ADAMTS-5 Deficiency Does Not Block Aggrecanolysis at Preferred Cleavage Sites in the Chondroitin Sulfate-rich Region of Aggrecan
2007; Elsevier BV; Volume: 282; Issue: 12 Linguagem: Inglês
10.1074/jbc.m605750200
ISSN1083-351X
AutoresCharlotte J. East, Heather Stanton, Suzanne B. Golub, Fraser M. Rogerson, Amanda Fosang,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoIn the mouse, proteolysis in the aggrecan interglobular domain is driven by ADAMTS-5, and mice deficient in ADAMTS-5 catalytic activity are protected against aggrecan loss and cartilage damage in experimental models of arthritis. Here we show that despite ablation of ADAMTS-5 activity, aggrecanolysis can still occur at two preferred sites in the chondroitin sulfate-rich region. Retinoic acid was more effective than interleukin-1α (IL) in promoting cleavage at these sites in ADAMTS-5-deficient cartilage. These results suggest that cleavage at preferred sites in the chondroitin sulfate-rich region is mediated by ADAMTS-4 or an aggrecanase other than ADAMTS-5. Following retinoic acid or IL-1α stimulation of cartilage explants, aggrecan fragments in medium and extracts contained SELE1279 or FREEE1467 C-terminal sequences. Some SELE1279 and FREEE1467 fragments were retained in the cartilage, with intact G1 domains. Other SELE1279 fragments were released into the medium and co-migrated with the 374ALGS neoepitope, indicating they were aggrecanase-derived fragments. In contrast none of the FREEE1467 fragments released into the medium co-migrated with the 374ALGS neoepitope, suggesting that, despite their size, these fragments were not products of aggrecanase cleavage in the interglobular domain. ADAMTS-5, but not ADAMTS-1, -4, or -9, was up-regulated 8-fold by retinoic acid and 17-fold by IL-1α treatment. The data show that whereas ADAMTS-5 is entirely responsible for cleavage in the interglobular domain, cleavage in the chondroitin sulfate-rich region is driven either by ADAMTS-4, which compensates for loss of ADAMTS-5 in this experimental system, or possibly by another aggrecanase. The data show that there are differential aggrecanase activities with preferences for separate regions of the core protein. In the mouse, proteolysis in the aggrecan interglobular domain is driven by ADAMTS-5, and mice deficient in ADAMTS-5 catalytic activity are protected against aggrecan loss and cartilage damage in experimental models of arthritis. Here we show that despite ablation of ADAMTS-5 activity, aggrecanolysis can still occur at two preferred sites in the chondroitin sulfate-rich region. Retinoic acid was more effective than interleukin-1α (IL) in promoting cleavage at these sites in ADAMTS-5-deficient cartilage. These results suggest that cleavage at preferred sites in the chondroitin sulfate-rich region is mediated by ADAMTS-4 or an aggrecanase other than ADAMTS-5. Following retinoic acid or IL-1α stimulation of cartilage explants, aggrecan fragments in medium and extracts contained SELE1279 or FREEE1467 C-terminal sequences. Some SELE1279 and FREEE1467 fragments were retained in the cartilage, with intact G1 domains. Other SELE1279 fragments were released into the medium and co-migrated with the 374ALGS neoepitope, indicating they were aggrecanase-derived fragments. In contrast none of the FREEE1467 fragments released into the medium co-migrated with the 374ALGS neoepitope, suggesting that, despite their size, these fragments were not products of aggrecanase cleavage in the interglobular domain. ADAMTS-5, but not ADAMTS-1, -4, or -9, was up-regulated 8-fold by retinoic acid and 17-fold by IL-1α treatment. The data show that whereas ADAMTS-5 is entirely responsible for cleavage in the interglobular domain, cleavage in the chondroitin sulfate-rich region is driven either by ADAMTS-4, which compensates for loss of ADAMTS-5 in this experimental system, or possibly by another aggrecanase. The data show that there are differential aggrecanase activities with preferences for separate regions of the core protein. A feature of joint pathology in arthritis is destruction of articular cartilage. The major structural components of cartilage are type II collagen and the large aggregating