All animal procedures were carried out with approval by the University of California-San Francisco Institutional Animal Care and Use Committees. The experiments reported herein were conducted in compliance with the Animal Welfare Act and in accordance with the principles set forth in the National Research Council's Guide for the Care and Use of Laboratory Animals.

To determine whether the reduced Klk4 expression in the presence of fluoride resulted in decreased KLK4 activity in the enamel matrix, 3-week-old female Wistar rats (Jackson Laboratory, Sacramento, CA) were divided into two groups, with the groups given either deionized drinking water or deionized drinking water supplemented with 100 ppm (5.3 mM) fluoride as sodium fluoride (Sigma-Aldrich, St. Louis, MO) ad libitium for 4 weeks. After 4 weeks, the rats were euthanized, and the mandibular incisors were dissected to allow access to separately dissected the enamel matrix and enamel organ for KLK4 activity assays, and quantitative real-time polymerase chain reaction (qPCR) of the relative expression of Klk4 mRNA.

Three-week-old C57BL/6J female mice (Jackson Laboratory) were divided into two groups, with the groups given either deionized drinking water or deionized drinking water supplemented with 50 ppm (2.6 mM) fluoride as sodium fluoride (Sigma-Aldrich) ad libitium for 4 weeks. After 4 weeks, the mice were euthanized, and mandibles were obtained for morphology, immunohistochemical, and qPCR analyses.

Rat mandibular incisors were removed from the alveolar bone and enamel matrix was dissected from the underlying dentin surface at the early-maturation stage. This includes enamel matrix underlying the distal root of the first molar and continuing until the enamel was too hard to dissect (Stahl et al., 2015). Extracts were homogenized in 100 μl of 5% TCA, and incubated at room temperature for 30 min. After centrifugation at 10,000 × g for 10 min, the supernatants were removed and pellets were re-suspended in 200 μl of ammonium bicarbonate. The total protein concentration of each sample was measured by Bradford assay. KLK4 activity was analyzed by using Boc-Val-Pro-Arg-AMC fluorogenic peptide substrate (R&D Systems Inc., Minneapolis, MN). Each reaction in 96-well black plates contained 150 μl of protein extracts, 5 μl of peptide and 40 μl of 5x reaction buffer (250 mM Tris–HCl, 250 mM NaCl, 50 mM CaCl). The fluorescence signal was detected using a spectrometer at 37°C (excitation at 380 nm and emission at 460 nm), and measured every 20 min for 180 min. For each sample, the fluorescence data was normalized to the protein concentration of a given sample. For each sample, we used R software environment with drc package (Ritz and Streibig, 2005; Team, 2014) to generate an averaged 4-parametric regression curve of the treatment groups. Significance of differences at 60, 120, and 180 min were determined by independent Student's t-test.

Total RNA was isolated from (rat and mouse) mandibular maturation-stage incisor enamel organs that were dissected according to landmarks described previously (Stahl et al., 2015), and also from ameloblast lineage cells (ALCs; detail in Cell Culture) using RNeasy Mini kit (Qiagen, Germantown, MD). Conversion of mRNA to cDNA was obtained by reverse transcription of the mRNA using Superscript III First-Strand Synthesis Supermix for qRT-PCR (Life Technologies, Carlsbad, CA).

Expression of mRNAs was examined by qPCR with FastStart Universal SYBR Green Master Kit (Roche Diagnostics, Indianapolis, IN) using primer sets for Klk4, Ccnd1, Ar, Tgfbr2, and Tgfb1 (Elim Biopharmaceuticals, Hayward, CA). Mrpl19 was used as a reference gene for enamel organ samples and Eef1a1 for ALCs. Primer sequences are listed in Table 1. The relative expression level of target genes was analyzed by the ΔΔCt method (Livak and Schmittgen, 2001). Expression of each gene was calculated as a relative expression level (fold change) compared with WT (mice samples) or untreated controls (cell culture). Significance of differences was determined using ΔCt values by the two-tailed multiple t-test with Benjamini & Hochberg correction following ANOVA (Benjamini and Hochberg, 1995).

