title: Biochemical and molecular characterization of stromelysin synthesized by human osteoarthritic chondrocytes stimulated with recombinant human interleukin-1.
abstract: 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.
authors.affiliation: Research Department, Ciba-Geigy Corporation, Summit, New Jersey.
authors.forename: V S
authors.initials: VS
authors.lastname: Ganu
authors.forename: S I
authors.initials: SI
authors.lastname: Hu
authors.forename: R
authors.initials: R
authors.lastname: Melton
authors.forename: C
authors.initials: C
authors.lastname: Winter
authors.forename: V M
authors.initials: VM
authors.lastname: Goldberg
authors.forename: T M
authors.initials: TM
authors.lastname: Haqqi
authors.forename: C J
authors.initials: CJ
authors.lastname: Malemud
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002356:Cartilage / Q000378:metabolism* / Q000473:pathology; D003094:Collagen / Q000235:genetics; D004792:Enzyme Precursors / Q000302:isolation & purification; D006801:Humans; D007375:Interleukin-1 / Q000145:classification / Q000494:pharmacology*; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D010003:Osteoarthritis / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D013373:Substance P / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004792:Enzyme Precursors; D007375:Interleukin-1; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013373:Substance P; D003094:Collagen; D008666:Metalloendopeptidases; C048047:prostromelysin; D019278:Matrix Metalloproteinase 3