Fluoride Alters Gene Expression via Histone H3K27 Acetylation in Ameloblast-like LS8 Cells

Abstract

Excessive fluoride ingestion during tooth development can cause dental fluorosis. Previously, we reported that fluoride activates histone acetyltransferase (HAT) to acetylate p53, promoting fluoride toxicity in mouse ameloblast-like LS8 cells. However, the roles of HAT and histone acetylation status in fluoride-mediated gene expression remain unidentified. Here, we demonstrate that fluoride-mediated histone modification causes gene expression alterations in LS8 cells. LS8 cells were treated with or without fluoride followed by ChIP-Seq analysis of H3K27ac. Genes were identified by differential H3K27ac peaks within ±1 kb from transcription start sites. The levels of mRNA of identified genes were assessed using rea-time PCR (qPCR). Fluoride increased H3K27ac peaks associated with Bax, p21, and Mdm2 genes and upregulated their mRNA levels. Fluoride decreased H3K27ac peaks and p53, Bad, and Bcl2 had suppressed transcription. HAT inhibitors (Anacardic acid or MG149) suppressed fluoride-induced mRNA of p21 and Mdm2, while fluoride and the histone deacetylase (HDAC) inhibitor sodium butyrate increased Bad and Bcl2 expression above that of fluoride treatment alone. To our knowledge, this is the first study that demonstrates epigenetic regulation via fluoride treatment via H3 acetylation. Further investigation is required to elucidate epigenetic mechanisms of fluoride toxicity in enamel development.

Keywords: ChIP-Seq; HAT; HDAC; ameloblasts; amelogenesis; dental fluorosis; epigenetics; histone modification.

Figure 1

Analysis of gene expressions associated with acetylation of H3K27 via fluoride in LS8 cells. LS8 cells were treated with sodium fluoride (NaF) (0 to 5 mM) for 24 h. (A) Integrative Genomics Viewer (IGV) images show H3K27ac patterns of Bax, Mdm2, and p21 from ChIP-Seq. Y-axis denotes ChIP signal amplitude, while x-axis indicates genome positions. The blue dotted box shows the increase in the peak area in the promoter regions induced with 5 mM NaF treatment. (B) The mRNA levels of Bax, p21, and Mdm2 were analyzed using real-time PCR (qPCR) (N = 4/group). Data are presented as means ± SD. ** p < 0.01, NS; no significant differences.

Figure 2

Analysis of gene expressions associated with deacetylation of H3K27 via fluoride in LS8 cells. LS8 cells were treated with NaF (0 to 5 mM) for 24 h. (A) Integrative Genomics Viewer (IGV) images show H3K27ac patterns of Bcl2, Bad, and p53 from ChIP-Seq. Y-axis denotes ChIP signal amplitude, while x-axis indicates genome positions. The blue dotted box shows the reduced peak area in the promoter regions induced via 5 mM NaF treatment. (B) The mRNA levels of Bcl2, Bad, and p53, and (C) the Bax/Bcl2 mRNA ratio were analyzed using qPCR (N = 4/group). Data are presented as means ± SD. ** p < 0.01.

Figure 3

Effects of histone acetyltransferase (HAT) inhibitors (Anacardic acid; AA and MG149) on gene expressions associated with histone H3K27 acetylation via fluoride. LS8 cells were treated with 50 μM AA or 50 μM MG149 for 1 h prior to 5 mM NaF treatment for 24 h. mRNA levels were evaluated via qPCR (N = 5/group for AA, N = 4/group for MG149). (A) Addition of AA to NaF significantly decreased mRNA of p21 and the Bax/Bcl2 mRNA ratio compared to NaF treatment alone, but other genes were not changed with the addition of AA. (B) Addition of MG149 to NaF significantly decreased mRNA of p21 and Mdm2 and the Bax/Bcl2 mRNA ratio compared to NaF treatment alone, but other genes were not changed by MG149 addition. Control (Ctrl) and NaF included vehicles (DMSO 0.1% for AA and 0.04% for MG149). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, NS; no significant differences.

Figure 4

The effect of fluoride on class I, II, and III histone deacetylases (HDACs) in LS8 cells. LS8 cells were treated with 5 mM NaF for 24 h. The protein levels of phosphorylated HDACs, p-HDAC2 (Ser394) (62 kDa), p-HDAC3 (Ser424) (49 kDa), p-HDAC7 (Ser155) (124 kDa), and p-SIRT1 (Ser47) (82 kDa) were detected via western blot (WB). (A) Fluoride treatment attenuated the protein levels of class I HDACs (p-HDAC2 and p-HDAC3) and class II HDAC, p-HDAC7. (B) Class III HDAC, p-SIRT1, expression was increased with fluoride treatment. α-Tubulin (52 kDa) and β-actin (44 kDa) were used as loading controls. Representative images are shown. Quantification and statistical analyses of relative protein levels are shown in Supplementary Figure S1A.

