1 Molecular Pathology Section, Division of Biomedical Sciences, Imperial College School of Medicine, South Kensington, London SW7 2AZ,
2 Royal Veterinary College, Royal College Street, London NW1 0TU, UK and
3 Sankyo Co., Ltd, Tokyo, Japan
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Abstract |
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Method. An explant culture system was used to investigate the release of the G1 domain of aggrecan from porcine articular cartilage treated with retinoic acid or interleukin 1ß and to study how the activity of these agents is modified by the proteinase inhibitor, batimastat (BB94).
Results. Retinoic acid and interleukin 1ß induced both enzyme activities and the release of the G1 domain into the culture medium. Proteinase activity was significantly reduced when the tissue was incubated in the presence of BB94. The functional properties of the enzyme-generated G1 domain were studied using large-pore, agarose/polyacrylamide gel electrophoresis, and it was shown to interact with hyaluronan and link protein.
Conclusions. The results show that there must be a mechanism for removing a functional G1 domain from aggrecan during tissue turnover using this culture system.
KEY WORDS: Cartilage, Aggrecanase, Metalloproteinase, Aggrecan, Neo-epitopes.
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Introduction |
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Although aggrecan is cleaved by a number of matrix metalloproteinases (MMPs) between Asn341 and Phe342 in the interglobular domain of the molecule, the major fragments of aggrecan detected in arthritic synovial fluid result from cleavage at the Glu373Ala374 site [1]. This bond is cleaved by aggrecanase and the activity of this enzyme(s) is believed to be central for aggrecan breakdown in vivo [2]. It was shown that aggrecanase activity can be induced by agents such as retinoic acid and the proinflammatory cytokine interleukin (IL)-1 in vitro, and that it induces the degradation of aggrecan at the Glu373Ala374 site within the interglobular domain of the core protein of the molecule [2]. However, the relative involvements of MMPs and aggrecanase appear to differ between species [3], making inter-species comparisons difficult and adding little to our understanding of human aggrecanolysis.
Two different aggrecanases have been identified recently and are members of the ADAMts family. Both aggrecanase-1 (ADAMts-4) and aggrecanase-2 (ADAMts-5) have been detected in arthritic tissue [4, 5]. This group of enzymes have a very similar domain structure to the ADAMs (a disintegrin and metalloproteinase), but they contain a thrombospondin type I motif rather than transmembrane and cytoplasmic regions. This motif is believed to bind aggrecanase to the glycosaminoglycan side-chains of the aggrecan substrate.
The present study was carried out to characterize two new polyclonal antisera raised against peptide sequences corresponding to the neo-C-terminal epitopes generated by cleavage of aggrecan within its interglobular domain by MMPs (anti-FVDIPEN; for a definition of FVDIPEN see below) and by aggrecanase (anti-NITEGE; for a definition of NITEGE see below). These antisera were then used to determine the action of IL-1ß and retinoic acid on the degradation and release of porcine articular aggrecan and link protein from the tissue.
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Materials and methods |
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Analytical methods
The glycosaminoglycan content of the culture medium and tissue was determined by the dye binding method of Farndale et al. [6]. Cartilage was dispersed by digesting with papain before analysis. The incorporation of 35S-sulphate into cartilage was determined using the method described by Maroudas and Evans [7].
Large-pore agarosepolyacrylamide gel electrophoresis
Large-pore gels were prepared and electrophoresis and Western blotting were performed as described previously [8].
Preparation of antisera, SDSPAGE and Western blotting
The method for preparation of antisera was as described by Sugimoto et al. [9]. Peptide antisera were raised in rabbits against the following antigens: (i) a peptide sequence, CYTGEDFVDIPEN, linked to keyhole limpet haemocyanin (KLH) (FVDIPEN is the neo-C terminal amino acid sequence generated by the action of MMPs on the interglobular domain of aggrecan); (ii) a peptide sequence, LPLPRNITEGE, linked to KLH (NITEGE is the neo-C-terminal amino acid sequence generated by the action of aggrecanase on the interglobular domain of aggrecan). Both of the antisera were affinity-purified using columns to which the purified peptide had been attached. The original and modified peptides were checked for purity and their molecular weights by MALDI-TOF mass spectrometry. A monoclonal antibody, 8A4, that recognizes link protein was also used to localize the protein on Western blots. Immunoreactive proteins were visualized using the enhanced chemiluminescence (ECL) reagent obtained from Amersham Pharmacia Biotech, Amersham, UK.
