Immunological and Zymographic Analyses of MMP-2 and -9 in Dentin

Objective: Matrix metalloproteinases are involved in several developmental and disease-associated processes (Mannello et al., Curr Cancer Drug Targets 5:285,2005).  They have been suggested as playing key roles on the degradation of resin-dentin matrices.  Purpose: The purpose of this study was to identify if MMPs are present in human dentin.  Methods: Extracts of dentin from freshly extracted human teeth were electrophoresed by gelatin zymography (identifying all gelatinolytic (pro-)MMP enzymes), and the isoforms immunologically identified by western blotting (WB) using monoclonal antibodies against MMP-2 and -9 (Martin-De Las Heras et al., Arch Oral Biol 45:757,2000; Mannello et al., Clin Chem 50:1715,2004).  Results: Demineralized dentin matrix contained several isoforms of gelatinolytic enzymes with M_r of 72, 92 and 200 kDa, corresponding to proMMP-2, proMMP-9 and complexed MMP-2, respectively. Activated MMP isoforms with lower molecular weights (at 66, 84 and 195 kDa, respectively) were also detected, suggesting an autolytic activation of dentin MMPs. Gelatinolytic acitivity was Ca/Zn-dependent, fully inhibited by both calcium and zinc chelators (as well as by specific MMP inhibitor BB-94) and specifically activated by APMA, through a time- and dose-dependent fashion. Furthermore, WB identified them as MMP-2 and MMP-9 isoforms (72 and 200, and 92 kDa respectively).  Conclusions: The role and function of dentin MMPs are not well understood, but they may contribute to the degradation of the organic matrix of dentin (Pashley et al., JDR 83:216,2004). The balance between proteolytic degradation of dentin collagen by MMP-2 and -9 and their inhibition by specific TIMPs may explain the involvement of matrixins in dentin matrix degradation. Since their activity seems correlated with the effects of etching agents or carious demineralization, the role and functions of MMPs should be further validated by more in vivo investigations (Hebling et al., J Dent Res 84:741, 2005).  Supported by R01DE015306 from NIDCR (P.I. D.Pashley).