(A) Schematic diagrams of the plasmids that were used to transfect NIH 293 cells. Plasmids pGL3-basic and pGL3-control served as a promoterless negative and positive control for the luciferase reporter gene assay. A DNA fragment containing 1855 bp 5′ flanking sequence upstream of the initiating ATG of human MMP-20 gene was cloned into pGL3-basic vector in the sense orientation, and designated as pGL3-MMP20. (B) Twenty-four hours after transfection, cells transfected with pGL3-control and pGL3-MMP20 displayed large amounts of luciferase activity compared to cells transfected with a promoterless pGL3-basic plasmid. Addition of fluoride to the pGL3-MMP20 transfected cells decreased their luciferase activity by 35%. One way ANOVA shows p < 0.05, Tukey’s multiple comparison post-test shows * is significantly different from the rest of samples (n=3). Fluoride had no effect on the cells transfected with pGL3-control.

(A) Total proteins of ameloblast lineage cells exposed to 10 μM of fluoride for 0, 15 min, 30 min or 60 min time course were analyzed by Western blot assay. Actin served as a loading control. (B) Densitometry analyses of the immunoreactive bands show that fluoride did not have a significant effect on pan JNK and c-Jun protein levels. However, phosphorylation levels of JNK and c-Jun in the ameloblast lineage cells were significantly decreased after 30 min and 60 min duration. One way ANOVA shows p < 0.05, Tukey’s multiple comparison post-test shows * is significantly different from the 0 min control (n=3).

(A) A schematic diagram shows the sequences and location of four potential AP-1 binding motifs within the human MMP-20 promoter, predicted by computer analysis and published report. The numbers indicate the distance from the transcription initiation site to the most distal sequence of the AP-1 binding motif within the promoter of the human MMP-20 gene. (B) Immobilized biotinylated double-stranded oligonucleotides containing the sequences of each of AP-binding motif and flanking region were used to precipitate proteins from the total cell lysate of ameloblast lineage cells. Western blot analysis shows that oligonucleotides representing the -112AP-1, -711AP-1 or -1456 AP-1 binding motif precipitated c-Jun protein from total cell lysate. (C) Immobilized rabbit anti-human pan c-Jun antibody or rabbit non-immune IgG was incubated with cross-linked and fragmented DNA/protein complex isolated from ameloblast lineage cells treated by PMA. The precipitated DNA/protein complex was reverse cross-linked and the DNA was extracted. PCR was performed to amplify sequences flanking individual AP-1 binding site within the MMP-20 promoter by using extracted DNA as template and oligonucleotides complimentary with the end of the flanking sequence of -112AP-1, -711AP-1 or -1456 AP-1 binding motif as primers (product was designated as c-Jun). DNA/protein complex precipitated by immobilized c-Jun antibody served as a negative control (−), and DNA extracted from reverse cross-linked of fragmented total DNA/protein complex served as a positive control (+). PCR products at length 176 bp, 230 bp or 162 bp containing sequences flanking -112AP-1, -711AP-1 or -1456 AP-1 binding motif were visualized on a 1.2% agarose gel. The results suggest that c-Jun binds to the AP-1 sites on the promoter of the MMP-20 gene as MMP-20 was induced.

(A) Western blot analyses for the amounts of amelogenin, MMP-20 and TIMP-2 after 24 h of fluoride exposure. Actin served as a loading control. (B) The graph shows the relative intensity of each immunoreactive band measured with NIH Image software. Fluoride significantly decreased MMP-20 levels by 21% compared to the untreated sample as determined by t-test, *, P < 0.05 (n=3). Amounts of amelogenin and TIMP-2 in ameloblast lineage cells were not significantly affected by fluoride.

(A) Western blot was performed to assess the expression of MMP-20 after 24 h of incubation with MAPK inhibitors specific to p38, MEK1/2 or JNK subgroup. Expression of actin served as a loading control. (B) Densitometry analyses of the immunoreactive bands show only SP600125, the JNK specific inhibitor had an effect. It lowered MMP-20 protein levels by 24% compared to control. The difference is significant based on one-way ANOVA followed by Tukey’s multiple comparison post-test (n=3).

(A) Total proteins of the ameloblast lineage cells exposed to 400 nM of PMA for either 0, 15 min, 30 min or 60 min were resolved on a 10% SDS-PAGE gel, then transferred to nitrocellulose membrane for Western blot assay. Actin served as a loading control. (B) Densitometry analyses of the immunoreactive bands shows PMA increased the phosphorylation of JNK or c-Jun at 15 min or 30 min duration. One way ANOVA shows p < 0.05, Tukey’s multiple comparison post-test shows * is significantly different from the 0 min control (n=3). (C) Total proteins from ameloblast lineage cells incubated with 400 nM of PMA for 24 h, were subjected to Western blot analysis to asses the expression of MMP-20. Actin served as loading control. (D) Densitometry analyses of the immunoreactive bands show PMA up-regulated the expression of MMP-20 in those cells. * versus control, p < 0.05, determined by t-test (n=3).

(A) Schematic diagrams show the structure of the plasmid carrying luciferase reporter gene driven by either a wild type or mutated MMP-20 promoter. QuikChange II site–directed mutagenesis kit was used to delete the various AP-1 binding sites at a time on the pGL3-MMP20 construct. Plasmids were used to transfect NIH 293 cells and its luciferase reporter activity was assessed. (B) Luciferase activity measured 24 h after transfection, dramatically decreased by 85%, 44% or 58% in NIH 293 cells transfected with mutated plasmid with a deletion of either -112AP-1, -711AP-1 or -1456AP-1 binding site respectively compared to cells transfected with the pGL3-MMP20 construct. One way ANOVA shows p < 0.05, Tukey’s multiple comparison post-test shows * is significantly different from the rest of the samples (n=3).