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ERS Mediated by GRP-78/PERK/CHOP Signaling Is Involved in Fluoride-Induced Ameloblast Apoptosis

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Abstract

Fluoride can be widely ingested from the environment, and its excessive intake could result in adverse effects. Dental fluorosis is an early sign of fluoride toxicity which can cause esthetic and functional problems. Though apoptosis in ameloblasts is one of the potential mechanisms, the specific signal cascade is in-conclusive. High-throughput sequencing and molecular biological techniques were used in this study to explore the underlying pathogenesis of dental fluorosis, for its prevention and treatment. A fluorosis cell model was established. Viability and apoptosis rate of mouse ameloblast-derived cell line (LS8 cells) was measured using cell counting kit-8 (CCK-8) assay and flow cytometry analysis. Cells were harvested with or without 2-mM sodium fluoride (NaF) stimulation for high-throughput sequencing. Based on the sequencing data, subcellular structures, endoplasmic reticulum stress (ERS), and apoptosis related biomarkers were verified using transmission electron microscopy, quantitative real-time polymerase chain reaction, and Western blotting techniques. Expression of ERS markers, apoptosis related proteins, and enamel formation enzymes were detected using Western blotting after addition of 4-phenylbutyrate (4-PBA). NaF-inhibited LS8 cells displayed time- and dose- dependent viability. Additionally, apoptosis and morphological changes were observed. RNA-sequencing data showed that protein processing in endoplasmic reticulum was obviously affected. ERS and apoptosis were induced by excessive NaF. Downregulation of kallikrein-related peptidase 4 (KLK4) was also observed. Inhibition of ERS by 4-PBA rescued the apoptotic and functional protein changes in cells. Excessive fluoride induces apoptosis by activating ERS, which is mediated by GRP-78/PERK/CHOP signaling. Key proteinase is present in maturation-stage enamel; KLK4 was also affected by fluoride, but rescued by 4-PBA. This study presents a possibility for therapeutic strategies for dental fluorosis, while further exploration is required.

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Data Availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

4-PBA:

4-phenylbutyric acid

ATF:

activating transcription factor

Bcl-2:

B-cell lymphoma-2

CHOP:

C/EBP homologs protein

eIF2α:

eukaryotic initiation factor 2α

ERS:

endoplasmic reticulum stress

GADD34:

growth arrest and DNA damage inducible gene 34

GO:

Gene Ontology

GRP-78:

glucose-regulated protein 78

IRE1:

inositol-requiring enzyme 1

KEGG:

Kyoto Encyclopedia of Genes and Genomes

KLK4:

kallikrein-related peptidase 4

MMP20:

matrix metalloproteinase 20

PERK:

protein kinase PKR-like ER kinase

RIDD:

Regulated IRE1-Dependent Decay

UPR:

unfolded protein response

XBP1:

X-box-binding protein 1

References

  1. Revelo-Mejia IA, Hardisson A, Rubio C, Gutierrez AJ, Paz S (2021) Dental fluorosis: the risk of misdiagnosis. Biol Trace Elem Res 199(5):1762–1770

    Article  CAS  PubMed  Google Scholar 

  2. Peckham S, Awofeso N (2014) Water fluoridation: a critical review of the physiological effects of ingested fluoride as a public health intervention. Sci World J 2014:293019

    Article  Google Scholar 

  3. Walsh T, Worthington HV, Glenny AM, Marinho VC, Jeroncic A (2019) Fluoride toothpastes of different concentrations for preventing dental caries. CDSR 3:CD007868

    PubMed  Google Scholar 

  4. Green R, Lanphear B, Hornung R, Flora D, Martinez-Mier EA, Neufeld R et al (2019) Association between maternal fluoride exposure during pregnancy and IQ scores in Offspring in Canada. JAMA pediatrics 173(10):940–948

    Article  PubMed  PubMed Central  Google Scholar 

  5. Hewavithana PB, Jayawardhane WM, Gamage R, Goonaratna C (2018) Skeletal fluorosis in Vavuniya District: an observational study. Ceylon Med J 63(3):139–142

    Article  CAS  PubMed  Google Scholar 

  6. Dharmaratne RW (2019) Exploring the role of excess fluoride in chronic kidney disease: a review. Hum Exp Toxicol 38(3):269–279

