EGF mRNA and EGFR protein levels were increased in enterocytes of CD patients with villous atrophy and of GFD CD patients.

<p>Example of selected crypt enterocytes from 5-micron sections of intestinal biopsies frozen and air dried before and after capture. For each sample, 300 crypt epithelial cells were captured. B) Semiquantitative PCR analysis of a biopsy from a CD patient with villous atrophy, a CD patient on GFD and a control. (a) PCR of GADPH, used as a loading control, one representative experiment is shown, c is the control lane without mRNA. (b) PCR of EGF, one representative experiment is shown, c is the control lane without mRNA. (c) statistical analysis of data obtained from 3 CD patients with villous atrophy, 3 CD patients on gluten-free diet (GFD) and 3 controls with gastro-esophageal reflux. Columns represent the mean, bars the standard deviation, * = P<0.05; ** = P<0.01 (Student's t-test). C. Immunofluorescence of CD patients and controls' biopsies stained with anti-EGFR antibody. 40× objective. C = control without primary antibody.</p

Proliferation of crypt enterocytes was increased in CD.

<p>A. Immunofluorescence images of crypts from duodenal biopsies from a control, from a CD patient with villous atrophy, from a potential CD patient who were on a gluten-containing diet and from a GFD CD patient. Biopsies were cultured for 24 h with BrdU and then stained for cytokeratin to identify epithelial cells (red) and for BrdU (green) to identify proliferating cells. One representative experiment is shown. B. Quantitation of BrdU incorporation in intestinal biopsies. More than 300 cytokeratin-positive cells were counted in several fields in each sample; the number of BrdU- positive cells was expressed as a proportion of the total cytokeratin-positive cells. Columns represent the mean, bars the standard deviation, N. is the number of biopsies tested * = P<0.05; *** = P<0.001 (Student's t-test). One-way analysis of variance (ANOVA): P value = 0.0037 (4 groups, F = 5.437, R squared = 0.3242).</p

Phosphorylation of EGFR, ERK and total proteins was increased in skin fibroblasts of CD patients.

<p>Staining of total phosphorylated proteins in CD and controls fibroblasts. (a) Immunofluorescence of double staining with phalloidin (red) and anti-phosphotyrosin (green). Images obtained using a 63× objective (2× digital zoom) are shown. (b) Statistical analysis of fluorescence intensity/cell. For 5 patients and 4 controls, 3 independent experiments were done; in each experiment, the fluorescence intensity of 10 cells in random fields was measured. Columns represent means and bars standard deviation. * = P<0.05 (Student's t-test). B. Western blot analysis of total phosphorylated proteins in skin fibroblasts from CD patients on GFD and from controls. Phosphoproteins from CD patients and controls fibroblasts were lysates and immunoprecipitated (Ip), blotted and stained with anti-phosphotyrosine antibodies (blot anti-pY). The blots were stained again with anti-EGFR (blot anti-EGFR) and anti-ERK (blot anti-ERK) antibodies to identify the corresponding phosphorylated proteins. One representative experiment of 3 independent ones is shown for each subject (4 controls and 5 patients). C. Western blot analysis of phosphorylated ERK and EGFR in skin fibroblasts from CD patients on a GFD and from controls. (a) Western blot analysis of skin fibroblasts from CD patients and controls stained with anti-pY-ERK, anti-ERK and anti-tubulin antibodies. (b) Statistical analysis of WB obtained from 5 CD patients and 4 controls. Columns represent the mean, bars the standard deviation of the relative intensity of pY-ERK respect to total ERK protein. *** = P<0.001 (Student's t-test). (c) Western blot analysis of EGFR immunoprecipitated from skin fibroblasts and stained with anti-pY antibody. (d) Statistical analysis of WB obtained from 5 CD patients and 4 controls. Columns represent the mean, bars the standard deviation of the relative intensity of pY-EGFR respect to total EGFR protein** = P<0.01 (Student's t-test).</p

Increased proliferation of crypt enterocytes in CD depended on ERK activation.

