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• W1985247208 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, GADD45α), 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 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, GADD45α), 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. Fluoride is an effective caries prophylactic. However, acute or chronic exposure to fluoride can result in enamel (1DenBesten P.K. Community Dent. Oral Epid. 1999; 27: 41-47Crossref PubMed Scopus (97) Google Scholar) and skeletal fluorosis (2Boivin G. Chavassieux P. Chapuy M.C. Baud C.A. Meunier P.J. Bone (NY). 1989; 10: 89-99Crossref PubMed Scopus (108) Google Scholar), renal toxicity (3Zager R.A. Iwata M. Am. J. Pathol. 1997; 150: 735-745PubMed Google Scholar), and epithelial lung cell toxicity (4Thrane E.V. Refsnes M. Thoresen G.H. Lag M. Schwarze P.E. Toxicol. Sci. 2001; 61: 83-91Crossref PubMed Scopus (99) Google Scholar). Fluoride is present in fresh water at concentrations of less than 0.1 ppm to >100 ppm, and concentrations of ∼1.6–1.8 ppm in drinking water are the threshold for fluorosis risk among the population (5Whitford G.M. Myers H.M. The Metabolism and Toxicity of Fluoride. 2nd Ed. Karger, New York1996: 1-156Google Scholar). Fluoride ingestion between the ages of 15 and 30 months may be the most critical for fluorosis of the esthetically important human maxillary central incisors (6Evans R.W. Stamm J.W. J. Public Health Dent. 1991; 51: 251-259Crossref PubMed Scopus (107) Google Scholar, 7Evans R.W. Darvell B.W. J. Public Health Dent. 1995; 55: 238-249Crossref PubMed Scopus (93) Google Scholar). It is during this time that dental enamel forms on the unerupted permanent teeth. Rodents, including mice and rats, have continuously erupting incisors that manifest each developmental stage of enamel formation (amelogenesis). Moving from the distal tip of the incisor back to where the incisor grows beneath the molars, the developmental stages become progressively less mature. The initial stage of enamel development is the secretory stage. The columnar ameloblast cells of the enamel organ are responsible for dental enamel development. During the secretory stage the ameloblasts are tall, contain an extensive endoplasmic reticulum (ER), 1The abbreviations used are: ER, endoplasmic reticulum; BiP, binding protein; CHOP, C/EBP homologous protein; CA-VI, carbonic anhydrase VI; GADD, growth arrest and DNA damage-inducible; MEF, mouse embryonic fibroblast; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; UPR, unfolded protein response; Xbp-1, X-box-binding protein-1; ELISA, enzyme-linked immunosorbent assay; z-, benzyloxycarbonyl; siRNA, short interfering RNA; TUNEL, terminal dUTP nick-end labeling; EO, enamel organ; CMV, cytomegalovirus. 1The abbreviations used are: ER, endoplasmic reticulum; BiP, binding protein; CHOP, C/EBP homologous protein; CA-VI, carbonic anhydrase VI; GADD, growth arrest and DNA damage-inducible; MEF, mouse embryonic fibroblast; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; UPR, unfolded protein response; Xbp-1, X-box-binding protein-1; ELISA, enzyme-linked immunosorbent assay; z-, benzyloxycarbonyl; siRNA, short interfering RNA; TUNEL, terminal dUTP nick-end labeling; EO, enamel organ; CMV, cytomegalovirus. and secrete large amounts of protein into the enamel matrix. During the maturation stage the ameloblasts are short, and in contrast to the secretory stage, they absorb proteins from the enamel matrix (8Smith C.E. Crit. Rev. Oral Biol. Med. 1998; 9: 128-161Crossref PubMed Scopus (542) Google Scholar). In 1977 Smith and Warshawsky (9Smith C.E. Warshawsky H. Anat. Rec. 1977; 187: 63-98Crossref PubMed Scopus (107) Google Scholar) demonstrated that during the transition