Analysis of ADAMTS4 and MT4-MMP indicates that both are involved in aggrecanolysis in interleukin-1-treated bovine cartilage

OBJECTIVE: To investigate the mechanism of aggrecanolysis in interleukin-1 (IL-1)-treated cartilage tissue by examining the time course of aggrecan cleavages and the tissue and medium content of membrane type 4-matrix metalloproteinases (MT4-MMP) and a disintegrin and metalloproteinase with thrombospondin type I motifs (ADAMTS)4. METHODS: Articular cartilage explants were harvested from newborn bovine femoropatellar groove. The effects of IL-1 treatment with or without aggrecanase blockade were investigated by Western analysis of aggrecan fragment generation, ADAMTS4 species (p68 and p53), and MT4-MMP, as well as by realtime PCR (polymerase chain reaction) for ADAMTS4 and 5. Aggrecanase was blocked with mannosamine (ManN), an inhibitor of glycosylphosphatidylinositol anchor synthesis, and esculetin (EST), an inhibitor of MMP-1, MMP-3, and MMP-13 gene expression. RESULTS: IL-1 treatment caused a major increase in MT4-MMP abundance in the tissue and medium. ADAMTS4 (p68) was abundant in fresh cartilage and this was retained in the tissue in untreated cartilage. IL-1 treatment for 6 days caused a marked loss of p68 from the cartilage and the appearance of p53 in the medium. Addition of either 1.35 mM ManN or 31-500 microM EST blocked IL-1-mediated aggrecanolysis and this was accompanied by nearly complete inhibition of the MT4-MMP increase, the p68 loss and the formation of p53. IL-1 treatment increased mRNA abundance for ADAMTS4 ( approximately 3-fold) and ADAMTS5 ( approximately 10-fold) but this was not accompanied by a marked change in enzyme protein abundance. CONCLUSION: These studies support a central role for MT4-MMP in IL-1-induced cartilage aggrecanolysis and are consistent with the identification of p68 as the aggrecanase that cleaves within the CS2 domain, and of p53 as the aggrecanase that generates G1-NITEGE. Since the induction by IL-1 was not accompanied by marked changes in total ADAMTS4 protein, but rather in partial conversion of p68 to p53 and release of both from the tissue, we conclude that aggrecanolysis in this model system results from MT4-MMP-mediated processing of a resident pool of ADAMTS4 and release of the p68 and p53 from their normal association with the cell surface.