survival [6,9,10]. The catalytic activity of tTG is implicated in the pathogenesis of several human diseases, including CD [10]. In celiac patients, tTG deamidates specific gliadin glutamines, thus generating a series of gliadin peptides that bind to HLA-DQ2 and DQ8 molecules with high affinity. The resulting HLA-DQ2 (DQ8)-gliadin peptide interaction triggers the proinflammatory T cell response [1]. Moreover, in accordance with the upregulation of tTG in intestinal inflamed sites, tTG may generate additional antigenic epitopes by cross-linking gliadin peptides to itself or to other cellular proteins. Gliadin-tTG complexes may elicit an immune response to tTG by stimulating normally silent auto- reactive B-cells [11,12]. In fact, active CD is associated with serum antibodies against tTG. The exact location at which deamidation of immunogenic gliadin peptides and formation of gliadin–tTG complexes take place is not clear. Although little is known about the processing of gliadin peptides, there is evidence that they enter enterocytes [13,14]. However, do tTG-induced gliadin modifications in CD patients occur in enterocytes and/or in other antigen-presenting cells, or in the extracellular matrix? It has been demonstrated that extracellular tTG is inactive in the intestinal mucosa in the resting state and it is only transiently activated after some inflammatory stimuli and tissue injury [15]. Moreover, under normal conditions, tTG in the intracellular environment is a latent protein due to a low Ca2+ concentration and inhibition by GTP/GDP. However, under extreme conditions of cell stress or trauma, and after disturbance or loss of Ca2+ homeostasis, tTG may be activated and cause cross-linking of intracellular proteins, as observed during apoptosis or necrosis [16,17]. It has been reported that p31–43 causes increased production of reactive oxygen species, which leads to tTG overexpression and activation in intestinal epithelial cells; active tTG then induces ubiquitination and degradation of the peroxisome proliferator-activated receptor (PPAR)c [18], thus contributing to the inflammatory response. Although Ca2+ is a prerequisite for activation of tTG, and for the production of reactive oxygen species, the role of this important cellular ionic mediator in the pathogenesis of CD remains unknown and unexplored. We carried out the present study to determine the effect of gliadin peptides on Ca2+ homeostasis in the attempt to gain additional insights into the possible role exerted by gliadin peptides in the molecular mechanisms of tTG activation. Here we demonstrate that dietary wheat gliadin is able to regulate intracellular Ca2+ homeostasis. In fact, gliadin peptides rapidly mobilize Ca2+ ions from intracellular stores in a cell model of intestinal epithelial cells. However, p31–43 at a low concentration mobilizes calcium from the endoplasmic reticulum (ER), whereas both p31–43 and a higher concentration of p57–68 mobilize Ca2+ from mitochondria. We also show that Ca2+ ions released from intracellular stores as a consequence of gliadin peptide stimulation, are able to activate cytosolic and nuclear tTG. Finally, we found that p31–43, but not p57–68, increases the expression of tTG. It also increased the expression of glucose-regulated protein (GRP)- 78 and of CCAAT/enhancer binding protein-homologous protein (CHOP) thereby implicating the ER-stress pathway in CD mucosal damage. Methods Peptides Synthetic peptides were from Inbios (Naples, Italy). The sequences of p31–43 and p57–68 from A-gliadin were LGQQQPFPPQQPY and QLQPFPQPQLPY, respectively. In some experiments, we used N-terminal-biotin-labeled p31–43 and Ca2+ Mobilization Induced by Gliadin p57–68. Three synthetic peptide were used as irrelevant control peptides: QQPQDAVQPF from durum wheat (pCTR) [19]; PLIRPLLARPAK, which represents the 537–548 region of the human thyroid peroxidase (pHTP) [2]; LPQFEEIRNLALQTL- PAM, which represents the C-terminal sequence of A-gliadin (p229–246) [20]. Cell Culture Caco-2 cells were obtained from Interlab Cell Line Collection (Centro di Biotecnologie Avanzate, Genoa, Italy). Caco-2 cells were cultured in 100610-mm Petri dishes containing Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) non-essential amino acids, 0.2 mM L- glutamine, 50 units/ml penicillin and 50 mg/ml streptomycin (Invitrogen SRL, Milan, Italy). Cells were maintained at 37uC in a 5% CO2, 95% air-humidified atmosphere and passaged twice a week. Treatments were generally performed after 72–96 h after seeding. Intracellular Ca2+ Concentration Measurement Intracellular Ca2+ concentration ([Ca2+] ) was measured in i Caco-2 cells by single cell computer-assisted videoimaging using the Ca2+ indicator Fura-2 acetoxymethyl ester, as previously described [21,22]. Gliadin peptides were loaded at concentrations between 0.2 and 20 mg/ml for 30 min at 37uC in normal Krebs solution containing the following (in mM): 5.5 KCl, 160 NaCl, 1.2 MgCl2, 1.5 CaCl2, 10 glucose, and 10 Hepes-NaOH, pH 7.4. To selectively deplete intracellular Ca2+ stores, experiments were performed in the presence of thapsigargin (THP) (1 mM), an irreversible and selective inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase, and in the presence of the mitochondrial uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (300 nm). In situ tTG Assay and Quantification of tTG Transamidating Activity In situ tTG activity was visualized by using the tTG substrate pentylamine-biotin (Euroclone, Milan, Italy) as reported elsewhere [22]. Caco-2 cells were treated for 30 min with 10 mM ionomycin (Sigma-Aldrich, Milan, Italy), or with different amounts of gliadin peptides, or with 1 mM THP, in the presence of 0.5 mM pentylamine-biotin, before being processed for microscopy obser- vation. To visualize biotin-labeled p31–43 and p57–68 in the cells, peptides needed to be used at a concentration of 0.2 mM. Tissue TG localization in permeabilized cells was revealed by incubating cells with the mouse anti-tTG antibody, clone CUB7402 (Bioptica, Milan, Italy), diluted 1:200, and a secondary TRITC-conjugated antibody (1:200). Pentylamine-biotin was also used to quantify the tTG transamidating activity by a microplate assay [22]. Briefly, after 30 min of treatment with 20 mg/ml gliadin peptides, or with 1 mM THP, cells were lysed and proteins (25 mg) were coated into the wells of a 96-well plate. The wells were blocked with 10% bovine serum albumin then incubated with 1:5000 peroxidase- conjugated streptavidin (Euroclone) in 5% bovine serum albumin. To reveal peroxidase activity, 3,39,5,59-tetramethylbenzidine (Sigma-Aldrich, Milan, Italy) was added to each well and, after stopping the reaction with H2SO4, absorbances were read at 450 nm. RNA Extraction and Real-time RT-PCR Caco-2 cells were treated with different amounts of p31–43 or p57–68, or alternatively with 1 mM THP for 24 or 48 h. Total RNA was extracted with the Tri-Reagent (Sigma-Aldrich) PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45209