proteoglycan, aggrecan. In healthy cartilage, type II collagen and aggrecan confer strength and compliance that enables this tissue to resist compressive forces. In arthritic diseases, the progressive degradation of aggrecan and type II collagen leads to cartilage erosion. Aggrecan has two globular domains, G1 and G2 at the N terminus, and a third globular domain, G3 at the C terminus. An extended sequence between the G2 and G3 domains is heavily substituted with keratan sulfate and chondroitin sulfate chains, organized into a distinct keratan sulfate-rich region and chondroitin sulfate-1 (CS-1) 2The abbreviations used are: CS-1, chondroitin sulfate-1 domain; CS-2, chondroitin sulfate-2 domain; IGD, interglobular domain; MMP, matrix metalloproteinases; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motif; GdnHCl, guanidine HCl; IL, interleukin. and chondroitin sulfate-2 (CS-2) domains. An interglobular domain (IGD) of ∼150 amino acids separates G1 from G2. The aggrecan IGD is highly sensitive to proteinases. Proteolysis in the IGD releases the entire chondroitin sulfate and keratan sulfate-rich regions essential for the biomechanical properties of aggrecan, and is therefore thought to be the most detrimental for cartilage function. In pathology, proteolysis is driven mainly by aggrecanases, but there may also be some involvement of matrix metalloproteinases (MMPs) in late stage disease (1van Meurs J.B. van Lent P.L. van de Loo A.A. Holthuysen A.E. Bayne E.K. Singer I.I. Van Den Berg W.B. Ann. Rheum. Dis. 1999; 58: 350-356Crossref PubMed Scopus (30) Google Scholar, 2van Meurs J.B. van Lent P.L. Holthuysen A.E. Singer I.I. Bayne E.K. Van Den Berg W.B. Arthritis Rheum. 1999; 42: 1128-1139Crossref PubMed Scopus (115) Google Scholar, 3van Meurs J.B. van Lent P.L. Singer I.I. Bayne E.K. van de Loo F.A. Van Den Berg W.B. Arthritis Rheum. 1998; 41: 647-656Crossref PubMed Scopus (52) Google Scholar). “Aggrecanase” was first identified as a novel activity that cleaved the aggrecan core protein at the E373↓374A bond in the IGD (4Sandy J.D. Neame P.J. Boynton R.E. Flannery C.R. J. Biol. Chem. 1991; 266: 8683-8685Abstract Full Text PDF PubMed Google Scholar, 5Loulakis P. Shrikhande A. Davis G. Maniglia C.A. Biochem. 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Ther. 2005; 7: 160-169Crossref PubMed Scopus (154) Google Scholar). ADAMTS-5 has recently been identified as the major aggrecanase in mouse cartilage (17Glasson 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, 18Stanton 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 (757) Google Scholar). Aggrecan cleavage at the E373↓374A bond in the IGD is the signature activity of the aggrecanases and is widely reported in humans and animals as a marker of aggrecanase activity. However, cleavage at E373↓374A is not the preferred action of these enzymes. In vitro, recombinant ADAMTS-4 and -5 preferentially cleave aggrecan in the CS-2 domain (19Tortorella M.D. Pratta M. Liu R.Q. Austin J. Ross O.H. Abbaszade I. Burn T. Arner E. J. Biol. Chem. 2000; 275: 18566-18573Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 20Tortorella M.D. Liu R.Q. Burn T. Newton R.C. Arner E. Matrix Biol. 2002; 21: 499-511Crossref PubMed Scopus (120) Google Scholar). The two most preferred cleavage sites in bovine aggrecan are at KEEE1666↓1667GLGS, followed by GELE1480↓1481GRGT. Thereafter, further cleavages occur at TAQE1771↓1772AGEG and VSQE1871↓1872LGQR in the CS-2 region and at NITEGE373↓374ARGS in the IGD. A similar hierarchy of cleavage preferences is shown by native aggrecanases in other species (21Sandy J.D. Thompson V. Doege K. Verscharen C. Biochem. J. 2000; 351: 1-166Crossref PubMed Google Scholar, 22Sandy J.D. Verscharen C. Biochem. J. 2001; 358: 615-626Crossref PubMed Scopus (166) Google Scholar). We recently published that ablation of ADAMTS-5 protects against aggrecan loss and cartilage erosion in a mouse model of inflammatory arthritis (18Stanton 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 (757) Google Scholar). This study was complementary to a study by Glasson et al. (17Glasson 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) who showed that the ADAMTS-5-deficient mouse was also