Fluoride induced endoplasmic reticulum stress and calcium overload in ameloblasts.
OBJECTIVE
The aim of the study was to evaluate the involvement of endoplasmic reticulum stress and intracellular calcium overload on the development of dental fluorosis.


METHODS
We cultured and exposed rat ameloblast HAT-7 cells to various concentrations of fluoride and measured apoptosis with flow cytometry and intracellular Ca2+ changes using confocal microscopy, investigated the protein levels of GRP78, calreticulin, XBP1 and CHOP by western blotting, and their transcriptional levels with RT-PCR. We also created an in vivo model of dental fluorosis by exposing animals to various concentrations of fluoride. Subsequently, thin dental tissue slices were analyzed with H&E staining, immunohistochemical staining, and transmission electron microscopy, TUNEL assay was also performed on dental tissue slices for assessment of apoptosis.


RESULTS
High fluoride concentration was associated with decreased ameloblast proliferation, elevated ameloblast apoptosis, and increased intracellular Ca2+ in vitro. The translation and transcription of the proteins associated with endoplasmic reticulum stress were significantly elevated with high concentrations of fluoride. Based on immunohistochemical staining, these proteins were also highly expressed in animals exposed to high fluoride concentrations. Histologically, we found significant fluorosis-like changes in tissues from animals exposed to high fluoride concentrations. Transmission electron microscopy cytology indicated significant apoptotic changes in tissues exposed to high concentrations of fluoride.


CONCLUSIONS
These results indicate that exposure to high levels of fluoride led to endoplasmic reticulum stress which induced apoptosis in cultured ameloblasts and in vivo rat model, suggesting an important role of calcium overload and endoplasmic reticulum stress triggered by high concentrations of fluoride in the development of dental fluorosis.
Shanghai Stomatology Hospital, Department of Preventive Dentistry, Fudan University, Shanghai, China. Electronic address: zhangyingcmu@vip.163.com.
Ying
Y
Zhang
Department of Preventive Dentistry, School of Stomatology, China Medical University, Shenyang, Liaoning, China; Liaoning Institute of Dental Research, China.
KaiQiang
K
Zhang
Department of Preventive Dentistry, School of Stomatology, China Medical University, Shenyang, Liaoning, China; Liaoning Institute of Dental Research, China.
Lin
L
Ma
Department of Preventive Dentistry, School of Stomatology, China Medical University, Shenyang, Liaoning, China; Liaoning Institute of Dental Research, China.
HeFeng
H
Gu
Department of Preventive Dentistry, School of Stomatology, China Medical University, Shenyang, Liaoning, China; Liaoning Institute of Dental Research, China.
Jian
J
Li
Department of Preventive Dentistry, School of Stomatology, China Medical University, Shenyang, Liaoning, China; Liaoning Institute of Dental Research, China.
Shuang
S
Lei
D000565:Ameloblasts / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D037282:Calreticulin / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003743:Dental Enamel / Q000187:drug effects; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D005459:Fluorides / Q000008:administration & dosage / Q000494:pharmacology*; D009050:Fluorosis, Dental / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000032:analysis / Q000235:genetics; D007150:Immunohistochemistry; D051381:Rats; D017208:Rats, Wistar; D012969:Sodium Fluoride / Q000008:administration & dosage / Q000494:pharmacology; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism; D000071717:X-Box Binding Protein 1 / Q000235:genetics / Q000378:metabolism
D037282:Calreticulin; C497251:Ddit3 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D000071717:X-Box Binding Protein 1; C000605545:Xbp1 protein, rat; D051742:Transcription Factor CHOP; D012969:Sodium Fluoride; D005459:Fluorides; D002118:Calcium
Apoptosis; Cell signaling; Enamel; Gene expression; Protein expression: amelogenin