TMEM203 expression activates calcineurin dependent transcription factors by elevating basal calcium levels
Previously we described a high content microscopy based cDNA screen to identify genes that induced nuclear translocation of the CREB coactivator, CRTC1 [8]. This and other work demonstrated translocation of the CRTC1, is induced by elevations in cAMP or intracellular calcium through rapid calcineurin dependent dephosphorylation of CRTC1 [6,8]. In addition to known regulators of calcium and cAMP signaling, several novel proteins were identified and retested here for the ability to induce nuclear translocation of CRTC1. Of the genes analyzed, TMEM203 transfection resulted in efficient CRTC1 translocation without inducing gross morphologic and/or apoptotic changes (S1 Table). As shown in Fig 1A, exogenous expression of TMEM203 in CRTC1-eGFP expressing HeLa cells resulted in nuclear translocation which was blocked by the calcineurin inhibitors Cyclosporine A (CsA) and FK506. Addition of the calcium chelator, EGTA, also blocked TMEM203 induced nuclear CRTC1, and this inhibition was reversed by the addition of excess calcium (data not shown). In addition expression of TMEM203-FLAG induced nuclear localization of NFAT1(1–402)-GFP in cotransfected HeLa cells (Fig 1B). TMEM203 expression resulted in production of de-phosphorylated NFAT-GFP which was blocked by CsA or FK506 (Fig 1C).
10.1371/journal.pone.0127480.g001 Fig 1  TMEM203 expression drives calcineurin dependent transcription factor activation by elevating the basal cytosolic calcium levels in HeLa cells.
(A) Stably expressed CRTC1-GFP localization was visualized using fluorescent microscope in HeLa-CRTC1-GFP cell line transiently expressing TMEM203–FLAG for 48 hrs. CRTC1-GFP (green) nuclear translocation was induced in cells co-expressing TMEM203-Flag (red). Nuclei (blue) were visualized with Hoechst. Nuclear translocation was inhibited by treatment with 5nM Cyclosporine A or 10nM FK506 for 2 hour prior to fixing the cells. Scale bars = 15 μm. (B) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector. 48 hours later the cells were visualized using fluorescent microscope. Scale bars = 15 μm. (C) HeLa cells were co-transfected with NFAT2 (1–402)-GFP and TMEM203-FLAG or empty vector as indicated. 48 hours later the cells were treated with 5nM Cyclosporine A (CsA) or 10nM FK506 for 2 hours and total cell lysates were prepared. The lysates were subjected to immunoblotting with indicated antibodies. (D) TMEM203-mcherry or mcherry transfected HeLa cells were seeded onto coverslips and single cell Fura-2 fluorescence based calcium measurements were performed. The measurements showed elevated basal calcium levels in TMEM203-mcherry expressing cells. (Mean; +/- SE; n = 64 cells (mcherry); 55 cells (TMEM203-mcherry) from multiple coverslips; p value = 4.06719E-30). Consistent with activation of calcineurin, TMEM203 over-expression increased intracellular calcium. After transfection of an m-cherry-TMEM203 construct, HeLa cells maintained significantly higher basal cytosolic calcium levels as compared to m-cherry transfected cells (Fig 1D). TMEM203 expression induced NFAT dependent transcription as indicated by an NFAT dependent reporter gene assay in the presence or absence of PMA (S1 Fig). Reporter induction was inhibited by CsA as well as by SKF96365, a potent inhibitor of extracellular calcium entry thought to inhibit ORAI1. These observations strongly suggest that TMEM203 expression leads to SOCE, increased cytoplasmic calcium concentrations, calcineurin activation and subsequent dephosphorylation and nuclear translocation of CRCT1 and NFAT.