Ca2+ Mobilization Induced by Gliadin Figure 4. Microscopic visualization of tTG transamidating activity in Caco-2 cells. (A) Microscopic visualization of pentylamine-biotin incorporation in situ, 640, in the presence of a complete medium. Peptides were used at 20 mg/ml, ionomycin at 10 mM, and THP at 1mM. Where indicated, the tTG inhibitor cystamin (200 mM) was added 5 min before treatment. (B) Microscopic visualization of p31–43-biotin and p57–68-biotin incorporation in situ, x40, in the presence of ionomycin. (C) Confocal images of pentylamine-biotin incorporation in situ, x63 (LSM 510 Zeiss microscope), in the absence or presence of p31–43 20 mg/ml. (D) tTG localization (red) in ionomycin-treated cells and superimposition (merge) with intracellular tTG activity (green). Nuclei are in blue (Hoescht staining). Arrows indicate nuclei in which tTG is increased (E) Microscopic visualization of pentylamine-biotin incorporation in situ, 640, in the presence of a Ca2+-free medium. doi:10.1371/journal.pone.0045209.g004 (Figure 4E). This finding confirms that extracellular Ca2+ ions were not involved in tTG activation within the cells and that tTG activation was due to Ca2+ mobilization from intracellular stores. Finally, by treating cells with THP, both in the presence and in the absence of extracellular Ca2+ ions, we found that Ca2+ release from the ER was sufficient to activate intracellular tTG (Figure 4A and E). To quantify the increase of tTG activity, we carried out a microplate assay on cell homogenates obtained after treatment with p31–43 or p57–68. We found that tTG activity was more than 50% higher in p31–43-treated cells and about 40% higher in p57–68-treated cells than in cells exposed to pentylamine-biotin alone (Figure 5A). Tissue TG activation was similar in THP- treated cells, whereas control peptides had no effect. As expected, we observed a significant reduction of tTG activity, induced by ionomycin or p31–43, when we pretreated cells with cystamin (Figure 5B). Finally, we evaluated the effect of prolonged treatment with gliadin peptides on tTG expression. As shown in Figure 6, we recorded a moderate increase of tTG mRNA and protein after 24 h of treatment with p31–43 but not with p57–68. However, treatment with p31–43 for 48 h induced a four-fold increase of tTG mRNA expression and a two-fold increase of tTG protein. Gliadin Peptide 31–43 Induces GRP78 and CHOP Expression Since p31–43 can mobilize Ca2+ ions from the ER, we asked whether persistent stimulation with p31–43 could induce the ER stress response in Caco-2 cells. To test this hypothesis, we analysed PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45209