The Regulatory Effects Of TGF-β1/TGFBR1/Smad3 Signaling On The Expression Of AMTN Gene In Ameloblasts

AIM:To investigate the regulatory of TGF-β1/TGFBR1/Smad3 signaling on the expression of AMTN gene in ameloblasts, and provide an ideal experimental platform for studying the effects of TGF-β1/TGFBR1/Smad3 signaling in dental enamel developing。METHODS:1 TGF-β1 regulates AMTN gene expression through transcription factor Smad3 in ameloblastsWe use Reverse transcription-polymerase chain reaction (RT-PCR) to investigate the effects of TGF-β1 and Smad3 on the expression of AMTN gene in ameloblasts. Western blot was used to analyzed phosphorylation kinetics of Smad3 in response to TGF-β1 stimulation. we used Smad3 siRNA, specific inhibitors of Smad3 and TGFBRI to evaluate their effect on TGF-β1-stimulated expression of AMTN.2 The regulatory effects of TGF-β1/Smad3 signaling on the promoter activity of AMTN gene in ameloblastsDual luciferase analysis was used to observe the effects of TGF-β1 and Smad3 on the transcriptional activity of AMTN promoter in ameloblasts.The Smad3 binding sites on the AMTN promoter are mutated. Finally Dual lueiferase analysis was used to observe the effects of TGF-β1 and Smad3 on the transcriptional activity of the mutant or wild type AMTN promoter.3 Smad3 interacts with Smad binding site within the AMTN promoter.We performed electrophoretic mobility shifting assay (EMSA) and Chromatin Immunoprecipitation (ChIP) assay to indicated that Smad3 interacted with Smad binding site within the AMTN promoter.Results:1 After ameloblasts are stimulated by 5ng/ml TGF-β1 2.5 hours, Real-time RT-PCR analysis demonstrated the strongest induction of AMTN mRNA expression. TGF-β1 significantly enhanced the phosphorylation of Smad3 as early as 15 mins after TGF-β1 treatment and peaked at 30min (p<0.05). The group treated by TGFBRI inhibitor SB431542 revealed a significant up-regulation of Smad3 phosphorylation as early as 15 min but the level of p-Smad3 expression was consistant from 15 min to 45 min after TGF-β1 treatment.After Smad3 siRNA, specific inhibitors of Smad3 and TGFBRI were used to ameloblasts, AMTN expression induced by TGF-β1 were significantly abolished (p<0.05). The specific inhibitors of Smad3 also inhibited the expression of AMTN which significantly enhanced by overexpression of pcaTGFBRI (p<0.05).2 We found a putative Smad-binding element "AGAC" at the position-1418/-1415 of AMTN promoter and the "AGAC" were mutated to "TGTC".Dual luciferase analysis demonstrated that transcriptional activity of the AMTN promoter was significantly promoted by the effects of TGF-β1. Smad3 and overexpression of pcaTGFBRI. Mutation of the Smad-binding element inhibited the transcriptional activity of the mouse AMTN promoter.3 Electrophoretic mobility shifting assay(EMSA) and Chromatin Immunoprecipitation (ChIP)assay suggested that transcription factor Smad3 could bind to SBE-(-1418/-1415) "AGAC" of Amtn promoter.Conclutions:TGF-β1 regulates AMTN gene expression through transcription factor Smad3 in ameloblasts, Smad3 gene silencing significantly inhibit TGF-β1-induced AMTN gene expression; We found a Smad-binding element "AGAC" which interact with Smad3 in Amtn promoter, TGF-β1/TGFBRI/Smad3 signaling could mediate the AMTN gene expression via the Smad-binding element "AGAC".