Dentin phosphophoryn activates Smad protein signaling through Ca2+-calmodulin-dependent protein kinase II in undifferentiated mesenchymal cells.

Dentin phosphophoryn (DPP) is a major noncollagenous protein in the dentin matrix. In this study, we demonstrate that pluripotent stem cells such as C3H10T1/2 and human bone marrow cells can be committed to the osteogenic lineage by DPP. Treatment with DPP can stimulate the release of intracellular Ca(2+). This calcium flux triggered the activation of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII induced the phosphorylation of Smad1 and promoted nuclear translocation of p-Smad1. Inhibition of store Ca(2+) depletion by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or down-regulation of CaMKII by KN-62, a selective cell-permeable pharmacological inhibitor or a dominant negative plasmid of CaMKII, blocked DPP-mediated Smad1 phosphorylation. Activation of Smad1 resulted in the expression of osteogenic markers such as Runx2, Osterix, DMP1, Bone sialoprotein, Osteocalcin, NFATc1, and Schnurri-2, which have been implicated in osteoblast differentiation. These findings suggest that DPP is capable of triggering commitment of pluripotent stem cells to the osteogenic lineage.