TMEM203 is required for maintaining normal ER calcium level and calcium dependent gene expression
TMEM203 deficient mice were created (See S4 Fig and Methods) and ER calcium levels in Tmem203 deficient and wild type (WT) MEF cells were compared. Surprisingly, TG-induced total calcium release from the ER was substantially lower in Tmem203 null MEF cells as compared to Het or WT MEFs (Fig 3A). Treatment with m-3mFBS, a PLC agonist which depletes ER by the activation of the IP3R, or ionomycin, also produced much lower calcium release from the Tmem203 null MEF cells (Fig 3B and 3C). These data suggest that TMEM203 deficiency also resulted in lower ER calcium stores. The role of TMEM203 on ER calcium stores in human HEK293 cells was also examined by measuring ER calcium stores directly using the D1ER calcium sensor after siRNA knockdown of TMEM203. As shown in Fig 3D, TMEM203 siRNA treatment significantly lowered basal calcium levels. Treatment with TG further reduced ER calcium with similar kinetics and magnitude in TMEM203 and control siRNA treated cells suggesting that the steady state levels of ER calcium were reduced by TMEM203 inhibition but SERCA refilling was unaffected. Inhibition of TMEM203 expression by siRNA was confirmed by QPCR and the extent of reduction of TMEM203 expression with multiple siRNAs correlated well with the observed reduced constitutive ER calcium levels (data not shown).
10.1371/journal.pone.0127480.g003 Fig 3  Altered calcium homeostasis in Tmem203 deficient Mouse Embryonic Fibroblast cells.
(A) Cytoplasmic calcium flux were measured by flow cytometry from MEF cells derived from Tmem203—WT (Blue), HET (Green) and null (Red) mice. Treatment with 1 μM TG and EGTA in calcium free buffer showing ER-released calcium flux. Data are representative of at least 3 experiments from multiple MEF derived from littermates. (B) As described in (A), the calcium flux were measured upon treatment with 50 μM m-3M3FBS. (C) As described in (A), the calcium flux were measured upon treatment with 5 μM Ionomycin. (D) Single cell fluorescent microscopy based direct ER calcium measurements in D1ER-HEK293 cells transfected with non-targeting or TMEM203 specific siRNA. Basal ER calcium levels and ER calcium release upon TG treatment was monitored in siRNA or control siRNA transfected D1ER-HEK293 cells. The measurements showed reduced calcium levels in TMEM203 knock down cells but similar TG induced calcium leak kinetics. [Mean; +/- SE; n = 47 (non targeting siRNA) & n = 54 (TMEM203 siRNA)]. Further, we looked at the expression of calcium/NFAT dependent genes—calreticulin (Carl) and calcitonin receptor (Calcr) [41,42] upon stimulation with TG or Ionomycin in MEF cells. In response to ER calcium depletion by TG or Ionomycin treatment, the expression of Carl and Calcr were induced as previously reported. Induction of Carl and Calcr were significantly reduced in Tmem203 null MEF cells as compared to WT-MEF cells (S5 Fig) indicating that Tmem203 affects store depletion-induced calcium/NFAT dependent gene expression.