    Article  CAS  PubMed  Google Scholar 

  7. Marshall TA, Levy SM, Warren JJ, Broffitt B, Eichenberger-Gilmore JM, Stumbo PJ (2004) Associations between Intakes of fluoride from beverages during infancy and dental fluorosis of primary teeth. J Am Coll Nutr 23(2):108–116

    Article  PubMed  Google Scholar 

  8. Erdal S, Buchanan SN (2005) A quantitative look at fluorosis, fluoride exposure, and intake in children using a health risk assessment approach. Environ Health Perspect 113(1):111–117

    Article  CAS  PubMed  Google Scholar 

  9. Johnston NR, Strobel SA (2020) Principles of fluoride toxicity and the cellular response: a review. Arch Toxicol 94(4):1051–1069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wei W, Pang S, Sun D (2019) The pathogenesis of endemic fluorosis: research progress in the last 5 years. J Cell Mol Med 23(4):2333–2342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wang L, Zhu Y, Wang D (2016) High-dose fluoride induces apoptosis and inhibits ameloblastin secretion in primary rat ameloblast. Biol Trace Elem Res 174(2):402–409

    Article  CAS  PubMed  Google Scholar 

  12. Wang L, Zhu Y, Wang D (2016) High-fluoride acitivates the FasL signalling pathway and leads to damage of ameloblast ultrastructure. Arch Oral Biol 71:31–37

    Article  CAS  PubMed  Google Scholar 

  13. Zhang YYQ, Li W, DenBesten PK (2006) Fluoride down regulates the expression of matrix metalloproteinase-20 in human fetal tooth ameloblast-lineage cells in vitro. Eur J Oral Sci 114(suppl):105–110

    Article  CAS  PubMed  Google Scholar 

  14. Tye CE, Antone JV, Bartlett JD (2011) Fluoride does not inhibit enamel protease activity. J Dent Res 90(4):489–494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Suzuki M, Shin M, Simmer JP, Bartlett JD (2014) Fluoride affects enamel protein content via TGF-beta1-mediated KLK4 inhibition. J Dent Res 93(10):1022–1027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Le MH, Nakano Y, Abduweli Uyghurturk D, Zhu L, Den Besten PK (2017) Fluoride alters Klk4 expression in maturation ameloblasts through androgen and progesterone receptor signaling. Front Physiol 8:925

    Article  PubMed  PubMed Central  Google Scholar 

  17. Yang T, Zhang Y, Li Y, Hao Y, Zhou M, Dong N et al (2013) High amounts of fluoride induce apoptosis/cell death in matured ameloblast-like LS8 cells by downregulating Bcl-2. Arch Oral Biol 58(9):1165–1173

    Article  CAS  PubMed  Google Scholar 

  18. Mao X, Cai T, Olyarchuk JG, Wei L (2005) Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 21(19):3787–3793

    Article  CAS  PubMed  Google Scholar 

  19. Zhao L, Li J, Su J, Snead ML, Ruan J (2016) LS8 cell apoptosis induced by NaF through p-ERK and p-JNK - a mechanism study of dental fluorosis. Acta Odontol Scand 74(7):539–549

    Article  CAS  PubMed  Google Scholar 

  20. Kubota K, Lee DH, Tsuchiya M, Young CS, Everett ET, Martinez-Mier EA et al (2005) Fluoride induces endoplasmic reticulum stress in ameloblasts responsible for dental enamel formation. J Biol Chem 280(24):23194–23202

    Article  CAS  PubMed  Google Scholar 

  21. Kolb PS, Ayaub EA, Zhou W, Yum V, Dickhout JG, Ask K (2015) The therapeutic effects of 4-phenylbutyric acid in maintaining proteostasis. Int J Biochem Cell Biol 61:45–52

    Article  CAS  PubMed  Google Scholar 

  22. McIlwain DR, Berger T, Mak TW (2013) Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol 5(4):a008656

    Article  PubMed  PubMed Central  Google Scholar 

  23. Chen Y, Feng X, Hu X, Sha J, Li B, Zhang H et al (2018) Dexmedetomidine ameliorates acute stress-induced kidney injury by attenuating oxidative stress and apoptosis through inhibition of the ROS/JNK signaling pathway. Oxid Med Cell Longev 2018:4035310

