Tumor necrosis factor-α stimulates human amelotin gene transcription in gingival epithelial cells

• Published in: Inflammation research Vol. 67; no. 4; pp. 351 - 361
• Main Authors: Yamazaki, Mizuho, Iwai, Yasunobu, Noda, Keisuke, Matsui, Sari, Kato, Ayako, Takai, Hideki, Nakayama, Yohei, Ogata, Yorimasa
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.04.2018; Springer Nature B.V
• Subjects: 1-Phosphatidylinositol 3-kinase; Allergology; Ameloblasts; Assaying; Basal lamina; Biomedical and Life Sciences; Biomedicine; CCAAT/enhancer-binding protein; Chromatin; Dermatology; EBP1 protein; Electrophoretic mobility; Enamel; Epithelial cells; Epithelium; Gene expression; Gene regulation; Gingiva; Immunology; Immunoprecipitation; Inflammation; Kinases; Mutation; Necrosis; Neurology; NF-κB protein; Original Research Paper; Pharmacology/Toxicology; Protein kinase A; Protein-tyrosine kinase; Proteins; Rheumatology; Transcription factors; Transfection; Tumor necrosis factor-TNF; Tumor necrosis factor-α; Tyrosine; Western blotting; YY1 protein; Junctional epithelium; Transcription; Inflammation; Tumor necrosis factor-α; Gene promoter; Amelotin; Gingival epithelial cells
• ISSN: 1023-3830; 1420-908X
• DOI: 10.1007/s00011-017-1126-3

Objective Amelotin (AMTN) is an enamel protein that is localized in the basal lamina of ameloblasts in their maturation stage and the internal basal lamina of junctional epithelium (JE) and it is suggested that AMTN could be involved in the dentogingival attachment. To elucidate the transcriptional regulation of human AMTN gene in inflamed gingiva, we have analyzed the effect of tumor necrosis factor-α (TNF-α) on the expression of AMTN gene in Ca9-22 and Sa3 human gingival epithelial cells. Materials and methods Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10 ng/ml). AMTN mRNA and protein levels were measured by real-time PCR and Western blotting. Transient transfection analyses were completed using the various lengths of human AMTN gene promoter constructs with or without TNF-α. Gel mobility shift and chromatin immunoprecipitation assays were performed to investigate the transcription factors bindings to the human AMTN gene promoter by TNF-α. Results TNF-α (10 ng/ml) increased AMTN mRNA and protein levels after 12 h. TNF-α induced luciferase activities of human AMTN gene promoter constructs (− 211AMTN, − 353AMTN, and − 501AMTN). TNF-α-induced luciferase activities were partially inhibited in the mutation − 353AMTN constructs that included 3-bp mutations in CCAAT enhancer-binding protein 1 (C/EBP1), C/EBP2 and Ying Yang 1 (YY1) elements. Transcriptional activities induced by TNF-α were inhibited by protein kinase A, Src-tyrosine kinase, MEK1/2, p38 kinase, NF-κB, and PI3-kinase inhibitors. Gel shift assays showed that TNF-α increased nuclear proteins binding to two types of C/EBP elements (C/EBP1 and C/EBP2) and YY1 element. The results of the chromatin immunoprecipitation assays showed that C/EBPβ binding to C/EBP1 and C/EBP2, and YY1 binding to YY1 were increased by TNF-α. Conclusions These findings demonstrated that TNF-α stimulates AMTN gene transcription in human gingival epithelial cells via C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter.