Department of Orthodontics, Faculty of Medicine, Justus Liebig University Giessen, 35392 Giessen, Germany.
Department of Periodontology, Faculty of Medicine, Justus Liebig University Giessen, 35392 Giessen, Germany.
Int J Mol Sci. 2022 Aug 18;23(16):9311. doi: 10.3390/ijms23169311.
In animal models, the administration of ciliary neurotrophic factor (CNTF) was demonstrated to reduce bone mass and to participate in bone remodeling. Cementoblasts, a cell type embedded in the cementum, are the main cells to produce and mineralize the extracellular matrix. The effect of CNTF on cementoblasts has not yet been addressed. Thus, the goal of this in vitro study was to investigate possible influences of exogenous CNTF on cementogenesis, as well as autophagy regulation and subsequent mechanisms in cementoblasts. Cementoblasts (OCCM-30) were stimulated with exogenous CNTF. Alizarin Red staining was performed to analyze the functional differentiation (mineralization) of OCCM-30 cells. The release of OPG was quantified by ELISA. The expression of cementogenesis markers (, , , , and ) was evaluated by RT-qPCR. Western blotting (WB) was performed for the protein expression of STAT3, COX-2, SHP-2, cPLAα, cPLAβ; ERK1/2, P38, and JNK. The autophagic flux was assessed using WB and RT-qPCR analysis of LC3A/B, Beclin-1, and Atg-5, and the autophagosome was investigated by immunofluorescence staining (IF). The ERK1/2 (FR180204) or STAT3 (sc-202818) antagonist was added, and the cellular response was analyzed using flow cytometry. Exogenous CNTF significantly attenuated mineralized nodule formation, impaired OPG release, and downregulated the mRNA levels of , , , and . Moreover, CNTF induced the phosphorylation of STAT3 and activated a transient activation of SHP-2, cPLAβ, ERK1/2, P38, and JNK protein. CNTF also induced autophagosome formation and promoted autophagy-associated gene and protein expressions. Additionally, the inhibition of ERK1/2 or STAT3 reversed a CNTF-induced mineralization impairment and had regulatory effects on CNTF-induced autophagosome formation. Our data revealed that CNTF acts as a potent inhibitor of cementogenesis, and it can trigger autophagy, in part by ERK1/2 and STAT3 commitment in the cementoblasts. Thus, it may play an important role in inducing or facilitating inflammatory root resorption during orthodontic tooth movement.
在动物模型中，已证实睫状神经营养因子 (CNTF) 的给药可减少骨量并参与骨重塑。成牙骨质细胞是一种嵌入牙骨质的细胞类型，是产生和矿化细胞外基质的主要细胞。CNTF 对成牙骨质细胞的影响尚未得到研究。因此，本体外研究的目的是探讨外源性 CNTF 对成牙骨质细胞的成牙骨质作用以及自噬调节和随后的机制。用外源性 CNTF 刺激成牙骨质细胞（OCCM-30）。通过茜素红染色分析 OCCM-30 细胞的功能分化（矿化）。通过 ELISA 定量测定 OPG 的释放。通过 RT-qPCR 评估成牙骨质标志物（、、、和）的表达。通过 Western blot（WB）测定 STAT3、COX-2、SHP-2、cPLAα、cPLAβ 的蛋白表达；ERK1/2、P38 和 JNK。通过 WB 和 LC3A/B、Beclin-1 和 Atg-5 的 RT-qPCR 分析评估自噬流，并通过免疫荧光染色（IF）研究自噬体。添加 ERK1/2（FR180204）或 STAT3（sc-202818）拮抗剂，通过流式细胞术分析细胞反应。外源性 CNTF 显著减弱矿化结节形成，损害 OPG 释放，并下调、、、和的 mRNA 水平。此外，CNTF 诱导 STAT3 磷酸化并激活瞬时 SHP-2、cPLAβ、ERK1/2、P38 和 JNK 蛋白激活。CNTF 还诱导自噬体形成并促进自噬相关基因和蛋白表达。此外，ERK1/2 或 STAT3 的抑制逆转了 CNTF 诱导的矿化损伤，并对 CNTF 诱导的自噬体形成具有调节作用。我们的数据表明，CNTF 是成牙骨质作用的有效抑制剂，它可以通过 ERK1/2 和 STAT3 在成牙骨质细胞中的作用触发自噬。因此，它可能在正畸牙齿移动过程中诱导或促进炎症性牙根吸收中发挥重要作用。