Figure 5

The effect of sodium butyrate (SB) on class I, II, and III HDACs in LS8 cells. LS8 cells were treated with 5 mM NaF for 24 h. The protein levels of phosphorylated HDACs, p-HDAC2 (Ser394) (62 kDa), p-HDAC3 (Ser424) (49 kDa), p-HDAC7 (Ser155) (124 kDa), and p-SIRT1 (Ser47) (82 kDa) were detected via WB. SB attenuated the protein levels of p-HDAC2, p-HDAC7 (A), and p-SIRT1 (B). SB did not change p-HDAC3 (A). α-Tubulin (52 kDa) and β-actin (44 kDa) were used as loading controls. Representative images are shown. Quantification and statistical analyses of relative protein levels are shown in Supplementary Figure S1B.

Figure 6

Increase in histone acetylation (H3ac and H3K27ac) via sodium butyrate in LS8 cells. LS8 cells were treated with 1 mM SB with or without 5 mM NaF for 24 h. (A) H3ac (17 kDa) levels were detected via WB. SB increased the protein level of H3ac dose-dependently. β-Actin (44 kDa) was used as a loading control. Representative images are shown. Quantification and statistical analyses of relative protein levels are shown in Supplementary Figure S2. (B) SB treatment for 24 h increased the signal of H3ac in nuclei compared to control (Ctrl). Nucleus (DAPI; blue), H3ac (red), and Actin (green) were detected via immunofluorescence. Representative images are shown. Scale bars; 50 μm. (C) H3K27ac (17 kDa) expressions were detected via WB. SB increased the protein levels of H3ac and H3K27ac with or without fluoride. β-Actin (44 kDa) was used as a loading control. Representative images are shown. Quantification and statistical analyses of relative protein levels are shown in Supplementary Figure S3.

Figure 7

The effect of sodium butyrate on identified genes’ mRNAs which are associated with histone H3K27 deacetylation via fluoride treatment. LS8 cells were treated with 1 mM SB for 1 h prior to 5 mM NaF treatment for 24 h. qPCR results showed that addition of SB significantly increased mRNA levels of Bcl2 and Bad compared to NaF alone, but p53 mRNA level was not changed with SB addition. Data are presented as means ± SD. (N = 4/group). ** p < 0.01, NS; no significant differences.

Figure 8

Sodium butyrate suppresses fluoride-mediated apoptosis in LS8 cells. LS8 cells were treated with 1 mM SB for 1 h prior to 5 mM NaF for 24 h. (A) qPCR results showed that addition of SB significantly decreased the mRNA levels of p21 and Mdm2 but did not change Bax mRNA. (B) SB addition suppressed the mRNA ratio of Bax/Bcl2 compared to NaF treatment alone. Data are presented as means ± SD. (N = 4/group). ** p < 0.01, NS; no significant differences. (C) Cleaved-caspase-3 (17 kDa) was detected via WB. SB with fluoride significantly decreased the protein level of cleaved-caspase-3 compared to fluoride treatment alone. β-Actin (44 kDa) was used as a loading control. Representative images are shown. Quantification and statistical analyses of relative protein levels are shown in Supplementary Figure S5.

Figure 9

Schema of fluoride-mediated gene alterations via H3K27 acetylation in LS8 cells. Fluoride activates HATs and upregulates acetylation of H3K27 near transcription start sites (TSS) of p21, Mdm2, and Bax genes. The mRNA levels of p21 and Mdm2 are increased, but Bax mRNA levels are not changed. HAT inhibitors AA (CBP/P300 and PCAF inhibitor) or MG149 (Tip60/KAT5 inhibitor) suppress the transcription of p21 and Mdm2 associated with acetylation of H3K27. Fluoride suppresses active form class I HDAC (p-HDAC2 and p-HDAC3) and class II HDAC (p-HDAC7), which is involved in the increase in histone acetylation. On the other hand, fluoride activates class III HDAC (p-SIRT1) which is involved in deacetylation of H3K27 to suppress mRNA levels of Bcl2, Bad, and p53. HDAC inhibitor sodium butyrate (SB) suppresses fluoride-mediated SIRT1 activation to increase the mRNA levels of Bcl2 and Bad. Up arrows: increase, Down arrows: decrease, Right arrows: no change.