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

Abstract

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.

Keywords: Akt; Canonical Wnt signaling; Dental follicle cells; Mineralization; NF-κB; Osteogenic differentiation; Protein kinase C; β-catenin.

Fig. 1

Inhibitory role of classical PKCs during osteogenic differentiation of DFCs and Akt activity during differentiation and after inhibition of classical PKCs. a Protein expression of PKCα after 1, 7, 14, or 28 days cultivation in control medium (DMEM), osteogenic differentiation medium (ODM), or BMP2 containing differentiation medium determined by Western blot analysis. b DFCs were cultivated for 28 days in ODM and concurrently treated with 100 nM inhibitor of classical PKCs Gö6976 for either the whole 28 days or only during the first, second, third, or fourth week of differentiation. Cells cultivated in DMEM were used as control. Mineralization of extracellular matrix was determined by Alizarin Red staining. Microscopic photographs (total width of each photograph corresponds to 1.24 mm) of stained cells are shown below the relative quantification results. c Protein expression of P-Akt (Ser473) in DFCs as an indicator of Akt activity after 7, 14, or 28 days cultivation in control or differentiation media as above, determined by Western blot analysis. Expression of total Akt was determined as control. d, e Protein expression of P-Akt (Ser473) in DFCs after cultivation in control or differentiation media as above for 1 (d) or 7 (e) days and simultaneous treatment with 100 nM classical PKC inhibitor Gö6976, determined by Western blot analysis. Bar charts show means + SD (n = 3). One-way ANOVA was performed to compare different media at the same time point including Tukey’s post hoc tests comparing the individual differentiation media with the control medium (a, c) or to compare all groups including Tukey’s post hoc tests comparing differences between continuous Gö6976 treatment in ODM and other groups in ODM (b). Student’s t test was performed to determine statistically significant differences between the control and treatment group for each medium (d, e). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Mineralization of DFCs after treatment with Akt activators/inhibitors and interactions with PKC and BMP signaling. a–d DFCs were cultivated for 28 days in control medium (DMEM), osteogenic differentiation medium (ODM) or BMP2 containing differentiation medium, and concurrently treated with either 10 μM Akt activator SC-79 (a), 200 nM Akt inhibitor MK2206 (b), or 100 nM classical PKC inhibitor Gö 6976 alone or in combination with SC-79 (c, d). Mineralization of extracellular matrix was determined by Alizarin Red staining. Microscopic photographs (total width of each photograph corresponds to 1.24 mm) of stained cells and relative quantification results are shown. e DFCs were treated with 10 μM Akt activator SC-79 for 15, 30, and 60 min. Protein expression of P-SMAD 1/5 (Ser463/465) was determined by Western blot analysis. Bar charts show means + SD (n = 3). Student’s t test was performed to determine statistically significant differences between control and treatment group for each medium (a, b). One-way ANOVA was performed to compare all groups including Tukey’s post hoc tests comparing different groups in the same medium pairwise (c–e) or DMEM to ODM/BMP2 control groups (c, d). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Regulation of canonical Wnt signaling pathway by PKC/Akt axis. a–d DFCs were treated with 10 μM Akt activator SC-79 (a, c) or 200 nM Akt inhibitor MK2206 (b, d) for 15, 30, and 60 min. Expression of P-Akt (Ser473) (a, b) as control and P-GSK3β (Ser9) (c, d) was determined by Western blot analysis. e, f DFCs were cultivated in control medium (DMEM), osteogenic differentiation medium (ODM), or BMP2 containing differentiation medium with and without simultaneous treatment with 100 nM classical PKC inhibitor Gö6976 for 3 days. Expression of active β-catenin was determined in Western blot analysis after enrichment of cytoplasmic (e) or nuclear (f) proteins. Bar charts show means + SD (n = 3). One-way ANOVA was performed to compare all groups including Tukey’s post hoc tests comparing different groups in the same medium pairwise (a–d). Student’s t test was performed to determine statistically significant differences between the control and treatment group for each medium (e, f). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Regulation of NF-κB during osteogenic differentiation and impact of NF-κB inhibition on mineralization. a, b Expression of NF-κB (p65 subunit, a) and P-NF-κB (p65 subunit, Ser536, b) in DFCs after cultivation in control medium (DMEM), osteogenic differentiation medium (ODM) or BMP2 containing differentiation medium for 1, 7, 14 or 28 days was determined by Western blot analysis. The phospho-ratio (b) was calculated as ratio between phosphorylated and total NF-κB. c, d DNA binding activity of NF-κB subunits p50 (c) or p65 (d) in DFCs after 7 days cultivation in control or differentiation media as above, measured by NF-κB activity assay. e DFCs were cultivated in the control medium (DMEM) or osteogenic differentiation medium (ODM) and simultaneously treated with either 200 nM PKC activator PMA alone or in combination with 500 nM NF-κB inhibitor CID2858522 for 28 days. Mineralization was determined by Alizarin Red staining. Bar charts show means + SD (n = 3). One-way ANOVA was performed to compare different media at the same time point including Tukey’s post hoc tests comparing the individual differentiation media with the control medium (a–d) or to compare all groups including Tukey’s post hoc tests comparing the different groups in ODM pairwise or DMEM to ODM control group (e). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Regulation of NF-κB pathway after inhibition of PKC and Akt. DFCs were cultivated in control medium (DMEM), osteogenic differentiation medium (ODM), or BMP2 containing differentiation medium with or without simultaneous treatment with 100 nM classical PKC inhibitor Gö6976 for 7 days (a, c, e, g, i) or 200 nM Akt inhibitor MK2206 for 8 days (b, d, f, h, j). Expression of NF-κB (p65 subunit, a, b), P-NF-κB (p65 subunit, Ser536, c, d), IκBα (e, f), IKKα (g, h), and IKKβ (I, J) was determined by Western blot analysis. Phospho-ratios (c, d) were calculated as ratios between phosphorylated and total NF-κB. Bar charts show means + SD (n = 3). Student’s t test was performed to determine statistically significant differences between the control and treatment group for each medium. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Activation of PKC and Akt by Wnt5a and PTHrP. Undifferentiated DFCs were transfected with specific siRNA against WNT5A (Wnt5a, a, b) or PTHLH (PTHrP, c–e) or control siRNA for 3 days. Expression of P-PKC (phosphorylation sites according to Ser660 on PKC βII, a, c), P-Akt (Ser473, b, d), and NF-κB (e) was determined by Western blot analysis. Bar charts show means + SD (n = 3). Student’s t test was performed to determine statistically significant differences in comparison to control siRNA. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

Graphical summary of the investigated signaling pathways during osteogenic differentiation of DFCs. The osteogenic differentiation in DFCs can be induced by dexamethasone or BMP2. Induction by dexamethasone leads to downregulation of classical PKCs, while induction by BMP2 inhibits both classical PKCs and Akt. Activity of classical PKCs is sustained by PTHrP and can be inhibited by Wnt5a. In osteogenic differentiating cells, classical PKCs inhibit the activity of Akt, while Akt was shown to inhibit the NF-κB pathway and sustain the activity of β-catenin by phosphorylation and thereby inhibition of GSK3β. Classical PKCs stimulate the NF-κB pathway. Eventually, the transcription factors NF-κB and β-catenin modulate the osteogenic differentiation of DFCs. Furthermore, the BMP2/Smad signaling pathway, which is pivotal for BMP2-induced osteogenic induction, is disturbed by Akt