Enamel formation begins with the secretion of enamel matrix proteins during the secretory stage of enamel development. Together, these proteins form a matrix that organizes the hydroxyapatite crystals of the enamel. Once the crystals reach their full length, ameloblasts secrete KLK4 to degrade the matrix proteins, allowing the crystals to grow in width and thickness. The degraded proteins are then resorbed by ameloblasts, leaving behind fully mature, hardened enamel that has a mineral content greater than 96%. Compared to normal enamel, fluorosed enamel has a lower mineral content and a higher protein content 
and therefore, has reduced hardness. Retention of the matrix proteins is thought to be responsible for the higher protein content of fluorosed enamel 
. It was previously suggested that F−
decreases KLK4 activity, resulting in increased protein retention 
. However, mechanisms leading to reduced KLK4 activity are not known.
Ameloblasts are unique because, during the maturation stage of enamel formation, they are in direct contact with the acidic mineralizing enamel matrix (pH<6.0) 
. They are not as well-protected as other cells exposed to low pH, such as the cells lining the stomach. The latter are sheltered by a bicarbonate-rich mucus barrier that neutralizes the acid produced during digestion 
, and are continually replaced every 3–5 days. Ameloblasts, on the other hand, do not have any protective barriers and are not regenerated. Therefore, maturation stage ameloblasts may be directly exposed to F−
under low pH conditions.
Several reports point toward a relation between F−
and pH. For example, a decrease in pH facilitated the entry of F−
into L929 fibroblasts 
. In addition, F−
-mediated cytotoxicity in osteosarcoma cells was enhanced by low pH 
uptake in micro-organisms also occurs as a function of the culture medium pH gradient 
-resistant mutants become more sensitive to effects of F−
at low pH 
. In vivo
absorption rate from the stomach increased as the gastric pH decreased 
. Similarly, a decrease in serum pH increased F−
absorption in the hamster cheek pouch and in the renal tubules of rat 
, rabbit 
, dog 
and human 
. Conversely, less fluoride was excreted as the urinary pH decreased 
. Significantly, rats rendered acidotic by treatment with NH4
Cl retain increased quantities of F−
in their dental enamel 
. Therefore, the more acidic the extracellular fluid, the greater the tissue fluoride concentration 
Here, we propose a novel, integrated mechanism based on pH and cell stress to explain the development of dental fluorosis. We hypothesize that F− is converted to HF during the acidic maturation stage of enamel development and that HF flows down a steep pH concentration gradient from the enamel matrix into the ameloblast cytosol. The neutral pH inside the cell reverts HF to F−. Excess F− within the cell interferes with ER homoestasis, inducing ER stress and activation of the UPR (), resulting in compromised ameloblast function.
Schematic showing our postulated mechanism for maturation stage ameloblast sensitivity to fluoride.
We validate our hypothesis by demonstrating that low pH enhanced F−
-mediated stress in vitro
and in vivo
. Phosphorylation of eIF2α was observed in the papillary layer as well as in the maturation stage ameloblasts. The complete absence of staining in the control (untreated) maturation stage ameloblasts as well as the papillary layer suggests that the staining is specific. However, the results are not surprising. Maturation stage ameloblasts are in contact with the papillary layer near the basal terminal bars 
. Ameloblasts and papillary layer cells are extensively interconnected by several large gap junctions 
. The presence of numerous coated vesicles and also microvilli in the papillary cells suggest that they function similar to ameloblasts in the transport of ions, water and small nutrients during maturation 
. Therefore, it is possible that fluoride ions within the ameloblast could reach the papillary cells through the gap junctions. This would result in papillary cell stress and consequently, lead to the phosphorylation of eIF2α. Moreover, carbon-dioxide produced within the ameloblasts during metabolism can lead to the formation of bicarbonate ions and hydrogen ions, catalyzed by carbonic anhydrases (as shown below):
Ameloblasts contain at least 2 different carbonic anhydrases, CA2 and CA6 
. Because the blood capillary-rich papillary layer is in close proximity with the ameloblasts, it is likely that the H+
ions are pumped to the capillaries and that this will cause a local decrease in the extracellular pH of the papillary layer as well.
We also showed that F−
inhibited cell function (Gluc secretion) in a pH-dependent manner. Indeed, F−
-mediated decrease in protein synthesis and/or secretion has been well-documented 
. Importantly, we demonstrated a decrease in enamel matrix transcripts during the maturation stage.
Taken together, our data show that F− can regulate KLK4 activity by at least 3 different mechanisms. First, F− can decrease KLK4 synthesis through stress-mediated phosphorylation of the translation initiation factor, eIF2α. This results in transient attenuation of global translation. Second, F− can also decrease KLK4 secretion from ameloblasts. Third, F− can decrease the steady state levels of mRNAs expressed during the maturation stage. While this can occur for all proteins that pass through the secretory pathway, it is especially important for Klk4. Reduced Klk4 expression may hinder enamel matrix protein degradation and their removal. These mechanisms of F− action provide an explanation for the higher protein content in fluorosed enamel as compared to normal enamel.
In conclusion, our research points toward a novel mechanism to explain fluorosis – namely, that the low pH environment of the maturation stage ameloblasts renders them more susceptible to F− toxicity and that pH could be a defining factor in determining sensitivity of tissues to fluoride.