Mouse mandibles were fixed in 4% paraformaldehyde in 0.06 M sodium cacodylate buffer (pH 7.3) at 4°C for 24 h. After decalcification in 8% EDTA (pH 7.3), samples were processed for routine paraffin embedding and sagittally sectioned. The sections were incubated with 10% swine and 5% goat sera followed by incubation with rabbit anti-human AR (1:75; Novus Biologicals, Littleton, CO, NB100-91658), rabbit anti-mouse TGFBR2 (1:100; Santa Cruz Biotech, Santa Cruz, CA, sc-1700), rabbit anti-human TGFB1 (1:50; Abcam PLC, Cambridge, MA, ab92486), and rabbit anti-mouse PR (Santa Cruz Biotech, sc-166170) antibodies respectively overnight at room temperature. A biotinylated swine anti-rabbit IgG F(ab')2 fraction (Dako, Carpinteria, CA) was used as the secondary antibody for 1 h at room temperature incubation. Following incubation with alkaline phosphatase conjugated streptavidin (Vector Laboratories Inc., Burlingame, CA) for 30 min, immunoreactivity was visualized using a Vector® Red kit (Vector Laboratories) resulting in pink/red color for positive staining. Counter-staining was performed with methyl green (Dako). Negative control was done with normal rabbit sera.

Mouse ameloblast-lineage cells (ALCs, a kind gift from Dr. Toshihiro Sugiyama, Akita University, Japan and Dr. John Bartlett, Forsyth Institute, Boston, Massachusetts) (Nakata et al., 2003) were cultured in DMEM (UCSF Cell Culture Facility, San Francisco, CA) supplemented with 10% Fetal Bovine Serum (FBS) (Gemini Bio-Products, West Sacramento, CA) and 1% penicillin-streptomycin. ALCs were seeded into 6-well plates (2.0 × 10⁵ cells/cm²) or 8-well chamber slides (1.5 × 10⁵ cells/cm²). After 24 h, the cells were treated with fluoride (0 or 1 mM) as sodium fluoride (Sigma-Aldrich), followed by total RNA extraction. Other cells, initially cultured in the medium with FBS for 24 h, were exposed to 2% TCM® serum replacement (MP Biomedicals, Santa Ana, CA) for 24 h. After 24 h, cells were transfected with antisense oligonucleotides targeting AR, LNA™ GapmeRs (Exiqon Incorporated, Woburn, MA) to silence Ar mRNA, using Lipofectamine 2000 Transfection Reagent (ThermoFisher Scientific, Waltham, MA). Cells were then harvested for total RNA and extracts were analyzed for Klk4, Ar, Pgr, Tgfb1, Tgfbr2, and Ccnd1 expression. For immunofluorescent staining, some cells were treated with fluoride as described above. Other cells were initially cultured in FBS containing medium for 24 h. After culturing in the medium with 2% TCM® serum replacement for another 24 h, cells were treated with 0 or 1 μM dihydrotestosterone/DHT (Cerilliant Corporation, Round Rock, TX). Some of the DHT treated cells were further exposed to aluminum (0, 10, or 100 μM) as aluminum chloride (Sigma-Aldrich) and/or fluoride (0 or 1 mM) as sodium fluoride (Sigma-Aldrich). Aluminum concentration was equivalent to the range of those in calf serum (Tomza-Marciniak et al., 2011)

Cells grown in 8-well chamber slides were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature and washed with PBS. They were then permeablized with 0.25% Triton-X 100 and incubated with 10% swine and 5% goat sera to block non-specific binding. Cells were then simultaneously incubated with AR (Novus Biologicals) and HSP90 (Santa Cruz Biotech) antibodies that were fluorescently tagged with Zenon Rabbit IgG Labeling Kit (Molecular Probes, Eugene, OR) per manufacturer's instructions. AR antibody was labeled with Alexa Fluor 594 and HSP90 antibody was labeled with Alex Fluor 488. After antibody incubation, cells were washed with PBS, then counterstained with DAPI and mounted with mounting medium.

