulated expression of chondrocyte TLR2 and TLR3 in OA cartilage, compared to normal, may reflect a response to repair cartilage or prevent further extracellular matrix destruction. These data suggest modulation of TLR-mediated signalling as a potential therapeutic strategy for the treatment of OA.

Structure of full-length porcine synovial collagenase reveals a C-terminal domain containing a calcium-linked, four-bladed beta-propeller.

BACKGROUND: The collagenases are members of the family of zinc-dependent enzymes known as the matrix metalloproteinases (MMPs). They are the only proteinases that specifically cleave the collagen triple helix, and are important in a large number of physiological and pathological processes. Structures are known for the N-terminal catalytic' domain of collagenases MMP-1 and MMP-8 and of stromelysin (MMP-3). This catalytic domain alone, which comprises about 150 amino acids, has no activity against collagen. A second domain, of 200 amino acids, is homologous to haemopexin, a haem-binding glycoprotein. RESULTS: The crystal structure of full-length MMP-1 at 2.5 A resolution gives an R-factor of 21.7%. Two domains are connected by an exposed proline-rich linker of 17 amino acids, which is probably flexible and has no secondary structure. The catalytic domain resembles those previously observed, and contains three calcium-binding sites. The haemopexin-like domain contains four units of four-stranded antiparallel beta sheet stabilized on its fourfold axis by a cation, which is probably calcium. The domain constitutes a four-bladed beta-propeller structure in which the blades are scarcely twisted. CONCLUSIONS: The exposed linker accounts for the difficulty in purifying full-length collagenase. The C-terminal domain provides a structural model for haemopexin and its homologues. It controls the specificity of MMPs, affecting both substrate and inhibitor binding, although its role remains obscure. These structural results should aid the design of site-specific mutants which will reveal further details of the specificity mechanism.

The activation of latent pig synovial collagenase.

Latent pig synovial collagenase (EC 3.4.24.7) can be activated by a variety of different treatments to give an active enzyme form of lower molecular weight which rapidly degrades collagen. Trypsin and plasmin effectively activated the latent collagenase whilst elastase and cathepsin G degraded most of the latent enzyme before it was activated. A number of mercurials were compared and maximum activation was achieved using 4-aminophenylmercuric acetate and phenylmercuric chloride. The latent collagenase bound to a mercurial-Sepharose column and was eluted in the active form with NaCl. The latent collagenase also activated spontaneously and the conditions which encouraged and prevented this activation were studied. High NaCl concentration, diisopropylphosphofluoridate, soybean trypsin inhibitor, low Zn2+ concentration and high and low pH all prevented the spontaneous activation of latent pig synovial collagenase.

Activation of the matrix metalloproteinase inhibitor complex by a low molecular weight angiogenic factor.

Tissue inhibitor of matrix metalloproteinases is the major inhibitor of the collagenolytic enzymes and the inhibitory complex has been thought to be irreversible. In this paper we show that a low molecular weight non-protein endothelial cell stimulating angiogenic factor is able to reactivate the enzyme from the inhibitor complex and liberate free inhibitor. The importance of an angiogenic factor able to initiate limited degradation of extra-cellular matrix such that space is created for new capillary growth is discussed.

Modulation of plasminogen activator production by interleukin 1: differential responses of fibroblasts derived from human skin and rheumatoid and non-rheumatoid synovium.

Rheumatoid synovial fibroblasts were treated with purified porcine interleukin 1 alpha and recombinant human interleukin 1B, and the production of secreted and cell-associated plasminogen activator activity was measured. No stimulation of plasminogen activator activity was seen in response to either preparation of interleukin 1, and in more than half of the cell cultures interleukin 1 caused a significant decrease in the secreted levels of PA activity. Increased levels of prostaglandin E were produced in the same experiments, indicating that the cells were responsive to the interleukin 1 preparations. Both retinoic acid and unfractionated monocyte conditioned medium were able to stimulate the production of PA activity by the rheumatoid synovial fibroblast cultures. The rheumatoid synovial fibroblasts produced two species of plasminogen activator as indicated by SDS polyacrylamide gel electrophoresis, with apparent Mr of approx. 50,000 and 100,000. The Mr = 50,000 species co-migrates with urokinase-type plasminogen activator. No species is produced which co-migrates with tissue type plasminogen activator. Studies with antibodies also indicate that the activity produced is urokinase-type plasminogen activator. The Mr = 100,000 species may be an enzyme-inhibitor complex. Two non-rheumatoid synovial fibroblast cultures and two out of six human skin fibroblast cultures did produce elevated levels of plasminogen activator activity in response to recombinant human interleukin 1B. The results suggest that fibroblast populations may differ in their response to interleukin 1, in terms of production of plasminogen activator activity.

