Liu Hangbo, Yu Miao, Sun Kai, Zheng Jinglei, Wang Jiayu, Liu Haochen, Feng Hailan, Liu Yang, Han Dong
Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing, China.
J Cell Physiol. 2024 Dec;239(12):e31437. doi: 10.1002/jcp.31437. Epub 2024 Sep 19.
Enamel protects teeth from external irritation and its formation involves sequential differentiation of ameloblasts, a dental epithelial cell. Keratinocyte differentiation factor 1 (KDF1) is important in the development of epithelial tissues and organs. However, the specific role of KDF1 in enamel formation and corresponding regulatory mechanisms are unclear. This study demonstrated that KDF1 was persistently expressed in all stages of ameloblast differentiation, through RNAscope in situ hybridization. KDF1 expression in the mouse ameloblast cell line LS8 was demonstrated via immunofluorescence assay. KDF1 was knocked out in LS8 cells using the CRISPR/Cas-9 system or overexpressed in LS8 cells through lentiviral infection. In vitro ameloblast differentiation induction, quantitative reverse transcription PCR, western blot analysis, and alkaline phosphatase (ALP) assay indicated that knockout or overexpression of KDF1 in LS8 cells decreased or increased the mRNA and protein levels of several key amelogenesis markers, as well as ALP activity. Furthermore, liquid chromatography-mass spectrometry and co-immunoprecipitation analyses revealed that KDF1 can interact with the IKK complex, thereby inhibiting the NF-κB pathway. Suppressing NF-κB activity partially recovered the decreased ameloblast differentiation in LS8 cells induced by KDF1-knockout. This study demonstrated that KDF1 can promote ameloblast differentiation of LS8 cells by inhibiting the IKK/IκB/NF-κB axis, and is a potential target for functional enamel regeneration.
牙釉质保护牙齿免受外部刺激，其形成涉及成釉细胞（一种牙上皮细胞）的顺序分化。角质形成细胞分化因子1（KDF1）在上皮组织和器官的发育中起重要作用。然而，KDF1在牙釉质形成中的具体作用及相应调控机制尚不清楚。本研究通过RNAscope原位杂交表明，KDF1在成釉细胞分化的所有阶段持续表达。通过免疫荧光测定证实了KDF1在小鼠成釉细胞系LS8中的表达。使用CRISPR/Cas-9系统在LS8细胞中敲除KDF1，或通过慢病毒感染在LS8细胞中过表达KDF1。体外成釉细胞分化诱导、定量逆转录PCR、蛋白质免疫印迹分析和碱性磷酸酶（ALP）测定表明，LS8细胞中KDF1的敲除或过表达降低或增加了几种关键成釉蛋白标志物的mRNA和蛋白质水平，以及ALP活性。此外，液相色谱-质谱联用和免疫共沉淀分析表明，KDF1可与IKK复合物相互作用，从而抑制NF-κB途径。抑制NF-κB活性部分恢复了KDF1敲除诱导的LS8细胞中成釉细胞分化的降低。本研究表明，KDF1可通过抑制IKK/IκB/NF-κB轴促进LS8细胞的成釉细胞分化，是功能性牙釉质再生的潜在靶点。
