Adhesive and signaling properties of Dsg2 in intestinal epithelial barrier regulation

Ungewiß, Hanna (2019): Adhesive and signaling properties of Dsg2 in intestinal epithelial barrier regulation. Dissertation, LMU München: Fakultät für Biologie

Abstract

The human gastrointestinal tract is covered by a simple epithelium that operates as a selective permeable barrier and allows the uptake of essential nutrients while simultaneously preventing the entry of macromolecules and pathogens from the gut lumen. Several types of intercellular junctions tightly connect the epithelial cells and thus establish a functional barrier. While tight junctions (TJ) as well as adherens junctions (AJ) have been studied extensively, less information is available for desmosomes. For a long time, desmosomes were considered to provide primarily the mechanical strength to intercellular cohesion. However, growing evidence suggest a role in regulating signaling cascades. Desmosomal cadherins constitute the adhesive core of desmosomes with extracellular domains (ED) binding to adjacent cadherins and a cytoplasmic tail that anchors the junctional complex to the intermediate filament cytoskeleton. In the human intestine, only two desmosomal cadherins are present, desmoglein 2 (Dsg2) and desmocollin 2 (Dsc2), of which Dsg2 has been reported to be crucial for barrier function and to play a critical role in the pathogenesis of inflammatory bowel disease (IBD). The main objective of this study was to investigate the adhesive and signaling functions of Dsg2 in human intestinal cells. At first, I characterized cultured enterocytes regarding their ability to establish a functional barrier that is suitable as model for the intestinal epithelium. Here, cultured cells showed mature barrier properties with fully formed apical junctional complexes and characteristic microvilli on the cell surface similar as observed in human tissue. Furthermore, I identified that Dsg2 is present additionally outside of desmosomes on the surface of polarized enterocytes. To characterize Dsg2 binding properties, I established atomic force microscopy (AFM) on living enterocytes, which was then used to investigate the effect of several signaling mediators on Dsg2 binding. An antibody targeting the ED of Dsg2 was able to inhibit binding events in AFM measurements and activated p38MAPK but did not influence cell cohesion under same conditions. To elucidate the signaling properties of Dsg2 more in detail, I compared WT and Dsg2-deficient enterocytes and found a deregulated p38MAPK signaling pathway. Using transepithelial resistance (TER) measurements, I further showed that the interrelationship of p38MAPK and Dsg2 regulates barrier properties. IV Summary Moreover, I identified EGFR as direct interaction partner of Dsg2 and that this interaction inhibits the proliferative function of EGFR. In Dsg2-deficient cells, EGFR is absent at cell-cell borders and proliferation is increased. Co-localization of Dsg2 and EGFR was also observed in human tissue samples, indicating that this newly discovered mechanism is universal. Further, I characterized the heterophilic binding and demonstrated that EGFR mediators inhibit the interaction. In addition, EGFR mediators impaired barrier establishment and reduced cell adhesion, suggesting that the Dsg2-EGFR complex regulates barrier properties of intestinal epithelial cells. Since impaired barrier function and in particular Dsg2 function is implicated in the pathogenesis of Crohn’s disease (CD), I participated in studies investigating the mechanisms leading to loss of barrier function in IBD. Here, I analyzed human tissue samples from CD patients and showed changes in desmosome ultrastructure. Furthermore, comparison of Dsg2 and Dsc2 deficient cells revealed a differential biological relevance of these cadherins. More specifically, Dsg2 had a more profound impact on barrier properties than Dsc2. In addition, I investigated the effect of the neurotrophic factor GDNF on Dsg2 binding, because GDNF has been reported to have a protective function in IBD patients. Here, using AFM, I showed that GDNF increases the binding properties of Dsg2 at cell borders and protects Dsg2 from cytokine-induced reduction at cell borders.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Desmosomes, Intestinal epithelial barrier, Desmoglein, barrier regulation
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 11. Januar 2019
• 1. Bericht­erstatter:in: Leonhardt, Heinrich