CHONDROCYTE CULTURES EXPRESS MATRIX METALLOPROTEINASE MESSENGER-RNA AND IMMUNOREACTIVE PROTEIN - STROMELYSIN-1 AND 72-KDA GELATINASE ARE LOCALIZED IN EXTRACELLULAR-MATRIX VESICLES

Previous studies have shown that costochondral cartilage cell cultures produce extracellular matrix vesicles which contain metalloproteinase activity. In the present study, we examined whether two matrix metall oproteinases (MMPs) known to be present in cartilage, stromelysin-1 an d 72 kDa gelatinase, are expressed by fourth passage resting zone and growth zone costochondral chondrocytes and whether they are specifical ly incorporated into matrix vesicles produced by the cells. We also ex amined whether the cells synthesize tissue inhibitor of metalloprotein ase-1 and -2 (TIMP-1 and TIMP-2). Oligonucleotide primers for stromely sin-1, 72 kDa gelatinase, tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2), and GAPDH were synthesized and optimized for use in the reverse transcription-polymerase chain reaction (RT-PCR). I t was found that both resting zone and growth zone chondrocytes produc ed mRNA for both MMPs and the two TIMPs. Further, immunostaining of ce ll layers with antibodies to 72 kDa gelatinase and stromelysin-1 showe d that both cell types produced these MMPs in culture. Substrate gel e lectrophoresis and Western analysis were used to characterize MMP acti vity in matrix vesicles, media vesicles, or plasma membranes as well a s in conditioned media produced by the chondrocyte cultures. It was fo und that matrix vesicles but not plasma membranes or media vesicles we re selectively enriched in stromelysin-1. Also, 72 kDa gelatinase was found in matrix vesicles, but to a lesser extent than seen in media ve sicles. The relative activity of each enzyme detected was cell maturat ion-dependent. No MMP activity was detected in conditioned media produ ced by either cell type. The results of this study show that MMPs are expressed by resting zone and growth zone chondrocytes in culture and differentially distributed among three different membrane compartments . This suggests that, in addition to the well-known activators and inh ibitors of MMP activity in the matrix, differential membrane distribut ion may enable more precise control over the site, rate, and extent of matrix degradation by the cell. (C) 1996 Wiley-Liss, Inc.