The secretion of the tissue inhibitor of metalloproteinases (TIMP) by human synovial fibroblasts is modulated by all-trans-retinoic acid.

The matrix metalloproteinases are a family of enzymes involved in the turnover of the connective tissues. The regulation of these enzymes is complex, involving the control of synthesis, the activation of proenzyme forms and the presence of specific inhibitors. Retinoids have been reported to inhibit the production of metalloproteinases by human and rabbit synovial fibroblasts and by human skin fibroblasts. The production of the highly specific tissue inhibitor of metalloproteinases (TIMP) by connective tissue cells may be crucial in the regulation of connective tissue breakdown and this present study was undertaken to determine if retinoic acid (RA) could modulate TIMP and collagenase production by synovial fibroblasts. The results show that RA at concentrations from 10(-7) to 10(-5) M significantly stimulated the secretion of TIMP by two of three human synovial cell lines. The effect of mononuclear cell factor (MCF) on TIMP and collagenase levels was also investigated. The apparent reduction of collagenase levels in the presence of RA, could result from a failure to accurately measure this enzyme in the presence of increased levels of TIMP.

Transforming growth factor beta stimulates the production of the tissue inhibitor of metalloproteinases (TIMP) by human synovial and skin fibroblasts.

IL-1 stimulates the secretion of metalloproteinases by a variety of connective tissue cells and is thought to be the primary inducing agent of connective tissue breakdown in rheumatoid arthritis. Transforming growth factor-beta (TGF-beta) is known to be capable of inhibiting the synthesis of metalloproteinases and to be able to partially inhibit interleukin-1 (IL-1) induced cartilage degradation. The present paper examines the ability of TGF-beta to modulate the action of IL-1 on fibroblasts of synovial and skin origin and investigates the secretion of the tissue inhibitor of metalloproteinases (TIMP) by these cells after exposure to TGF-beta and IL-1. The principal findings are that when four out of five fibroblast lines were exposed to TGF-beta and IL-1 in combination they displayed a significant increase in TIMP secretion; furthermore, in two of these cell lines a significant stimulation of TIMP secretion was induced by TGF-beta alone.

Fragments of human fibroblast collagenase: interaction with metalloproteinase inhibitors and substrates.

On purification, active human fibroblast collagenase breaks down by an autolytic mechanism into two major forms (M(r) 22,000 and M(r) 27,000) and one minor form (M(r) 25,000). The ability of human collagenase to bind to the tissue inhibitor of metalloproteinases (TIMP) and to TIMP-2 resides mainly in the active site area of the 22,000 M(r) N-terminal domain of the molecule, but the 27,000 M(r) C-terminal domain also has a role in stabilizing these interactions. The 22,000 M(r) fragment is able to form a complex with TIMP and TIMP-2 which is stable to gel filtration in a similar manner to the whole molecule, but no such complexes are formed by the 27,000 M(r) fragment. Complex formation with the whole molecule is prevented by EDTA and by 1,10-phenanthroline demonstrating the importance of the active site; additionally TIMP and TIMP-2 will compete with a reversibly bound peptide hydroxamic acid inhibitor for the active site. The inhibition of enzyme activity by TIMP and TIMP-2 is less pronounced in the 22,000 M(r) fragment when compared to the whole molecule and a similar effect is seen with the peptide hydroxamic acid inhibitor and also with alpha 2-macroglobulin, suggesting a role for the C-terminal domain in interacting with these inhibitors. Whole molecule collagenase and the 27,000 M(r) fragment bind to type 1 collagen-Sepharose while the 22,000 M(r) fragment exhibits no such binding, suggesting that the C-terminal domain has an important role in the binding of enzyme to substrate.

Identification of plasma kallikrein as an activator of latent collagenase in rheumatoid synovial fluid.

Rheumatoid synovial fluid contains an activator of latent collagenase from culture medium of pig synovium. The activator was purified by gel chromatography on Ultrogel AcA 44 and affinity chromatography on soybean trypsin inhibitor coupled to Sepharose 4B. The purified material was homogeneous on SDS-polyacrylamide gel electrophoresis with Mr 88 000. The activator had limited proteolytic activity against azo-casein, but showed amidase activity on Pro-Phe-Arg-NMec, Z-Phe-Arg-NMec, D-Val-Leu-Arg-NPhNO2 and D-Pro-Phe-Arg-NPhNO2, with an optimum at pH 8.0. Activity was completely inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, leupeptin and Pro-Phe-Arg-CH2Cl, whereas lima bean trypsin inhibitor, Tos-Lys-CH2Cl, a specific inhibitor of factor XIIa from maize, EDTA and iodoacetate were not inhibitory. These properties of the activator suggested that it might be plasma kallikrein (EC 3.4.21.34), and the possibility was further examined. The activator was treated with [3H]diisopropyl fluorophosphate, and run in SDS-polyacrylamide gel electrophoresis with reduction; a radioautograph of the gel showed a pair of [3H]diisopropyl phosphoryl-labelled bands (Mr 36 000 and 34 000) identical to those obtained with authentic plasma kallikrein. Double immunodiffusion with monospecific antiserum against human plasma kallikrein confirmed the identification. This is the first demonstration of collagenase-activating activity of plasma kallikrein, and raises the possibility that activation of prokallikrein in the inflamed joint space may contribute to the disease process not only by the production of bradykinin, but also by activating latent collagenase.

