Figure 1.
Effect of p31–43 and p57–68 on [Ca2+]i in Caco-2 cells.
(A) and (B) Single-cell traces representative of the effect of 20 µg/ml p31–43 and p57–68, respectively, on [Ca2+]i. Starting time of perfusion is indicated by the arrows. Ionomycin (1µM) was added as control (grey arrows) at the end of the experiment. (C) and (D) Dose-dependent effect of p31–43 and p57–68, respectively, on [Ca2+]i increase. PCTR (20 µg/ml) served as control. From 40–65 cells were monitored in each experiment. Each bar represents the mean ± SEM of data obtained in three independent experimental sessions. *p<0.05 versus its respective control (basal values); **p<0.05 versus previous concentrations and control.
Figure 2.
Effect of Ca2+-free and THP on [Ca2+]i increase induced by gliadin peptides in Caco-2 cells.
(A) and (B) Superimposed single-cell traces representative for the effect of 20 µg/ml p31–43 and p57–68, respectively, in a Ca2+-free buffer, or in a Ca2+-free buffer plus 1 µM THP, on [Ca2+]i. Before peptide perfusion (black arrows), cells were preincubated with THP for 10 min (grey arrows), to deplete ER. (C) Quantification of the effect of the treatments reported in (A) and (B) on [Ca2+]i. Each bar represents the mean ±SEM of data obtained in three independent experimental sessions. *p<0.05 versus its respective control; **p<0.05 versus peptide alone.
Figure 3.
Effect of FCCP on [Ca2+]i increase induced by gliadin peptides in Caco-2 cells.
(A) and (B) Superimposed single-cell traces representative of the effect of 20 µg/ml p31–43 and p57–68, respectively, in a Ca2+-free buffer, and in a Ca2+-free buffer plus 1µM THP and 300 nM FCCP, on [Ca2+]i. Before peptide perfusion (black arrows), cells were preincubated with FCCP and THP for 10 min (grey arrows). (C) Quantification of the effect of the treatments reported in (A) and (B) on [Ca2+]i. Each bar represents the mean ± SEM of data obtained in three independent experimental sessions. *p<0.05 versus peptide alone; **p<0.05 versus peptide plus THP.
Figure 4.
Microscopic visualization of tTG transamidating activity in Caco-2 cells.
(A) Microscopic visualization of pentylamine-biotin incorporation in situ, ×40, in the presence of a complete medium. Peptides were used at 20 µg/ml, ionomycin at 10 µM, and THP at 1µM. Where indicated, the tTG inhibitor cystamin (200 µM) was added 5 min before treatment. (B) Microscopic visualization of p31–43-biotin and p57–68-biotin incorporation in situ, x40, in the presence of ionomycin. (C) Confocal images of pentylamine-biotin incorporation in situ, x63 (LSM 510 Zeiss microscope), in the absence or presence of p31–43 20 µg/ml. (D) tTG localization (red) in ionomycin-treated cells and superimposition (merge) with intracellular tTG activity (green). Nuclei are in blue (Hoescht staining). Arrows indicate nuclei in which tTG is increased (E) Microscopic visualization of pentylamine-biotin incorporation in situ, ×40, in the presence of a Ca2+-free medium.
Figure 5.
Quantification of tTG activity by the microplate assay.
(A) The microplate assay was performed on 25 µg of cell lysates obtained after treatment. Values are the means ± SD of at least 3 independent experiments in triplicate. *p<0.05 versus tTG basal activity. (F) Inhibition by cystamin of tTG activity induced by ionomycin and p31–43. Values are the means ± SD of three independent experiments in triplicate. *p<0.05 versus the respective activity in the absence of the inhibitor.
Figure 6.
Analysis of p31–43 induced tTG expression in Caco-2 cells.
(A) Quantification of tTG mRNA by real-time RT-PCR after 24 and 48 h of treatment with 20 µg/ml p31–43 and p57–68. The amount of mRNA of tTG is normalized to that of GAPDH. Values are the means ± SD of three independent experiments. *p<0.05 versus untreated. (B) Western blot analysis of tTG protein level after 48 h of treatment with 20 µg/ml p31–43 and p57–68. The blot shown is representative of three independent experiments. (C) Densitometric analysis (means of three independent western blot experiments) of protein levels after 24 and 48 h of treatments. The amount of tTG is normalized to that of tubulin. Values are the means ± SD *p<0.05 versus untreated.
Figure 7.
Analysis of GRP78 expression in Caco-2 cells.
(A) Quantification of GRP78 mRNA by real-time RT-PCR after 24 h of treatment with 1 µM THP, or 20 µg/ml p31–43 and p57–68. The amount of mRNA of GRP78 is normalized to that of GAPDH. Values are the means ± SD of at least three independent experiments. *p<0.05 versus untreated. (B) Western blot analysis of GRP78 protein level after 24 and 48 h of treatments with 20 µg/ml p31–43 and p57–68. Cells were exposed to THP (1 µM) for 24 h only. The blot shown is representative of three independent experiments. (C) Densitometric analysis (means of three independent western blot experiments). The amount of GRP78 is normalized to that of tubulin. Values are the means ± SD *p<0.05 versus untreated.
Figure 8.
Analysis of CHOP expression in Caco-2 cells.
(A) Quantification of CHOP mRNA by real-time RT-PCR after 24 h of treatment with 1 µM THP, or 20 µg/ml p31–43 and p57–68. The amount of CHOP mRNA is normalized to that of GAPDH. Values are the means ± SD of three independent experiments. *p<0.05 versus untreated. (B) Western blot analysis of CHOP protein level after 24 h of treatments. The blot shown is representative of three independent experiments. (C) Densitometric analysis (means of three independent western blot experiments). The amount of CHOP is normalized to that of tubulin. Values are the means ± SD *p<0.05 versus untreated.
Figure 9.
A model of the possible relationship between gliadin peptides, tTG activation, ER-stress and the inflammatory response.
Toxic and immunogenic gliadin peptides penetrate inside the cells through vesicular trafficking (1) [14], [18], [20] and rapidly induce Ca2+ release from the ER (2) and mitochondria (3). The increased [Ca2+]i activates normally silent tTG (4), which in turn deamidates gliadin peptides (5) and/or produces cross-links between the peptides and the tTG itself (6) or between the peptides and other cellular proteins [4], [5], [11], [12]. In addition, active tTG transamidates IκBα [38], PPARγ [18], and Rho A [36], which are key regulators of the inflammatory response. Persistent stimulation with toxic gliadin peptides (p31–43) can also trigger an ER-stress response (8) that, in turn, can modulate the expression of inflammatory genes (9) [45].