<p>Quantitation of BrdU incorporation in intestinal biopsies. More than 300 cytokeratin-positive cells were counted in several fields in each sample; the number of BrdU- positive cells was expressed as a proportion of the total cytokeratin-positive cells. Dots represent single patients before (UN) and after ERK inhibitor PD98059, treatment. The horizontal bar is the mediane. ** = P<0.01 (Mann Whitney Test).</p

A Celiac Cellular Phenotype, with Altered LPP Sub-Cellular Distribution, Is Inducible in Controls by the Toxic Gliadin Peptide P31-43

<div><p>Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP) gene was identified as strongly associated with CD using genome-wide association studies (GWAS). The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD) and controls, without and with treatment with A-gliadin peptide P31-43. We observed a “CD cellular phenotype” in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis.</p></div

ERK was more phosphorylated in CD enterocytes.

<p>Immunohistochemical images of crypts and villi of intestinal biopsies from CD patients and controls stained with an antibody that recognizes the phosphorylated form of ERK 1/2 (pY-ERK) and with hematoxylin/eosin. One representative experiment out of 5 independent experiments is shown. B. Statistical analysis of pY-ERK positive nuclei with respect to total nuclei in the enterocytes of the crypts and villi of 5 CD patients for each group and 5 controls. More than 300 pY-ERK- positive nuclei were counted in several fields in each sample on several slides. Columns represent means and bars standard deviation. * = P<0.05; *** = P<0.001 (Student's t-test). C. (a) Western blot analysis of biopsies from CD patients and controls stained with anti-pY-ERK and anti-ERK antibodies. Similar results were obtained in 5 subjects in each group. (b) Statistical analysis of WB of biopsies from 5 subjects for each group. Columns represent the mean, bars the standard deviation of the relative intensity of pY-ERK respect to total ERK protein. * = P<0.05; ** = P<0.01 (Student's t-test).</p

Analysis of LPP levels and sub-cellular distribution in CD and controls fibroblasts before and after P31-43 treatment.

<p>A. Increased focal adhesion localization of LPP in CD fibroblasts with respect to controls, as revealed through paxillin co-staining. a) Confocal immunofluorescence images of fibroblasts from CD patients and controls treated or not with P31-43 for 30 min and stained with antibodies against paxillin (red) and LPP (green); the merge of the red and green fields is shown in yellow. Representative fields. b) Statistical analysis of the number of LPP/paxillin-merged positive focal adhesions per cell. Focal adhesions of 30 fibroblasts from several fields from 3 independent experiments with 5 patients and 5 controls were counted. Columns represent the means, and the bars represent the standard deviations. Student's <i>t</i>-test. * = <i>p</i><0.05;*** = <i>p</i><0.001. B. LPP protein levels were not increased in the fibroblasts from CD patients with respect to the controls and did not vary after 24 h treatment with P31-43. a) Western blot analysis of fibroblasts protein lysates from controls and CD patients treated or not with P31-43. The upper line was stained for LPP, and the lower line was stained for tubulin. Representative experiment. b) Densitometric analysis of western blots stained for LPP in fibroblasts from 5 CD patients and 5 controls treated or not with P31-43. LPP levels were normalized to tubulin levels. Columns represent the means, and the bars represent the standard deviations. Student's <i>t</i>-test. C. Decreased localization of LPP in the nuclear fraction of CD fibroblasts. a) Western blot analysis of LPP after separating the nuclear and cytosolic protein fractions. The upper lines were stained for LPP, and the lower lines were stained for tubulin and lamin A/C, which were used as loading controls for the cytosol and nuclear fractions, respectively. Representative experiment. b) Densitometric analysis of western blots stained for LPP in fibroblasts from 5 CD patients and 5 controls. The LPP levels were normalized in each protein fractions to the loading controls. Columns represent the means, and the bars represent the standard deviation. Student's <i>t</i>-test. * = p<0.05; ** = <i>p</i><0.01.</p

Cell shape and area of fibroblasts from CD patients and controls before and after P31-43 treatment.