between the secretory and maturation stages (transition stage) an approximate 25% loss of rat incisor ameloblasts occurs with another 25% loss before the completion of the maturation stage. Subsequent studies confirmed the presence of apoptotic ameloblasts associated with normal rodent incisor development (10Bronckers A.L. Lyaruu D.M. Goei W. Litz M. Luo G. Karsenty G. Woltgens J.H. D'Souza R.N. Eur. J. Oral Sci. 1996; 104: 102-111Crossref PubMed Scopus (54) Google Scholar, 11Bronckers A.L. Goei S.W. Dumont E. Lyaruu D.M. Woltgens J.H. van Heerde W.L. Reutelingsperger C.P. van den Eijnde S.M. Histochem. Cell Biol. 2000; 113: 293-301Crossref PubMed Scopus (36) Google Scholar, 12Kondo S. Tamura Y. Bawden J.W. Tanase S. Arch. Oral Biol. 2001; 46: 557-568Crossref PubMed Scopus (27) Google Scholar, 13Nishikawa S. Sasaki F. Histochem. Cell Biol. 1995; 104: 151-159Crossref PubMed Scopus (49) Google Scholar). Although enamel fluorosis research has spanned seven decades (14Schour I. Smith M. J. Am. Dent. Assoc. 1935; : 796-813Google Scholar), no consensus exists as to the mechanism of its cause. One school of thought presents compelling evidence that fluorosis is caused primarily by the presence of excessive fluoride within the forming enamel. The fluoride ions are postulated to adversely affect the precipitation of hydroxyapatite that forms the enamel structure (15Aoba T. Fejerskov O. Crit. Rev. Oral Biol. Med. 2002; 13: 155-170Crossref PubMed Scopus (335) Google Scholar). Although this postulate has merit, it does not adequately explain why different inbred strains of mice had different susceptibilities/resistance to enamel fluorosis, whereas the overall levels of fluoride present in their erupted incisors did not differ significantly (16Everett E.T. McHenry M.A. Reynolds N. Eggertsson H. Sullivan J. Kantmann C. Martinez-Mier E.A. Warrick J.M. Stookey G.K. J. Dent. Res. 2002; 81: 794-798Crossref PubMed Google Scholar). Additionally, fluoride concentrations in enamel from unerupted human molars did not correlate positively with fluorosis severity, and it was concluded that individual genetic variation likely plays a role in fluorosis susceptibility (17Vieira A.P. Hancock R. Limeback H. Maia R. Grynpas M.D. J. Dent. Res. 2004; 83: 76-80Crossref PubMed Scopus (53) Google Scholar). We examined the possibility that fluoride can cause an endoplasmic reticulum (ER) stress response. ER stress occurs when nascent proteins are not folded properly and/or are misfolded, leading to the initiation of the unfolded protein response (UPR). As the unfolded proteins accumulate in the ER, the chaperone-binding protein BiP is released into the lumen and binds to hydrophobic regions on the surface of the unfolded proteins to facilitate proper folding (18Kaufman R.J. J. Clin. Investig. 2002; 110: 1389-1398Crossref PubMed Scopus (1072) Google Scholar). The UPR can activate three different primary ER stress response pathways (18Kaufman R.J. J. Clin. Investig. 2002; 110: 1389-1398Crossref PubMed Scopus (1072) Google Scholar, 19Harding H.P. Calfon M. Urano F. Novoa I. Ron D. Annu. Rev. Cell Dev. Biol. 2002; 18: 575-599Crossref PubMed Scopus (795) Google Scholar, 20Oyadomari S. Mori M. Cell Death Differ. 