Synovial fluids from infected joints contain metalloproteinase--tissue inhibitor of metalloproteinase (TIMP) complexes.

Samples of synovial fluids aspirated from patients with septic arthritis prior to the commencement of any treatment contained active metalloproteinases but no proteinase inhibitory activity. We therefore assayed these samples for proteinase-inhibitor complexes. Although no biologically active alpha 2-macroglobulin or tissue inhibitor of metalloproteinase (TIMP) was present in the fluids, immunoassay of the samples clearly showed that high molecular weight proteinase-TIMP complexes were present. It is proposed that high levels of active metalloproteinases are released from neutrophils into septic synovial fluids and that these proteinases complex all the available TIMP, forming metalloproteinase-TIMP complexes.

Interleukin-4 blocks the release of collagen fragments from bovine nasal cartilage treated with cytokines.

Interleukin-1 (IL-1) in combination with other cytokines can induce a reproducible release of collagen fragments from bovine nasal cartilage in culture. Over 70% of the total collagen is released by day 14 and this release is accompanied by the appearance of collagenolytic activity in the medium that cleaves collagen specifically at the one quarter/three quarter position. Interleukin-4 is able to prevent the release of collagen fragments from the tissue and this is accompanied by a reduced secretion and activation of collagenase (MMP-1) with an increase in tissue inhibitor of metalloproteinases-1 (TIMP-1). IL-4, especially in the presence of IL-1, increased TIMP secretion by bovine nasal cartilage in culture. These results suggest that IL-4 is able to specifically block cartilage collagen resorption by down-regulating the production of collagenase (MMP-1) and up-regulating TIMP-1 by chondrocytes within the cartilage.

Purification and secondary structural analysis of tissue inhibitor of metalloproteinases-1.

In connective tissue diseases such as rheumatoid arthritis, the matrix metalloproteinases are the primary enzymes involved in tissue degradation. Tissue inhibitor metalloproteinases-1 (TIMP-1) is a specific inhibitor of these enzymes, which is thought to regulate their action in vivo. The structure and function of TIMP-1 may therefore be important as the basis for the rational design of therapeutic agents. This paper describes a simple and effective method for the purification of sufficient quantities of TIMP-1 for spectroscopic studies. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy have, together, showed TIMP-1 to be mostly in a beta-sheet conformation, with significant amounts of alpha-helix and beta-turn. Two-dimensional nuclear magnetic resonance spectroscopy indicated a correspondingly high proportion of beta-sheet. CD and FTIR have also shown TIMP-1 to have high thermostability.

Metalloproteinase inhibitors and the prevention of connective tissue breakdown.

The primary agents responsible for cartilage and bone destruction in joint diseases are active proteinases degrading collagen and proteoglycan. All four main classes of proteolytic enzymes are involved in either the normal turnover of connective tissue or its pathological destruction. These proteinases are made by different cells found within the joints. Both extracellular and intracellular pathways exist, and individual enzymes can be inhibited by specific proteinaceous inhibitors that block their activity. Recent research has implicated the matrix metalloproteinases in many of the processes involved in joint diseases. Conventional treatments do little to affect the underlying disease processes, and recently, the use of proteinase inhibitors has been suggested as a new therapeutic approach. A large variety of different synthetic approaches have been used and highly effective metalloproteinase inhibitors have been designed, synthesised and tested. These metalloproteinase inhibitors can prevent the destruction of animal cartilage in model systems and slow the progression of human tumours. Future patient trials will test the effectiveness of these compounds in vivo for the treatment of joint diseases.

Crystallization and preliminary X-ray analysis of porcine synovial collagenase.

Crystals of porcine synovial collagenase suitable for an X-ray structure analysis have been obtained. The crystals belong to space group I4, with unit cell dimensions a = b = 160.0 A, c = 53.1 A, with one molecule in the asymmetric unit. Diffraction extends beyond 3 A perpendicular to the c axis but along the 4-fold axis, the intensities are measurable only to 4 A.

Production in Escherichia coli of porcine type-I collagenase as a fusion protein with beta-galactosidase.

