ODAM 通过激活牙釉质细胞样细胞系 ALC 中的 WNT1 信号通路促进连接上皮相关基因表达。
ODAM promotes junctional epithelium-related gene expression via activation of WNT1 signaling pathway in an ameloblast-like cell line ALC.
机构信息
Department of Prosthodontics, School and Hospital of Stomatology, Peking University, Beijing, China.
National Clinical Research Center for Oral Diseases, National Engineering Laboratory for Digital and Material Technology of Stomatology, Beijing Key Laboratory of Digital Stomatology, Beijing, China.
出版信息
J Periodontal Res. 2021 Jun;56(3):482-491. doi: 10.1111/jre.12848. Epub 2021 Jan 16.
OBJECTIVE
In this study, we investigated the potential and mechanism of odontogenic ameloblast-associated protein (ODAM) in the promoting junctional epithelium-related gene expression in an ameloblast-like cell line ALC.
BACKGROUND
ODAM is expressed in ameloblasts and JE and acts as a component of the inner basal lamina (IBL) and intercellular matrix of JE. ODAM KO mice showed destruction of the integrity of the JE, which detaches from teeth. ODAM was confirmed to regulate the cytoskeleton through the ODAM-ARHGEF5-RhoA signaling pathway of the JE. Whether ODAM contributes to the regulation of ameloblast differentiation in JE remains unclear. After the formation of enamel, the ameloblast undergoes a series of morphological changes. Whether ODAM will affect the biological behavior of ameloblasts making them have the characteristics of JE is unclear.
METHODS
A murine ameloblast-like cell line, ALC, was used to investigate the effects of ODAM on the JE-like changes of ALC cells in an epithelium-induced environment by generating ODAM overexpression and ODAM knockdown cells through a lentivirus transduction approach. The biomarkers of junctional epithelium CK19, SLPI, and ODAM and the potential regulatory gene WNT1 were investigated by real-time PCR, western blot, immunocytochemistry, immunostaining, luciferase reporter, and rescue assays.
RESULTS
ODAM, CK19, and SLPI were significantly upregulated after epithelial induction. Overexpression of ODAM in ALC cells markedly increased CK19 and SLPI expression, while knockdown of ODAM in ALC cells clearly decreased CK19 and SLPI expression. A reporter luciferase assay showed that ODAM activated the WNT signaling pathway, especially through WNT1. Exogenous overexpression of ODAM upregulated WNT1 expression, while knockdown of ODAM reversed this effect. The WNT1 inhibition assay further confirmed the above results and showed that the WNT1 pathway was positively correlated with biomarkers of junctional epithelium CK19 and SLPI expression. Rescue studies showed that knocking down WNT1 in the ODAM-overexpressing ALC cells decreased the expression of CK19 and SLPI. Immunocytochemistry showed that ODAM colocalized with CK19, SLPI, and WNT1 in the cells.
CONCLUSION
In conclusion, the research work showed that ODAM promotes junctional epithelium-related gene expression in ALC via the ODAM-WNT1 axis, which may provide new insight into the function of ODAM and JE formation.
目的
在这项研究中，我们研究了牙源性釉细胞蛋白（ODAM）在促进成釉细胞相关基因表达中的潜力和机制，该蛋白在成釉细胞样细胞系 ALC 中表达。
背景
ODAM 表达于成釉细胞和连接上皮（JE）中，作为 JE 内层基底膜（IBL）和细胞间基质的组成部分。ODAM KO 小鼠的 JE 完整性遭到破坏，JE 从牙齿上脱落。ODAM 被证实通过 JE 中的 ODAM-ARHGEF5-RhoA 信号通路调节细胞骨架。ODAM 是否有助于 JE 中成釉细胞分化的调节尚不清楚。在釉质形成后，成釉细胞经历一系列形态变化。ODAM 是否会影响成釉细胞的生物学行为，使它们具有 JE 的特征尚不清楚。
方法
利用小鼠成釉细胞样细胞系 ALC，通过慢病毒转导方法生成 ODAM 过表达和 ODAM 敲低细胞，研究 ODAM 在上皮诱导环境下对 ALC 细胞 JE 样变化的影响。通过实时 PCR、western blot、免疫细胞化学、免疫染色、荧光素酶报告和挽救实验检测连接上皮标志物 CK19、SLPI 和 ODAM 以及潜在的调节基因 WNT1。
结果
上皮诱导后，ODAM、CK19 和 SLPI 表达显著上调。ODAM 在 ALC 细胞中的过表达显著增加 CK19 和 SLPI 的表达，而 ODAM 在 ALC 细胞中的敲低则明显降低 CK19 和 SLPI 的表达。报告基因荧光素酶实验表明，ODAM 激活了 WNT 信号通路，特别是通过 WNT1。ODAM 的过表达上调 WNT1 的表达，而 ODAM 的敲低则逆转了这一效应。WNT1 抑制实验进一步证实了上述结果，并表明 WNT1 通路与连接上皮标志物 CK19 和 SLPI 的表达呈正相关。挽救研究表明，在 ODAM 过表达的 ALC 细胞中敲低 WNT1 降低了 CK19 和 SLPI 的表达。免疫细胞化学显示 ODAM 在细胞中与 CK19、SLPI 和 WNT1 共定位。
结论
综上所述，研究工作表明，ODAM 通过 ODAM-WNT1 轴促进 ALC 中与连接上皮相关的基因表达，这可能为 ODAM 和 JE 形成的功能提供新的见解。
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