protected against aggrecan loss and cartilage erosion in a surgically induced model of arthritis, more like human osteoarthritis. Here, we present new data on aggrecanase cleavage at preferred sites in the mouse CS-2 domain that were not examined in our earlier study. We find that unlike cleavage in the IGD, cleavage in the CS-2 domain at FREEE1467 and SELE1279 is not blocked in the ADAMTS-5-Δcat mouse. Our results highlight the recent realization that an accurate readout of aggrecanolysis requires analysis of both IGD and CS-2 domain cleavage. Generation of ADAMTS-4 and ADAMTS-5-Δcat Mice—The generation of ADAMTS-4 and -5-Δcat mice by Cre-mediated excision of floxed exons encoding the catalytic sites has been described previously (18Stanton 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 (757) Google Scholar). In the present study, cartilage taken for in vitro experiments was harvested from mice in which the Cre transgene, previously present on one allele of chromosome X, was out-bred. Cre was removed to avoid the possibility of Cre-mediated recombination between cryptic pseudo-loxP sites and the effects reported in vitro (23Loonstra A. Vooijs M. Beverloo H.B. Allak B.A. van Drunen E. Kanaar R. Berns A. Jonkers J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9209-9214Crossref PubMed Scopus (454) Google Scholar) and in vivo (24Schmidt E.E. Taylor D.S. Prigge J.R. Barnett S. Capecchi M.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13702-13707Crossref PubMed Scopus (267) Google Scholar). Age-matched mice from wild type breeders on a C57/129 mixed background were used as controls for mutant mice from Δcat breeders. Cartilage Explant Cultures—Femoral head (hip) cartilage was harvested from 3-week-old mice. These explants contain a small amount of growth plate cartilage as reported previously (25Little 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 (93) Google Scholar) but no bone, periosteum, or synovium. Remnants of ligament (ligament teres femoralis) are rarely present. Hypertrophic chondrocytes and mineral are present in a central region of calcified cartilage that separates the growth plate from the articular surface. However, because the femoral head does not become vascularized, it does not form a true secondary center, marrow cavity, or bone. The explants were initially cultured at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 units/ml penicillin, 100 units/ml streptomycin, 2 mm l-glutamine, and 20 mm HEPES. After 2 days, the explants were washed in serum-free medium and placed in a 48-well culture dish with fresh serum-free medium containing either 10–5 m retinoic acid (Sigma) or 10 ng/ml human recombinant IL-1α (Peprotech, NJ) and cultured for a further 3 days. Medium and explants were collected at days 0, 1, 2, and 3 after changing to serum-free medium. Aggrecan and aggrecan fragments were extracted from the explants for 48 h at 4 °C with 4 m guanidine hydrochloride (GdnHCl), 50 mm sodium acetate, pH 5.8, 10 mm EDTA, and 0.1 m caproic acid. Residual, non-extractable aggrecan remaining in the cartilage was recovered by digesting overnight at 60 °C with 3.5 units/ml papain in buffer containing 0.1 m sodium acetate, pH 5.5, 5 mm cysteine, 5 mm EDTA. Quantitation of Aggrecan—The 4 m GdnHCl cartilage extracts were dialyzed against ultrapure water containing 10 mm EDTA at 4 °C. The concentration of sulfated glycosaminoglycan (as a measure of aggrecan) in extracts, medium, and papain digests was determined using the 1,9-dimethylmethylene blue assay (26Farndale R.W. Sayers C.A. Barrett A.J. Connect. Tissue Res. 1982; 9: 247-248Crossref PubMed Scopus (1159) Google Scholar). Total aggrecan was calculated as the sum of glycosaminoglycan in the conditioned media, GdnHCl extracts, and papain digests. The cumulative release of aggrecan into the conditioned media was expressed as a percentage of total aggrecan. Western Blotting—Prior to SDS-PAGE, aliquots of conditioned media and dialyzed extracts were digested for 6 h at 37 °C with 0.01 units of chondroitinase ABC (Seikugaku, Japan) per 10 μg of glycosaminoglycan in 0.1 m Tris acetate, pH 6.5, containing proteinase inhibitors E-64 (10 μg/ml), 4-(2-aminoethyl)benzenesulfonyl