Lysosomal accumulation of gliadin p31-43 peptide induces oxidative stress and tissue transglutaminase-mediated PPARgamma downregulation in intestinal epithelial cells and coeliac mucosa.
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.
Luciani
Alessandro
A
Institute of Pediatrics, University of Foggia, viale Pinto 1, Foggia 71100, Italy. maiuri@unina.it
Villella
Valeria Rachela
VR
Vasaturo
Angela
A
Giardino
Ida
I
Pettoello-Mantovani
Massimo
M
Guido
Stefano
S
Cexus
Olivier N
ON
Peake
Nick
N
Londei
Marco
M
Quaratino
Sonia
S
Maiuri
Luigi
L
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
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