Gliadin Peptides Induce Tissue Transglutaminase 2+ Activation and ER-Stress through Ca Mobilization in Caco-2 Cells Ivana Caputo1,2*, Agnese Secondo 3 , Marilena Lepretti1, Gaetana Paolella1, Salvatore Auricchio2, Maria Vittoria Barone2,4, Carla Esposito1,2 1 Department of Chemistry and Biology, University of Salerno, Fisciano, Salerno, Italy, 2 European Laboratory for the Investigation of Food-Induced Diseases (ELFID), University Federico II, Naples, Italy, 3 Department of Neurosciences, University Federico II, Naples, Italy, 4 Department of Pediatrics, University Federico II, Naples, Italy Abstract Background: Celiac disease (CD) is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG) seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca2+ homeostasis and tTG activity. Methods/Principal Findings: We studied Ca2+ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31–43 and 57–68 rapidly mobilized Ca2+ from intracellular stores. Specifically, peptide 31–43 mobilized Ca2+ from the endoplasmic reticulum (ER) and mitochondria, whereas peptide 57–68 mobilized Ca2+ only from mitochondria. We also found that gliadin peptide-induced Ca2+ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31–43, but not peptide 57–68, induces an increase of tTG expression. Finally, we monitored the expression of glucose- regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31–43, but not of peptide 57–68, induces the expression of both genes. Conclusions: By inducing Ca2+ mobilization from the ER, peptide 31–43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31–43 and 57–68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin–tTG crosslinking in enterocytes and specialized antigen-presenting cells. Citation: Caputo I, Secondo A, Lepretti M, Paolella G, Auricchio S, et al. (2012) Gliadin Peptides Induce Tissue Transglutaminase Activation and ER-Stress through Ca2+ Mobilization in Caco-2 Cells. PLoS ONE 7(9): e45209. doi:10.1371/journal.pone.0045209 Editor: Maria Gasset, Consejo Superior de Investigaciones Cientificas, Spain Received April 2, 2012; Accepted August 16, 2012; Published September 25, 2012 Copyright: 2012 Caputo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from ‘‘Fondi di Ateneo per la Ricerca di Base (FARB)’’ 2009, ORSA090313. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: icaputo@unisa.it Introduction Celiac disease (CD) is a complex inflammatory condition of the proximal small intestine caused by a specific response to peptides derived from ingested gliadin [1]. Immunotoxic gliadin peptides initiate a deleterious adaptive and innate immune response in the intestinal epithelium of CD patients. A-gliadin peptide 31-43/49 (p31–43) is the prototype of peptides that modulate the innate response [2], whereas peptide 57–68 (p57–68), which binds to HLA-DQ2/8 molecules, is one of the dominant epitopes recognized by T cells isolated from the intestine of CD patients [3]. However, the innate and adaptive immune systems may respond synergistically to gliadin peptides [1]. The role of post- translational modifications of gliadin peptides catalyzed by tissue transglutaminase (tTG) is thought to play a crucial role in CD [4,5]. Tissue TG is a Ca2+-dependent enzyme that catalyzes the formation of isopeptide linkages between the c-carboxamide group of protein-bound glutamine residues and the e-amino group of protein-bound lysine residues [6]. Glutamine residues can be deamidated to glutamic acid as a side-reaction in the absence of suitable amines or at low pH. Furthermore, tTG also binds and GTP; hence the enzyme can function as a cell signal transducer in association with the a1b-adrenoreceptor [7]. Tissue TG is predominantly an intracellular protein localized in the cytosol, mitochondria, nucleus, and cell membrane compartments [6], but it is also secreted extracellularly even though it lacks a signal leader peptide. Recently, Zemskov et al. described secretion of tTG that involves phospholipid-dependent delivery into recycling endo- somes [8]. Various functions have been ascribed to tTG in both the intra- and extracellular environment: in fact, it plays a role in matrix stabilization, cell adhesion and migration, and in cell death and PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45209