Fluoride Exposure Significantly Reduces Protein Secretion in Cultured LS8 Cells

Exposure to excessive amounts of fluoride (F) can trigger dental and skeletal fluorosis in susceptible individuals. We hypothesize that F causes cell stress in ameloblasts that are responsible for enamel formation and that this stress prohibits the ameloblasts from functioning efficiently. Previously, we demonstrated that high dose F induces a stress response in cultured cells and in ameloblasts of mice provided with fluoridated drinking water (J. Biol. Chem. 24:23194-23202, 2005). Objective: Here we ask if low-dose F causes cultured cells to function less efficiently and therefore lead to the initiation of a stress response to alleviate the F-induced stress. Methods: The ameloblast-derived LS8 cell line was stably transfected with secreted alkaline phosphatase (SEAP). SEAP is a highly sensitive reporter protein that traverses the secretory pathway. SEAP activity can be easily assayed from culture supernates and was previously demonstrated to be an excellent indicator of endoplasmic reticulum (ER) stress. Results: We demonstrate that F (2.4 ppm or above) directly inhibits SEAP secretion from LS8 cells in a linear, dose-dependent manner. This decrease is accompanied by a corresponding increase in SEAP intracellular accumulation within the ER. Furthermore, we demonstrate a concurrent induction of the stress-induced chaperone BiP and phosphorylation of the translation initiation factor eIF2a that, with the exception of selected stress response genes, down-regulates overall translation. We also show that the observed reduction of SEAP secretion is not associated with F-induced inhibition of phosphatase activity. Conclusions: These data suggest that F inhibits protein secretion and causes the intracellular accumulation of proteins, thereby initiating an ER-stress response. Thus, F exposure may cause cell stress in ameloblasts which prohibits these cells from functioning efficiently during enamel development. Supported in part by NIDCR grant DE016276.