Ca2+ Mobilization Induced by Gliadin Figure 5. Quantification of tTG activity by the microplate assay. (A) The microplate assay was performed on 25 mg of cell lysates obtained after treatment. Values are the means 6 SD of at least 3 independent experiments in triplicate. *p,0.05 versus tTG basal activity. (F) Inhibition by cystamin of tTG activity induced by ionomycin and p31–43. Values are the means 6 SD of three independent experiments in triplicate. *p,0.05 versus the respective activity in the absence of the inhibitor. doi:10.1371/journal.pone.0045209.g005 the expression of two well known biochemical marker of ER stress, GRP78 and CHOP ([26,27]. We treated Caco-2 cells for 24 h with 20 mg/ml of p31–43 or with 1 mM of THP, which is considered an ER stress-inducing agent. We found that, in the presence of THP, GRP78 mRNA expression increased about 30-fold with respect to basal GRP78 expression in untreated cells (Figure 7A). Importantly, in the presence of p31–43, GRP78 mRNA expression increased about nine-fold (Figure 7A), which indicates that p31–43 triggered an ER-stress response in Caco-2 cells. Interestingly, p31–43 induced a very similar GRP78 mRNA increase when used at lower concentrations (i.e., 10 and 5 mg/ml). However, under the afore- mentioned experimental conditions, p57–68 also induced a small but significant increase in GRP78 mRNA expression. We are unable to explain this finding. As shown in Figure 7B and C, the expression of the GRP78 protein increased by about 50% after treatment for 24 h with 20 mg/ml of p31–43. In addition, GRP78 protein expression was doubled after treatment for 48 h with p31– 43. Under the same conditions, p57–68 did not induce an increase of GRP78 protein expression. As expected, after treatment with THP for 24 h, GRP78 protein expression was increased about five-fold. We next measured CHOP expression and found that treatment with THP for 24 h caused an increase of about 14-fold and three- fold in CHOP mRNA and protein expression, respectively (Figure 8). Treatment with p31–43 increased CHOP mRNA expression four-fold, and CHOP protein expression two-fold; p57– 68 did not modify CHOP expression (Figure 8). Discussion In CD, the catalytic activity of tTG seems to be crucial for the deamidation of immunogenic gliadin peptides as well as for the formation of gliadin-tTG complexes [1,4]. Moreover, the catalytic activity of tTG has been associated with an increased inflamma- tory response to gliadin [18]. However, it is not clear where tTG is activated at cellular and tissue level. Inside cells, tTG is normally latent because of the low Ca2+ concentration and the inhibitory PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45209