The overall goal of this project is to define the role of endoplasmic reticulum (ER) stress in Dental fluorosis by identifying genes and molecular pathways that respond to fluoride (F) exposure. The ER mediates protein synthesis, protein folding, and post-translational modification. Perturbations in ER homeostasis can interfere with these processes resulting in the accumulation of unfolded or misfolded proteins that cause ER distention and trigger ER-stress. ER-stress activates specific signaling pathways, termed the unfolded protein response (UPR). The UPR induces: I) chaperone expression to help fold the accumulated proteins, II) reduces overall protein synthesis to allow the ER to cope with the existing proteins, III) directs the degradation of misfolded proteins, and IV) initiates apoptosis. Previously we have shown that F activates the UPR in vivo and in vitro (1). These data corroborate prior studies demonstrating F-induced ER distention (2) and defective ER to Golgi protein transport (3). Also, UPR-mediated reduction in overall protein synthesis may preclude F exposed ameloblasts from actively removing enamel matrix proteins from maturation stage enamel. This could cause the increased protein content observed in fluorosed enamel. We posit that the UPR pathways initiated in response to F-induced ER-stress play a role in Dental fluorosis. Therefore, Aim 1 is to identify UPR genes induced by F and determine if these genes play a role in fluorosis. In this project, we will determine if gene mutations in specific UPR pathways make cultured cells more susceptible to F or make mice more susceptible or resistant to fluorosis. Studies will be performed in: the ameloblast-derived LS8 cell line;LS8 cells carrying the dominant-negative XBP1 expression plasmid;Xbp1+/+, Xbp1+/-, Xbp1-/-;Perk+/+ and Perk-/- mouse embryo fibroblasts (MEFs);and Xbp1+/+, Xbp1+/-, Perk+/+ and Perk+/- mice.
Aim 2 is to characterize the contribution of extracellular pH to F susceptibility and to determine if low-dose F causes ER-stress when ameloblasts are present in an acid environment. We posit that acidification of the enamel matrix during the maturation stage of tooth development drives F into ameloblasts and that this increased concentration of intracellular F induces ER stress that culminates in Dental fluorosis. The preliminary studies demonstrate that an acidic environment reduces the threshold F dose required to: a) inhibit proliferation and induce toxicity in vitro, b) activate UPR pathway genes in cultured cells, and c) activate UPR pathway genes in mouse ameloblasts in vivo. In this project, we will determine if cells are more sensitive to ER-stress at low pH, determine if the UPR pathways are altered as a function of low pH, and confirm the cell culture results in ameloblasts from mice with induced acidosis. Public Health Significance: If ER-stress plays a major role in Dental fluorosis, chemical treatments may be available to help ameloblasts properly fold their ER proteins and prevent fluorosis. Chemical treatment of mice for ER-stress improves diabetic glucose homeostasis (4;5) and protects against cerebral ischemic injury (6). Project Narrative The prevalence of fluorosis among the population is increasing (7) yet we currently know very little about what causes fluorosis. We have previously demonstrated that fluoride elicits a cell stress response which in turn, activates genes to help the cell cope with the stress (1). This application seeks to identify those stress response genes and determine if they play a role in causing Dental fluorosis.
|Suzuki, Maiko; Everett, Eric T; Whitford, Gary M et al. (2017) 4-phenylbutyrate Mitigates Fluoride-Induced Cytotoxicity in ALC Cells. Front Physiol 8:302|
|Faibish, D; Suzuki, M; Bartlett, J D (2016) Appropriate real-time PCR reference genes for fluoride treatment studies performed in vitro or in vivo. Arch Oral Biol 62:33-42|
|Suzuki, Maiko; Bandoski, Cheryl; Bartlett, John D (2015) Fluoride induces oxidative damage and SIRT1/autophagy through ROS-mediated JNK signaling. Free Radic Biol Med 89:369-78|
|Suzuki, Maiko; Sierant, Megan L; Antone, Jerry V et al. (2014) Uncoupling protein-2 is an antioxidant that is up-regulated in the enamel organ of fluoride-treated rats. Connect Tissue Res 55 Suppl 1:25-8|
|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|
|Sierant, Megan L; Bartlett, John D (2012) Stress response pathways in ameloblasts: implications for amelogenesis and dental fluorosis. Cells 1:631-45|
|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|