Gliadin Peptides Induce Tissue Transglutaminase Activation and ER-Stress through Ca2+ Mobilization in Caco-2 Cells

Abstract

BackgroundCeliac 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 FindingsWe 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.ConclusionsBy 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.

Biochemical modifications of gliadins induced by microbial transglutaminase on wheat flour

“…Subsequent studies showed that p31-43 altered the trafficking of vesicular compartments in the intestinal Caco-2 cell line leading to overexpression of IL-15/IL5R alpha complex and delaying the degradation of the active epidermal growth factor receptor (EGFR) that induces cell proliferation of cell lines and, possibly, crypts hyperplasia in CD [35]. More recently, gliadin peptide p31-43 was reported to induce tTGase activation in Caco-2 cells, an event mediated by the release of Ca 2+ ions from intracellular stores [24]. Enhancement of tTGase activity could contribute to the inflammatory response in CD.…”

“…It was previously shown that Ca 2+ mobilization induced by gliadin peptide p31-43 activated the crosslinking function of intracellular tTGase in Caco-2 cells [24]. Therefore, we tested the ability of p31-49 to activate tTGase in this cell line.…”

“…Therefore, we tested the ability of p31-49 to activate tTGase in this cell line. We used pentylamine-biotin as tTGase substrate and quantify the intracellular transamidating activity in Caco-2 cells by a microplate assay on cell homogenates [24] obtained after incubation with different amounts of p31-49. As previously reported for p31-43 [24], we found that tTGase activity was higher in p31-49-treated cells than in cells exposed to pentylamine-biotin alone (Fig.…”

Role of Corticotropin-releasing Factor in Gastrointestinal Permeability

“…Increased permeability and breakdown of intestinal barrier have been implicated in the origin of gastrointestinal and liver disorders, including celiac disease, 113 132 and cellular stress, through MHC class I chain related genes A and B and endoplasmic reticulum stress pathways, is linked to the disregulation of mucosal homeostasis. 133,134 Early in the 80s, celiac patients were shown to display increased intestinal permeability that normalized after several months on a gluten-free diet. 113 More precise in vitro investigations revealed that although strict gluten withdrawal restored intestinal histology, a subjacent defect in mucosal permeability, measured by cellobiose/mannitol ratio, was transiently induced by short exposure to gluten, suggesting that increased intestinal permeability in celiac disease could be a primary defect.…”

4-Nonylphenol reduces cell viability and induces apoptosis and ER-stress in a human epithelial intestinal cell line

“…Immunocomplexes were revealed using a chemiluminescence detection kit (Euroclone) according to the manufacturer's instructions. To detect GRP78 protein expression, cells were treated for 24 or 72 h with different amounts of 4-NP, or with THP, then cell lysates were prepared and western blot analysis was performed as previously described (Caputo et al, 2012). GRP78 protein was detected by using a rabbit anti-GRP78 polyclonal antibody (H-129, 1:1,000; Santa Cruz).…”