To determine the ultimate effect of fluoride on KLK4 synthesis, we assessed synthesis of KLK4 in fluorosed rat enamel, by comparing KLK4 proteinase activity in the enamel matrix of rats given either 0 or 100 ppm fluoride in drinking water. Enamel matrix protein extracts of rats given 100 ppm F in drinking water had reduced matrix KLK4 proteolytic activity when compared with control rats (Figure 1A), and significance of the reduction was confirmed at three different time points of incubation (Figure 1B). Consistent with the reduced proteolytic activity in the enamel matrix, Klk4 mRNA expression was significantly reduced (Figure 1C). Similar to rats, fluorosed mice (given 50 ppm F in drinking water) also had reduced Klk4 expression (data not shown). To further investigate the effect of fluoride on molecular profiles in maturation ameloblasts, we therefore used the mouse model.

Mice given high levels of fluoride in drinking water had retained enamel matrix (Figures 2A,B), with no obvious morphological alteration in ameloblasts (Figures 2C,D). To investigate the association of AR with fluorosis, we examined the presence and localization pattern of AR in maturation stage ameloblasts. In control mice, most of the AR immunostaining was in the ameloblast nucleus (Figure 2E), whereas in mice ingesting fluoride, immunostaining showed AR mostly remained in the cytoplasm (Figure 2F). Similarly, in vitro, we found that ALCs grown in serum containing medium with 1 mM fluoride had significantly less Klk4 expression as compared to ALCs grown in medium without fluoride (Figure 3A). Under this condition, Ar expression (Figure 3A) and cell proliferation (data not shown) were not changed, but visibly less AR localized in the nuclei (Figure 3B). These in vivo and in vitro data together show that nuclear translocation of AR is inhibited by fluoride.

Nuclear translocation of steroid hormone receptors is well-known to be regulated by the heat shock proteins (HSPs) (Azad et al., 2015). HSP90 plays a major role in the activation process of the steroid hormone receptors by interacting with the unliganded steroid hormone receptors to open the steroid-binding cleft to allow access by a steroid (Pratt et al., 2004). Once the ligand binds to the steroid hormone receptors, HSP90 disassociates from the receptors, allowing the nuclear translocation of the steroid receptors to function as a transcription factor (Azad et al., 2015). To determine if the dissociation of HSP90 from AR is altered by fluoride, we co-immunostained AR and HSP90, and observed co-localization of HSP90 and AR remained in the cytoplasm under the influence of fluoride. We found that HSP90 and AR co-localized in the cytoplasm particularly in the perinuclear region (Figure 3C). In the presence of fluoride, mRNA transcription and protein synthesis of HSP90 were unchanged (data not shown).

Previously, Suzuki et al. reported that fluoride treated ALCs had decreased mRNA expression of Tgfβ1 and Klk4 (Suzuki et al., 2014). In normal and oncogenic prostate cells, AR signaling is known to cross-talk with the TGF-β signaling pathway (Gerdes et al., 1998; Bruckheimer and Kyprianou, 2001; Chipuk et al., 2002; Kang et al., 2002; Pratt et al., 2004; Yang et al., 2014). Furthermore, activation of TGF-β signaling is also regulated by HSP90, and inhibition of HSP90 induces degradation of TGF-β receptors (Wrighton et al., 2008).

