One of the best studied animal models of
osteoarthritis is a dog model in which the anterior cruciate ligament of the hind limb stifle joint is transected (Pond-Nuki model). To determine whether
stromelysin might play a role in this model, it was necessary to purify the
enzyme for production of suitable probes. In the present study, dog synovial fibroblasts were stimulated to express a
metalloproteinase that was demonstrated to be canine
prostromelysin by Northern blot,
protein purification and amino-terminal sequence analyses. Unlike rabbit synoviocytes, passaged dog synoviocytes did not express
stromelysin mRNA in response to recombinant human
IL-1, but expressed
stromelysin mRNA only upon treatment with dog monocyte-
conditioned medium (
dMCM). The aminophenylmercuric
acetate (APMA)-activatable
metalloproteinase present in the culture supernatants of stimulated dog synoviocytes was purified using a combination of ion-exchange and
dye matrix affinity chromatography. The purified canine
metalloproteinase co-migrated on reducing SDS-PAGE with recombinant human prostromelysin-1 as a doublet with apparent molecular masses of 54 and 56 kDa. Similar to APMA-activated human prostromelysin-1, the APMA-activated canine
metalloproteinase was inhibited by
1,10-phenanthroline or recombinant human
tissue inhibitor of metalloproteinase (TIMP). The amino-terminal sequences of the canine pro- and APMA-activated
enzymes were compared with those of human, rabbit and rat
stromelysin. The striking homologies among the sequences demonstrated that the purified canine
metalloproteinase was indeed canine
prostromelysin. A rabbit anti-canine
prostromelysin polyclonal antiserum was generated and used to localize the
enzyme within cultured dog synoviocytes and articular cartilage stimulated with
dMCM. The
reagents developed in this study should be useful for examining the expression and distribution of
prostromelysin in canine models of
osteoarthritis.