ODAM promotes junctional epithelium-related gene expression via activation of WNT1 signaling pathway in an ameloblast-like cell line ALC
ODAM promotes junctional epithelium-related gene expression via activation of WNT1 signaling pathway in an ameloblast-like cell line ALC
复制标题
ODAM 通过激活成釉细胞样细胞系 ALC 中的 WNT1 信号通路促进交界上皮相关基因表达
DOI：
10.1111/jre.12848
复制
发表时间：
2021
影响因子：
3.5
通讯作者：
Wang L.
中科院分区：
文献类型：
--
作者：
Song D;Yang S;Tan T;Wang R;Ma Z;Wang Y;Wang L.
本研究探讨成牙本质成釉细胞相关蛋白(ODAM)在成釉细胞样细胞系ALC中促进连接上皮相关基因表达的作用及其机制。奥达姆KO小鼠表现出乙脑的完整性被破坏，乙脑从牙齿上脱落。ODAM被证实通过ODAM-ARHGEF5-RhoA信号通路调节JE的细胞骨架。ODAM是否对乙脑成釉细胞分化的调节作用尚不清楚。釉质形成后，成釉细胞经历了一系列的形态变化。ODAM是否会影响成釉细胞的生物学行为，使其具有乙脑的特性尚不清楚。方法采用小鼠成釉细胞样细胞系ALC，通过慢病毒转导产生ODAM过表达和ODAM基因敲除细胞，观察ODAM在上皮诱导的环境中对ALC细胞JE样改变的影响。采用实时荧光定量聚合酶链式反应、免疫印迹、免疫细胞化学、免疫组织化学、荧光素酶报告基因和挽救实验等方法，对连接上皮细胞CK19、SLPI、ODAM和潜在调控基因WNT1的表达进行检测。ODAM在ALC细胞中的过表达显著增加CK19和SLPI的表达，而ODAM在ALC细胞中的敲除明显降低CK19和SLPI的表达。记者的荧光素酶分析表明，ODAM激活了WNT信号通路，特别是通过WNT1。外源过表达ODAM可上调WNT1的表达，而敲除ODAM则逆转了这一作用。WNT1抑制实验进一步证实了上述结果，并表明WNT1通路与连接上皮细胞CK19和SLPI的表达呈正相关。救援研究表明，在ODAM过表达的ALC细胞中敲除WNT1会降低CK19和SLPI的表达。免疫细胞化学显示ODAM与CK19、SLPI和WNT1共定位于细胞内。结论研究表明ODAM通过ODAM-WNT1轴促进ALC中连接上皮相关基因的表达，这可能为研究ODAM和JE的形成提供新的思路。
ObjectiveIn 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.BackgroundODAM 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.MethodsA 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.ResultsODAM, 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.ConclusionIn 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.