ADAMTS-5 Deficiency Does Not Block Aggrecanolysis at Preferred Cleavage Sites in the Chondroitin Sulfate-rich Region of Aggrecan

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-1␣ (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-1␣ 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-1␣ 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) 2 and chondroitin sulfate-2 (CS-2) domains. An interglobular domain (IGD) of ϳ150 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...

Proteomic characterization of mouse cartilage degradation in vitro

“…Differential proteomic profiling of femoral head cartilage extracts and explant culture media. Previous studies using mouse femoral head cartilage to investigate aggrecanolysis used DMEM containing FCS prior to treatment with IL-1 and RetA (9,15,22). In this study, the omission of FCS was essential to identify proteins and fragments released from cartilage into the culture media and to avoid adding nonuniform levels of growth factors and cytokines.…”

Distinguishing Aggrecan Loss from Aggrecan Proteolysis in ADAMTS-4 and ADAMTS-5 Single and Double Deficient Mice

“…In addition, recombinant ADAMTS-1, -4, and -5 cleave aggrecan at 5 sites: one within the interglobular domain and four in the CS attachment region generating aggrecan fragments that match those characterized previously in bovine cartilage and synovial fluid (3,18,19). The processing of aggrecan by aggrecanases does not appear to be species-specific because the same cleavages have been reported in bovine, equine, porcine, murine, and human aggrecan (3,6,9,(22)(23)(24). The aggrecanases are initially synthesized as latent enzymes that require proteolytic modification by autolysis or the action of other proteinases as well as interaction with other matrix macromolecules to gain activity (15,(25)(26)(27)(28).…”

“…⌬cat Mice-The generation of ADAMTS-4, ADAMTS-5, and ADAMTS-4/-5 ⌬cat mice by Cre-mediated excision of floxed exons encoding the catalytic sites has been described previously (9,22,35).…”

“…Cartilage Cultures-Femoral head (hip) cartilage free of bone and adhering fibrous connective tissues (9,22,34) was isolated from 3-week-old mice and cultured in Hepes-, Bes-, and Tesbuffered Dulbecco's modified Eagle's medium (DMEM) containing 20% newborn calf serum for 24 h at 37°C in screwcapped tubes (37). The tissue was then incubated with [ 35 S]sulfate (200 Ci/ml) (PerkinElmer Life Sciences) under the same conditions for 6 h at 37°C.…”

Emerging roles of ADAM and ADAMTS metalloproteinases in cancer

“…ADAM-8 expression is increased in an animal model of asthma following allergen exposure [51] and in the bronchi of human asthmatics [48,52]. ADAMTS-4 and TS-5 are involved in the turnover of aggrecan from cartilage resulting in loss of functionality of tissue and joint disability [53][54][55][56]. Studies have indicated that ADAMTS-5 is likely the major aggrecanase in cartilage metabolism and pathology [56].…”