CHARACTERIZATION AND IDENTIFICATION OF PROTEINASES AND PROTEINASE-INHIBITORS SYNTHESIZED BY TEMPOROMANDIBULAR-JOINT DISC CELLS

The adult mammalian temporomandibular joint (TMJ) disc is a fibrocarti laginous tissue that undergoes normal developmental remodeling, requir ing removal of the existing extracellular matrix and its replacement b y new matrix macromolecules. This remodeling is probably mediated by m atrix-degrading enzymes, but to date none has been demonstrated in ass ociation with the TMJ disc. We characterized, identified, and determin ed the regulation of proteinases and proteinase inhibitors (PIs) synth esized by TMJ disc cells in organ and cell cultures. TMJ discs were re trieved from 14-week-old male NZW rabbits and both tissue- and disc-de rived cells were cultured in serum-free medium. The conditioned media were retrieved at 12-hour intervals and assayed for proteinases and PI s in gelatin- and casein-impregnated polyacrylamide gels. Three protei nases with gelatinolytic activities at 92 kDa, 72 kDa, and 53/57 kDa a nd one caseinolytic activity at 51/54 kDa were detected. AU were inhib ited by 1,10-1 phenanthroline, thus characterizing these enzymes as ma trix metalloproteinases (MMPs), most likely 92-kDa gelatinase (proMMP- 9), 72-kDa gelatinase (proMMP-2), procollagenase (proMMP-1), and prost romelysin (proMMP-3). The identity of the latter two MMPs was confirme d by Western blots. Two PIs of 30 kDa and 20 kDa, probably tissue inhi bitors of metalloproteinase (TIMP) and TIMP-2, were observed on revers e zymograms. TPA, a protein kinase-C agonist, increased the expression of 92-kDa gelatinase and 30-kDa PI by both explanted discs and isolat ed disc cells. The profile of MMPs constitutively expressed by disc ce lls is similar to that of synovial fibroblasts but different from that of chondrocytes. These findings implicate MMPs and PIs in the normal remodeling of the TMJ disc, and potentially in the pathologic degradat ion of the disc during various arthritides.