Evidence of a novel aggrecan-degrading activity in cartilage - Studies of mice deficient in both ADAMTS-4 and ADAMTS-5

Objective. To characterize aggrecan catabolism and the overall phenotype in mice deficient in both ADAMTS-4 and ADAMTS-5 (TS-4/TS-5 Delta-cat) activity. Methods. Femoral head cartilage from the joints of TS-4/TS-5 Delta-cat mice and wild-type mice were cultured in vitro, and aggrecan catabolism was stimulated with either interleukin-1 alpha (IL-1 alpha) or retinoic acid. Total aggrecan release was measured, and aggrecanase activity was examined by Western blotting using neoepitope antibodies for detecting cleavage at EGE(373-374)ALG, SELE(1279-1280)GRG, FREEE(1467-1468)GLG, and AQE(1572-1573)AGEG. Aggrecan catabolism in vivo was examined by Western blotting of cartilage that had been extracted immediately ex vivo. Results. TS-4/TS-5 Delta-cat mice were viable, fertile, and phenotypically normal. TS-4/TS-5 Delta-cat cartilage explants did not release aggrecan in response to IL-1 alpha, and there was no detectable increase in aggrecanase neoepitopes. TS-4/TS-5 Delta-cat cartilage explants released aggrecan in response to retinoic acid. There was no retinoic acid-stimulated cleavage at either EGE(373-374)ALG or AQE(1572-1573)AGEG. There was a low level of cleavage at SELE(1279-1280)GRG and major cleavage at FREEE(1467-1468)GLG. Ex vivo, cleavage at FREEE(1467-1468)GLG was substantially reduced, but still present, in TS-4/TS-5 Delta-cat mouse cartilage compared with wild-type mouse cartilage. Conclusion. An aggrecanase other than ADAMTS-4 and ADAMTS-5 is expressed in mouse cartilage and is up-regulated by retinoic acid but not IL-1 alpha. The novel aggrecanase appears to have different substrate specificity from either ADAMTS-4 or ADAMTS-5, cleaving E-G bonds but not E-A bonds. Neither ADAMTS-4 nor ADAMTS-5 is required for normal skeletal development or aggrecan turnover in cartilage.