    PubMed  PubMed Central  Google Scholar 

  24. Luo X, Lin B, Gao Y, Lei X, Wang X, Li Y et al (2019) Genipin attenuates mitochondrial-dependent apoptosis, endoplasmic reticulum stress, and inflammation via the PI3K/AKT pathway in acute lung injury. Int Immunopharmacol 76:105842

    Article  CAS  PubMed  Google Scholar 

  25. Li J, Kou M, Cui M, Ruan J, Cheng Z (2022) NaF reduces KLK4 expression by decreasing Foxo1/Runx2 expression in LS8 cells. Arch Oral Biol 133:105311

    Article  CAS  PubMed  Google Scholar 

  26. Li J, Zhao L, Zhao X, Wang P, Liu Y, Ruan J (2018) Foxo1 attenuates NaF-induced apoptosis of LS8 cells through the JNK and mitochondrial pathways. Biol Trace Elem Res 181(1):104–111

    Article  CAS  PubMed  Google Scholar 

  27. Scott D, Roberts SA (1987) Extrapolation from in vitro tests to human risk: experience with sodium fluoride clastogenicity. Mutat Res 189(1):47–58

    Article  CAS  PubMed  Google Scholar 

  28. Yang T, Zhang Y, Zheng D, Hao Y, Snead ML, Duan X (2015) High-fluoride promoted phagocytosis-induced apoptosis in a matured ameloblast-like cell line. Arch Oral Biol 60(1):84–90

    Article  CAS  PubMed  Google Scholar 

  29. Mazat JP, Devin A, Ransac S (2020) Modelling mitochondrial ROS production by the respiratory chain. Cell Mol Life Sci 77(3):455–465

    Article  CAS  PubMed  Google Scholar 

  30. Senft D, Ronai ZA (2015) UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci 40(3):141–148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sovolyova N, Healy S, Samali A, Logue SE (2014) Stressed to death - mechanisms of ER stress-induced cell death. Biol Chem 395(1):1–13

    Article  CAS  PubMed  Google Scholar 

  32. Fernandez A, Ordonez R, Reiter RJ, Gonzalez-Gallego J, Mauriz JL (2015) Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis. J Pineal Res 59(3):292–307

    Article  CAS  PubMed  Google Scholar 

  33. Iurlaro R, Muñoz-Pinedo C (2016) Cell death induced by endoplasmic reticulum stress. Febs j 283(14):2640–2652

    Article  CAS  PubMed  Google Scholar 

  34. Moriguchi M, Watanabe T, Fujimuro M (2019) Capsaicin induces ATF4 translation with upregulation of CHOP, GADD34 and PUMA. Biol Pharm Bull 42(8):1428–1432

    Article  CAS  PubMed  Google Scholar 

  35. Liu X, Huang R, Gao Y, Gao M, Ruan J, Gao J (2021) Calcium mitigates fluoride-induced kallikrein 4 inhibition via PERK/eIF2alpha/ATF4/CHOP endoplasmic reticulum stress pathway in ameloblast-lineage cells. Arch Oral Biol 125:105093

    Article  CAS  PubMed  Google Scholar 

  36. Li Y, Jiang W, Niu Q, Sun Y, Meng C, Tan L et al (2019) eIF2alpha-CHOP-BCl-2/JNK and IRE1alpha-XBP1/JNK signaling promote apoptosis and inflammation and support the proliferation of Newcastle disease virus. Cell Death Dis 10(12):891

    Article  PubMed  PubMed Central  Google Scholar 

  37. Su CL, Huang LL, Huang LM, Lee JC, Lin CN, Won SJ (2006) Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells. FEBS Lett 580(13):3185–3191

    Article  CAS  PubMed  Google Scholar 

  38. Cheng Jiang ZW, Ganther H, Junxuan L (2001) Caspases as key executors of methyl selenium-induced apoptosis (Anoikis) of DU-145 prostate cancer cells. Cancer Res 61(7):3062–3070

    Google Scholar 

  39. Suzuki M, Everett ET, Whitford GM, Bartlett JD (2017) 4-Phenylbutyrate mitigates fluoride-induced cytotoxicity in ALC cells. Front Physiol 8:302

