Ca2+ Mobilization Induced by Gliadin Figure 6. Analysis of p31–43 induced tTG expression in Caco-2 cells. (A) Quantification of tTG mRNA by real-time RT-PCR after 24 and 48 h of treatment with 20 mg/ml p31–43 and p57–68. The amount of mRNA of tTG is normalized to that of GAPDH. Values are the means 6 SD of three independent experiments. *p,0.05 versus untreated. (B) Western blot analysis of tTG protein level after 48 h of treatment with 20 mg/ml p31–43 and p57–68. The blot shown is representative of three independent experiments. (C) Densitometric analysis (means of three independent western blot experiments) of protein levels after 24 and 48 h of treatments. The amount of tTG is normalized to that of tubulin. Values are the means 6 SD *p,0.05 versus untreated. doi:10.1371/journal.pone.0045209.g006 effect of GTP/GDP. When activated, tTG can interact with various intracellular proteins and so alter their structure, function, and/or stability [28–30]. Interestingly, in a previous proteomic study, we identified many tTG-modified protein targets in human intestinal epithelial cells by using both an acyl-acceptor tTG substrate (pentylamine-biotin) and an acyl-donor tTG substrate (the hexapeptide TVQQEL) [31]. Here we report that treatment of Caco-2 cells with gliadin peptides p31–43 and p57–68 activated intracellular tTG by causing a rapid increase of Ca2+ in the cytosol. However, the effect induced by p31–43 was more pronounced than the effect induced by p57–68. Treatment of cells with irrelevant control peptides did not induce tTG activation. Interestingly, as a consequence of tTG activation, p31–43 and p57–68 appear to be cross-linked to cellular acyl-acceptor proteins. This finding is in line with our previous result that tTG mediates p31–43 incorporation into CD mucosal enterocytes in situ [23]. The observation that active tTG can recognize and modify gliadin peptides inside the cells led to the notion that also tTG-mediated deamidation could be an intracellular event. Our finding that the intracellular Ca2+ concentration increased in Caco-2 cells also in the absence of extracellular Ca2+ ions demonstrates that gliadin peptides induced Ca2+ mobilization from intracellular Ca2+ deposits. Consequently, we used THP and FCCP, which specifically deplete the ER and mitochondria, respectively, to identify the intracellular Ca2+ stores involved in the mechanism of Ca2+ release induced by p31–43 and p57–68. Our data reveal two main pathways of Ca2+ release, namely, p31–43 modulates intracellular Ca2+ homeostasis by mobilizing Ca2+ ions from the ER and mitochondria, whereas p57–68 mobilizes Ca2+ ions only from mitochondria. Peptides 31–43 and p57–68 can enter cells and localize at vesicular level. In particular, p31–43 localizes in the early vesicular compartment when p57–68 progresses to the late compartment [14,20]. Peptide 31–43 is delayed in the early compartment because it deranges the correct localization of the hepatocyte growth factor-regulated tyrosine kinase substrate [14]. Indeed, the hepatocyte growth factor- regulated tyrosine kinase substrate is a key regulator of endocytic maturation that acts at the early vesicular compartment. Thus, the PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45209