<p>A. Cell shape and area are altered in fibroblasts from CD patients with respect to controls (CTR). Confocal immunofluorescence images of fibroblasts from CD patients and controls stained with Phalloidin-FITC to highlight F-actin. White lines identify the cell area. Representative fields. B. Treatment with P31-43 alters the shape and area of control fibroblasts. Confocal immunofluorescence images of fibroblasts from CD patients and controls treated with P31-43 for 30 min and stained with Phalloidin-FITC to highlight F-actin. White lines identify the cell area. Representative fields. C. Statistical analysis of 30 fibroblasts from several fields of 3 independent experiments from 6 patients and 6 controls. The area of the cells was analyzed using LSM-Zeiss confocal software. Columns represent the means, and the bars represent the standard deviation of the fibroblasts area. The area of the cells was analyzed using LSM-Zeiss confocal software. Columns represent the means, and the bars represent the standard deviation. Student's <i>t</i>-test. * = <i>p</i><0.05; ** = <i>p</i><0.01.</p

Fibronectin adhesion of CD fibroblasts respect to controls.

<p>A. CD fibroblasts adhered more than controls to fibronectin. a) Transmitted light images of crystal violet stained fibroblasts from CD patients and controls, seeded for 1 h on increasing concentrations of fibronectin, as indicated. Representative fields. b) Statistical analysis of the number of adherent fibroblasts from CD patients and controls seeded on different concentrations of fibronectin, as indicated. For each fibronectin concentration, the fibroblasts of 5 fields from 3 independent experiments with 5 patients and 5 controls were counted. Columns represent the means, and the bars represent the standard deviations. Student's <i>t</i>-test. * = <i>p</i><0.01. B. P31-43 increased adhesion to fibronectin. Statistical analysis of the number of adherent fibroblasts from CD patients and controls seeded on 50 µg of fibronectin after P31-43 treatment for 1 h. Fibroblasts in 5 fields from 3 independent experiments with 5 patients and 5 controls were counted. Columns represent the means, and the bars represent the standard deviations. Student's <i>t</i>-test.</p

Analysis of the focal adhesion number and paxillin protein levels in CD and controls fibroblasts before and after P31-43 treatment.

<p>A. Increase of the focal adhesion number in CD, as revealed through paxillin staining before and after P31-43 treatment. a) Confocal immunofluorescence images of fibroblasts from CD patients and controls stained with antibodies against paxillin. Representative fields. b) Confocal immunofluorescence images of fibroblasts stained with antibodies against paxillin from CD patients and controls treated with P31-43 for 30 min. Representative fields. c) Statistical analysis of the number of paxillin-positive focal adhesions per cell before and after P31-43 treatment. Focal adhesions of 30 fibroblasts of several fields from 3 independent experiments from 6 patients and 6 controls were counted. Columns represent the means, and the bars represent the standard deviations. Student's <i>t</i>-test. ** = <i>p</i><0.01; *** = <i>p</i><0.001. B. Paxillin protein levels are increased in fibroblasts from CD patients with respect to controls. a) Western blot analysis of fibroblast protein lysates from controls and CD patients. The upper line is stained for paxillin, and the lower line is stained for tubulin. Representative experiment. b) Densitometric analysis of western blots stained for paxillin in fibroblasts from 6 CD patients and 6 controls. Paxillin levels were normalized to the tubulin levels. Columns represent the means, and the bars represent the standard deviation. Student's <i>t</i>-test. *** = <i>p</i><0.001. C. Paxillin phopshorylation is increased in CD fibroblasts and is further increased after P31-43 treatment for 30′. a) Western blot analysis of fibroblast protein lysates from controls and CD patients. The upper line is stained for pY-paxillin, the middle line is stained for paxillin, and the lower line is stained for tubulin. Representative experiment. b) Densitometric analysis of western blots stained for pY-paxillin and paxillin in fibroblasts from 5 CD patients and 5 controls. pY-paxillin levels were normalized to the paxillin levels. Columns represent the means, and the bars represent the standard deviation. Student's <i>t</i>-test. * = <i>p</i><0.05; ** = <i>p</i><0.01.</p