Distinguishing Aggrecan Loss from Aggrecan Proteolysis in ADAMTS-4 and ADAMTS-5 Single and Double Deficient Mice
2007; Elsevier BV; Volume: 282; Issue: 52 Linguagem: Inglês
10.1074/jbc.m703184200
ISSN1083-351X
AutoresMirna Z. Ilic, Charlotte J. East, Fraser M. Rogerson, Amanda Fosang, Christopher J. Handley,
Tópico(s)Chemokine receptors and signaling
ResumoAggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Δcat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Δcat mice were cultured with or without interleukin (IL)-1α or retinoic acid and analyzed for (i) the kinetics of 35S-labeled aggrecan loss, (ii) the pattern of 35S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Δcat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1α and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Δcat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Δcat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1α, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Δcat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Δcat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Δcat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis. Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Δcat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Δcat mice were cultured with or without interleukin (IL)-1α or retinoic acid and analyzed for (i) the kinetics of 35S-labeled aggrecan loss, (ii) the pattern of 35S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Δcat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1α and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Δcat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Δcat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1α, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Δcat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Δcat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Δcat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis. Aggrecan catabolism has been studied extensively in the past 30–40 years as part of the metabolic processes important in normal functional cartilage and in cartilage pathology. The major enzymes involved in degradation of aggrecan in normal and pathological cartilage are aggrecanases members of the ADAMTS 2The abbreviations used are: ADAMTSa disintegrin and metalloproteinase with thrombospondin motifsDMEMDulbecco's modified Eagle's mediumCSchondroitin sulfateBes2-[bis(2-hydroxyethyl)amino]ethanesulfonic acidTes2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidILinterleukin subfamily of adamalysin (M12) metalloproteinases (1Abbaszade I. Liu R.-Q. Yang F. Rosenfeld S.A. Ross O.H. Link J.R. Ellis D.M. Tortorella M.D. Pratta M.A. Hollis J.M. Wynn R. Duke J.L. George H.J. Hillman Jr., M.C. Murphy K. Wiswall B.H. Copeland R.A. Decicco C.P. Bruckner R. Nagase H. Itoh Y. Newton R.C. Magolda R.L. Trzaskos J.M. Burn T.C. J. Biol. Chem. 1999; 274: 23443-23450Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 2Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. 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In mouse models of osteoarthritis and inflammatory arthritis, the absence of ADAMTS-5 activity confers partial protection against aggrecan loss and cartilage erosion (5Glasson S.S. Askew R. Sheppard B. Carito B. Blanchet T. Ma H.L. Flannery C.R. Peluso D. Kanki K. Yang Z. Majumdar M.K. Morris E.A. Nature. 2005; 434: 644-648Crossref PubMed Scopus (1010) Google Scholar, 9Stanton H. Rogerson F.M. East C.J. Golub S.B. Lawlor K.E. Meeker C.T. Little C.B. Last K. Farmer P.J. Campbell I.K. Fourie A.M. Fosang A.J. Nature. 2005; 434: 648-652Crossref PubMed Scopus (758) Google Scholar). Furthermore the analysis of aggrecan degradation in ADAMTS-1-, -4-, and -5-deficient mice showed that ADAMTS-5 is the main aggrecanase (5Glasson S.S. Askew R. Sheppard B. Carito B. Blanchet T. Ma H.L. Flannery C.R. Peluso D. Kanki K. Yang Z. Majumdar M.K. Morris E.A. Nature. 2005; 434: 644-648Crossref PubMed Scopus (1010) Google Scholar, 9Stanton H. Rogerson F.M. East C.J. Golub S.B. Lawlor K.E. Meeker C.T. 