Retinoic acid-induced type II collagen degradation does not correlate with matrix metalloproteinase activity in cartilage explant cultures

OBJECTIVE: To determine the role of matrix metalloproteinases (MMPs) in retinoic acid (RetA)-induced degradation of type II collagen in cartilage. METHODS: Bovine nasal cartilage explants were cultured with 1 microM RetA or in 3 nM interleukin-1alpha (IL-1alpha). Release of proteoglycan and type II collagen into the medium was measured by colorimetric assay and immunoassay, respectively. MMP activity in the medium was determined using a quenched fluorescent substrate assay, while specific collagenases were identified by Western immunoblotting. In some cases the effects of low molecular mass synthetic MMP inhibitors and serum on collagen degradation were studied. RESULTS: RetA promoted maximal breakdown of type II collagen after 4 or 5 weeks in culture, compared with 3 weeks in culture with IL-1alpha. In IL-1alpha-stimulated cultures, collagen degradation was coincident with a large increase in MMP activity in the culture medium, whereas in RetA-stimulated cultures, there was only a small increase. In Western immunoblots of culture media containing RetA, prointerstitial collagenase and active collagenase 3 were sometimes detected, but not in all experiments. In IL-1alpha cultures, active interstitial collagenase was always detected, and active collagenase 3 was detectable in some experiments. Neutrophil collagenase was not detected in any cultures. IL-1alpha-stimulated collagen degradation was effectively inhibited by a potent, broad-spectrum inhibitor of MMPs, whereas it was poorly inhibited by a weak MMP inhibitor. The same 2 compounds were both only weak inhibitors of RetA-induced collagen degradation. When fetal calf serum was included in cartilage cultures, MMP activity in the culture medium was reduced to low levels. This resulted in a marked inhibition of IL-1alpha-induced type II collagen degradation, whereas there was no inhibition of RetA-induced collagen degradation. CONCLUSION: Unlike IL-1alpha, RetA induces degradation of type II collagen in cartilage explants by a mechanism that is mainly independent of those MMPs that can be detected in the culture medium.