Hydrostatic pressure promotes odontoblast differentiation via PIEZO1-dependent activation of RUNX2 and WNT16 in SHED

Mechanical stimulation plays a crucial role in odontoblast differentiation. However, the underlying molecular mechanisms remain unclear. We have previously shown that hydrostatic pressure (HP) applied to stem cells from human exfoliated deciduous teeth (SHED) promotes odontoblast differentiation by translocating RUNX2 and increasing WNT16 expression through PIEZO1 signaling. In this study, we further explored the downstream signaling cascade linking PIEZO1 activation and odontoblast differentiation. HP stimulation increased the expression of odontoblast differentiation markers PANX3 and DSPP, as shown by qPCR, and enhanced Alizarin Red staining-results significantly suppressed by siRNA targeting either PIEZO1 or WNT16. RT-PCR analysis revealed that, among the two known human WNT16 isoforms, only WNT16b was expressed in SHED. qPCR demonstrated that HP-induced WNT16 expression was reduced by siPIEZO1 and further decreased by siRUNX2. Promoter analysis identified four RUNX2-binding sites within the upstream region of WNT16. A luciferase reporter assay using plasmids containing the WNT16 promoter showed that RUNX2 overexpression in HEK293 cells significantly increased luciferase activity. Moreover, HaloChIP assays with a HaloTag-RUNX2 expression vector confirmed RUNX2's binding to the WNT16 promoter. These findings suggest that PIEZO1-mediated mechanical stress promotes odontoblast differentiation through the RUNX2-dependent transcriptional activation of WNT16.