Abstract
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-1alpha (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-1alpha stimulation of cartilage explants, aggrecan fragments in medium and extracts contained SELE(1279) or FREEE(1467) C-terminal sequences. Some SELE(1279) and FREEE(1467) fragments were retained in the cartilage, with intact G1 domains. Other SELE(1279) fragments were released into the medium and co-migrated with the (374)ALGS neoepitope, indicating they were aggrecanase-derived fragments. In contrast none of the FREEE(1467) fragments released into the medium co-migrated with the (374)ALGS 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-1alpha 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.
Highlights
8632 JOURNAL OF BIOLOGICAL CHEMISTRY pressive forces
This study was complementary to a study by Glasson et al [17] who showed that the ADAMTS-5deficient mouse was protected against aggrecan loss and cartilage erosion in a surgically induced model of arthritis, more like human osteoarthritis
Given the urogential phenotype of the ADAMTS-1 null mouse [30, 31], we examined the kidney, ovary, and testis of the ADAMTS-5-⌬cat mice and found that these tissues were indistinguishable from wild types
Summary
Generation of ADAMTS-4 and ADAMTS-5-⌬cat Mice—The generation of ADAMTS-4 and -5-⌬cat mice by Cre-mediated excision of floxed exons encoding the catalytic sites has been described previously [18]. 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 [25] but no bone, periosteum, or synovium. 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. Total aggrecan was calculated as the sum of glycosaminoglycan in the conditioned media, GdnHCl extracts, and papain digests. 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 g of glycosaminoglycan in 0.1 M Tris acetate, pH 6.5, containing proteinase inhibitors E-64 (10 g/ml), 4-(2-aminoethyl)benzenesulfonyl fluoride (0.5 mM), pepstatin (5 g/ml), and EDTA (10 mM). 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
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