No abstract

Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell-cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2; DSC2). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2, but not DSC2 mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2 mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2 animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2 mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2 mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.

Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells, and are essential for the mechanical integrity of mammalian tissues. Mutations in desmosomal proteins cause severe human pathologies including epithelial blistering and heart muscle dysfunction. However, direct evidence for their load-bearing nature is lacking. Here we develop Förster resonance energy transfer (FRET)-based tension sensors to measure the forces experienced by desmoplakin, an obligate desmosomal protein that links the desmosomal plaque to intermediate filaments. Our experiments reveal that desmoplakin does not experience significant tension under most conditions, but instead becomes mechanically loaded when cells are exposed to external mechanical stresses. Stress-induced loading of desmoplakin is transient and sensitive to the magnitude and orientation of the applied tissue deformation, consistent with a stress absorbing function for desmosomes that is distinct from previously analyzed cell adhesion complexes.

Intestinal epithelial barrier properties are maintained by a junctional complex consisting of tight junctions (TJ), adherens junctions (AJ) and desmosomes. Desmoglein 2 (Dsg2), an adhesion molecule of desmosomes and the only Dsg isoform expressed in enterocytes, is required for epithelial barrier properties and may contribute to barrier defects in Crohn’s disease. Here, we identified extradesmosomal Dsg2 on the surface of polarized enterocytes by Triton extraction, confocal microscopy, SIM and STED. Atomic force microscopy (AFM) revealed Dsg2-specific binding events along the cell border on the surface of enterocytes with a mean unbinding force of around 30pN. Binding events were blocked by an inhibitory antibody targeting Dsg2 which under same conditions activated p38MAPK but did not reduce cell cohesion. In enterocytes deficient for Dsg2, p38MAPK activity was reduced and both barrier integrity and reformation were impaired. Dsc2 rescue did not restore p38MAPK activity indicating that Dsg2 is required. Accordingly, direct activation of p38MAPK in Dsg2-deficient cells enhanced barrier reformation demonstrating that Dsg2-mediated activation of p38MAPK is crucial for barrier function. Collectively, our data show that Dsg2, beside its adhesion function, regulates intestinal barrier function via p38MAPK signalling. This is in contrast to keratinocytes and points towards tissue-specific signalling functions of desmosomal cadherins.

Rapidly renewing epithelial tissues such as the intestinal epithelium require precise tuning of intercellular adhesion and proliferation to preserve barrier integrity. Here, we provide evidence that desmoglein 2 (Dsg2), an adhesion molecule of desmosomes, controls cell adhesion and proliferation via epidermal growth factor receptor (EGFR) signaling. Dsg2 is required for EGFR localization at intercellular junctions as well as for Src-mediated EGFR activation. Src binds to EGFR and is required for localization of EGFR and Dsg2 to cell-cell contacts. EGFR is critical for cell adhesion and barrier recovery. In line with this, Dsg2-deficient enterocytes display impaired barrier properties and increased cell proliferation. Mechanistically, Dsg2 directly interacts with EGFR and undergoes heterotypic-binding events on the surface of living enterocytes via its extracellular domain as revealed by atomic force microscopy. Thus, our study reveals a new mechanism by which Dsg2 via Src shapes EGFR function towards cell adhesion.

No abstract

Rapidly renewing epithelial tissues such as the intestinal epithelium require precise modulation of intercellular adhesion to preserve barrier integrity during cell division and differentiation. Previously, we have shown that Desmoglein 2 (Dsg2), an adhesion molecule of desmosomes, beside its adhesion function, regulates intestinal barrier properties via p38MAPK signaling. Here we show that Dsg2 and epidermal growth factor receptor (EGFR) co‐localize at the apical part of cell‐cell contacts in polarized enterocytes using confocal and stimulated emission depletion (STED) microscopy. DLD1 cells deficient for Dsg2 display EGFR mislocalization and reduced protein levels of activated as well as total EGFR. Atomic force microscopy (AFM) revealed EGFR‐Dsg2 interaction both under cell‐free conditions as well as on living enterocytes, which was confirmed by co‐immunoprecipitation. Moreover, EGFR inhibition reduced cell cohesion and barrier reformation. In line with this, we demonstrate that Src activity is required for barrier formation and localization of EGFR and Dsg2 to cell‐cell contacts. Thus, our study reveals a new mechanism by which Dsg2 regulates barrier properties via EGFR.Support or Funding InformationThis work was funded by grants of the Deutsche Forschungsgemeinschaft: the priority program DFG_SPP1782This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.