Production of gelatin-degrading matrix metalloproteinases ('type IV collagenases') and inhibitors by articular chondrocytes during their dedifferentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor alpha

Abstract

The gelatin-degrading matrix metalloproteinase (MMP) activities and their inhibitors produced by rabbit articular chondrocytes have been characterized by gel substrate analysis ('zymography') after electrophoresis on non-reducing sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Differentiated chondrocytes in confluent primary culture produced constitutively only one gelatinase which presented the main characteristics of proMMP-2 ('72 kDa type IV procollagenase'). It had an apparent Mr of 66,000 (unreduced), which was partially or totally converted to 61,000 by, respectively, trypsin or APMA treatment; exogenous TIMP (tissue inhibitor or metalloproteinases) inhibited the conversion triggered by APMA but not that induced by trypsin. This proMMP-2 was also the predominant gelatinase found, together with its 61 kDa activation product, in extracts of articular cartilage. Differentiated chondrocytes simultaneously produced MMP inhibitors which on reverse zymograms were distributed over two bands with Mr of 27,500 and 20,400, resistant to both pH 2 and 100 degrees C, corresponding, respectively, presumably, to TIMP and TIMP-2. Interleukin-1 (IL1) and tumor necrosis factor alpha (TNF alpha) did not affect the production of the proMMP-2 nor of the two species of TIMP. However, IL1 induced the coordinated production of 91 and 55 kDa gelatinases. The 91 kDa activity is likely to correspond to proMMP-9. It could be converted to a 81 kDa gelatinase by trypsin or APMA treatment, in a process that was inhibited in both cases by exogenous TIMP. The 55 kDa gelatinolytic activity most probably represents the sum of the activities of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). It was sequentially converted to lower size forms (49 to 35 kDa) by either trypsin or APMA; that conversion was inhibited by TIMP, with the exception, however, of the first steps (from 55 to 49, then to 42 kDa) induced by trypsin. The 55 kDa and its conversion forms were all active on both gelatin and casein. TNF alpha did also stimulate the production of proMMP-9, although less efficiently than IL1, but it did not induce, or very poorly, that of the 55 kDa proMMP-1/proMMP-3 activity. Low levels of proMMP-9 and of its 81 kDa product of activation were also found in extracts of cartilage. With increasing passage number and cell dedifferentiation, confluent chondrocytes produced increasing amounts of proMMP-2 and of the two species of TIMP. A spontaneous low production of proMMP-9 and proMMP-1/proMMP-3 was only occasionally observed in cultures of dedifferentiated chondrocytes, accompanying a spontaneous low production of procollagenase.(ABSTRACT TRUNCATED AT 400 WORDS)