PMCA Ca2+ clearance in dental enamel cells depends on the magnitude of cytosolic Ca2+

Enamel formation (amelogenesis) is a two-step process whereby crystals partially grow during the secretory stage followed by a significant growth expansion during the maturation stage concurrent with an increase in vectorial Ca2+ transport. This requires tight regulation of cytosolic Ca2+ (cCa2+) concentration in the enamel forming ameloblasts by controlling Ca2+ influx (entry) and Ca2+ extrusion (clearance). Gene and protein expression studies suggest that the plasma membrane Ca2+-ATPases (PMCA1-4) are likely involved in cCa2+ extrusion in ameloblasts, yet no functional analysis of these pumps has been reported nor whether their activity changes across amelogenesis. PMCAs have high Ca2+ affinity and low Ca2+ clearance which may be a limiting factor in their contribution to enamel formation as maturation stage ameloblasts handle high Ca2+ loads. We analyzed PMCA function in rat secretory and maturation ameloblasts by blocking or potentiating these pumps. Low/moderate elevations in cCa2+ measured using the Ca2+ probe Fura-2-AM show that secretory ameloblasts clear Ca2+ faster than maturation stage cells through PMCAs. This process was completely inhibited by an external alkaline (pH 9.0) solution or was significantly delayed by the PMCA blockers vanadate and caloxin 1b1. Eliciting higher cCa2+ transients via the activation of the ORAI1 Ca2+ channel showed that the PMCAs of maturation ameloblasts were more efficient. Inhibiting PMCAs decreased the rate of Ca2+ influx via ORAI1 but potentiation with forskolin had no effect. Our findings suggest that PMCAs are functional Ca2+ pumps during amelogenesis regulating cCa2+ upon low and/or moderate Ca2+ stimulus in secretory stage, thus participating in amelogenesis.

Table S1.Rat primer sequences used for qRT-PCR.

Fig. S1. Confirmation of the purity of the enamel organ cell dissections. (A)
Representative fluorescence images of primary ameloblast cultures labelled using a CD90 antibody for fibroblasts (red left panels) or loaded with the ratiometric Ca 2+ probe Fura-2-AM (green center panels).Red and green images were merged (right panels) and the overlapping cells were not included in the analysis.Scale bars: 100 μm.A rise in cCa 2+ elicited by ATP (3 μM, 10 μM and 30 μM) in secretory (SEC) and maturation (MAT) ameloblast stages compared to levels evoked by SOCE stimulation.Histograms show the delta (∆) Ca 2+ peak.Data represent the mean ± SEM of ≥40 cells from 3-5 independent experiments analyzed by one-way ANOVA followed by Tukey's multiple comparison post-hoc test.*P < 0.05 vs. comparing maturation relative to secretory stage under the same conditions.#P < 0.05 and denotes comparison between SEC stage cells from 3 μM and 10 μM ATP, between 10 μM and 30 μM ATP.&P < 0.05 denotes comparisons across MAT for subsequent treatments (ATP 3 μM vs 10 μM, 10 μM vs 30 μM, and 30μM ATP vs SOCE).n.s., non-significant.showing cCa 2+ transients recorded after pre-incubation with thapsigargin (TG,15 min, 2 μM) followed by perfusion of Ca 2+ -free Ringer's (60 to 300s).Ca 2+ clearance via PMCAs was monitored in the absence or presence of forskolin (10 µM, for 10 min).Quantification of the rate of Ca 2+ efflux is shown in (C).Data represent the mean ± SEM of ≥121 cells from 3-5 independent experiments analyzed by one-way ANOVA followed by Tukey's multiple comparison post-hoc test.*P < 0.05 vs. #p < 0.05 (#denotes differences between SEC and MAT).n.s.non-significant.

Fig. S1 .
Fig. S1.Purity of the enamel ameloblasts culture confirmed using a PE-conjugated monoclonal anti-rat CD90 antibody and Enam and Odam expression by RT-PCR in secretory (SEC) and maturation (MAT) ameloblasts.

Figure S4 .
Fig. S3.Ringer's solution replacement does not affect cCa2+ concentrations in ameloblasts.(A) Changes in external solutions with or without Ca 2+ does not alter cCa 2+ in secretory (SEC) or maturation (MAT) stage ameloblasts.We evaluated cCa 2+ levels in Ca 2+ -free Ringer's solution followed by a re-addition of 2.5 mM of extracellular Ca 2+ in normal Ringer's.(B) Delta (∆) Ca 2+ peak.Data represent the mean ± SEM of 3 independent experiments analyzed by two-tailed unpaired Student's t-test at n.s., non-significant.

Table S1 .
Rat primers sequences used for qRT-PCR.