Porcine type-I collagenase (Colg-1) was produced as a fusion protein in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to Colg-1. Recombinant collagenase (reColg-1) was biologically active in the form of a fusion protein and could be released by treatment with factor Xa to yield Colg-1 with the authentic N terminus (phenylalanine) found in vivo. The results show that reColg-1 produced in E. coli is folded correctly, cleaves type-I collagen into 1/4 and 3/4 fragments at the characteristic Colg-sensitive site, and is produced at high enough levels to generate a source of recombinant enzyme for x-ray crystallography studies.

Preferential binding of collagenase to alpha 2-macroglobulin in the presence of the tissue inhibitor of metalloproteinases.

The binding of collagenase to both alpha 2-macroglobulin and the tissue inhibitor of metalloproteinases was studied using purified materials. Collagenase bound preferentially to alpha 2-macroglobulin although no transfer of collagenase to alpha 2-macroglobulin occurred if the enzyme was first mixed with the tissue inhibitor of metalloproteinases. The sequences of amino acids in both inhibitors likely to be responsible for the binding of collagenase are discussed and compared to the cleavage site in the collagen molecule.

A rapid and reproducible method for the analysis of immune complexes using affinity chromatography and Western blotting.

A new procedure which couples different analytical techniques in a format permitting the rapid analysis of immune complex components is described. Complexes obtained from sera by polyethylene glycol (PEG) precipitation were resuspended and then added, using a batch method, to antibody coupled to Sepharose beads. Antibody directed against either human C1q or human C3c were used in the present study. Bound immune complexes were washed and then eluted from the Sepharose by sodium dodecyl sulphate (SDS) treatment and simultaneously reduced with dithiothreitol. Individual components were separated by SDS gradient polyacrylamide gel electrophoresis and then transferred to nitrocellulose by Western blotting. Individual strips of nitrocellulose were investigated using specific antisera and a radiolabelled probe. Immune complexes (IC) isolated from the sera of 7 rheumatoid arthritis (RA) patients were analysed using this method and the results obtained for both affinity adsorbents compared.

The platelet reactivity of collagen type I: evidence for multiple platelet-reactive sites in the type I collagen molecule.

In this study, the ability of peptides, obtained by fragmentation of the collagen type I molecule, to induce platelet aggregation has been examined. In order to satisfy requirements for tertiary and quaternary structure, peptides were first renatured (where necessary) to restore triple-helical configuration and then polymerised. Fragmentation with mammalian collagenase indicated the presence of platelet-reactive sites in both the N-terminal three-quarter and C-terminal one quarter fragment of the collagen molecule. Cleavage with cyanogen bromide indicated the presence in the constituent alpha 1(I)-chain of at least four platelet-reactive sites. Our results suggest a relatively wide distribution of platelet-binding sites situated throughout the length of the collagen (type I) molecule, each probably of relatively low affinity and low structural specificity, at least in terms of amino acid sequence, and probably of a similar nature to those that might be expected to exist in any collagen-like species.

Matrix metalloproteinases and TIMPs: properties and implications for the treatment of chronic obstructive pulmonary disease.

The matrix metalloproteinases (MMPs) are a unique family of metalloenzymes that, once activated, can destroy connective tissue. The active enzymes are all inhibited by tissue inhibitors of metalloproteinases (TIMPs). The relative amounts of active MMPs and TIMPs are important in determining whether tissues are broken down in disease. Although elastase is often regarded as the target enzyme in chronic obstructive pulmonary disease (COPD), both the neutrophils and macrophages in the lung contain metalloproteinases and both collagen and elastin are degraded in disease. Transgenic studies have shown that when MMP1 is over-expressed, pulmonary emphysema develops in mice, while MMP12 knockout mice do not develop pulmonary emphysema when exposed to cigarette smoke. New drugs that can specifically block active MMPs are now available. These potent inhibitors are effective in vitro and prevent the destruction of tissue in animal models. Future patient trials will test the effectiveness of these compounds in preventing tissue destruction.

The regulation of MMPs and TIMPs in cartilage turnover.

The treatment of cartilage with mediators initiates the breakdown of proteoglycan followed by collagen. This is accompanied by the modulation of different proteinases and inhibitors that include members of the MMP family and TIMPs. We have evidence that a chondrocyte membrane-associated metalloproteinase cleaves aggrecan. This activity is rapidly induced after stimulation with IL-1 and OSM and is not inhibited by TIMPs-1 and -2 but is inhibited by synthetic MMP inhibitors. This same combination of cytokines also upregulates the collagenases with the subsequent release of collagen fragments, and there is a close correlation between the amount of collagen released and collagenase activity produced. Collagen release can be prevented after treatment with specific inhibitors of MAP kinases, inhibitors of MMP transcription, synthetic metalloproteinase inhibitors, TIMPs and treatment of cartilage with agents that upregulate TIMPs. The results from bovine cartilage culture models show that collagen release occurs when TIMP levels are low, collagenases are upregulated and then subsequently activated.