Fluoride Induces Endoplasmic Reticulum Stress in Ameloblasts Responsible for Dental Enamel Formation

Abstract

The mechanism of how fluoride causes fluorosis remains unknown. Exposure to fluoride can inhibit protein synthesis, and this may also occur by agents that cause endoplasmic reticulum (ER) stress. When translated proteins fail to fold properly or become misfolded, ER stress response genes are induced that together comprise the unfolded protein response. Because ameloblasts are responsible for dental enamel formation, we used an ameloblast-derived cell line (LS8) to characterize specific responses to fluoride treatment. LS8 cells were growth-inhibited by as little as 1.9 -3.8 ppm fluoride, whereas higher doses induced ER stress and caspase-mediated DNA fragmentation. Growth arrest and DNA damage-inducible proteins (GADD153/CHOP, GADD45␣), binding protein (BiP/glucose-responsive protein 78 (GRP78), the non-secreted form of carbonic anhydrase VI (CA-VI), and active X-box-binding protein-1 (Xbp-1) were all induced significantly after exposure to 38 ppm fluoride. Unexpectedly, DNA fragmentation increased when GADD153 expression was inhibited by short interfering RNA treatment but remained unaffected by transient GADD153 overexpression. Analysis of control and GADD153 ؊/؊ embryonic fibroblasts demonstrated that caspase-3 mediated the increased DNA fragmentation observed in the GADD153 null cells. We also demonstrate that mouse incisor ameloblasts are sensitive to the toxic effects of high dose fluoride in drinking water. Activated Ire1 initiates an ER stress response pathway, and mouse ameloblasts were shown to express activated Ire1. Ire1 levels appeared induced by fluoride treatment, indicating that ER stress may play a role in dental fluorosis. Low dose fluoride, such as that present in fluoridated drinking water, did not induce ER stress.

The Kinase Inhibitor Sorafenib Induces Cell Death through a Process Involving Induction of Endoplasmic Reticulum Stress

“…Unexpectedly, results from studies employing siRNA argue against a functional role for GADD153 in sorafenib-mediated lethality. In this regard, results from a recent report suggest a protective role for GADD153 in cell death mediated by fluoride, an agent that induces ER stress in the mouse ameloblast-derived cell line LS8 as well as in MEF cells (29). Finally, induction of constitutively active MEK1 failed to prevent eIF2␣ phosphorylation or GADD153 accumulation.…”

Fluoride Affects Enamel Protein Content via TGF-β1-mediated KLK4 Inhibition

“…Previously, we demonstrated that F -causes endoplasmic reticulum stress in ameloblasts responsible for enamel formation (Kubota et al, 2005); that F --induced endoplasmic reticulum stress inhibits protein synthesis and secretion (Sharma et al, 2008); that, in the presence of F -, culture medium at a moderately low pH increases the level of the stress response (Sharma et al, 2010); and, recently, that Sirt1 and autophagy protect cells from F --induced cell stress . Additionally, we have focused on determining if MMP20 or KLK4 plays a role in dental fluorosis.…”

From acute ER stress to physiological roles of the Unfolded Protein Response

“…It was reported that the UPR plays a role in the ameloblast, especially for its susceptibility to the toxic effects of fluoride exposure. 99 The UPR also plays an essential role in neurons, especially in a large class of conformational diseases associated with accumulation of abnormal protein aggregates, which is discussed in detail by Lindholm in this perspective series.…”