DOP51 Investigation on the role of Par4-associated cell polarity and associated barrier defects in inflammatory bowel diseases

Abstract Background In inflammatory bowel diseases (IBD), epithelial barrier defects occur as a consequence of chronic inflammation. Recent research suggested that cell polarity alterations may be upstream of barrier defects and additionally play a role in IBD-associated carcinogenesis (colitis-associated and small intestinal carcinoma). Par4 is a gene encoding a protein crucial in the development of cell polarity. LKB1, its human homologue, is mutated in Peutz–Jeghers syndrome (PJS), a genetic condition characterised by a higher risk of epithelial cancers. While the pivotal role of Par4/LKB1 in the development of epithelial cell polarity is established, its involvement in IBD-associated barrier defects and carcinogenesis is yet to be defined. Methods Endoscopic bowel mucosa samples from patients with Crohn’s disease, ulcerative colitis, PJS and controls were analysed to assess expression and localisation of Par4/LKB1. Cryosections were immunostained for Par4/LKB-1 along with a diverse set of markers of cell polarity and intercellular adhesion. The analysis was performed by confocal laser scanning microscopy in order to compare the expression of mucosal as well as the subcellular localisation of Par4/LKB1 in the gut mucosa. A quantitative analysis of protein expression with western blotting was performed as well. The function of Par4/LKB1 in intestinal epithelial cells (IEC) was evaluated by means of a Par4-deficient IEC model as well as in an IEC Par4-overexpression model. Moreover, a preliminary assessment of the possible role of Par4/LKB1-related polarity processes in IBD-associated carcinomas was performed through scanning genetic data from colitis-associated carcinomas for potential Par4-related mutations and altered Par4/LKB1 expression. Results The immunofluorescent staining allowed visualisation of intracellular expression of Par4 in epithelia from PJS, IBD patients and controls. In PJS-polyps, despite the alteration of regular tissue architecture typical of these lesions, the polarity of epithelial cells was maintained - contrary to control tissue, a punctate pattern of the Par4 staining was shown. In IBD tissue, no relevant differences in Par4 expression at confocal microscopy, as well as a quantitative assessment, were observed as compared with controls. No differences were observed in PJS or IBD samples as regards the expression of cell polarity markers such as Par3, CD71, crb3 or adhesion molecules such as ZO-1, e-cadherin, occludin, JAM-A. As