Research ArticleCalcium signaling

Fluoride exposure alters Ca2+ signaling and mitochondrial function in enamel cells

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Science Signaling  18 Feb 2020:
Vol. 13, Issue 619, eaay0086
DOI: 10.1126/scisignal.aay0086
  • Fig. 1 Fluoride leads to loss Ca2+ in internal stores only in enamel cells.

    (A to C) Ca2+ levels in internal stores and SOCE upon re-addition of external Ca2+ in isolated primary enamel organ (EO) cells from secretory (SEC) (A) and maturation (MAT) (B) stages and LS8 cells (C) incubated with NaF (1 mM) for 24 hours and treated with the SERCA inhibitor thapsigargin (TG) or tBHQ. (D) Ca2+ levels in internal stores and SOCE upon re-addition of external Ca2+ in fluoride-treated HEK-293 incubated with NaF (1 mM) for 24 hours and treated with the SERCA inhibitor tBHQ. (E and F) Quantification of area under the curve (A.U.C.) (E) (between 2 and 9 min) and peak of SOCE (F) from (A) to (D). Data in (A) and (B) represent the mean ± SEM of three independent experiments analyzed using unpaired Student’s t test (*P < 0.01 and **P < 0.001). EO cells were obtained from six rats. Data in (C) and (D) represent the mean ± SEM of four to six independent experiments using unpaired Student’s t test (**P < 0.001); ns, nonsignificant.

  • Fig. 2 Fluoride, but not bromide, affects ER Ca2+.

    (A) ER Ca2+ in LS8 cells transfected with the genetically encoded ER Ca2+ probe CEPIA-red stimulated with thapsigargin and exposed to fluoride for 30 min (gray trace), 1 hour (blue trace), and 2 hours (green trace). Data represent the mean ± SEM of three independent experiments analyzed using Dunnett’s multiple comparisons test (**P < 0.001) of 30 min, 1 hour, and 2 hours compared to controls. (B) ER Ca2+ in primary secretory cells treated with fluoride (24 hours, 1 mM NaF) loaded with the ER Ca2+ probe Mag-Fura-2 and stimulated with thapsigargin. Data represent the mean ± SEM of three independent experiments analyzed using Student’s t test (black compared to red: ***P < 0.001). EO cells obtained from three rats. (C) ER Ca2+ levels in untreated LS8 cells (black trace) or cells treated for 1 hour with NaBr (1 mM) (orange trace). (D) SOCE in untreated LS8 (black trace) or cells treated with NaBr (1 mM) (orange trace) for 1 hour after maximally depleting the stores with tBHQ (5 μM) before re-addition of 2 mM Ca2+. (E and F) Same as (C) and (D) with cells treated for 24 hours. Data in (C) to (F) represent the mean ± SEM of three independent experiments.

  • Fig. 3 RNASeq analyses of fluoride-treated LS8 and HEK-293 cells.

    (A and B) Volcano plots of statistical significance (shown as the negative log base 10 of the P value on the y axis) compared to the magnitude of differential gene expression (shown as the log base 2 of magnitude of mean expression difference on the x axis) between control and NaF (1 mM) treatment for murine LS8 (A) and HEK-293 (B) cells. Data in (A) and (B) were obtained from four sets of untreated and three sets of NaF-treated cells. (C) Heat map of transcript abundance using one-way hierarchical clustering of untreated and 1 mM NaF–treated LS8 cells for 24 hours, showing the differential expression of genes encoding proteins associated with cell stress and UPR. Red indicates up-regulation. (D) RT-PCR showing the mRNA expression of the ER stress marker GRP78 in LS8 and HEK-293 cells in response to NaF (red boxes). Data represent the mean ± SEM of three independent experiments (*P < 0.05, ANOVA). (E) Differential expression of genes encoding proteins associated with the biosynthesis of mitochondrial complexes in LS8 cells. (F) Differential expression of genes encoding proteins associated with mitoribosome biosynthesis in HEK-293 cells. (E and F) Blue indicates down-regulation.

  • Fig. 4 IP3R and SERCA functions are modified by fluoride in enamel cells.

    (A to C) Permeabilized LS8 cells (A), primary secretory (B), and maturation (C) stage cells loaded with the ER Ca2+ indicator Mag-Fura-2 were stimulated with IP3 to induce Ca2+ release from the ER. Each stimulus resulted in ER Ca2+ decrease. Representative tracings of ~100 cells per cell type. (D) Release from ER Ca2+ pools in permeabilized LS8 cells loaded with the ER Ca2+ dye Mag-Fura-2 and transiently stimulated with fluoride at 10 μM (green trace) or 1 mM (red trace). Representative of ~140 cells per group. (E) ER Ca2+ release by IP3 stimulation was measured in permeabilized LS8 cells loaded with Mag-Fura-2 pretreated with 10 μM NaF (green trace) and 1 mM NaF (red trace). Representative of ~140 cells per group. (F) Kinetics of release from ER Ca2+ pools in cells in (E). Data represent the mean ± SEM of three independent experiments using Tukey’s multiple comparisons test (black compared to red: **P < 0.0001; black compared to green: **P < 0.001; red compared to green: ns). (G and H) Kinetics of SERCA refilling measured in permeabilized untreated LS8 cells after tBHQ stimulation (black trace) and exposure to NaF (10 μM; green trace, and 1 mM; red trace). Data represent the mean ± SEM of three independent experiments using Tukey’s multiple comparisons test (black compared to red: ****P < 0.0001; black compared to green: *P < 0.0235; red compared to green: ****P < 0.0001).

