Identification of the metalloproteinase stromelysin in the physis

For long bone growth to occur, calcification of the matrix must commence in the lower hypertrophic zone of the growth plate. It is generally accepted that physeal proteoglycans help regulate mineralization, and that at least in vitro, intact proteoglycans can inhibit mineralization. Thus degradation of proteoglycan may be a necessary step prior to calcification. Previous work in our laboratory has demonstrated the presence of neutral metallo-proteases in the growth plate with highest levels in the hypertrophic zone, where calcification occurs. Stromelysin (MMP-3) is a connective tissue matrix-degrading enzyme. It was formerly known as proteoglycanase and is generally considered to be one of the major proteoglycan degrading enzymes in cartilage. Stromelysin is implicated in cartilage destruction in osteoarthritis and may also be involved in tissue remodeling in the physis. Our goal was to determine if the neutral protease previously reported by the authors in the physis was stromelysin. In this study we used Western blots and antibodies to stromelysin and to the stromelysin cleavage site in aggrecan, the most common form of proteoglycan, to demonstrate the presence of stromelysin in the bovine physis. When an antibody raised against the stromelysin cleavage site of aggrecan (FVDIPEN) was incubated with a Western blot, which had been run with aggrecan extracted from bovine physes, a positive reaction resulted. This suggests that there is stromelysin degradation in vivo in the physis. Two different polyclonal antibodies to stromelysin gave positive results on Western blots of purified media from growth plate cultures indicating that stromelysin is produced in vitro in culture. These antibodies also reacted with active stromelysin. The presence of stromelysin in the physis implicates it in physeal physiology. The concentration of its activity in the lower hypertrophic zone and zone of provisional calcification suggests that it may be particularly important in mineralization.