2004; 11: 381-389Crossref PubMed Scopus (2178) Google Scholar). The only pathway that is conserved among all eukaryotic cells is that initiated by Ire1 (21Liu C.Y. Kaufman R.J. J. Cell Sci. 2003; 116: 1861-1862Crossref PubMed Scopus (159) Google Scholar). Ire1 is both a kinase and an endoribonuclease. ER stress initiates Ire1 autophosphorylation and subsequent RNase activity specific for spliceosome-independent processing of Xbp1 mRNA. Processing of the Xbp1 mRNA by Ire1 results in a translation frameshift that allows encoding of active Xbp1 (22Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Crossref PubMed Scopus (2067) Google Scholar, 23Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2870) Google Scholar). Active Xbp1 is a basic leucine zipper (bZIP) transcription factor that can bind to and initiate transcription from both the ER stress response element (ERSE) and the UPR element (23Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2870) Google Scholar, 24Wang Y. Shen J. Arenzana N. Tirasophon W. Kaufman R.J. Prywes R. J. Biol. Chem. 2000; 275: 27013-27020Abstract Full Text Full Text PDF PubMed Google Scholar). The UPR will also adapt to ER stress by translation attenuation, by protein degradation, or finally, by apoptosis. Although GADD45α is not known to be part of the UPR, it is a cell cycle checkpoint protein that arrests cells at G2/M phase (25Jin S. Antinore M.J. Lung F.D. Dong X. Zhao H. Fan F. Colchagie A.B. Blanck P. Roller P.P. Fornace Jr., A.J. Zhan Q. J. Biol. Chem. 2000; 275: 16602-16608Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). GADD153/CHOP is an ER stress response gene that can also be induced by other stressors such as amino acid deprivation and exposure to oxidants (26Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2273) Google Scholar, 27Jousse C. Bruhat A. Harding H.P. Ferrara M. Ron D. Fafournoux P. FEBS Lett. 1999; 448: 211-216Crossref PubMed Scopus (79) Google Scholar). The GADD153/CHOP gene encodes a bZIP C/EBP homologous protein that forms heterodimers with other CCAAT enhancer-binding proteins (C/EBP) (28Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (969) Google Scholar). When phosphorylated by p38 mitogen-activated protein kinase, GADD153 becomes a more potent inducer of apoptosis. GADD34 was recently demonstrated to be directly activated by GADD153 (29Marciniak S.J. Yun C.Y. Oyadomari S. Novoa I. Zhang Y. Jungreis R. Nagata K. Harding H.P. Ron D. Genes Dev. 2004; 18: 3066-3077Crossref PubMed Scopus (1441) Google Scholar). Even so, the downstream effects of GADD153 expression are not well characterized (20Oyadomari S. Mori M. Cell Death Differ. 2004; 11: 381-389Crossref PubMed Scopus (2178) Google Scholar). In this study we utilize the LS8 ameloblast cell line that was derived from mouse enamel organ (30Chen L.S. Couwenhoven R.I. Hsu D. Luo W. Snead M.L. Arch. Oral Biol. 1992; 37: 771-778Crossref PubMed Scopus (96) Google Scholar) to assess whether fluoride can induce ER stress and initiate the UPR. We present evidence that fluoride induces an ER stress response involving caspases and increased expression levels of Bip, Xbp-1, GADD153, GADD45α, Ire1, and the non-secreted form of carbonic anhydrase VI (CA-VI). Cell Culture—The mouse ameloblast-derived cell line (LS8) and the CHOP-/- and CHOP+/+ (referred to as GADD153-/- or -+/+) mouse embryo fibroblasts were maintained in α minimal essential medium (Invitrogen) supplemented with fetal bovine serum (10%), penicillin (50 units/ml), and streptomycin (50 μg/ml). Sodium Fluoride Treatment and Determination of Cell Growth and Viability—To assess cell viability and growth, survival and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed. For survival assays, LS8 cells were plated at a density of 1000 cells in T-25 cm2 flasks for 18 h and exposed to concentrations of NaF (stock, 100× in water) from between 0 and 2 mm for a period of 24 h. Cells were washed with phosphate-buffered saline and allowed to grow in fresh medium for ∼8–9 days. The resulting colonies were stained with 0.5% methylene blue in 50% methanol and counted. Percent cell survival was then calculated (31Bartlett J.D. Luethy J.D. Carlson S.G. Sollott S.J. Holbrook N.J. J. Biol. Chem. 1992; 267: 20465-20470Abstract Full Text PDF PubMed Google Scholar, 32Bertrand R. Kerrigan D. Sarang M. Pommier Y. Biochem. Pharmacol. 