ABSTRACTS: Oral Presentation Abstracts
Introduction: Specific ‘immunogenic’ regions of gliadin are generally thought to induce a dysregulated immune response in celiac disease (CD), but additional pleiotropic non-immune effects of the protein have been described in cultured cell lines and intestine preparations, that can be mapped to a ‘toxic’ peptide, P31–43, also found to damage mucosa from CD patients in vivo (1). To evaluate direct gliadin effects on cytoskeleton and cell cycle independently from T-cell immunogenic response, we used this ‘toxic’ peptide, and a mixture of peptic-tryptic gliadin peptides (PTG), in several cell systems.
Methods: Phalloidin-tex-red was used to stain actin cytoskeleton in Caco2, MCF7 and NIH3T3 cell lines treated or not with PTG, PTL (peptic-tryptic lactoalbumin peptides), P31–43 and the ‘T-cell immunogenic’ P57–68. Bromodeoxiuridine (BrdU) incorporation revealed the number of cells in S-phase of the cell cycle. ZD1839 was employed to inhibit Epidermal Growth Factor Receptor (EGFR) activity. Western blot analysis of EGFR and EGF-fitc allowed to follow EGFR-endocytosis both biochemically and by immunofluorescence microscopy.
Results: Crude gliadin peptic-tryptic peptides (PTG), or P31–43 alone, but not PTL or P57–68, induce in Caco2, MCF7 and NIH3T3 cell lines, after only 15 min, a rearrangement of actin citoskeleton very similar to that induced by EGF. ZD 1893, an inhibitor of EGFR kinase activity, prevents PTG induced actin rearrangements. PTG and P31–43 induce other EGF effects such as G0>S trasversion of cell cycle in arrested NIH3T3, that can also be prevented with EGFR inhibitor ZD1893, suggesting that, at least for these two assays, actin modification and cell cycle, PTG and P31–43 can act as EGF-like factors. A data bank search for structural analogs of peptide P31–43, revealed a strong similarity with HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), a protein essential to EGFR and other growth factors inactivation through endocytosis. Synergistic PTG-EGF effects on cell cycle, gliadin-induced delay in EGFR degradation and HRS ability to prevent PTG effects, propose a new role for gliadin peptides in EGFR, and possibly other receptors, endocytosis.
Conclusion: A model is proposed where gliadin fragments interfering with endocytosis could amplify the effects on actin rearrangements and cell cycle of trace amounts of EGF by prolonging receptor activation.
1. Marsh MN, Morgan S, Ensary A, Wardle T, Lobley R, Mills C, Auricchio S. Gastroenterology 1995,108: A871