fluoride (0.5 mm), pepstatin (5 μg/ml), and EDTA (10 mm). The total amount of aggrecan (medium + extract + residue) was calculated for each sample. We found that total aggrecan was a more accurate denominator than tissue wet weight for normalizing samples for Western blot analysis because the weight of a single mouse hip is so small (between 0.5 and 1.0 mg). The volume of medium or extract loaded onto the gel was determined empirically for each neoepitope. Samples analyzed for 374ALGS and NVTEGE373 neoepitopes were electrophoresed on 7.5% SDS-polyacrylamide gels containing 2 m urea, under reducing conditions and then transferred to polyvinylidene difluoride membranes (Millipore). Membranes were probed with antibodies that recognize neoepitopes created by aggrecanase activity and include anti-NITEGE373 (27Mercuri F.A. Maciewicz R.A. Tart J. Last K. Fosang A.J. J. Biol. Chem. 2000; 275: 33038-33045Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), anti-FREEE1467, anti-SELE1279, and anti-374ALGS. The anti-NITEGE373 antibody recognized the mouse NVTEGE373 neoepitope after aggrecanase cleavage. After immunodetection with anti-NITEGE373 or anti-374ALGS, the blots were stripped with Re-blot Plus (Chemicon International) and reprobed with monoclonal antibody 2B6 that recognizes stubs of chondroitin 4-sulfate chains on aggrecan after digestion with chondroitinase ABC. Samples analyzed for SELE1279 and FREEE1467 neoepitopes were electrophoresed on 5% gels. Primary antibody binding was detected using horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) and enhanced chemiluminescence (ECL Plus, Amersham Biosciences). Western blot membranes for direct comparison with each other were developed together. In most cases, blots were developed with ECL reagent until the most strongly detected band in the series approached saturation. Generation of the Anti-374ALGS, Anti-FREEE1467, and Anti-SELE1279 Neoepitope Antibodies—Synthetic peptides with the sequences ALGSVILTAGGC, CGGPTTFREEE, and CGGATTSSELE (Auspep, Australia) were conjugated to ovalbumin using bromoacetic acid N-hydroxysuccinimide ester (28Hughes C. Caterson B. White R.J. Roughley P.J. Mort J.S. J. Biol. Chem. 1992; 267: 16011-16014Abstract Full Text PDF PubMed Google Scholar). Polyclonal rabbit antiserum was raised against the ovalbumin-conjugated peptides at the Institute for Medical and Veterinary Science (Adelaide, Australia) and screened against the same peptide immunogens conjugated to bovine serum albumin. Polyclonal rabbit antiserum against the identical ovalbumin-conjugated 374ALGS immunogen was also raised by Chemicon International. The anti-374ALGS, anti-FREEE1467, and anti-SELE1279 IgGs were purified from rabbit sera by protein-A affinity chromatography. The titer and specificity of the purified anti-374ALGS IgG from the Institute for Medical and Veterinary Science and Chemicon were the same. RNA Isolation and Real-time Reverse Transcriptase-PCR Analyses—Total RNA was extracted from Δcat and wild type cartilage using the RNeasy kit (Qiagen) and isolated according to the manufacturer's instructions. Reverse transcription was done on 100 ng of total RNA using murine leukemia virus reverse transcriptase (Applied Biosystems) and random hexamer primers. The reaction was done for 1 h at 42 °C, after which the enzyme was inactivated by incubation for 5 min at 99 °C. TaqMan® real-time reverse transcriptase-PCR was used to measure the mRNA levels of ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-9. Primer and probe sets for the detection of each ADAMTS mRNA (18Stanton 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 (757) Google Scholar) were designed and synthesized using the ABI “Primer-by-Design” service and reactions were done using the ABI Prism 7700 Sequence Detection System (PerkinElmer Life Sciences). Multiplex PCR was done with primer/probe sets to the target (ADAMTS) and reference (18 S rRNA) in the same reaction. mRNA as quantitated using the comparative CT method (29Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25582) Google Scholar), using 18 S rRNA as the reference. Preliminary experiments showed that the efficiencies of target and reference