1991; 42: 77-85Crossref PubMed Scopus (97) Google Scholar). For MTT assays, cells were plated in 96-well plates, and the experiments were performed after 18 h. The indicated concentrations of NaF were added to the wells. After 24 and 48 h, cell growth was determined by measuring MTT (Sigma) reductase activity. Briefly, MTT (0.5 mg/ml final concentration) was added and incubated for 3.5 h. After removal of the medium, crystals were dissolved in dimethyl sulfoxide (Me2SO) (Sigma), and optical density was measured at 550 nm using a microplate reader (HTS 7000 Bio Assay Reader, PerkinElmer Life Sciences). Six wells were analyzed, and the mean value was calculated for each NaF concentration. Experiments were performed in triplicate. DNA Fragmentation ELISA Assay—DNA fragmentation was assayed using the Cell Death Detection ELISA kit (Roche Diagnostics). Cells were incubated in the presence or absence of either 50 μm z-VAD-fluoromethyl ketone (Promega) or 2 μm z-DEVD-fluoromethyl ketone (BioVision) for 1 h at 37 °C and then treated with NaF for 24 or 48 h followed by processing for the Cell Death ELISA assay. The procedure was performed according to the manufacturer's instructions. All assays were performed in triplicate. Reverse Transcriptase-Polymerase Chain Reaction—Total RNA was extracted from LS8 cells with Trizol reagent (Invitrogen), and cDNA was prepared using the SuperScript first-strand synthesis system (Invitrogen). PCR primers were: CA-VI Type A (sense), 5′-AGTGCTGGGCTTAGTTTAGAGCTTTCC-3′, CA-VI Type B (sense), 5′-TCCTGCATTCAGGGCTACAGCATCTG-3′, and CA-VI type A and B (antisense), 5′-AGATCGATCGATACTGTGTGTCCGT-3′. Northern Blot Analysis—The GADD153, BiP, GADD45α, XBP-1, β-actin, and CA-VI type B cDNA fragments were labeled with [α-32P]dCTP (6000 Ci/mmol) (PerkinElmer Life Sciences) using Prime-It RmT random primer labeling kit (Stratagene). In brief, 50 ng of PCR product was added to the reagent mix (containing random primers and dNTPs) and boiled for 5 min. Ten μl of [α-32P]dCTP and 3 μl of magenta DNA polymerase were added to the boiled mix and incubated at 37 °C for 10 min. The labeled cDNA fragment was denatured and added to the hybridization solution. Ten μg of total RNA was run on a formaldehyde-agarose gel and transferred to a Hybond-N nylon membrane (Amersham Biosciences). The membrane was prehybridized in hybridization solution with 0.1 mg/ml heat-denatured salmon sperm DNA (Invitrogen) at 65 °C for 1 h. 32P-Labeled cDNA was added, and the membrane was incubated at 65 °C overnight. The membrane was washed (0.1 × SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate), 0.5% SDS) at 65 °C 3 times for 30 min each. Membranes were stripped and re-probed for the indicated mRNAs. Extraction and Treatment of Primary Porcine Enamel Organ Epithelial Cells—Third molar tooth buds were removed from the mandible of a 6-month-old pig and placed in Hanks' balanced salt solution supplemented with 0.02% EDTA for 30 min. Enamel organs were dissected from mineralizing tooth cusps and pulp organs. The enamel organs were dissociated into a single cell suspension as previously described (33Young C.S. Kim S.W. Qin C. Baba O. Butler W.T. Taylor R.R. Bartlett J.D. Vacanti J.P. Yelick P.C. Arch. Oral Biol. 2005; 50: 259-265Crossref PubMed Scopus (41) Google Scholar) with the modification that any undissociated tissue pieces were eliminated by passing the suspension through a 40-μm cell sieve (BD Biosciences). Approximately 3 × 106 cells were obtained per enamel organ. Enamel organ cells were grown in Primaria T-75 flasks in LHC-8 medium (BIOSOURCE) supplemented with 10% fetal bovine serum, penicillin/streptomycin/amphotericin B, and 0.5 μg/ml epinephrine at 37 °C in 5% CO2. After 