DMP-1 Signals Through MAPK in hMSC, MDPC23 and MC3T3 Cells

Objectives: Our goal is to study the signaling pathway of DMP-1 in human mesenchymal stem cells, mouse preosteoblast MC3T3 and mouse odontoblasts MDPC23.

Methods: The phosphorylation of MEK1/2, Erk, Jnk, c-Jun (ser63 and ser73) and FAK were determined by western blot. c-Jun transcriptional activity was assessed by quantifying the luciferase expression following the transfection of c-Jun-Gal and Gal-Luciferase plasmids. Anti-avß3 integrin antibodies were used to determine the role of integrin in DMP1 signaling. 10uM U0126 and 30uM SP600125 were used to block the MEK1/2 and JNK respectively 1h before the cells were treated with DMP1. Erk and Jnk translocation, the formation of focal adhesion points and the FAK phosphorylation were examined by immunocytochemistry.

Results: The data shows that DMP-1 signals through integrin avß3 and MAPK. After treating cells with DMP-1 for 1 or 3 hours, Erk and Jnk were phosphorylated respectively. c-Fos, a downstream component of p-Erk, was activated following the activation of Erk. c-Jun (ser63 and ser73) was phosphorylated by the p-Jnk . c-Jun activation was also demonstrated by luciferase expression following the transfection of the cells with c-Jun-gal and gal-luciferase plasmids. When using MAPK inhibitors (U0126 and SP600125), the p-Erk, p-Jnk and p-c-Jun were strongly inhibited. In addition, the phosphorylation of ERK and JNK were blocked by the anti-avß3 integrin antibody. Immunocytochemistry showed the FAK was phosphorylated and the p-Jnk and p-Erk translocated to the nucleus following DMP-1 treatment.

Conclusion: We have demonstrated that DMP-1 binds to the integrin and signals through the MAPK-Erk and Jnk-AP1pathways in hMSC, MC3T3 and MDPC23.