Classical isoforms of protein kinase C (PKC) and Akt regulate the osteogenic differentiation of human dental follicle cells via both β-catenin and NF-κB

Background Human dental follicle cells (DFCs) are the precursor cells of the periodontium with a high potential for regenerative therapies of (alveolar) bone. However, the molecular mechanisms of osteogenic differentiation are inadequately understood. Classical isoforms of protein kinase C (PKC) are reported to inhibit osteogenesis of stem/precursor cells. This study evaluated the role of classical PKCs and potential downstream targets on the osteogenic differentiation of DFCs. Methods DFCs were osteogenic differentiated with dexamethasone or bone morphogenetic protein 2 (BMP2). Expression of PKC and potential upstream/downstream regulators was manipulated using activators, inhibitors, and small interfering ribonucleic acid (siRNA). Expression of proteins was examined by Western blot analysis, while the activation levels of enzymes and transcription factors were examined by their phosphorylation states or by specific activation assays. Expression levels of osteogenic markers were examined by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) analysis. Activity of alkaline phosphatase (ALP) and accumulation of calcium nodules by Alizarin Red staining were measured as indicators of mineralization. Results Classical PKCs like PKCα inhibit the osteogenic differentiation of DFCs, but do not interfere with the induction of differentiation. Inhibition of classical PKCs by Gö6976 enhanced activity of Akt after osteogenic induction. Akt was also regulated during differentiation and especially disturbed BMP2-induced mineralization. The PKC/Akt axis was further shown to regulate the canonical Wnt signaling pathway and eventually nuclear expression of active β-catenin during dexamethasone-induced osteogenesis. Moreover, the nuclear factor “kappa-light-chain-enhancer” of activated B cells (NF-κB) pathway is regulated during osteogenic differentiation of DFCs and via the PKC/Akt axis and disturbs the mineralization. Upstream, parathyroid hormone-related protein (PTHrP) sustained the activity of PKC, while Wnt5a inhibited it. Conclusions Our results demonstrate that classical PKCs like PKCα and Akt regulate the osteogenic differentiation of DFCs partly via both β-catenin and NF-κB.

CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

Chromodomain helicase DNA-binding protein 7 (CHD7) is an ATP-dependent chromatin remodeling enzyme, functioning as chromatin reader to conduct epigenetic modification. Its effect on osteogenic differentiation of human dental follicle cells (hDFCs) remains unclear. Here, we show the CHD7 expression increases with osteogenic differentiation. The knockdown of CHD7 impairs the osteogenic ability of hDFCs, characterized by reduced alkaline phosphatase activity and mineralization, and the decreased expression of osteogenesis-related genes. Conversely, the CHD7 overexpression enhances the osteogenic differentiation of hDFCs. Mechanically, RNA-seq analyses revealed the downregulated enrichment of PTH (parathyroid hormone)/PTH1R (parathyroid hormone receptor-1) signaling pathway after CHD7 knockdown. We found the expression of PTH1R positively correlates with CHD7. Importantly, the overexpression of PTH1R in CHD7-knockdown hDFCs partially rescued the impaired osteogenic differentiation. Our research demonstrates that CHD7 regulates the osteogenic differentiation of hDFCs by regulating the transcription of PTH1R.