Generation and novel distribution of matrix metalloproteinase-derived aggrecan fragments in porcine cartilage explants

We have studied aggrecan catabolism mediated by matrix metalloproteinases ( MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN341 and (342)FFGVG neoepitopes, we have detected MMP-derived fragments in conditioned medium and cultured cartilage, by radioimmunoassay, Western blotting, and immunolocalization. Radioimmunoassay revealed that the amoun t (pmol of epitope/mg of total glycosaminoglycan) of (342)FFGVG epitope rel eased from cartilage remained constant over a 5-day culture period and was not increased by IL-1 alpha or retinoate. However, the proportion (pmol of epitope/mg of released glycosaminoglycan) of (342)FFGVG epitope released wa s 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 exper iments showed that DIPEN341 and ITEGE(373) epitopes were increased by treat ment with IL-1 alpha and retinoate. Confocal microscopy revealed that ITEGE (373) epitope was largely intracellular but with matrix staining in the sup erficial zone, whereas DIPEN341 epitope was cell-associated and widely dist ributed in the matrix. Surprisingly, the majority of (342)FFGVG epitope, de termined by radioimmunoassay and Western blotting, was retained in the tiss ue despite the absence of a G1 domain anchor. Interleukin-1 alpha stimulati on caused a marked increase in tissue DIPEN341 and (342)FFGVG epitope, and the (342)FFGVG fragments retained in the tissue were larger than those rele ased into the medium. Active porcine aggrecanase was unable to cleave (342) FFGVG fragments at the down arrow Glu(373) down arrow Ala(374) bond but cle aved intact aggrecan at this site, suggesting that (342)FFGVG fragments are not substrates for aggrecanase, The apparent retention of large (342)FFGVG fragments within cartilage, and their resistance to N-terminal cleavage by aggrecanase suggests that (342)FF6V6 fragments may have a role in cartilag e homeostasis.