DMP-1 Signaling Role in hMSC, MC3T3 and MDPC23 cells

Objectives: Determine the DMP-1 signaling pathway(s) and the mechanisms involved in dentin/bone gene activation in hMSC, MC3T3 and MDPC23.

Methods: Several molecular and cell biology techniques were used to determine the signaling role of DMP-1: 1) Western blots to detect the phosphorylation of MEK1/2, Erk, Jnk, c-Jun (ser63 and ser73) and c-Fos; 2) c-Jun transcriptional activity was assessed by quantifying the luciferase expression following the transfection of c-Jun-Gal and Gal-Luciferase plasmids. 3) anti-αvβ3 integrin antibodies were used to determine the role of integrin in DMP1 signaling; 4) specific drugs to block the MAPK pathway; 5) Immunocytochemical analysis to detect Erk and Jnk translocation as well as the formation of focal adhesion points.

Results: Our data show the MAPK pathway is involved in DMP-1 signaling. This was demonstrated by the following data: 1) Erk and Jnk were phosphorylated following DMP1 treatment. We should note that Erk and Jnk phosphorylation could be observed up to 1 and 3 hours, respectively, following the addition of DMP-1. 2) c-Fos, a downstream component of p-Erk, was also activated following the activation of Erk. 3) c-Jun (ser63 and ser73) was phosphorylated by the p-Jnk . 4) c-Jun activation was also demonstrated by luciferase expression following cell transfection with c-Jun-gal and gal-luciferase plasmids. 5) When using MAPK inhibitors such as 5uM U0126 (MEK1/2 inhibitor) or 30uM Sp600125 (JNK inhibitor) 1h before the cells were treated with DMP1, the p-Erk , p-Jnk and p-c-Jun were strongly inhibited. In addition, the phosphorylation of ERK and JNK was also blocked by the addition of anti-αvβ3 integrin antibody. 6) Immunohistochemistry shows the translocation of p-Jnk and pErk to the nucleus following DMP-1 treatment.

Conclusion: These findings demonstrate that DMP-1 regulates target gene expression via binding to the integrin and activation of the MAPK-Erk and Jnk-AP1 signaling pathway.