amplifications were similar in all cases, thus validating the use of this protocol. The relative amount of mRNA expression in samples taken at days 1, 2, or 3, compared with samples taken immediately ex vivo (day 0), was determined by the formula 2(–ΔΔCT), where ΔΔCT was calculated by subtracting the ΔCT value for day 0 from the ΔCT value for days 1, 2, or 3. The experiment was done once with pooled cartilage from 5 to 7 mice for each genotype, at each time point, and it required 70 mice. For the present study, we generated new lines of Δcat/Δcat and control mice that lacked the Cre transgene. Heterozygous, Cre-negative ADAMTS-4-Δcat breeders on a mixed C57/129 background produced litters of normal size and gender distribution (390 pups from 55 litters) with the expected Mendelian ratio of wild type, heterozygous, and homozygous Δcat pups (24, 49, and 27%, respectively). Heterozygous Cre-negative ADAMTS-5-Δcat breeders also produced litters of normal size and gender distribution (450 pups from 60 litters) with the expected ratio of wild type, heterozygous, and homozygous Δcat pups (28, 48, and 24%, respectively). Phenotypic Analysis—Homozygous ADAMTS-4- and ADAMTS-5-Δcat mice were phenotypically normal with no morphological differences between mutant mouse lines and wild type mice in heart, kidney, lung, liver, spleen, thymus, and small intestine (data not shown). Given the urogential phenotype of the ADAMTS-1 null mouse (30Mittaz L. Russell D.L. Wilson T. Brasted M. Tkalcevic J. Salamonsen L.A. Hertzog P.J. Pritchard M.A. Biol. Reprod. 2004; 70: 1096-1105Crossref PubMed Scopus (148) Google Scholar, 31Shindo T. Kurihara H. Kuno K. Yokoyama H. Wada T. Kurihara Y. Imai T. Wang Y. Ogata M. Nishimatsu H. Moriyama N. Oh-hashi Y. Morita H. Ishikawa T. Nagai R. Yazaki Y. Matsushima K. J. Clin. Investig. 2000; 105: 1345-1352Crossref PubMed Scopus (272) Google Scholar), we also examined the kidney, ovary, and testis of the ADAMTS-5-Δcat mice and found that these tissues were indistinguishable from wild types (data not shown). Glasson et al. (32Glasson S.S. Askew R. Sheppard B. Carito B.A. Blanchet T. Ma H.L. Flannery C.R. Kanki K. Wang E. Peluso D. Yang Z. Majumdar M.K. Morris E.A. Arthritis Rheum. 2004; 50: 2547-2558Crossref PubMed Scopus (255) Google Scholar) have published the normal histology of tissues in the ADAMTS-4-Δcat mouse (32Glasson S.S. Askew R. Sheppard B. Carito B.A. Blanchet T. Ma H.L. Flannery C.R. Kanki K. Wang E. Peluso D. Yang Z. Majumdar M.K. Morris E.A. Arthritis Rheum. 2004; 50: 2547-2558Crossref PubMed Scopus (255) Google Scholar). Aggrecanase activity and ADAMTS-4 protein are present in mouse growth plate (17Glasson 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, 32Glasson S.S. Askew R. Sheppard B. Carito B.A. Blanchet T. Ma H.L. Flannery C.R. Kanki K. Wang E. Peluso D. Yang Z. Majumdar M.K. Morris E.A. Arthritis Rheum. 2004; 50: 2547-2558Crossref PubMed Scopus (255) Google Scholar, 33Mort J.S. Flannery C.R. Makkerh J. Krupa J.C. Lee E.R. Biochem. Soc. Symp. 2003; : 107-114PubMed Google Scholar), so ablation of the ADAMTS catalytic sites might have been expected to disturb or delay normal skeletal growth. We found that the average body weights for wild type, ADAMTS-4-, and ADAMTS-5-Δcat mice were indistinguishable at all ages, indicating that neither ADAMTS-4 nor ADAMTS-5 catalytic deficiency interferes with mouse growth and development (data not shown). There were no detectable differences in tibiofemoral growth plates at any age between 3 days and 6 months. The width of tibial growth plates did not vary with genotype, and there was no apparent accumulation of aggrecan, as assessed by toluidine blue staining (data not shown). In combination, the growth curves and the growth plate histology indicate that ADAMTS-4 and -5 have negligible roles in aggrecan resorption during endochondral ossification. Retinoic Acid and IL-1α Induce Aggrecanase-mediated Aggrecan Loss from Mouse Cartilage Explants—Previously we examined the effect of IL-1α on the release of aggrecan from mouse cartilage after 3 days in culture (18Stanton 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 (757) Google Scholar). In the present