SDSPAGE (sodium dodecyl sulphatepolyacrylamide gel electrophoresis) and Western blotting were carried out as described by Bird et al. [10]. The volume of sample loaded into each well of the gel, whether it was culture medium or cartilage extract, was calculated from the original weight of tissue cultured, so that a comparison could be made of the immunoreactivity within each set of cultures.
Explant culture of porcine articular cartilage and extraction of cartilage
Articular cartilage was removed under sterile conditions from the metacarpophalangeal joints of pig trotters (animals 36 months of age) obtained from the abattoir. The subsequent culture conditions depended on the measurement that was performed. When the rate of 35S-sulphate incorporation was determined, triplicate cultures [50 mg wet weight cartilage/ml DMEM (Dulbecco's Modified Eagle Medium)] were incubated for 4 h with 10 µCi/ml 35S-sulphate and the incorporation of radiolabel into macromolecules was measured as described by Brocklehurst and Bayliss [11]. When the release of glycosaminoglycan and protein fragments was investigated, cultures of cartilage (200 mg wet weight) were incubated for up to 10 days in 2 ml DMEM containing either 1 µM all-trans retinoic acid or 10 ng/ml IL-1ß, changing the medium every 2 days. The ability of the proteinase inhibitor BB94 (10 µM) to ameliorate the action of retinoic acid and IL-1ß was also investigated in this culture system. The IC50 (median inhibitory concentration) data for batimastat show it to be a potent inhibitor of MMP1, 2, 3, 7, 8 and 9 in the nanomolar range, but it is not known if it will inhibit all MMPs and the two aggrecanases [12]. Aliquots of culture medium were digested with chondroitinase ABC and keratanase I and II prior to electrophoresis in SDSpolyacrylamide gels. When extracts of the tissue and culture medium were electrophoresed in agarosepolyacrylamide gels, the chondroitinase ABC and keratanase treatment of the sample was not carried out.
The cartilage at the end of culture was extracted with 4 M guanidine HCl and was stored at -20°C until it was required, when aliquots were dialysed against distilled water and freeze-dried. Digestion of the extracts with chondroitinase ABC was carried out as described above.
Recombination of aggrecan, hyaluronan and link protein
Aggrecan (A1D1) and link protein from porcine articular cartilage were prepared as described by Bonnet et al. [13]. The preliminary experiment carried out to test the activity of the commercial source of stromelysin was performed after activating the proteinase (1 µg/ml) with 2 mM APMA and using it to digest porcine A1D1. The recombination experiment shown in Fig. 6 was carried out by recombining either porcine A1D1 or the culture medium retrieved at day 6 with 0.1 mg/ml hyaluronan and 2.5 mg/ml porcine link protein, before testing the ability of the G1 domain in each preparation to form aggregates that were excluded from agarose/polyacrylamide gels.
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Results |
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Release of glycosaminoglycan-containing fragments from porcine articular cartilage by IL-1ß and retinoic acid
Retinoic acid induced a much greater release of glycosaminoglycan than IL-1ß and maximum release was obtained at days 24 (Fig. 2a). The metalloproteinase inhibitor BB94 inhibited glycosaminoglycan release induced by either retinoic acid or IL-1ß, and it also inhibited release from explants treated with the carrier (DMSO) (Fig. 2b
). The rate of sulphate incorporation by explants treated with either retinoic acid or IL-1ß was measured after 2 days of culture and confirmed that these agents inhibited uptake of the isotope. However, it should be noted that the uptake of 35S-sulphate by porcine articular cartilage incubated under control conditions was 10 times less on day 2 of culture than that measured at the beginning of the experiment (results not shown).