    Article  PubMed  PubMed Central  Google Scholar 

  40. Hardwick JM, Chen YB, Jonas EA (2012) Multipolar functions of BCL-2 proteins link energetics to apoptosis. Trends Cell Biol 22(6):318–328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Saghiri MA, Asatourian A, Gurel Z, Sorenson CM, Sheibani N (2017) Bcl-2 expression is essential for development and normal physiological properties of tooth hard tissue and saliva production. Exp Cell Res 358(2):94–100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kondo STY, Bawden JW, Tanase S (2001) The immunohistochemical localization of Bax and Bcl-2 and their relation to apoptosis during amelogenesis in developing rat molars. Arch Oral Biol 46(6):557–568

    Article  CAS  PubMed  Google Scholar 

  43. Wei Wei YG, Wang C, Zhao L, Sun D (2013) Excessive fluoride induces endoplasmic reticulum stress and interferes enamel. Environ Toxicol 28(6):332–341

    Article  CAS  PubMed  Google Scholar 

  44. Lu Y, Papagerakis P, Yamakoshi Y, Hu JC, Bartlett JD, Simmer JP (2008) Functions of KLK4 and MMP-20 in dental enamel formation. Biol Chem 389(6):695–700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. DenBesten PK, Yana Y, JFH JDBF, Smith CE, Lia W (2002) Effects of fluoride on rat dental enamel matrix proteinases. Arch Oral Biol 47:763–770

    Article  CAS  PubMed  Google Scholar 

  46. Li J, Wang P, Gao J, Fei X, Liu Y, Ruan J (2018) NaF reduces KLK4 gene expression by decreasing Foxo1 in LS8 cells. Biol Trace Elem Res 186(2):498–504

    Article  CAS  PubMed  Google Scholar 

  47. Sharma R, Tye CE, Arun A, MacDonald D, Chatterjee A, Abrazinski T et al (2011) Assessment of dental fluorosis in Mmp20 +/- mice. J Dent Res 90(6):788–792

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chen LS, Couwenhoven RI, Hsu D, Luo W, Snead ML (1992) Maintenance of amelogenin gene expression by transformed epithelial cells of mouse enamel organ. Arch Oral Biol 37(10):771–779

    Article  CAS  PubMed  Google Scholar 

  49. Sharma R, Tsuchiya M, Skobe Z, Tannous BA, Bartlett JD (2010) The acid test of fluoride: how pH modulates toxicity. PLoS One 5(5):e10895

    Article  PubMed  PubMed Central  Google Scholar 

  50. Vega H, Agellon LB, Michalak M (2016) The rise of proteostasis promoters. IUBMB Life 68(12):943–954

    Article  CAS  PubMed  Google Scholar 

  51. Hetz C, Chevet E, Harding HP (2013) Targeting the unfolded protein response in disease. Nat Rev Drug Discov 12(9):703–719

    Article  CAS  PubMed  Google Scholar 

  52. Zeng W, Guo YH, Qi W, Chen JG, Yang LL, Luo ZF et al (2014) 4-Phenylbutyric acid suppresses inflammation through regulation of endoplasmic reticulum stress of endothelial cells stimulated by uremic serum. Life Sci 103(1):15–24

    Article  CAS  PubMed  Google Scholar 

  53. Hong L, Xu Y, Wang D, Zhang Q, Li X, Xie C et al (2023) Sulforaphane ameliorates bisphenol A-induced hepatic lipid accumulation by inhibiting endoplasmic reticulum stress. Sci Rep 13(1):1147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Iannitti T, Palmieri B (2011) Clinical and experimental applications of sodium phenylbutyrate. Drugs R D 11(3):227–249

    Article  PubMed  Google Scholar 

  55. Collins AF, Pearson HA, Giardina P, McDonagh KT, Brusilow SW, Dover GJ (1995) Oral sodium phenylbutyrate therapy in homozygous beta thalassemia: a clinical trial. Blood 85(1):43–49

    Article  CAS  PubMed  Google Scholar 

  56. Odièvre MH, Brun M, Krishnamoorthy R, Lapouméroulie C, Elion J (2007) Sodium phenyl butyrate downregulates endothelin-1 expression in cultured human endothelial cells: relevance to sickle-cell disease. Am J Hematol 82(5):357–362

