Research ArticleCalcium signaling

Differential regulation of Ca2+ influx by ORAI channels mediates enamel mineralization

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Science Signaling  23 Apr 2019:
Vol. 12, Issue 578, eaav4663
DOI: 10.1126/scisignal.aav4663
  • Fig. 1 Amelogenesis imperfecta in a patient with del541C null mutation in the ORAI1 gene.

    (A and B) Dental radiographs of an ~8-month-old healthy control compared with a patient homozygous for a del541C null mutation in ORAI1 (D and E) of similar age. ORAI1 null mutation was associated with “pitted” enamel with hypoplasia of the primary central and lateral incisors (D) and the primary mandibular molars (E). The pitted areas are indicated by white arrows. (C and F) Dental radiograph of the same patient at ~6 years of age (F) and an age-matched healthy control (C). Arrows indicate hypoplasia and associated dental caries of the mandibular permanent molar. The crowns of the primary molar teeth were covered with stainless steel crowns as indicated by asterisks.

  • Fig. 2 Abnormal SOCE in primary ameloblasts from ORAI1- and ORAI2-deficient mice.

    (A) Immunofluorescence staining for ORAI1 and ORAI2 of ameloblasts from WT, Orai1K14, and Orai2−/− mice. Nuclear staining by 4′,6-diamidino-2-phenylindole (DAPI) is shown in blue. Images are representative of n = 3 mice for each genotype. Scale bars, 20 μm. (B and C) Analysis of (B) Orai1 and (C) Orai2 gene expression in ameloblast cells isolated from Orai1K14 and Orai2−/− mice, respectively. Data represent the mean ± SEM of three independent experiments with ameloblasts obtained from five to seven mice for each genotype. (D to G) Measurements of [Ca2+]i (shown as F340/F380 ratio) in ameloblasts of WT, Orai1K14 (D and E), and Orai2−/− (F and G) mice after thapsigargin (TG) stimulation. The peaks of F340/F380 corrected for baseline levels (ΔPeak F340/F380) and the slope are quantified in (E) and (G). Data represent the mean ± SEM of three independent experiments, with ameloblasts obtained from four to five mice for each genotype. (H and I) RT-PCR analysis of enamel gene expression in ameloblasts of Orai1K14 (H) and Orai2−/− (I) mice. Data represent the mean ± SEM of three independent experiments, with ameloblasts obtained from five mice for each genotype. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using unpaired Welch’s t test (B and H), Mann-Whitney test (C, E, and G), or Student’s t test (I).

  • Fig. 3 Conditional deletion of Orai1 in murine ameloblast cells causes enamel defects.

    (A to D) Visual examination of teeth using a stereomicroscope reveals differences in the enamel of the incisors of WT (A) and Orai1K14 (C) mice. BSE-SEM micrographs of incisor cross sections showing enamel and underlying dentine. Images were taken 1 mm from the incisor tip of WT (B) and Orai1K14 (D) mice. (E and F) High-magnification micrographs of WT (E) and Orai1K14 (F) enamel after acid etching. (G and H) FE-SEM micrographs of WT (G) and Orai1K14 (H) enamel crystals. Images shown in (A) to (H) are representative of n = 4 mice for each genotype.

  • Fig. 4 Deletion of Orai1, Orai2, and Orai3 expression in the LS8 ameloblast cell line.

    (A to C) Expression pattern of Orai1 (A), Orai2 (B), and Orai3 (C) in LS8 cells compared to other murine tissues, including heart, lymph node, and brain measured by RT-PCR. Data in (A) to (C) represent averages (±SEM) of a minimum of three independent experiments. (D to F) RT-PCR analyses of Orai1, Orai2, and Orai3 deletion in LS8 cells after transduction with empty vector (EV ctrl.) or two independent shRNAs each against Orai1, Orai2, and Orai3. Data represent averages (±SEM) of a minimum of three independent experiments. (G to I) Evaluation of effects of Orai1, Orai2, and Orai3 deletion on cell size measured by flow cytometry (FSC-A) (G), proliferation measured using Ki67 staining by flow cytometry (H), and cell death measured using annexin V staining by flow cytometry (I). MFI, mean fluorescence intensity. Data in (G) to (I) represent four independent experiments (mean ± SEM). For all panels, *P < 0.05, **P < 0.01, ****P < 0.0001 using unpaired Welch’s t test [(A) and (B) with LS8 versus heart and LS8 compared to lymph node, (D) with EV ctrl. #2 compared to shOrai1 #2 (F)] or an unpaired Student’s t test (E).

  • Fig. 5 SOCE is mediated by ORAI1 and ORAI2 in LS8 cells.

