CaMKII Inhibition Enhances DPSC Odontoblastic Differentiation via TrkB Under TNFα-Induced Inflammation

Objectives: Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine-specific protein kinase that is thought to be an important mediator in normal and pathological conditions. However, limited information is available regarding roles of CaMKII in dentinogenesis, especially in inflammatory context. We previously demonstrated the crucial role of TrkB in the inflammation-induced dental pulp stem cells (DPSCs) odontoblastic differentiation. Here, we investigated the interaction between CaMKII and TrkB during DPSCs odontogenic differentiation.
Methods: DPSCs were cultured and knocked down for CAMKII using siRNA in antibiotic free media and then swapped with dentinogenic media. Cells were stimulated with TNFα at day 4 and 7 and treated with CAMKII inhibitor or CAMKII protein or TrkB inhibitor (CTX-B) at day 4, 7 and 10. Cells were stained using double immunofluorescence staining and/ or Alizarin Red Staining (ARS). PCR and ELISA were performed to detect expression levels of odontogenic and mineralization markers i.e., DSPP and DMP-1.
Results: Our data show that inhibition of CaMKII interacts with TrkB by increasing protein levels and enhances the transcriptional activity of TNFα-induced odontogenic differentiation. The CaMKII siRNA-mediated knockdown and CaMKII inhibitor increased the protein levels of DSPP (11.2 ± 1.3 vs control 3.3 ± 0.6, p < 0.05) and DMP-1 (5.6 ± 1.4 vs control 3.3 ± 0.6) while application of CaMKII protein decreased DSPP (2.3 ± 0.4, p < 0.01) and DMP-1 (2.02 ± 0.2, p < 0.05). Notably, treatment with CTX-B (TNFα+CaMKII-inhibitor vs TNFα+CaMKII-inhibitor+CTX-B) abolished DSPP (17.4 ± 2.8 vs 5.01 ± 1.2, p < 0.01) and DMP-1 (12.2 ± 2.01 vs 4.8 ± 1.3, p < 0.01) expression, respectively. Similarly, mRNA expression of DSPP and DMP-1 were reduced at day 10 (10.1 ± 3.2 vs 2.1±0.8, p < 0.05 & 0.7±0.13). ELISA and ARS results showed similar pattern among expression of DSPP and DMP-1.
Conclusions: Our data provide a novel mechanism of the role of CaMKII via TrkB in dentinogenesis that are required for successful DPSCs engineering strategies.