Sections were cut 5 mm thick and stained with hematoxylin and eosin or Pirosirius Red

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. It has been reported that p3143 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 c, 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, p3143 at a low concentration mobilizes calcium from the endoplasmic reticulum, whereas both p3143 and a higher concentration of p5768 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 p3143, but not p5768, increases the expression of tTG. It also increased the expression of glucose-regulated protein 78 and of CCAAT/enhancer binding protein-homologous protein thereby implicating the ER-stress pathway in CD mucosal damage. p5768. Three synthetic peptide were used as irrelevant control peptides: QQPQDAVQPF from durum wheat ; PLIRPLLARPAK, which represents the 537548 region of the human thyroid peroxidase ; LPQFEEIRNLALQTLPAM, which represents the C-terminal sequence of A-gliadin . Cell Culture Caco-2 cells were obtained from Interlab Cell Line Collection. Caco-2 cells were cultured in 100610-mm Petri dishes containing Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 0.2 mM Lglutamine, 50 units/ml penicillin and 50 mg/ml streptomycin. Cells were maintained at 37uC in a 5% CO2, 95% air-humidified atmosphere and passaged twice a week. Treatments were generally performed after 7296 h after seeding. Intracellular Ca2+ Concentration Measurement Intracellular Ca2+ concentration was measured in Caco-2 cells by single cell computer-assisted videoimaging using the Ca2+ indicator Fura-2 acetoxymethyl ester, as previously described. 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: 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 , an MedChemExpress Mertansine irreversible and selective inhibitor of the sarcoplasmic reticulum Ca2+ ATPase, and in the presence of the mitochondrial uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone . In situ tTG Assay and Quantification of tTG Transamidating Activity In situ tTG activity was visualized by using the tTG substrate pentylamine-biotin as reported elsewhere. Caco-2 cells were treated for 30 min with