    (A to F) [Ca2+]i measurements of LS8 cells transduced with empty vector (EV ctrl.) and either shOrai1 (A to C), shOrai2 (D to F), or shOrai3 (G to I). SOCE was analyzed after thapsigargin stimulation and readdition of 2 mM Ca2+. Quantification of SOCE corrected for baseline Ca2+ levels as ΔPeak F340/F380 and pooling of values from both shRNAs (B, E, and H). All [Ca2+]i measurements were done using a FlexStation 3 plate reader. (J to M) [Ca2+]i measurements in LS8 cells twice transduced with empty vector control plasmids, both shOrai1 and shOrai2, or both shOrai1 and shOrai3. Quantification of peak and rate of SOCE levels was corrected for baseline Ca2+ levels (ΔPeak F340/F380) in shOrai1/shOrai2 (K and L) and shOrai1/shOrai3 (M and N) cells. Ca2+ measurements were performed as described in (A). All data represent three independent experiments (mean ± SEM). **P < 0.01, ***P < 0.001, ****P < 0.0001 using Mann-Whitney test (B, C, F, I, K, and L) or unpaired Student’s t test (E and H).

  • Fig. 6 Increased mitochondrial function and Ca2+ reuptake into the ER in ORAI1-deficient LS8 cells.

    (A) Analysis of oxygen consumption rates (OCRs) in LS8 cells transduced with empty vector (EV ctrl.) or shRNAs against Orai1, Orai2, and Orai3 using a Seahorse flux analyzer. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; AA, antimycin A; R, rotenone. (B) Quantification of basal respiration, ATP production, maximal respiration, and spare respiratory capacity from experiments in (A). Data in (A) and (B) represent four independent experiments. (C) ATP measurements in LS8 cells transduced with empty vector or shOrai1. Luminescence intensities were measured using a FlexStation 3 plate reader. Data represent the mean ± SEM of five independent experiments. (D) Measurement of the GSH:GSSG ratio in empty vector and shOrai1 cells. Data represent the mean ± SEM of three independent experiments. (E) Relative mRNA expression of Atp2a2 (SERCA2) in control and shOrai1-transduced LS8 cells. Data represent the mean ± SEM of five independent experiments. (F) The refilling rate of ER Ca2+ was measured in Mag-Fura-2–loaded empty vector and shOrai1 cells after permeabilization. ER stores were depleted with the reversible SERCA inhibitor tBHQ in a solution containing 100 nM Ca2+ for 3 min without tBHQ to allow Ca2+ refilling (see also fig. S7). SERCA activity was also measured in cells pretreated with BSO to inhibit GSH synthesis (gray and red tracings) (see also fig. S9). Data represent averages (±SEM) of n = 3 independent experiments. **P < 0.01, ***P < 0.001 using Mann-Whitney test (B), Welch’s t test (C), or unpaired Student’s t test (E).

  • Fig. 7 Schematic representation of the effects of ORAI1 deficiency in enamel cells.

    In WT ameloblasts, ORAI1, the predominant subunit, and, to some extent, also ORAI2 and ORAI3 modulate Ca2+ influx. Under normal redox conditions, these cells have physiological levels of GSH and GSSG, normal mitochondrial respiration, and normal ATP production; also, S-glutathionylation is not activated. In enamel cells lacking ORAI1, there is a marked reduction in Ca2+ influx. Residual Ca2+ influx is likely mediated by ORAI2 and possibly ORAI3 subunits. In these conditions, the GSH/GSSG ratio decreases, indicative of altered redox status, specifically increased oxidizing conditions. This might be associated with the observed increase in mitochondrial respiration and elevated ATP production. Changes in redox also affect the activity of SERCA through S-glutathionylation, stimulating its pumping action.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/578/eaav4663/DC1

    Fig. S1. Unchanged expression of Ambn in ORAI-deficient cells.

    Fig. S2. Enamel cells of Orai1K14 mice do not show increased expression of an unfolded protein response marker.

    Fig. S3. Normal dental phenotype in ORAI2-deficient mice.

    Fig. S4. Transduction efficiency in LS8 cells.

    Fig. S5. Immunofluorescence analysis showing down-regulation of ORAI1 in GFP-positive shOrai1 cells.

    Fig. S6. Orai1 expression is not significantly altered in shOrai2 cells.

    Fig. S7. Apoptosis and cell death.

    Fig. S8. A small Ca2+ leak is present in shOrai1/Orai2 cells.

    Fig. S9. Increased extracellular acidification rate in shOrai1 cells.

    Fig. S10. Quantification of velocity (slope) of ER Ca2+ refilling.

    Table S1. Primers and shRNA sequences used in this study.

  • This PDF file includes:

    • Fig. S1. Unchanged expression of Ambn in ORAI-deficient cells.
    • Fig. S2. Enamel cells of Orai1K14 mice do not show increased expression of an unfolded protein response marker.
    • Fig. S3. Normal dental phenotype in ORAI2-deficient mice.
    • Fig. S4. Transduction efficiency in LS8 cells.
    • Fig. S5. Immunofluorescence analysis showing down-regulation of ORAI1 in GFP-positive shOrai1 cells.
    • Fig. S6. Orai1 expression is not significantly altered in shOrai2 cells.
    • Fig. S7. Apoptosis and cell death.
    • Fig. S8. A small Ca2+ leak is present in shOrai1/Orai2 cells.
    • Fig. S9. Increased extracellular acidification rate in shOrai1 cells.
    • Fig. S10. Quantification of velocity (slope) of ER Ca2+ refilling.
    • Table S1. Primers and shRNA sequences used in this study.

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