study, we examined the kinetics of aggrecan loss by analyzing samples on each of days 1, 2, and 3 of culture following treatment with retinoic acid, and compared them with IL-1α-treated samples. The results show that for both retinoic acid and IL-1α, the rate of aggrecan loss from wild type cartilage is non-linear. The rate of loss is greatest on day 1, and progressively less on days 2 and 3 (Fig. 1, a and b). Aggrecan loss from ADAMTS-4- and ADAMTS-5-Δcat cartilage is also non-linear, and is reduced after the first day in culture. Aggrecan loss from ADAMTS-4-Δcat cartilage was ∼15% less than from wild type cartilage following treatment with either agent. In contrast, aggrecan loss from ADAMTS-5-Δcat cartilage was substantially less than from wild type cartilage, and was ∼49% less than wild type in retinoic acid-treated cultures and ∼56% less than wild type in IL-1α-treated cultures. Il-1α promoted a greater release of aggrecan than retinoic acid for all genotypes. The Role of ADAMTS-4 and -5 in Retinoic Acid and IL-1α-induced Aggrecanolysis—To further examine the role of ADAMTS-4 and ADAMTS-5 in driving in vitro aggrecanolysis, we next examined aggrecanase cleavage at preferred sites in the CS-2 domain and compared it with IGD cleavage. Cleavage sites in the mouse IGD and CS-2 domains are highly conserved but not identical to other species (Fig. 2, b and c). Three new neoepitope antibodies, specific for the mouse sequences 374ALGS, SELE1279, and FREEE1467 (Fig. 2a), were generated in rabbits. To confirm the neoepitope specificity of the antibodies, aggrecan present in 4 m GdnHCl extracts of wild type mouse cartilage was incubated at 37 °C, with or without recombinant human ADAMTS-4 (p40; Chemicon International), and analyzed by Western blotting (Fig. 2, d–f). In each case, the antibodies failed to detect epitope in undigested cartilage extracts, but detected fragments of the predicted size in samples digested with ADAMTS-4. The results in Fig. 2 and subsequent figures confirm the neoepitope specificity of the antibodies used in this study. Retinoic Acid and IL-1α Induce ADAMTS-5 Cleavage in the Aggrecan IGD—Cleavage at the E373↓374A bond in the IGD was examined by Western blotting medium samples containing the 374ALGS neoepitope (Fig. 3, b and d), and cartilage extracts containing the NVTEGE373 neoepitope present on the G1 domain (Fig. 3, a and c). In retinoic acid-treated cultures, neither NVTEGE373 nor 374ALGS epitopes were detected on days 1 or 2 of culture, but were present by day 3 in both wild type and ADAMTS-4-Δcat cartilage. No IGD neoepitopes were detected in ADAMTS-5-Δcat cartilage treated with retinoic acid, in this system (Fig. 3, a and b). IL-1α treatment of wild type and ADAMTS-4-Δcat cartilage stimulated IGD cleavage commencing on day 1 of culture and increasing up to day 3, however, IGD fragments were not detected in cultures of ADAMTS-5-Δcat cartilage (Fig. 3, c and d). Overexposing the Western blots did not reveal NVTEGE373 or 374ALGS neoepitopes in either retinoic acid or IL-1α-treated ADAMTS-5-Δcat cartilage. Stripping the blots and reprobing with antibody 2B6 confirmed that samples were loaded in each lane (data not shown). These results strongly suggest that ADAMTS-5 is the major aggrecanase responsible for cleavage in the IGD of mouse aggrecan in response to treatment with both retinoic acid and IL-1α. ADAMTS-5 Deficiency Does Not Block Aggrecanase Cleavage in the CS-2 Domain of Aggrecan—Next we examined cleavage at E1279↓1280G and E1467↓1468G in the CS-2 domain using anti-SELE1279 and anti-FREEE1467 antibodies, respectively. We analyzed media and cartilage extracts and found that retinoic acid and IL-1α treatment generated SELE1279 and FREEE1467 epitopes in cartilage from all genotypes including ADAMTS-5-Δcat cartilage (Fig. 4). The epitopes were present on days 2 and 3 of culture and were detected in both the media and extracts. These results are consistent with our previous study (18Stanton 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 (757) Google Scholar) showing limited CS-2 domain cleavage at E1572↓1573A in ADAMTS-5-Δcat explants. IL-1α stimulated rel
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