Shikonin induces odontoblastic differentiation of dental pulp stem cells via AKT–mTOR signaling in the presence of CD44

In our previous study, we demonstrated that hyaluronan induces odontoblastic differentiation of dental pulp stem cells via interactions with CD44. However, it remains unclear whether CD44 expression by dental pulp stem cells is required for odontoblastic differentiation. Therefore, we searched for a compound that induces odontoblastic differentiation of dental pulp stem cells, regardless of the chemical structure and function of hyaluronan, and examined whether CD44 is involved in the induction of odontoblastic differentiation by the compound. Because vitamin K analogues can promote bone formation and tissue calcification, we focused on derivatives of naphthoquinone, the skeleton of vitamin K; we verified whether those compounds could induce odontoblastic differentiation of dental pulp stem cells. We found that dentin sialophosphoprotein, a marker of odontoblasts, was expressed in dental pulp stem cells after treatment with shikonin. The shikonin-induced expression of dentin sialophosphoprotein was inhibited by PI3K, AKT, and mTOR inhibitors. In addition, in dental pulp stem cells transfected with siRNA against CD44, the shikonin-induced expression of dentin sialophosphoprotein was inhibited. Thus, shikonin can stimulate dental pulp stem cells to undergo odontoblastic differentiation through a mechanism involving the AKT–mTOR signaling pathway and CD44. Hyaluronan stimulated dental pulp stem cells to undergo CD44-mediated odontoblastic differentiation in our previous study; the present study indicated that CD44 is necessary for dental pulp stem cells to undergo odontoblastic differentiation. Although expression of CD44 is important for inducing odontoblastic differentiation of dental pulp stem cells, the relationship between the AKT–mTOR signaling pathway and CD44 expression, in the context of shikonin stimulation, has not yet been elucidated. This study suggested that shikonin may be useful for inducing odontoblastic differentiation of dental pulp stem cells, and that it may have clinical applications, including protection of dental pulp.