Transcriptional regulation of human amelotin gene by interleukin‐1β

Abstract

One of the major causes of tooth loss is chronic inflammation of the periodontium, the tissues surrounding the tooth. Amelotin ( AMTN ) is a tooth enamel protein which is expressed in maturation‐stage ameloblasts and also in the internal basal lamina of junctional epithelium, a unique epithelial structure attached to the tooth surface which protects against the constant microbiological challenge to the periodontium. Localization of AMTN suggests that its function could be involved in the dentogingival attachment. The purpose of this study was to investigate the effect of interleukin‐1β ( IL ‐1β) on AMTN gene transcription in human gingival epithelial Ca9‐22 cells. IL ‐1β increased AMTN mRNA and protein levels at 3 h, and the levels reached maximum at 6 and 12 h. IL ‐1β induced luciferase activities of human AMTN gene promoter constructs (−211, −353, −501, −769, and −950AMTN), but these activities were partially inhibited in −353AMTN constructs that included 3‐bp mutations in CCAAT /enhancer binding protein 1 (C/ EBP 1), C/ EBP 2, and Ying Yang 1 ( YY 1) elements. Transcriptional activities induced by IL ‐1β were abrogated by protein kinase A ( PKA ), tyrosine kinase, mitogen‐activated protein kinase kinase ( MEK 1/2), and phosphatidylinositol 3‐kinase ( PI 3K) inhibitors. Gel shift and ChIP assays showed that IL ‐1β increased C/ EBP β binding to C/ EBP 1 and C/ EBP 2, and YY 1 binding to YY 1 elements after 3 h, and that these DNA –protein interactions were inhibited by PKA , tyrosine kinase, MEK 1/2, and PI 3K inhibitors. These results demonstrated that IL ‐1β increases AMTN gene transcription in human gingival epithelial cells mediated through C/ EBP 1, C/ EBP 2, and YY 1 elements in the human AMTN gene promoter.

C/EBPβ and YY1 bind and interact with Smad3 to modulate lipopolysaccharide‐induced amelotin gene transcription in mouse gingival epithelial cells

“…LPS could induce IL‐1β and TNF‐α production in THP‐1 cells , and IL‐1β and TNF‐α increased Amtn gene transcription in gingival epithelial cells . Similar to the results of these studies, we demonstrated that Pg LPS induced Amtn mRNA levels after 24 h treatment and upregulated mouse Amtn gene transcription via MEK1/2, PI3‐K and ALK5 signaling pathways in GE1 cells (Figs and ).…”

“…It is known that LPS upregulates ALK5 expression in the induction of TNF‐α biosynthesis . Regulatory mechanisms of Amtn gene transcription by Pg LPS are consistent with those for IL‐1β and TNF‐α, indicating that IL‐1β and TNF‐α induced transcription factor binding to C/EBP1, C/EBP2 and YY1 elements (Figs C, and ).…”

“…YY1 is a multifunctional zinc‐finger transcription factor belonging to the Polycomb protein family. This family is a group of homeobox gene receptors that regulate cell cycle and hematopoiesis, associated with cancer biology . YY1 is tightly associated with cancer; however, a relationship between Amtn and tumors has been reported in a few studies.…”

“…Their complementary oligonucleotide and Cy5‐labeled oligonucleotide were purchased (Sigma‐Aldrich Japan; Table ), and they were annealed under optimal conditions. The following steps were carried out as described in a recent study . Briefly, nuclear proteins (20 μg) and 0.1 p m Cy5‐labeled double‐stranded oligonucleotide were combined and incubated for 20 min at room temperature (RT) in the binding buffer; then, the DNA–nuclear protein complexes were separated by electrophoresis in 5% acrylamide gels.…”

“…LPS is one of the multiple virulence factors in the Gram‐negative bacterium and therefore might contribute to the initiation and progression of periodontal diseases . In our previous studies, tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) stimulate human Amtn gene transcription via CCAAT enhancer‐binding protein ( C/EBP ) and Ying Yang 1 ( YY1 ) elements . The relationship between C/EBPs and inflammation has been reported and YY1 is also a transcription factor associated with inflammation and cancer .…”