Biochemical and molecular characterization of stromelysin synthesized by human osteoarthritic chondrocytes stimulated with recombinant human interleukin-1.

OBJECTIVE: To study the biochemical and molecular characterization of stromelysin synthesized by human chondrocytes derived from osteofemoral heads.
METHODS: First passage human chondrocyte cultures were incubated with recombinant human interleukin-1 alpha or recombinant human interleukin-1 beta (10-1000 pg ml-1) for either 24 or 48 hrs. The medium compartment of these cultures was assayed for stromelysin activity. Total cellular RNA was used to determine: (i) the molecular structure of the stromelysin synthesized by these cells; and (ii) whether or not these chondrocytes expressed the Type II procollagen gene (COL2A1).
RESULTS: Human osteoarthritic chondrocytes released into the medium on enzyme requiring tryspin activation that possessed Substance P (SP) cleaving activity. SP cleaving activity was completely inhibited by EDTA. Casein zymography showed lysis zones produced by trypsin-activated chondrocyte culture medium that co-migrated with casein lysis zones produced by recombinant human prostromelysin. The majority of SP cleaving activity was eluted from a Zn-Sepharose column with 0.25 M glycine. Enzyme activity eluted from Zn-Sepharose produced casein lysis zones which co-migrated with lysis zones produced by recombinant human prostromelysin. Immunoblotting revealed the presence of prostromelysin (M(r), 55-57 kDa) in the pooled chondrocyte culture media applied to Zn-Sepharose and in the 0.25 M glycine eluate. Trypsin-activation converted prostromelysin to a mature stromelysin form (M(r), 45-47 kDa). Polymerase chain reaction (PCR) amplification of human chondrocyte cDNA demonstrated COL2A1 transcripts. A PCR product of expected size (680 bp) was produced by amplification of chondrocyte cDNA using stromelysin-1 oligonucleotide primers. The cloned and sequenced PCR product showed 100% homology between the chondrocyte stromelysin-1 mRNA-derived cDNA and the stromelysin-1 mRNA-derived cDNA of cultured human synovial, gingival and skin fibroblasts.
CONCLUSIONS: By several criteria, human osteoarthritic chondrocytes synthesized stromelysin which was biochemically and antigenically identical, and molecularly homologous with human fibroblast stromelysin-1. These results suggest that a quantitative imbalance between stromelysin-1 and endogenous stromelysin-1 inhibitors rather than the transcription of a new stromelysin gene is the mechanism underlying the increased proteoglycan degradation seen in osteoarthritic cartilage.