10–12 days in culture, a fast-growing fibroblast-like cell population grew to confluency and then expired. Slow-growing epithelial cells remained. These cells were polygonal and grew as tightly clustered colonies. The cells were 80–90% confluent after an additional 20 days in culture. Cells were harvested (trypsin) from flasks, counted, and inoculated into 6-well plates at a density of 2.5 × 105 cells per well. The next day duplicate wells were treated or not with 2 mm NaF for 48 h followed by collection of total RNA with Trizol® reagent (Invitrogen). This procedure was performed on EO cells from two different 6-month-old pigs. Real-time PCR Analysis of GADD153 and BiP Expression in Primary Porcine Enamel Organ Epithelial Cells—SuperScript III first-strand synthesis system for reverse transcription-PCR (Invitrogen) generated the cDNA for real-time PCR analysis. Primer sequence for expression analyses were: porcine BiP, forward, 5′-AAGACAAAGGTACAGGCAACAAAA-3′, reverse, 5′-CTCAGCAAACTTCTCAGCATCATT-3′; porcine GADD153, forward, 5′-CCTTGGGCTACTGCTGAC-3′, reverse, 5′-CATGAATAGAGGGGGTTGAG-3′. Internal control primers for porcine eEF1α1 were: forward, 5′-GATGGAAAGTCACCCGTAAAGATG-3′, and reverse, 5′-GTTGGACGAGTTGGTGGTAGAATG-3′. The PCR temperature profile was 3 min at 95 °C (initial melt) then 20 s at 95 °C, 30 s at 65 °C for 45 cycles, and 30 s at 95 °C for 1 cycle, and 1 min at 55 °C followed by stepwise temperature increases from 55 to 95 °C to generate the melt curve. Standard curves were generated with each primer set by use of untreated control cDNA preparations and a 10-fold dilution series ranging from 1000 ng/ml to 100 pg/ml. PCR efficiencies and relative expression levels of GADD153 and BiP as a function of eEF1α1 expression were calculated as previously described (34Pfaffl M.W. Nucleic Acids Res. 2001; 29: 2002-2007Crossref Scopus (24586) Google Scholar). Western Blot Analysis—LS8 cells were plated in 10-cm dishes and treated with 2 mm NaF or 0.5 μg/ml tunicamycin (Sigma) for 24 h. Nuclear proteins were extracted by resuspending washed cells in harvest buffer (10 mm HEPES, pH 7.9, 50 mm NaCl, 0.5 m sucrose, 0.1 mm EDTA, 0.5% Triton X-100, 1 mm dithiothreitol, 10 mm tetrasodium pyrophosphate, 100 mm NaF, 17.5 mm β-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 4 μg/ml aprotinin, and 2 μg/ml pepstatin A). After centrifugation, the precipitate was resuspended in buffer (10 mm HEPES, pH 7.9, 500 mm NaCl, 0.1 mm EDTA, 0.1 mm EGTA, 0.1% Nonidet P-40, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 4 μg/ml aprotinin, and 2 μg/ml pepstatin A). Insoluble debris was removed by centrifugation, and the protein concentration of nuclear extracts was determined by the BCA protein assay kit (Pierce). Thirty μg of protein was run on 12.5% SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad). Antibodies (Santa Cruz Biotechnologies) were specific for GADD153 (sc-7351, 1:2000) or Xbp-1 (M-186, 1:3000). After incubation with appropriate primary and horseradish peroxidase-conjugated secondary antibodies (anti-mouse IgG, 1:5000; anti-rabbit IgG, 1:5000, Cell Signaling Technology), specific protein bands were detected and analyzed by enhanced chemiluminescence substrate detection (ECL Western blotting analysis system, Amersham Biosciences). Immunocytochemistry—LS8 cells were plated in a four-well chamber slide (BD Biosciences). Cells were treated with or without 2 mm NaF for 24 h and fixed with 4% paraformaldehyde in phosphate-buffered saline. Primary antibody was either mouse monoclonal anti-GADD153 (sc-7351; Santa Cruz) or antisera specific for Ire1. 