PKC Inhibition by Gö6976 Promotes Osteogenic Differentiation of Dental Follicle Cells Involving Rho-Dependent Pathway Dynamics.

Background: Dental follicle cells (DFCs) are promising candidates for regenerative medicine due to their osteogenic potential. While the protein kinase C (PKC) inhibitor Gö6976 is known to enhance DFC differentiation, the underlying molecular mechanisms remain partially understood. Methods: A phosphoproteomic analysis of DFCs after 14 days of osteogenic induction was performed. Cells treated with osteogenic differentiation medium (ODM) were compared to those in control medium and ODM supplemented with Gö6976. Reactome pathway analysis identified the RhoGTPase signaling pathway as significantly regulated. This pathway was further validated using PCR arrays, Western blotting, and functional assays (ALP activity, Alizarin Red staining). The impact of RhoGTPase signaling was tested using inhibitors (NSC23766, Y27632, Rhosin) and the activator Geranylgeranyl pyrophosphate (GGPP). Results: Phosphoproteomic data highlighted RhoGTPase signaling as a regulatory node. While protein expression of RhoGTPases remained relatively stable, RhoA PCR arrays revealed significant transcriptional regulation after induction of osteogenic differentiation. However, functional inhibition via NSC, Y27632, or Rhosin did not significantly impair basal ODM-induced differentiation; however, Y27632 notably induced SOST expression. Conversely, activation of RhoGTPases via GGPP increased ALP activity and downregulated SOST, suggesting a supportive role of active Rho signaling during differentiation. Crucially, we demonstrated that Gö6976-enhanced mineralization is linked to the activation of RhoA and RhoB. This was confirmed by simvastatin-mediated regulation of Rho expression, which was fully reversed by simultaneous treatment with Gö6976. Furthermore, Rhosin effectively counteracted the pro-osteogenic effects of Gö6976 by inhibiting ALP activity and mineralization while inducing SOST, which is normally suppressed by Gö6976. Conclusions: These findings indicate that RhoGTPase signaling, particularly RhoA, is a critical downstream mediator required specifically for the Gö6976-enhanced osteogenic effect in DFCs. We conclude that Gö6976 exerts its stimulatory effect on mineralization by activating RhoGTPases and suppressing the osteogenesis inhibitor SOST, providing a context-dependent mechanism for accelerated differentiation rather than driving basal osteogenesis.