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Generation of G1 domain of aggrecan in explant cultures treated with IL-1ß and retinoic acid
Figure 3 shows Western blots of the culture medium collected at days 2, 4, 6 and 8. G1-domain neo-epitopes generated by the action of aggrecanase (NITEGE) and MMPs (FVDIPEN) were released into the culture medium by the action of either retinoic acid or IL-1ß. However, in keeping with the findings illustrated in Fig. 2a
, the amount of the G1 domain released was much greater and occurred earlier when the tissue was treated with retinoic acid. It is interesting to note that there was only a very small amount of aggrecanase-generated G1 domain released into the culture medium of control cultures on day 2. In contrast, MMP-generated G1 domain was detected in the culture medium of control cultures at high levels at this time (Fig. 3
). Once again, the proteinase inhibitor BB94 completely inhibited the release of both aggrecanase-generated and MMP-generated G1 domain from retinoic acid- and IL-1ß-treated cartilage (Fig. 4
). Immunoreactive fragments were still detected within the cartilage extracts with both anti-NITEGE and anti-FVDIPEN antiserum after treatment of the cultures with BB94, but these were at the same levels as those found in extracts of control tissue prior to culture (results not shown). These findings and those described above for the distribution of neo-epitopes in extracts of cartilage suggest that BB94 inhibits the release of physiological MMP-generated G1 domain from the tissue.
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Functional properties of the G1 domain released into the culture medium
In order to study the interaction of the G1 domain released from retinoic acid-treated cartilage with hyaluronan and link protein, we used electrophoresis of appropriate mixtures of the molecules in large-pore agarosepolyacrylamide gels. This gel system will retard aggrecan and its degradation products even if they have no glycosaminoglycans attached to them, but will exclude aggregates. The way in which proteoglycans and proteins are separated on these gels is poorly understood; therefore, a direct relationship between molecular weight and mobility does not apply here.
We carried out preliminary studies to identify the electrophoretic mobility of the G1 domain generated by the action of MMP3 (stromelysin) in this gel system. The proteinase obtained from the manufacturer was active in degrading A1D1 without the proteinase activator APMA being present (lane 2 in Fig. 6a; toluidine blue staining). Immunoreaction with the antiserum anti-FVDIPEN (MMPs) was only observed after aggrecan had been degraded, and the G1 domain generated by the action of stromelysin had a mobility that was less than that of intact aggrecan (lanes 5 and 6 in Fig. 6b
). These experiments showed us that the large-pore polyacrylamide gels could be probed with the antiserum, and also showed the mobility of the free G-domain that is generated by proteolytic action.
Western blots of media from cultures supplemented with retinoic acid were used to analyse the interaction of degraded aggrecan with hyaluronan and link protein, and were compared with native aggrecan (Fig. 6c and d). When porcine aggrecan was mixed with hyaluronan and link protein prior to electrophoresis, it formed a large complex that was excluded from the gel (lane 1 in Fig. 6c
). Moreover, because the preparation did not contain any proteinase-generated G1 domain, the antiserum anti-NITEGE did not react with this lane of the gel (results not shown). In contrast, the culture medium from retinoic acid-treated cartilage at day 6 (lane 2 in Fig. 6c
) contained a much more heterogeneous mixture of toluidine blue-stained material, as expected, and because it also contained aggrecanase-generated G1 domain the Western blot reacted positively with the anti-NITEGE antiserum (lane 1 in Fig. 6d
). When hyaluronan was added to the day 6 culture medium, there was only a slight change in the electrophoretic profile of the toluidine blue-staining material (lane 3 in Fig. 6c
), but immunoreactivity was considerably decreased (lane 2 in Fig. 6d
). Similarly, although immunoreactivity was also markedly reduced when link protein was included in the reaction mixture, a large proportion of the toluidine blue-staining material was now excluded from the gel (lane 4 in Fig. 6c
), together with the immunoreactive protein (lane 3 in Fig. 6d
). Moreover, the experiments described in Fig. 6c and d
were also carried out on aggrecan after it had been reduced and alkylated to avoid any non-specific interactions (results not shown). In this case, of course, there was no binding of the G1 domain to hyaluronan and link protein and all of it was retarded in the gel. The recombination experiments were also carried out using anti-FVDIPEN (results not shown) and the same effect was observed. They were also carried out using columns of Sepharose 6B (results not shown), which excludes the aggregates but retards the G1 domain and obviously uses much more material.
