Smad3 And C-Jun Control Transcription Activity Of Mmp20 In Ameloblasts

Objective To explore the effect of C-jun and Smad3 on tne Mmp20 gene expression by the analyses the AP-1-binding site and Smad-binding site of Mmp20 promoter.Methods1. Analyses of the Mmp20 promoterTo dissect the proximal promoter of the Mmp20 gene,we make five luciferase reporter gene constructs,designated as M1 to M4. Subsequently,Luciferase reporter gene activity assays analysed the transcriptional activity of different length of Mmp20 promoter,finding that the DNA sequence between -157 to -73 contains the cis-regulatory element to be positively regulated by Smad3. In addition,we performed a BLASTN search to find conserved sequences between the human and mouse Mmp20 proximal promoters and we have analyzed the promoter sequence using web software TFSEARCH (transcriptional factor search) and TESS (transcription element search system).2.The study of Smad3 enhancing Mmp20 gene expressionLuciferase reporter gene activity assays analyzed the effect of different dose of pSmad3 on transcription activity of Mmp20 promoter;Electrophoretic mobility shift assay(EMSA) analyzed the binding of Smad3 to Smad-binding site within Mmp20 promoter in vitro;We constructed mutant type of Mmp20 promoter by Smad-binding site or AP-1-binding site-directed mutation, and then Luciferase reporter gene activity assays studied the effects of Smad3 on transcription activity of wild or mutant type Mmp20 promoter.3.The effects of C-jun on the expression of Mmp20 geneLuciferase reporter gene activity assays analyzed the effect of different dose of pC-jun on transcription activity of Mmp20 promoter and the effects of C-jun on transcription activity of wild or mutant type Mmp20 promoter(M3-MT-SBS,M3-MT-ABS,M3-MT-DS);Electrophoretic mobility shift assay(EMSA) analyzed the binding of C-jun to Smad-binding site or AP-1-binding site within Mmp20 promoter in vitro;Luciferase reporter gene activity assays analyzed the effect of Smad3 and C-jun on the transcriptional activity of Mmp20 gene promoter.4.The expression of p-Smad3 and p-C-jun in ameloblastsRT-PCR studied the effect of different dose of pC-jun on Mmp20 gene expression in the presence of Smad3;immunohistochemical study indicated the expression of the phosphorylated Smad3 (p-Smad3)and c-Jun (p-c-Jun) in amelogenesis.5.The role of Smad3 signaling pathway and JNK signaling pathway in Smad3 and C-jun regulating Mmp20 gene expressionwe studied the role of C-jun in the Mmp20 gene expression in the presence of Smad3 inhibitor (SIS3) and the effect of Smad3 on the expression of Mmp20 gene in the presence of JNK inhibitor (SP600125)by RT-PCR.Results1.Luciferase reporter assays of transcription activity of different size of Mmp20 promoter indicate that the DNA sequence between-157 to -73 contains the cis-regulatory element to be positively regulated by Smad3;we performed a BLASTN search to find conserved sequences between the human and mouse Mmp20 promoters, and found that the sequences (-157 to -85) were>80% conserved between the species;We have analyzed the promoter sequence using web software TFSEARCH (transcriptional factor search) and TESS (transcription element search system), and found a 12 bp Smad/Ap-1 box (AGACATGAATCA) in the promoter. As a result,we can use mouse Mmp20 proximal promoter to study human Mmp20 gene expression,which is representative.2. The results of Luciferase reporter assays are shown that:high dose of pSmad3 significantly up-regulates luciferase activity in ALCs;EMSA shows complex formation of the oligonucleotide probe containing Smad-binding element and nuclear extracts of HEK 293T cells transfected with pSmad3. The results of supershift assay,mutagenesis analyses and cold competition demonstrate the specific binding between Smad3 and the identified responsive region within Mmp20 promoter;Subsequently,we perform site-directed mutagenesis analysis of the luciferase assay.Luciferase assays show that promoter activity is suppressed when Smad-binding site is mutated.So Smad-binding element plays an important role in Smad3 up-regulating expression level of Mmp20 and Smad3 up-regulates Mmp20 expression at transcriptional level through the sequence of SBE.3. Luciferase reporter assays indicate that low dose of pC-jun up-regulates Mmp20 promoter activity, while high dose of pC-jun down-regulates the promoter activity; Luciferase analysis shows that the inhibitory effect of c-Jun on Mmp20 promoter is minimally changed by mutation of the ABS in the promoter, while c-Jun significantly enhanced the promoter activity by mutation of the SBS in the promoter; double mutations of the SBS and ABS completely abolished the effects of c-Jun on Mmp20 promoter activity;EMSA shows that Smad3/C-jun complex can bind to the SBS within Mmp20 promoter,C-jun complex can bind to the ABS within Mmp20 promoter;Luciferase reporter assay indicates that Smad3 enhanced Mmp20 promoter activity and in the presence of C-jun,enhancement of Smad3 to Mmp20 promoter activity disappears.4.RT-PCR analyses shows that low dose of pC-jun elevates slightly expression of the Mmp20 gene with or without pSmad3 co-transfection.However,inhibition of relative high-dose pC-jun to Mmp20 expression is more enhanced with pSmad3 co-transfection than that of pC-jun without pSmad3 co-transfection;Immunohistochemistry study shows that p-c-Jun and p-Smad3 are co-expressed in ameloblasts during secretory stage of enamel development where Mmp20 is expressed; The expression of p-c-Jun, but not p-Smad3, significantly increased at transition stage where Mmp20 expression is eliminated in ameloblasts.Consequently,Smad3 is in fact involved in the regulation of high dose of pC-jun to the Mmp20 gene and low dose of pC-jun enhances Mmp20 gene expression indenpendently on Smad3.5.The result of RT-PCR indicated that Smad3 inhibitor augmented expression of Mmp20 gene induced by C-jun;Smad3 significantly enhanced Mmp20 expression in the presence of JNK inhibitor.Conclusion Smad3 enhances the expression of Mmp20 by Smad-binding site within Mmp20 promoter;Low dose of pC-jun up-regulates Mmp20 gene expression via AP-1-binding site;high dose of pC-jun suppresses Mmp20 gene expression by interacting with Smad3 via Smad-binding site within Mmp20 promoter.JNK signaling pathway takes part in the regulation of Smad3 to the Mmp20 gene expression;Smad3 signaling pathway is involved in the regulation of C-jun to the Mmp20 gene expression.