Ca2+ Mobilization Induced by Gliadin Figure 3. Effect of FCCP on [Ca2+]i increase induced by gliadin peptides in Caco-2 cells. (A) and (B) Superimposed single-cell traces representative of the effect of 20 mg/ml p31–43 and p57–68, respectively, in a Ca2+-free buffer, and in a Ca2+-free buffer plus 1mM THP and 300 nM FCCP, on [Ca2+]i. Before peptide perfusion (black arrows), cells were preincubated with FCCP and THP for 10 min (grey arrows). (C) Quantification of the effect of the treatments reported in (A) and (B) on [Ca2+]i. Each bar represents the mean 6 SEM of data obtained in three independent experimental sessions. *p,0.05 versus peptide alone; **p,0.05 versus peptide plus THP. doi:10.1371/journal.pone.0045209.g003 homeostasis. We next investigated whether the residual Ca2+ release induced by p31–43 was due to mitochondria, which are an important calcium store within the cell [24]. Using the mitochon- drial uncoupler FCCP (300 nM) together with THP, we found that the effect of p31–43 on [Ca2+]i was further reduced (Figure 3A and C). In addition, the effect of p57–68 was completely inhibited in the presence of FCCP (Figure 3B and C). These data suggest that p31–43 and p57–68 modulated intracellular Ca2+ homeosta- sis with different mechanisms: p31–43 mobilized Ca2+ from the ER and mitochondria, while p57–68 mobilized Ca2+ only from mitochondria. Gliadin Peptides Activate Intracellular tTG To investigate whether Ca2+ mobilization, induced by gliadin peptides, could activate the crosslinking function of intracellular tTG, we used pentylamine-biotin as tTG substrate to detect and quantify intracellular transamidating activity in Caco-2 cells. We first incubated cells in the presence of 20 mg/ml of p31–43 or p57– 68. In both cases, microscopy observation of fixed and permeabilized cells after incubation with FITC-conjugated streptavidin revealed a fluorescent signal, which indicates activa- tion of tTG crosslinking activity (Figure 4A). We obtained a similar fluorescence pattern when we treated cells with ionomycin, a Ca2+ ionophore that activates tTG intracellular activity [25]. To verify the specificity of tTG activity, we used cystamin, a well known tTG inhibitor, and found that cystamin inhibited the tTG activation induced by both ionomycin and p31–43 (Figure 4A). On the other hand, fluorescence intensity did not increase in cells treated with pCTR or with the other two irrelevant control peptides (pHTP and p229–246) (Figure 4A). Incubation with 5 mg/ml of p31–43 still produced a good fluorescence signal, whereas incubation with 5 mg/ml of p57–68 produced low fluorescence (not shown). Consequently, at a low concentration, p31–43 was more active than p57–68. We then explored the possibility that gliadin peptides could themselves be in situ tTG substrates. To this aim, we treated cells with biotinylated p31–43 or p57–68 for 30 min and we found that, in the presence of ionomycin, both peptides were able to cross-link to intracellular acyl-acceptor substrates (Figure 4B). After Ca2+ mobilization, we observed an increase in in- tracellular tTG transamidating activity in both the cytosol and nucleus of ionomycin- and peptide-treated cells; this is also evident in the confocal images of cells treated with p31–43 (Figure 4C). Moreover, tTG staining revealed that nuclear tTG, which normally constitutes a significant fraction of total tTG in Caco-2 cells, was often visibly increased in cells after Ca2+ mobilization and tTG activation (Figure 4D). Interestingly, we found that tTG was also activated when we stimulated cells with gliadin peptides in the presence of a Ca2+-free medium (i.e., phosphate-buffered saline without Ca2+ and Mg2+) PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45209