2K. Lipson, L. Nguyen, S. Fonseca, E. Foss, R. Bortell, A. Rossini, and F. Urano, submitted for publication. The Vector M.O.M. immunodetection kit (Vector) was used to detect GADD153, and the VectaStain Elite ABC kit (Vector) was used to detect Ire1. The staining procedure was performed according to the manufacturer's instructions. Inhibition of GADD153 with Short Interfering RNA (siRNA)—siRNA was used to down-regulate GADD153 expression (Qiagen). The siRNA sequence was AACAGAGGTCACACGCACATC. Briefly, LS8 cells were plated in 10-cm dishes (12 ml of medium without antibiotics) and transiently transfected with 0.4 μm siRNA in 3 ml of Opti-MEM with 30 μl of Lipofectamine 2000 (Invitrogen). For 96-well plates LS8 cells were plated at 100 μl/well and transiently transfected with 0.4 μm siRNA in 100 μl of Opti-MEM with 0.25 μl of Lipofectamine 2000. After incubation for 24 h, cells were treated with or without 5 mm NaF for 24 h. Nuclear proteins were extracted as described above and used for Western blot or DNA fragmentation analysis. Overexpression of GADD153—The mouse GADD153 cDNA was present or not (control) in the pcDNA3 vector (Invitrogen). The plasmids were transfected using Lipofectamine 2000 (Invitrogen). GADD153 protein expression was detected by Western blot analysis. Five μg of protein from whole cells were subjected to SDS-PAGE. In brief, cells were plated in 6-well plates and transiently transfected with 0.75 or 1.5 μg of DNA in 0.5 ml of Opti-MEM with 5 μl of Lipofectamine 2000. After 24 h cells were collected and lysed (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm NaF, 1 mm EDTA, 0.1% protease inhibitor mixture, 0.5 mm phenylmethylsulfonyl fluoride). Insoluble debris was removed by centrifugation, and the protein concentration was determined by the BCA protein assay kit (Pierce). DNA fragmentation analysis was performed as described above. In Vivo TUNEL Assay and Immunohistochemistry—All animals used in this work were housed in AAALAC-approved facilities, and all operations were performed in accord with protocols approved by the Institutional Animal Care and Use Committees at the Forsyth Institute. Six-week-old male C57BL/6J mice were purchased from Charles River Laboratories. Fluoride at a concentration of 0, 75, or 150 ppm as NaF was delivered ad libitum in the drinking water for 3–4 weeks. Fluoride concentration analyses of chow were performed in duplicate by a modification (36Rojas-Sanchez F. Kelly S.A. Drake K.M. Eckert G.J. Stookey G.K. Dunipace A.J. Community Dent. Oral Epid. 1999; 27: 288-297Crossref PubMed Scopus (73) Google Scholar) of the hexamethyldisiloxane (Sigma) microdiffusion method, and serum fluoride levels were performed by the method of Vogel et al. (37Vogel G.L. Carey C.M. Chow L.C. Brown W.E. J. Dent. Res. 1987; 66: 1691-1697Crossref PubMed Scopus (21) Google Scholar). Incisors were formalin-fixed, paraffin-embedded, and sectioned. For the TUNEL assay, the In Situ Cell Death Detection kit (Roche Applied Science) was used according to the manufacturer's instructions. The sections were incubated with anti-fluorescein antibody conjugated with horseradish peroxidase. For immunohistochemistry, sections were incubated in blocking agent (goat serum) for 20 min, in active Ire1α-specific antisera (1:100) overnight, in peroxidase-conjugated antibody (Vectastain Elite Reagent), and in Sigma Fast 3,3′-diaminobenzidine substrate. Sections were examined by light microscopy for the presence of fragmented DNA and for the presence of active Ire1α. NaF Inhibits LS8 Cell Growth—To establish the concentration of NaF necessary for toxicity of the ameloblast-derived LS8 cell line, we performed cell survival assays. LS8 cells were seeded into 25-cm2 flasks (1000 cells/flask) for 18 h before 24 h of treatment with NaF at concentrations of 0, 0.5, 1.0, 1.5, and 2.0 mm. After 8–9 days, the resulting colonies were stained with methylene blue and