Ca2+ Mobilization Induced by Gliadin Figure 8. Analysis of CHOP expression in Caco-2 cells. (A) Quantification of CHOP 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 CHOP mRNA 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 CHOP protein level after 24 h of treatments. The blot shown is representative of three independent experiments. (C) Densitometric analysis (means of three independent western blot experiments). The amount of CHOP is normalized to that of tubulin. Values are the means 6 SD *p,0.05 versus untreated. doi:10.1371/journal.pone.0045209.g008 found in ischemia-reperfusion injury, receptor over-stimulation and oxidative stress [44]. Depletion of Ca2+ from the ER, which is the most important intracellular store of Ca2+, can cause protein misfolding and ER-stress. ER-stress triggers a series of signaling and transcriptional events known as the unfolded protein response. The unfolded protein response attempts to restore homeostasis in the ER but, if unsuccessful, can trigger apoptosis in the stressed cells and local inflammation [45]. Chaperone production is upregulated in response to increased misfolding, and the magnitude of the increase in chaperone levels, particularly GRP78, is widely used as a marker of ER-stress [26,45]. Here we report that stimulation of Caco-2 cells with p31–43 increases GRP78 mRNA expression by about nine-fold, and GRP78 protein levels by about 50% versus basal GRP78 levels in untreated cells. We also evaluated whether prolonged treatment with gliadin peptides could activate CHOP, a transcription factor that primarily mediates stress-linked apoptosis in cells with an irrecoverable level of ER-stress [46]. We found a moderate but significant increase of CHOP expression (both as mRNA and as protein) in cells treated with p31–43, but not with p57–68. Interestingly, it was recently reported that ER-stress-induced CHOP can negatively modulate PPARc action, thus enhancing the pro-inflammatory response in human intestinal epithelial cells [47]. ER-stress has been associated with an increasing number and wide variety of human diseases, namely, cancer, diabetes, developmental disorders, and neurodegenerative, infectious and inflammatory diseases [48]. The concept that ER-stress, elicited by the gliadin peptide p31–43, could be involved in CD is intriguing also because unresolved ER-stress leads to intestinal epithelial cell dysfunctions typical of the entity inflammatory bowel disease [49]. In conclusion, given the direct links between ER-stress/un- folded protein response and local and systemic inflammation, we suggest that p31–43, which is responsible for innate immunity in CD, could promote an ER-stress pathway by inducing rapid Ca2+ mobilization from the ER, and so amplify a local inflammatory response (Figure 9). Moreover, by mobilizing Ca2+ from in- tracellular stores, both p31–43 and p57–68 could induce tTG- mediated modifications of several key regulators of the in- flammatory response. Finally, intracellular tTG activation could allow deamidation of immunogenic gliadin peptides and the formation of gliadin-tTG complexes (Figure 9) inside enterocytes and other specialized antigen-presenting cells, such as duodenal dendritic cells and macrophages, which contain large amounts of tTG. PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e45209