title: Lysosomal accumulation of gliadin p31-43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARgamma downregulation in intestinal epithelial cells and coeliac mucosa.
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, palpha-2 or palpha-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)gamma 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 palpha-2 or palpha-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 PPARgamma. Treatment with the antioxidant EUK-134 as well as TG2 gene silencing restored PPARgamma levels and reversed all monitored signs of innate activation, as indicated by the dramatic reduction of tyrosine and p42/p44 phosphorylation.
  
  
  CONCLUSIONS
  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 PPARgamma downregulation.
authors: Institute of Pediatrics, University of Foggia, viale Pinto 1, Foggia 71100, Italy. maiuri@unina.it
authors: Alessandro
authors: A
authors: Luciani
authors: Valeria Rachela
authors: VR
authors: Villella
authors: Angela
authors: A
authors: Vasaturo
authors: Ida
authors: I
authors: Giardino
authors: Massimo
authors: M
authors: Pettoello-Mantovani
authors: Stefano
authors: S
authors: Guido
authors: Olivier N
authors: ON
authors: Cexus
authors: Nick
authors: N
authors: Peake
authors: Marco
authors: M
authors: Londei
authors: Sonia
authors: S
authors: Quaratino
authors: Luigi
authors: L
authors: Maiuri
mesh_terms: D000293:Adolescent; D000328:Adult; D002446:Celiac Disease / Q000378:metabolism*; D015536:Down-Regulation / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism; D019204:GTP-Binding Proteins; D005903:Gliadin / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D009924:Organ Culture Techniques; D018384:Oxidative Stress / Q000502:physiology; D047495:PPAR gamma / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D017382:Reactive Oxygen Species / Q000378:metabolism; D011503:Transglutaminases / Q000502:physiology*; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism; D055815:Young Adult
chemical_list: D047495:PPAR gamma; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D025801:Ubiquitin; C549457:gliadin p31-43; D005903:Gliadin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D046988:Proteasome Endopeptidase Complex; D019204:GTP-Binding Proteins