Peptides Resulting from DMP1 Degradation by MMP2 Promote Cell Differentiation

Recent data had shown that DMP1 could act as a transcription factor if it transported to the nuclear compartment. Furthermore, DMP1 has the capacity to regulate the DSPP gene transcription during early odontoblast differentiation by binding to the promoter of this gene through its carboxyl end. Objectives: to study the degradation of DMP1 by MMP-2 and to analyze if the resulting peptide (s) could act as signaling molecules on human dental pulp stem cells (DPSCs). Methods: Activated recombinant MMP-2 (0.1 or 1 µg/ml ) was added to a 100 µg recombinant rat DMP1 at 37°C for 0 to 24 hours in a final volume of 100 µl of 100mM Tris buffer pH 7.2 containing 5Mm CaCl2 and 0.15 M NaCl. The resulting supernatant was run on 12% SDS PAGE for identifying peptides in the range 10kDa to 35kDa by Coomassie blue, stain's all or silver staining. The resulting bands were excised from the gels and analyzed by mass spectrometry after trypsin digestion. Results: MMP-2 at short time points (6 to 12 hours) and for both concentrations degraded DMP1 into two fragments; a major fragment (1-314, 39kDa) corresponded to the N terminus and a lower one to the C terminus (315-489) which could not be identified on various gels. At longer time points (16 to 24 hours), the N fragment is processed into 4 fragments and all could be identified. Results from the activity of two closely matching recombinant polypeptides demonstrated that the N-DMP1 polypeptide did not display any significant effect on DPSCs, whereas the C polypeptide had an effect on cell differentiation with an increased expression of DSPP. Conclusion: Our results suggest that MMP-2, by cleaving DMP1, generates peptides that can regulate differentiation of mesenchymal cells during dentinogenesis and reparative dentin formation.

Supported by NIH grant DE-11657 (AG)-----