Lysosomal accumulation of gliadin p31–43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARy downregulation in intestinal epithelial cells and coeliac mucosa

Maiuri, Luigi, Luciani, Alessandro, Villella, Valeria Rachela, Vasaturo, Angela, Giardino, Ida, Pettoello-Mantovani, Massimo, Guido, Stefano, Cexus, Oliver N., Peake, Nick, Londei, Marco and Quaratino, Sonia (2010) Lysosomal accumulation of gliadin p31–43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARy downregulation in intestinal epithelial cells and coeliac mucosa. Gut, 59 (3), 311-319. (doi:10.1136/gut.2009.183608).

Abstract

Background: an unresolved question in coeliac disease is to understand how some toxic gliadin peptides, in particular p31–43, can initiate an innate response and lead to tissue transglutaminase (TG2) upregulation in coeliac intestine and gliadin sensitive epithelial cell lines.

Aim: we addressed whether the epithelial uptake of p31–43 induces an intracellular pro-oxidative envoronment favouring TG2 activation and leading to the innate immune response.

Methods: the time course of intracellular delivery to lysosomes of p31–43, p?-2 or p?-9 gliadin peptides was analysed in T84 and Caco-2 epithelial cells. The effects of peptide challenge on oxidative stress, TG2 and peroxisome proliferator-activated receptor (PPAR)? ubiquitination and p42/44–mitogen activated protein (MAP) kinase or tyrosine phosphorylation were investigated in cell lines and cultured coeliac disease biopsies with/without anti-oxidant treatment or TG2 gene silencing by immunoprecipitation, western blot, confocal microscopy and Fluorenscence Transfer Resonance Energy (FRET) analysis.

Results: after 24 h of challenge p31–43, but not p?-2 or p?-9, is still retained within LAMP1-positive perinuclear vesicles and leads to increased levels of reactive oxygen species (ROS) that inhibit TG2 ubiquitination and lead to increases of TG2 protein levels and activation. TG2 induces cross-linking, ubiquitination and proteasome degradation of PPAR?. Treatment with the antioxidant EUK-134 as well as TG2 gene silencing restored PPAR? levels and reversed all monitored signs of innate activation, as indicated by the dramatic reduction of tyrosine and p42/p44 phosphorylation.

Conclusion: p31–43 accumulation in lysosomes leads to epithelial activation via the ROS–TG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPAR? downregulation

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