The long-term goal of this project is to identify genes that respond to F- exposure and protect against F- toxicity. Fluoride (F-) protects teeth from caries, but F- over-exposure causes dental fluorosis in a large and growing proportion of U.S. children. Importantly, dermal exposure of just 2.5% of body surface to F- as hydrofluoric acid (HF) is lethal. Yet, the molecular pathways and genes involved in the F- stress response are not well characterized. Previously studies demonstrated that F- induces phosphorylation of the stress-response mediator eIF2a (eIF2a-P). This occurs both in vitro and in vivo in mouse and rat incisor ameloblasts. This project tests the hypothesis that fluoride causes a stress response in ameloblasts to alleviate F- toxicity and that this stress response is mediated through eIF2a-P. eIF2a is a component of the ribosome that is necessary to translate proteins from mRNA templates. eIF2a phosphorylation inhibits overall protein translation, but will preferentially translate specific downstream stress response mRNAs that help cells to cope with a given stress. Depending upon the type of initiating stress, eIF2a can be phosphorylated by any one of four different kinases -- Gcn2 (Eif2ak4), Hri (Eif2ak1), Perk (Eif2ak3), and Pkr (Eif2ak2) - each of which responds to different stress stimuli. The objective of this project is to identify F--induced up- and downstream mediators of eIF2a-P so that a F-- induced stress response pathway can be elucidated.
AIM 1 is to identify upstream mediators of F- -induced eIF2a phosphorylation. We will identify the responsible kinase and determine if F- activates it directly or indirectly such as by inducing endoplasmic reticulum (ER) stress or oxidative stress.
AIM 2 is to identify downstream mediators of phosphorylated eIF2a (eIF2a-P) that alleviate stress. We will assess expression of genes such as Atf4 that are known to be regulated by eIF2a-P and will perform polysome profiling to identify previously unknown F--induced eIF2a-P regulated genes. We will isolate transcribed mRNAs in F--treated wild-type cells and in mutant cells that cannot phosphorylate eIF2a. Mutant cell polysome mRNAs will be eliminated from further analysis because they were not induced by eIF2a-P. Since eIF2a-P inhibits overall translation, microarray analysis should provide a manageable set of eIF2a-P regulated genes to characterize.
AIM 3 is to identify up- and downstream mediators of eIF2a-P in mouse ameloblasts in vivo. We will confirm that the F--induced eIF2a-P molecular pathway is the same in both cultured cells in vitro and in vivo in mouse ameloblasts responsible for enamel formation. Preliminary data suggest that F- activates Hri and that Hri phosphorylates eIF2a. Our colony of Hri null mice will be used to determine if Hri mediated phosphorylation of eIF2a protects mouse ameloblasts from F- toxicity. We predict that F- treated Hri-/- mice will have softer than normal enamel due to F- toxicity of ameloblasts. Completion of this study will provide a defined F--induced molecular pathway and will identify stress genes that protect cells from F-.

Public Health Relevance

The prevalence of dental fluorosis among the population is increasing. But, the genes that act to protect us from fluorosis remain unknown. Here we show that eIF2a-P is part of a fluoride-induced stress response pathway and our focus is to identify its up- and downstream components. Once this is accomplished, we will have defined a unique fluoride-induced stress response pathway that may be manipulated to prevent toxicity. The knowledge gained from this project could be used to prevent death from dermal exposure to hydrogen fluoride and from ensuing natural disasters such as the volcanic eruption in Iceland where people and livestock perished from hydrogen fluoride poisoning. Gene expression or lack thereof in this pathway may be diagnostic for individual fluoride susceptibility. Currently, we know little about how organisms respond to the toxic effects of fluoride, but that will change with the completion of this project. We are excited about the possibility of bringing to light a new and unique fluoride-mediated stress response pathway that may benefit those who are exposed to toxic concentrations of fluoride.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
2R01DE018106-03
Application #
8235253
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Wan, Jason
Project Start
2007-04-01
Project End
2017-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
3
Fiscal Year
2012
Total Cost
$594,208
Indirect Cost
$295,611
Name
Forsyth Institute
Department
Type
DUNS #
062190616
City
Cambridge
State
MA
Country
United States
Zip Code
02142
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Suzuki, M; Shin, M; Simmer, J P et al. (2014) Fluoride affects enamel protein content via TGF-?1-mediated KLK4 inhibition. J Dent Res 93:1022-7
Suzuki, Maiko; Bartlett, John D (2014) Sirtuin1 and autophagy protect cells from fluoride-induced cell stress. Biochim Biophys Acta 1842:245-55
Sharma, R; Tye, C E; Arun, A et al. (2011) Assessment of dental fluorosis in Mmp20 +/- mice. J Dent Res 90:788-92
Tye, C E; Antone, J V; Bartlett, J D (2011) Fluoride does not inhibit enamel protease activity. J Dent Res 90:489-94
Sharma, Ramaswamy; Tsuchiya, Masahiro; Skobe, Ziedonis et al. (2010) The acid test of fluoride: how pH modulates toxicity. PLoS One 5:e10895