    Article  PubMed  Google Scholar 

  57. Tanis RM, Piroli GG, Day SD, Frizzell N (2015) The effect of glucose concentration and sodium phenylbutyrate treatment on mitochondrial bioenergetics and ER stress in 3T3-L1 adipocytes. Biochim Biophys Acta 1853(1):213–221

    Article  CAS  PubMed  Google Scholar 

  58. Takeyari S, Kubota T, Ohata Y, Fujiwara M, Kitaoka T, Taga Y et al (2021) 4-Phenylbutyric acid enhances the mineralization of osteogenesis imperfecta iPSC-derived osteoblasts. J Biol Chem 296:100027

    Article  CAS  PubMed  Google Scholar 

  59. Kaur B, Bhat A, Chakraborty R, Adlakha K, Sengupta S, Roy S et al (2018) Proteomic profile of 4-PBA treated human neuronal cells during ER stress. Mol Omics 14(1):53–63

    Article  CAS  PubMed  Google Scholar 

  60. Wang L, Deng L, Lin N, Shi Y, Chen J, Zhou Y et al (2020) Berberine inhibits proliferation and apoptosis of vascular smooth muscle cells induced by mechanical stretch via the PDI/ERS and MAPK pathways. Life Sci 259:118253

    Article  CAS  PubMed  Google Scholar 

  61. Zhao J, Hai S, Chen J, Ma L, Rahman SU, Zhao C et al (2022) Zearalenone induces apoptosis in porcine endometrial stromal cells through JNK signaling pathway based on endoplasmic reticulum stress. Toxins (Basel) 14(11):758

    Article  CAS  PubMed  Google Scholar 

  62. Niu Q, Chen J, Xia T, Li P, Zhou G, Xu C et al (2018) Excessive ER stress and the resulting autophagic flux dysfunction contribute to fluoride-induced neurotoxicity. Environ Pollut 233:889–899

    Article  CAS  PubMed  Google Scholar 

  63. Estebanez B, de Paz JA, Cuevas MJ, Gonzalez-Gallego J (2018) Endoplasmic reticulum unfolded protein response, aging and exercise: an update. Front Physiol 9:1744

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Specially thanks to Professor Malcolm L. Snead (Department of Biomedical Sciences, University of Southern California) and Jian-ping Ruan (College of Stomatology, Xi’an Jiaotong University) for generous donation of LS8 cells. We would like to thank Editage (www.editage.cn) for English language editing.

Funding

This research was funded by the National Natural Science Foundation of China (no. 81801310), Fundamental Research Funds for the Central Universities (no. xzy012021068) and Shaanxi Province Youth Science and Technology New Star Project (2023KJXX-034).

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Authors and Affiliations

  1. Key laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, 710004, People’s Republic of China

    Li Jinyi, Dai Shanshan, Liu Ruirui, Guo Qingyu & Liu Fei

  2. Department of Pediatric Dentistry, College of Stomatology, Xi’an Jiaotong University, Xi Wu Road No.98, Xi’an 710004, 710041, People’s Republic of China

    Li Jinyi, Dai Shanshan, Guo Qingyu & Liu Fei

  3. National Regional Children’s Medical Center (Northwest), Xi’an Children’s Hospital, Affiliated Children’s Hospital of Xi’an Jiaotong University, Xi’an, 710003, People’s Republic of China

    Yang Keyu

  4. Faculty of dentistry, The university of Hong Kong, Hong Kong, SAR, People’s Republic of China

    He Shuyang

  5. Department of Prosthodontics, College of Stomatology, Xi’an Jiaotong University, Xi’an, 710004, People’s Republic of China

    Liu Ruirui

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  1. Li Jinyi
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Contributions

Liu Fei and Guo Qingyu: conceptualization and design of the study, supervision of the research, funding acquisition

Li Jinyi and Yang Keyu: performing the experiment, data collection and analysis, manuscript writing

Dai Shanshan, He Shuyang and Liu Ruirui: data analysis, review and editing the manuscript, critical revision.

All authors read and approved the final manuscript.

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Correspondence to Guo Qingyu or Liu Fei.

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Jinyi, L., Keyu, Y., Shanshan, D. et al. ERS Mediated by GRP-78/PERK/CHOP Signaling Is Involved in Fluoride-Induced Ameloblast Apoptosis. Biol Trace Elem Res 202, 1103–1114 (2024). https://doi.org/10.1007/s12011-023-03746-5

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