Ca2+ Mobilization Induced by Gliadin Figure 7. Analysis of GRP78 expression in Caco-2 cells. (A) Quantification of GRP78 mRNA by real-time RT-PCR after 24 h of treatment with 1 mM THP, or 20 mg/ml p31–43 and p57–68. The amount of mRNA of GRP78 is normalized to that of GAPDH. Values are the means 6 SD of at least three independent experiments. *p,0.05 versus untreated. (B) Western blot analysis of GRP78 protein level after 24 and 48 h of treatments with 20 mg/ml p31–43 and p57–68. Cells were exposed to THP (1 mM) for 24 h only. The blot shown is representative of three independent experiments. (C) Densitometric analysis (means of three independent western blot experiments). The amount of GRP78 is normalized to that of tubulin. Values are the means 6 SD *p,0.05 versus untreated. doi:10.1371/journal.pone.0045209.g007 different localization of the two gliadin peptides may explain why they mobilized Ca2+ ions from different compartments. We found that tTG was active in the cytosol and very active in the nucleus of Caco-2 cells treated with gliadin peptides, which indicates that tTG translocates into the nucleus in response to the elevated intracellular Ca2+ level. The presence of an enhanced level of active tTG enzyme in the nuclear fraction as a consequence of increased tTG import from the cytoplasm to the nucleus is not a novelty [25,32,33]. The molecular mechanisms underlying tTG translocation are poorly understood [33]. However, the impor- tance of nuclear tTG in regulating gene expression is well documented. For example, McConoughey et al. [34] reported that, in the nucleus, transamidation can dysregulate the expression of metabolic, chromatin, chaperone and cell death genes in Huntington’s disease. Moreover, Tatsukawa et al. [35] demon- strated that tTG activity is induced in the nuclei of ethanol-treated hepatocytes. Active tTG cross-links and inactivates the general transcription factor Sp1 thereby inducing hepatic apoptosis. In the cytosol, tTG-catalyzed transamidation of glutamine 63 of RhoA activates RhoA, which plays a key role in the signaling mechanism of the MAPK pathways [36]; it also increases endothelial permeability [37]. In addition, tTG participates in the activation of nuclear factor kappa (NF-k)B by cross-linking the inhibitory protein IkBa [38]. NF-kB is a transcription factor that is constitutively active in the intestinal mucosa of patients with untreated CD [39] and is considered central to intestinal inflammation [40–42]. Finally, it has been hypothesized that tTG-mediated downregulation of the PPARc signaling pathway in cells treated with p31–43 may contribute to NF-kB activation and therefore plays a pivotal role in CD-associated inflammation [18,43]. In line with this scenario, we observed that prolonged treatment of Caco-2 cells with p31–43, but not with p57–68, increased tTG mRNA and protein expression in Caco-2 cells. This finding supports the hypothesis that p31–43 could induce tTG- mediated pro-inflammatory modifications in cells. A large body of evidence suggests that Ca2+ is an important regulator of cell fate. An abrupt increase of intracellular Ca2+ is PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45209