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The use of wild type and ADAMTS-4-, -5-, and -4/-5-deficient mice together for the first time has allowed us to investigate the contribution of aggrecanase activity to aggrecan loss. This was done by determining the relationship between the kinetics of loss of newly synthesized 35S-labeled aggrecan from the matrix and the pattern and degree of proteolytic processing of radiolabeled and total aggrecan core protein in cartilage explant cultures maintained in the presence or absence of interleukin-1α (IL-1α) or retinoic acid. The radiolabeling techniques in conjunction with the use of anti-CS, anti-G3, and neoepitope antibodies have been used to overcome the limitations that arise from the low amounts of tissue available from mouse joints. This has also made it possible to obtain an overall picture of aggrecan degradation not restricted to the immunoanalysis of specific cleavage sites. Generation of ADAMTS-4, ADAMTS-5, and ADAMTS-4/-5 Δcat Mice—The generation of ADAMTS-4, ADAMTS-5, and ADAMTS-4/-5 Δcat mice by Cre-mediated excision of floxed exons encoding the catalytic sites has been described previously (9Stanton H. Rogerson F.M. East C.J. Golub S.B. Lawlor K.E. Meeker C.T. Little C.B. Last K. Farmer P.J. Campbell I.K. Fourie A.M. Fosang A.J. Nature. 2005; 434: 648-652Crossref PubMed Scopus (758) Google Scholar, 22East C.J. Stanton H. Golub S.B. Rogerson F.M. Fosang A.J. J. Biol. Chem. 2007; 282: 8632-8640Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 35Rogerson F. East C.J. Golub S.B. Stanton H. Fosang A.J. Trans. Orthop. Res. Soc. San Diego U. S. A. 2007; 32: 82Google Scholar). Cartilage Cultures—Femoral head (hip) cartilage free of bone and adhering fibrous connective tissues (9Stanton H. Rogerson F.M. East C.J. Golub S.B. Lawlor K.E. Meeker C.T. Little C.B. Last K. Farmer P.J. Campbell I.K. Fourie A.M. Fosang A.J. Nature. 2005; 434: 648-652Crossref PubMed Scopus (758) Google Scholar, 22East C.J. Stanton H. Golub S.B. Rogerson F.M. Fosang A.J. J. Biol. Chem. 2007; 282: 8632-8640Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 34Little C.B. Mittaz L. Belluoccio D. Rogerson F.M. Campbell I.K. Meeker C.T. Bateman J.F. Pritchard M.A. Fosang A.J. Arthritis Rheum. 2005; 52: 1461-1472Crossref PubMed Scopus (94) Google Scholar) was isolated from 3-week-old mice and cultured in Hepes-, Bes-, and Tes-buffered Dulbecco's modified Eagle's medium (DMEM) containing 20% newborn calf serum for 24 h at 37 °C in screw-capped tubes (37Handley C.J. Lowther D.A. Biochim. Biophys. Acta. 1979; 582: 234-245Crossref PubMed Scopus (22) Google Scholar). The tissue was then incubated with [35S]sulfate (200 μCi/ml) (PerkinElmer Life Sciences) under the same conditions for 6 h at 37 °C. At the end of the incubation period the tissue was washed with DMEM to remove unincorporated radiolabeled sulfate and cultured (2–4 femoral heads/ml medium) in serum-free DMEM in the presence or absence of 10 ng/ml human recombinant IL-1α (Peprotech) or 1 μm retinoic acid (Sigma) for 6 days with a change of medium every two days. The spent medium was stored at –20 °C in the presence of proteinase inhibitors (38Oegema Jr., T.R. Hascall V.C. Eisenstein R. J. Biol. Chem. 1979; 254: 1312-1318Abstract Full Text PDF PubMed Google Scholar). At the end of the culture period the tissue was extracted with 1 ml of 4 m guanidinium chloride buffered at pH 5.8 in the presence of proteinase inhibitors (38Oegema Jr., T.R. Hascall V.C. Eisenstein R. J. Biol. Chem. 1979; 254: 1312-1318Abstract Full Text PDF PubMed Google Scholar) for 48 h at 4 °C and then extracted with 1 ml of 0.5 m NaOH for 24 h. Measurement of Loss of 35S-Labeled Aggrecan from Cartilage Explants—The rate of loss of 35S-labeled aggrecan from the matrix of explant cultures was calculated from the amount of radiolabeled macromolecules appearing in the medium and remaining in the matrix at the end of the culture period (3Ilic M.Z. Handley C.J. Robinson H.C. Mok M.T. Arch. Biochem. Biophys. 1992; 294: 115-122Crossref PubMed Scopus (163) Google Scholar). The experiments were repeated twice using duplicate cultures. The variation in the rate of aggrecan loss from the two experiments for ADAMTS-4, -5, and -4/-5 Δcat mice was ≤5%. The wild-type control cultures were analyzed in parallel with ADAMTS-4-, -5-, and -4/-5-deficient cartilage, and the kinetic data show the outcome from three experiments. Wild-type cultures showed greater variation in the rate of aggrecan loss and represent the natural variation between individual animals on a mixed Ser-129/C57BL6 background. Analysis of Aggrecan Core Proteins—Aggrecan was isolated from pooled spent medium and guanidinium chloride tissue extracts by ion exchange chromatography as described previously (3Ilic M.Z. Handley C.J. Robinson H.C. Mok M.T. Arch. Biochem. Biophys. 