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Discussion |
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Retinoic acid and IL-1ß promote the degradation of aggrecan and inhibit its synthesis in many cartilage explant culture systems [14] and, as expected, our results indicate that this occurs in immature porcine articular cartilage. Although it is not possible to say with any certainty that the two enzymes are of similar importance in the degradation of aggrecan, we did find that MMPs are constitutively active in this cartilage and that retinoic acid and IL-1ß up-regulated the activity of both MMPs and ADAMts, fragments of aggrecan generated by these proteinases being released into the culture medium and being retained in the tissue. The avidity of the anti-NITEGE and anti-FVDIPEN for their epitopes is particularly important to know, because this will affect the strength of their binding and it may be that this interaction is affected by the glycosylation pattern in the sequence that is being degraded [5, 15]. This should be borne in mind when comparing the present results with others.
Little et al. [16], using monoclonal antibodies specific for the C- and N-terminal neo-epitopes generated by the action of aggrecanase and MMPs on the interglobular domain (IGD) of aggrecan core protein, showed that there was no evidence for the release of MMP-generated aggrecan metabolites into the culture medium, nor was there accumulation of MMP-generated catabolites within the tissue in either retinoic acid- or IL-1-treated explants of porcine cartilage. The studies by Hughes et al. [3], Mercuri et al. [17] and Fosang et al. [18] used a recombinant substrate to assay for enzyme activity released into the culture medium of porcine cartilage explants, but their results appear to contradict each other. Hughes et al. [3] showed that IL-1ß induced both aggrecanase and MMP activities, whereas retinoic acid induced only aggrecanase activity and inhibited MMP activity against the substrate. They concluded that aggrecanase and MMP catabolism of the IGD domain of aggrecan were independent and uncoupled. However, Mercuri et al. [17], using a different recombinant IGD substrate, found active soluble aggrecanase activity but no MMP activity after either IL-1 or retinoic acid treatment of the cartilage explants. The findings presented here are really only comparable to those of Little et al. [16], because our study and those described by Little et al. made an attempt to define the relative contributions of aggrecanase and MMPs to aggrecan turnover. However, no explanation can be given about the divergence of the results concerning MMP-generated fragments, except that in the present study polyclonal antisera were used rather than monoclonal antibodies. It is also interesting that the G1 domain generated by MMPs did not accumulate in the cartilage in the present study, unlike the G1 domain generated by aggrecanase. These findings are consistent with our observations of age-related accumulation of aggrecanase-generated free G1 domain in adult human articular cartilage [19].
The release of the G1 domain into the culture medium is noteworthy. Aggrecan is thought to be anchored in the cartilage matrix as proteoglycan aggregates [20]. Thus, if proteinases degrade aggrecan in the tissue, the G1 domain generated by them would be expected to remain attached to the hyaluronan backbone of the aggregate. However, because the G1 domain is released from the tissue there must be a biological mechanism for achieving this. Several possibilities should be considered, among which are the following: (i) there may be an enzymatic process that has not been recognized previously, although we are unsure what this may be; (ii) a change in the equilibrium of the components of the aggregate could occur which spontaneously releases G1 domain; or (iii) the G1 domain released from the tissue does not come from degradation of pre-existing aggregate but is a consequence of degradation of non-aggregated proteoglycan. A pool of proteoglycan that is non-aggregated has already been observed in articular cartilage [21, 22]. We therefore investigated the functional properties of the G1 domain released into the culture medium. Our experiments showed that the G1 domain released from tissue that was treated with retinoic acid was still functional and could bind to hyaluronan and link protein. Furthermore, the release of link protein from explants treated with retinoic acid or IL-1ß was also investigated. Most of the link protein extracted from porcine cartilage and released from it in culture was the high molecular weight form (48 kDa), although some of the lower molecular weight species (44 kDa) were present in tissue extracts.
The similarity in kinetics of release of link protein and the G1 domain, the partial inhibition of these processes by the proteinase inhibitor BB94 and the retention of some aggrecanase-generated G1 domain within the tissue suggest that further investigation of the mechanism(s) of aggrecan turnover in the tissue is necessary.
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Acknowledgments |
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Notes |
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References |
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