Amelogenin Is a Novel Substrate of MMP-9

MMP-9, a member of the matrix metalloproteinase family, is expressed in tooth tissues and plays important roles in extracellular matrix remodeling. Amelogenin comprises 90% of enamel organic matrix and is processed by proteinases into active forms during amelogenesis.  However, biological activities of amelogenin cleavage by MMP-9 are not fully understood.  Objectives: We investigated whether amelogenin is a substrate of MMP-9 and their expression pattern during tooth development.  Methods: Immunohistochemistry was performed on tooth tissue sections from embryonic day (E) 16.5 to postnatal day (PN) 18.5.  Expression of amelogenin and MMP-9 in mouse enamel organ epithelial (EOE-3M) cells was detected by RT-PCR, Western blotting, and gelatin zymography.  Radiology, histology, and immunohistochemistry were used to characterize the phenotypes and genotypes of MMP-9 knockout (KO) and wild-type mice.  Recombinant MMP-9 and amelogenin proteins were expressed and used for analysis of amelogenin and MMP-9 interaction and cleavage.  Results: Double-labeling immunohistochemistry showed that amelogenin and MMP-9 co-expression was seen within the cytoplasm of ameloblasts and in the enamel matrix from E 18.5 to PN 18.5. The two proteins were also detected in EOE-3M cells by RT-PCR, Western blotting, and gelatin zymography. MMP-9 bound to amelogenin by immunoprecipitation assays. Moreover, MMP-9 cleaved amelogenin into three major fragments as determined by SDS-PAGE and mass spectrometry analyses; however, mutant MMP-9 failed to catalyze amelogenin. Enzyme kinetics demonstrated a high catalytic efficiency of MMP-9 for amelogenin.  In MMP-9 KO mice, teeth exhibited delayed eruption and rapid attrition rates.  These mice had immature ameloblast differentiation and loss of ameloblast polarization.  Furthermore, high expression levels and different expression patterns of amelogenin were observed in MMP-9 KO mice.  Conclusions: This study has shown that amelogenin is an MMP-9 substrate, and MMP-9 may be involved in amelogenin processing and enamel formation.  This work was supported by NIH/DE019892.