1992; 294: 115-122Crossref PubMed Scopus (163) Google Scholar). Purified samples were concentrated and exchanged into H2O containing proteinase inhibitors using Amicon® Ultra-4 centrifugal filter devices with molecular weight cut-off of 10,000 (Millipore Corp., Bedford, MA) as described by the manufacturer, lyophilized and reconstituted in 0.1 m Tris/0.1 M sodium acetate, pH 7, and digested with chondroitinase ABC (0.025 units) (protease free from Proteus vulgaris; EC 4.2.2.20; ICN Biochemicals, Costa Mesa, CA) at 37 °C for 24 h in the presence of proteinase inhibitors (38Oegema Jr., T.R. Hascall V.C. Eisenstein R. J. Biol. Chem. 1979; 254: 1312-1318Abstract Full Text PDF PubMed Google Scholar). Digested samples were exchanged into H2O containing proteinase inhibitors using the filter devices and lyophilized. Samples were subjected to electrophoresis on 4–10% gradient polyacrylamide/SDS slab gels. Some gels were fixed and soaked in Amplify (Amersham Biosciences) for 20 min, dried, and exposed to Kodak BioMax Light film at –80 °C for 6–8 weeks. Other gels were electrotransferred onto polyvinylidene difluoride membranes (Immobilon P, Millipore Corp.) and probed with monoclonal antibody 2B6 (IgG) (kindly donated by Prof. B. Caterson, University of Wales, Cardiff, UK), which recognizes terminal unsaturated chondroitin 4-sulfated disaccharides (41Couchman J.R. Caterson B. Christner J.E. Baker J.R. Nature. 1984; 307: 650-652Crossref PubMed Scopus (323) Google Scholar), polyclonal antibodies anti-374ALGS, anti-SELE1279, and anti-FREEE1467, which react with the neoepitopes generated by cleavage of mouse aggrecan (42Walcz E. Deak F. Erhardt P. Coulter S.N. Fulop C. Horvath P. Doege K.J. Glant T.T. Genomics. 1994; 22: 364-371Crossref PubMed Scopus (58) Google Scholar) as described previously (22East C.J. Stanton H. Golub S.B. Rogerson F.M. Fosang A.J. J. Biol. Chem. 2007; 282: 8632-8640Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and polyclonal antibody anti-G3 (kindly provided by Dr. Jayesh Dudhia, Royal Veterinary College, Hatfield, UK). The primary antibody was detected with mouse or rabbit horseradish peroxidase-conjugated secondary antibodies (Chemicon International) using enhanced chemiluminescence (Chemicon International). All Western blot membranes were probed first by aggrecanase-specific neoepitope antibodies. The membranes were then stripped using mild antibody stripping solution (Re-Blot Plus, Chemicon International) according to the manufacturer's instructions and reprobed with the antibody 2B6. Rate of Loss of 35S-Labeled Aggrecan from Cartilage Explant Cultures—The kinetics of loss of 35S-labeled aggrecan in cartilage explant cultures examined in the presence or absence of IL-1α or retinoic acid is shown in Fig. 1. Approximately 18% of radiolabeled aggrecan was lost from the matrix of ADAMTS-4/-5 Δcat cartilage cultured with or without IL-1α after a 6-day culture period (Fig. 1A). In contrast, retinoic acid stimulated the loss of radiolabeled aggrecan to 56% from ADAMTS-4/-5 Δcat cartilage (Fig. 1B). Compared with ADAMTS-4/-5-deficient cartilage, a higher rate of loss of radiolabeled aggrecan was observed in unstimulated wild-type cartilage (41%) and in wild-type cartilage stimulated with IL-1α (65%) and retinoic acid (77%) (Fig. 1, A and B) after a 6-day culture period. The loss of radiolabeled aggrecan from unstimulated ADAMTS-4 Δcat and ADAMTS-5 Δcat cartilage cultures was similar to that in the wild type (Fig. 1, C and D, open symbols). In ADAMTS-4 Δcat cartilage, the loss of radiolabeled aggrecan was increased to 75% with Il-1α and to 73% with retinoic acid (Fig. 1, C and D). IL-1α did not promote loss of radiolabeled aggrecan from ADAMTS-5 Δcat cartilage, but there was a small stimulation to 53% by retinoic acid (Fig. 1, C and D). These results reporting the loss of newly synthesized (radiolabeled) aggrecan agree with those reported previously for the loss of total aggrecan from ADAMTS-4/-5 Δcat cartilage (35Rogerson F. East C.J. Golub S.B. Stanton H. Fosang A.J. Trans. Orthop. Res. Soc. San Diego U. S. A. 2007; 32: 82Google Scholar) and ADAMTS-4 Δcat and ADAMTS-5 Δcat cartilage (9Stanton H. Rogerson F.M. East C.J. Golub S.B. Lawlor K.E. Meeker C.T. Little C.B. Last K. Farmer P.J. Campbell I.K. Fourie A.M. Fosang A.J. Nature. 2005; 434: 648-652Crossref PubMed Scopus (758) Google Scholar, 22East C.J. Stanton H. Golub S.B. Rogerson F.M. Fosang A.J. J. Biol. 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