Abstract
Fluorosed maturation stage enamel is hypomineralized in part due to a delay in the removal of matrix proteins to inhibit final crystal growth. The delay in protein removal is likely related to reduced expression of kallikrein-related peptidase 4 (KLK4), resulting in a reduced matrix proteinase activity that found in fluorosed enamel. Klk4 transcription is known to be regulated in other cell types by androgen receptor (AR) and progesterone receptors (PR). In this study, we determined the possible role of fluoride in down-regulation of KLK4 expression through changes in AR and PR. Immunohistochemical localization showed that both AR and PR nuclear translocation was suppressed in fluoride exposed mice. However, when AR signaling was silenced in mouse ameloblast-lineage cells (ALCs), expression of both Pgr and Klk4 were increased. Similar to the effect from AR silencing, fluoride also upregulated Pgr in ALCs, but downregulated Klk4. This finding suggests that though suppression of AR transactivation by fluoride increases Prg expression, inhibition of PR transactivation by fluoride has a much greater effect, ultimately resulting in downregulation of Klk4 expression. These findings indicate that in ameloblasts, PR has a dominant role in regulating Klk4 expression. We found that when AR was retained in the cytoplasm in the presence of fluoride, that co-localized with heat shock protein 90 (HSP90), a well-known chaperone for steroid hormone receptors. HSP90 also known to regulate TGF-β signaling. Consistent with the effect of fluoride on AR and HSP90, we found evidence of reduced TGF-β signaling activity in fluorosed ameloblasts as reduced immunolocalization of TGFB1 and TGFBR-2 and a significant increase in Cyclin D1 mRNA expression, which also possibly contributes to the reduced AR signaling activity. In vitro, when serum was removed from the media, aluminum was required for fluoride to inhibit the dissociation of HSP90 from AR. In conclusion, fluoride related downregulation of Klk4 is associated with reduced nuclear translocation of AR and PR, and also reduced TGF-β signaling activity, all of which are regulated by HSP90. We suggest that a common mechanism by which fluoride affects AR, PR, and TGF-β signaling is through inhibiting ATP-dependent conformational cycling of HSP90.
Highlights
Enamel formed in the presence of high levels of fluoride has a delayed removal of matrix proteins (Den Besten, 1986; DenBesten and Thariani, 1992), most likely due to reduced proteolytic activity in the maturation stage fluorosed enamel (DenBesten et al, 2002)
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)
If KLK4 proteinase activity is reduced in the fluorosed enamel matrix, it should be due to reduced KLK4 synthesis
Summary
Enamel formed in the presence of high levels of fluoride has a delayed removal of matrix proteins (Den Besten, 1986; DenBesten and Thariani, 1992), most likely due to reduced proteolytic activity in the maturation stage fluorosed enamel (DenBesten et al, 2002). Reduced proteolytic activity in maturation stage fluorosed enamel is consistent with reduced expression of kallikrein-related peptidase 4 (KLK4) (Suzuki et al, 2014), the serine proteinase that is responsible for the final hydrolysis of matrix proteins, which allows enamel hydroxyapatite crystals to reach their full thickness. Binding of the ligand (i.e., androgenic hormones) promotes nuclear translocation and transactivation for transcription of the target genes (Azad et al, 2015). Jedeon and co-workers have reported that the AR is present in ameloblasts and when activated, regulates KLK4 expression (Jedeon et al, 2016)
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