Generation and Novel Distribution of Matrix Metalloproteinase-derived Aggrecan Fragments in Porcine Cartilage Explants

Abstract We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN341 and342FFGVG neoepitopes, we have detected MMP-derived fragments in conditioned medium and cultured cartilage, by radioimmunoassay, Western blotting, and immunolocalization. Radioimmunoassay revealed that the amount (pmol of epitope/mg of total glycosaminoglycan) of 342FFGVG epitope released from cartilage remained constant over a 5-day culture period and was not increased by IL-1α or retinoate. However, theproportion (pmol of epitope/mg of released glycosaminoglycan) of 342FFGVG epitope released was decreased upon stimulation, consistent with the involvement of a non-MMP proteinase, such as aggrecanase. The data suggest that in vitro MMPs may be involved in the base-line catabolism of aggrecan. Immunolocalization experiments showed that DIPEN341 and ITEGE373 epitopes were increased by treatment with IL-1α and retinoate. Confocal microscopy revealed that ITEGE373 epitope was largely intracellular but with matrix staining in the superficial zone, whereas DIPEN341epitope was cell-associated and widely distributed in the matrix. Surprisingly, the majority of 342FFGVG epitope, determined by radioimmunoassay and Western blotting, was retained in the tissue despite the absence of a G1 domain anchor. Interleukin-1α stimulation caused a marked increase in tissue DIPEN341 and342FFGVG epitope, and the 342FFGVG fragments retained in the tissue were larger than those released into the medium. Active porcine aggrecanase was unable to cleave342FFGVG fragments at the ↓Glu373↓Ala374 bond but cleaved intact aggrecan at this site, suggesting that 342FFGVG fragments are not substrates for aggrecanase. The apparent retention of large 342FFGVG fragments within cartilage, and their resistance to N-terminal cleavage by aggrecanase suggests that 342FF6V6 fragments may have a role in cartilage homeostasis.