  • Fig. 5 Fluoride modified mitochondrial function and SOCE.

    (A) Rhod-2–loaded control (untreated), LS8 (black trace), and fluoride-treated (24 hours) (red trace) cells transiently stimulated with ATP. Data represent the mean ± SEM of three independent experiments using Student’s t test (black compared to red: ****P < 0.0001). (B) Mitochondrial membrane potential measured in LS8 cells loaded with TMRM that were untreated and stimulated with FCCP (1.5 μM; black trace) or transiently stimulated with fluoride (1 mM) and FCCP (red trace). TMRM fluorescence was also monitored in cells not treated with FCCP (gray traces). Representative of 150, 100, and 120 cells. (C) Primary secretory cells analyzed as in (B). Representative of 90 untreated and stimulated with FCCP (black traces), 65 fluoride- and FCCP-treated cells (red traces), and 80 untreated cells (gray traces). (D) Oxygen consumption rate (OCR), basal respiration, ATP production, and maximal respiration in LS8 cells after 4 hours of NaF (1 mM) pretreatment. Data represent the mean ± SEM of three independent experiments using Student’s t test. (*P < 0.05 and ***P < 0.001). (E to G) Transmission electron micrographs of control LS8 cells (E) and fluoride-treated cells (F) (1 mM, 24 hours). Close-up of mitochondria (G) from (F) showing mitochondrial matrix with non–electron-dense matrix lacking cristae. Scale bars, 1 μm (E and F) and 0.5 μm (G). (H) SOCE measured after treating cells for 20 min with thapsigargin in untreated LS8 cells (black trace) or cells treated with NaF (1 mM) after 30 min, 60 min, and 24 hours. (I) [Ca2+] mit measured in Rhod-2–loaded LS8 cells after stimulation of SOCE as in (A). (J) SOCE measured after treating cells for 20 min with thapsigargin in untreated LS8 cells (black trace) or fluoride-treated (1 mM) cells (red trace) and cells treated with oligomycin (1 μM) and rotenone (2 μM) (green trace). Data in (H) to (J) represent the mean ± SEM of three independent experiments.

  • Fig. 6 Model for the effects of high fluoride in enamel cells.

    (A) In a healthy cell exposed to low fluoride, mitochondrial membrane potential (ΔΨm) and ATP production are normal, enabling SERCA to maintain its pumping function. SOCE is normal. (B) High fluoride exposure results in a decrease in the ΔΨm disrupting the proton motive force required to generate ATP. Decreased levels of ATP limit the capacity of SERCA to transfer Ca2+ into the ER lumen, resulting in increased Ca2+ leak. Cells respond by activating the UPR that allows the cells to survive, albeit in a low metabolic state. SOCE may be decreased because of low ATP or dysfunctional mitochondria.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/619/eaay0086/DC1

    Fig. S1. ER Ca2+ release and SOCE in LS8 cells treated with various concentrations of NaF.

    Fig. S2. Comparison of ER Ca2+ release by thapsigargin and tBHQ.

    Fig. S3. ATP stimulation of fluoride-treated LS8 cells.

    Fig. S4. Fluoride, but not chloride, affects Ca2+ homeostasis.

    Fig. S5. Cell death analyses.

    Fig. S6. Heat map of cell and ER stress genes in HEK-293 cells.

    Fig. S7. Expression of the ER stress marker GRP78 in cells exposed to various NaF concentrations.

    Fig. S8. Heat map of RP genes in HEK-293 cells.

    Fig. S9. IP3 uncaging in primary secretory stage enamel cells.

    Fig. S10. Transient fluoride application does not elicit changes in [Ca2+]cyt.

    Fig. S11. Western blot of SERCA in LS8 cells treated with fluoride.

    Fig. S12. Altering mitochondrial function by rotenone/oligomycin disrupts SOCE in LS8 cells.

    Table S1. DEGs in fluoride-treated LS8 cells.

    Table S2. DEGs in fluoride-treated HEK-293 cells.

  • This PDF file includes:

    • Fig. S1. ER Ca2+ release and SOCE in LS8 cells treated with various concentrations of NaF.
    • Fig. S2. Comparison of ER Ca2+ release by thapsigargin and tBHQ.
    • Fig. S3. ATP stimulation of fluoride-treated LS8 cells.
    • Fig. S4. Fluoride, but not chloride, affects Ca2+ homeostasis.
    • Fig. S5. Cell death analyses.
    • Fig. S6. Heat map of cell and ER stress genes in HEK-293 cells.
    • Fig. S7. Expression of the ER stress marker GRP78 in cells exposed to various NaF concentrations.
    • Fig. S8. Heat map of RP genes in HEK-293 cells.
    • Fig. S9. IP3 uncaging in primary secretory stage enamel cells.
    • Fig. S10. Transient fluoride application does not elicit changes in [Ca2+]cyt.
    • Fig. S11. Western blot of SERCA in LS8 cells treated with fluoride.
    • Fig. S12. Altering mitochondrial function by rotenone/oligomycin disrupts SOCE in LS8 cells.
    • Table S1. DEGs in fluoride-treated LS8 cells.
    • Table S2. DEGs in fluoride-treated HEK-293 cells.

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