To examine TGF-β signaling in ameloblasts, when fluoride inhibits AR activation in vivo, we immunostained maturation ameloblasts from fluoride treated and control mice for TGFB1 and TGF-β1 receptor type II (TGFR-2). In fluorosed ameloblasts, immunostaining for both TGFB1 and TGFR-2 was reduced (Figure 4A). Tgfβr2 transcription was also significantly reduced in fluorosed ameloblasts (Figure 4B), indicating a reduced TGF-β signaling activity. Cyclin D1 (CCND1) is a protein commonly known for regulating cell cycle dynamics, and expression of Ccnd1 is known to be negatively regulated by TGF-β1 signaling in intestinal epithelial cells (Ko et al., 1995). Moreover, CCND1 is known to further negatively regulate the AR function, not only by directly binding to AR to inhibit transactivation of AR, but also by attenuating AR-induced upregulation of Klk4 in prostate cancer (Knudsen et al., 1999; Comstock et al., 2011). Supporting our findings of reduced TGF-β signaling and inhibited AR activation in maturation ameloblasts, Ccnd1 expression in fluorosed ameloblast was significantly increased (Figure 4B). These results suggest that the previously described effects of fluoride on TGF-β signaling, as related to decreased Klk4 expression, are also possibly mediated by the effects of fluoride on HSP90.

AR is known to regulate Klk4 expression, but it does not directly interact with the Klk4 gene (Lai et al., 2009). PR induces Klk4 expression in breast cancer cells through direct binding to the Klk4 promoter (Lai et al., 2009). AR suppresses expression of PR-regulated genes in triple negative breast cancer (Tsang et al., 2014; Karamouzis et al., 2016), suggesting negative regulation of AR on Pgr expression. To understand how AR regulates Klk4 transcription in ameloblasts as well as the potential involvement of PR, we silenced Ar mRNA in ALCs and examined the expression of Klk4 and Pgr. The antisense oligonucleotides for Ar significantly reduced Ar mRNA expression in ALCs, but significantly upregulated both Klk4 and Pgr expression (Figure 5A). This suggests a negative regulation of Pgr by AR, while PR directly upregulates Klk4 expression.

To further examine the effect of fluoride induced suppression of AR activation on Pgr transcription, we examined Pgr expression in ameloblasts of fluorosed mice and in ALC culture. In both in vitro (Figure 5B), and in vivo (Figure 5C), fluoride exposure resulted in significantly increased Pgr expression. As PR nuclear translocation is also chaperoned by HSP90 (Dao-Phan et al., 1997), we examined the change of intracellular localization of PR in fluorosed maturation ameloblasts. Similar to the effects of fluoride on AR, nuclear translocation of PR was reduced in fluorosed ameloblasts as compared to controls (Figure 5D). Therefore, though fluoride increased Pgr expression, it inhibited nuclear translocation of both PR and AR, resulting in reduced expression of Klk4 (see diagram shown in Figure 5E).

Among the nuclear factor family steroid hormone receptors, whose activation strictly depends on the interaction with HSP90, glucocorticoid receptor is the most well-studied steroid hormone receptor (Grad and Picard, 2007). Housley et al. showed that fluoride requires aluminum to stabilize HSP90 binding to glucocorticoid receptor (Housley, 1990). To determine whether aluminum is also required for the fluoride related cytoplasmic stabilization of AR by HSP90 in ameloblasts, we cultured ALCs and examined the intracellular AR localization in presence of fluoride and aluminum. ALCs were grown in serum containing media for 24 h, after which the media was changed to one with an artificial serum substitute (TCM® serum replacement). We found that AR nuclear translocation was stimulated by dihydrotestosterone (DHT) in ALCs (Figure 6A). However, when nuclear translocation was stimulated by DHT, the addition of fluoride alone did not alter the intracellular localization of AR, whereas in the presence of both fluoride and aluminum, more AR remained in the cytoplasm (Figure 6B). Ar transcription was not significantly changed in the presence of aluminum and fluoride (data not shown). Further immunostaining showed that the increased cytoplasmic AR in the presence of both fluoride and aluminum co-localized with HSP90 (Figure 6B). An increased AR retention in the cytoplasm was observed in both 10 μM (data not shown) and 100 μM aluminum supplemented culture.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.