经典同工型蛋白激酶 C（PKC）和 Akt 通过β-连环蛋白和 NF-κB 调节人牙囊细胞的成骨分化。
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.
机构信息
Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany.
出版信息
Stem Cell Res Ther. 2021 Apr 14;12(1):242. doi: 10.1186/s13287-021-02313-w.
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.
背景
人牙周膜细胞（DFC）是牙周组织的前体细胞，具有再生治疗（牙槽）骨的巨大潜力。然而，成骨分化的分子机制尚未充分了解。蛋白激酶 C（PKC）的经典同工型被报道抑制干细胞/前体细胞的成骨分化。本研究评估了经典 PKC 及其潜在下游靶标在 DFC 成骨分化中的作用。
方法
用地塞米松或骨形态发生蛋白 2（BMP2）诱导 DFC 成骨分化。使用激活剂、抑制剂和小干扰核糖核酸（siRNA）来操纵 PKC 和潜在的上游/下游调节剂的表达。通过 Western blot 分析检查蛋白质的表达，通过磷酸化状态或特定的激活测定检查酶和转录因子的激活水平。通过 RT-qPCR（逆转录定量聚合酶链反应）分析检查成骨标志物的表达水平。通过茜素红染色测量碱性磷酸酶（ALP）的活性和钙结节的积累来衡量矿化程度。
结果
经典 PKC 如 PKCα 抑制 DFC 的成骨分化，但不干扰分化的诱导。Gö6976 抑制经典 PKC 可增强成骨诱导后 Akt 的活性。Akt 也在分化过程中受到调节，特别是干扰 BMP2 诱导的矿化。PKC/Akt 轴进一步显示调节经典 Wnt 信号通路，最终调节地塞米松诱导成骨过程中核内活性 β-连环蛋白的表达。此外，激活的 B 细胞核因子“κ-轻链增强子”（NF-κB）途径在 DFC 的成骨分化过程中受到调节，并通过 PKC/Akt 轴干扰矿化。上游，甲状旁腺激素相关蛋白（PTHrP）维持 PKC 的活性，而 Wnt5a 抑制其活性。
结论
我们的结果表明，经典 PKC 如 PKCα 和 Akt 通过β-连环蛋白和 NF-κB 部分调节 DFC 的成骨分化。
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