Low pH Enhances Fluoride-induced Endoplasmic Reticulum Stress in Ameloblasts

Enamel development has defined stages. Most mineral precipitates later during the maturation stage when the full-thickness enamel hardens. This mass precipitation of enamel mineral generates large quantities of acid and the enamel matrix can drop to a pH of 6.2 or less. Whitford postulated that fluoride (F) reversibly associates with hydrogen ion (H) to form HF and that intracellular F concentration is determined primarily by the diffusion equilibrium of HF. Since the pH of the cytosol is usually less than the extracellular pH, plasma F levels are typically higher than tissue levels. However, during the maturation stage when the ameloblasts are exposed to the acidic conditions of the enamel matrix, this situation is reversed. Endoplasmic reticulum (ER) stress occurs when proteins are not properly folded. This may result in a generalized reduction of protein synthesis through eIF2a phosphorylation (p-eIF2a) and may ultimately culminate in apoptosis. Objective: To characterize the contribution of extracellular pH to F susceptibility and ask if low-dose F causes ER-stress when ameloblasts are present in an acid environment. Methods: MTT assays, qPCR, Western blots, and immunohistochemistry were utilized to quantify the induction of ER-stress as a function of pH and/or F exposure. Results: F and low-pH significantly reduced cell proliferation when compared with either treatment alone. Low-pH alone significantly reduced expression of anti-apoptotic X-box-binding-protein-1 (Xbp1). However, phosphorylation of pro-apoptotic c-jun (p-c-jun) was significantly enhanced by low-pH and F, but not by either treatment alone. Only low-pH and F combined resulted in increased p-eIF2a levels. Additionally, mouse incisor ameloblasts from the maturation, but not earlier stages of development, stained positive for p-eIF2a in a F dose dependent-manner. Conclusions: The low pH of maturation stage enamel enhances fluoride-induced ER-stress in ameloblasts and may therefore be complicit in dental fluorosis. Supported in part by NIDCR grant DE14084.