肿瘤坏死因子-α对人成釉细胞牙本质相关蛋白基因的转录调控。

Transcriptional regulation of human odontogenic ameloblast-associated protein gene by tumor necrosis factor-α.

机构信息

Department of Periodontology, Nihon University School of Dentistry at Matsudo, Matsudo, Japan.

Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, Matsudo, Japan.

出版信息

Inflamm Res. 2022 Jan;71(1):119-129. doi: 10.1007/s00011-021-01523-5. Epub 2021 Nov 17.

DOI:10.1007/s00011-021-01523-5

PMID:34787682

Abstract

OBJECTIVE

Odontogenic ameloblast-associated protein (ODAM) is produced by maturation stage ameloblasts and junctional epithelium (JE). The function of ODAM is thought to be involved in the attachment of teeth and JE. To elucidate transcriptional regulation of human ODAM gene in inflamed gingiva, we have analyzed the effects of TNF-α on the expression of ODAM gene in Ca9-22 and Sa3 gingival epithelial cells.

MATERIALS AND METHODS

Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10 ng/ml). ODAM mRNA and protein levels were analyzed by qPCR and Western blotting. Luciferase (LUC) analyses were performed using LUC constructs inserted in various lengths of ODAM gene promoter. Gel shift and chromatin immunoprecipitation (ChIP) assays were carried out.

RESULTS

TNF-α increased ODAM mRNA and protein levels at 3 to 24 h. TNF-α induced LUC activities of the ODAM gene promoter constructs, and the activities were inhibited by protein kinase A, tyrosine kinase, MEK1/2, PI3-kinase and NF-κB inhibitors. Gel shift and ChIP assays revealed that TNF-α increased CCAAT/enhancer-binding protein (C/EBP) β and Yin Yang1 (YY1) binding to three kinds of C/EBPs and YY1 elements.

CONCLUSION

These results demonstrate that TNF-α stimulates ODAM gene transcription via C/EBPs and YY1 elements in the human ODAM gene promoter.

摘要

目的

牙源性上皮相关蛋白（ODAM）由成熟阶段成釉细胞和连接上皮（JE）产生。ODAM 的功能被认为涉及牙齿和 JE 的附着。为了阐明炎症性牙龈中人 ODAM 基因的转录调控，我们分析了 TNF-α 对 Ca9-22 和 Sa3 牙龈上皮细胞中 ODAM 基因表达的影响。

材料和方法

用 TNF-α（10ng/ml）刺激 Ca9-22 和 Sa3 细胞后提取总 RNA。通过 qPCR 和 Western blot 分析 ODAM mRNA 和蛋白水平。使用插入 ODAM 基因启动子不同长度的 LUC 构建体进行荧光素酶（LUC）分析。进行凝胶迁移和染色质免疫沉淀（ChIP）分析。

结果

TNF-α 在 3 至 24 小时内增加 ODAM mRNA 和蛋白水平。TNF-α 诱导 ODAM 基因启动子构建体的 LUC 活性，蛋白激酶 A、酪氨酸激酶、MEK1/2、PI3-激酶和 NF-κB 抑制剂抑制其活性。凝胶迁移和 ChIP 分析显示，TNF-α 增加了 CCAAT/增强子结合蛋白（C/EBP）β 和 Yin Yang1（YY1）与三种 C/EBP 和 YY1 元件的结合。