counted. Experiments were performed in triplicate, and colony counts from the treatment groups were compared with the untreated controls (Fig. 1A). Although the trend started at the lowest dose assayed (0.5 mm), significant levels of cell death were observed at the 1.5 (30%) and 2.0 mm (52%) NaF concentrations (Fig. 1A). Next we quantified LS8 cell proliferation by use of the tetrazolium salt MTT. MTT is reduced to an insoluble formazan dye by mitochondrial enzymes associated with metabolic activity, and the amount of dye formed correlates positively to the number of proliferating cells present. LS8 cells were treated with several concentrations of sodium fluoride of between 0 and 2 mm for either 24 or 48 h followed by assessment of cell proliferation by the MTT assay. Treatment with 0.1 mm NaF (1.9 ppm fluoride) reduced LS8 cell proliferation by about 4%, and a significant reduction in cell proliferation (∼10%) was observed after exposure to 0.2 mm NaF (3.8 ppm fluoride) for 24 h (Fig. 1B). After 48 h of fluoride treatment, the cells appeared to have time to recover from the low dose exposure (0.1, 0.2, and 0.5 mm) and proliferate at a slightly higher rate than the 24-h treatment groups. However, at the highest dose (2.0 mm), significantly less proliferation was apparent for the 48-h treatment compared with the 24-h treatment (Fig. 1B). NaF Induces Caspase-mediated DNA Fragmentation in LS8 Cells—DNA fragmentation was quantified by use of an ELISA-based TUNEL assay where adherent cells were assayed for DNA strand breaks. After 48 h of treatment with 2.0 mm NaF, a significant quantity of LS8 DNA strand breaks were observed (Fig. 2A). The addition of the general caspase inhibitor z-VAD eliminated the NaF-induced DNA strand breaks, demonstrating that caspases mediated the DNA fragmentation response. To determine whether z-VAD treatment protected the cells from NaF-induced cell death, we performed trypan blue dye exclusion assays in duplicate for each treatment in three different successive experiments (Fig. 2B). The results demonstrated that z-VAD did not significantly protect LS8 cells from the toxic effects of NaF, indicating that caspases are involved but are not essential for NaF-induced cell death. NaF Induces ER Stress—Because LS8 cells are more sensitive to the antiproliferative rather than the toxic effects of NaF, we asked if NaF induced a cell stress response. GADD genes are induced by growth arrest and DNA damage, so we assessed the expression levels of two GADD genes before and after NaF treatment. Northern blot analysis demonstrated that both GADD153 and GADD45α mRNAs were induced in LS8 cells after NaF exposure. The GADD153 induction was the strongest and increased in a time- and dose-dependent manner that peaked (40–60-fold induction) after 24 h of 2 mm NaF exposure (Fig. 3A). Because GADD153 expression may or may not stem from the ER stress response pathway (26Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. Stojdl D.F. Bell J.C. Hettmann T. Leiden J.M. Ron D. Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2273) Google Scholar, 27Jousse C. Bruhat A. Harding H.P. Ferrara M. Ron D. Fafournoux P. FEBS Lett. 1999; 448: 211-216Crossref PubMed Scopus (79) Google Scholar), we asked if the ER stress response gene BiP was also induced by exposure to NaF. BiP is an ER resident molecular chaperone that is thought to prevent protein aggregation while maintaining a protein-folding-competent state (38Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1" @default.
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• W1985247208 title "Fluoride Induces Endoplasmic Reticulum Stress in Ameloblasts Responsible for Dental Enamel Formation" @default.
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