Desmosomal cadherins mediate intercellular adhesion and provide mechanical strength to tissues. Intestinal epithelial cells express desmosomal cadherins Desmoglein‐2 (DSG‐2) and Desmocollin‐2 (DSC‐2). To explore the relative contribution of these cadherins in mediating intercellular adhesion of intestinal epithelial cells, we generated inducible intestinal‐epithelia specific (Villin‐CreERT2) Dsg‐2 and Dsc‐2 knockdown mice (Dsg‐2ERΔIEC; Dsc‐2ERΔIEC). While intestinal mucosal architecture was maintained in Dsg‐2ERΔIEC and Dsc‐2ERΔIEC mice, ultrastructural changes in desmosomal plaques and widened intercellular spaces were observed in these mice compared to littermate floxed control mice. Functional analysis revealed increased paracellular permeability to FITC dextran supporting compromised epithelial barrier function. Interestingly, Dsc‐2ERΔIEC mice showed reduction in desmosome length while Dsg‐2ERΔIEC animals exhibited a higher FITC dextran permeability, suggesting differential dominant roles of DSG‐2 in controlling intestinal epithelial barrier function and of DSC‐2 in regulating intercellular adhesion. Additionally, knockdown of these cadherins in‐vitro in epithelial cells resulted in decreased cell‐cell adhesion and compromised barrier function. Analysis of desmosome mechanical strain using DSG‐2 and DSC‐2 tension sensors revealed that loss of DSC‐2 increased intermediate filaments tension while the loss of DSG‐2 did not show any change in cytokeratin tension. These findings provide new insight on the relative contribution of DSG‐2 and DSC‐2 in controlling intestinal epithelial intercellular adhesion and barrier function.Support or Funding InformationExperiments that lead to this poster were supported by NIH RO1 DK059888, DK055679 to AN. The authors declare no conflict of interest.

Epithelial cells migrate and proliferate to cover denuded mucosal surfaces. The intestinal mucosal infiltrating immune cells and epithelium secrete an array of inflammatory mediators that have been proposed to modulate wound repair. Here, we report that Platelet Activating Factor (PAF), a molecule considered to be a pro-inflammatory mediator and its receptor (PAFR) are upregulated in healing colonic mucosal wounds. We show that in intestinal epithelial cells the PAFR up-regulation is downstream of TNF-α mediated signaling leading to enhanced wound repair after PAF treatment. In vitro PAF signaling promotes cell migration by activation of focal adhesion kinase that regulates dynamics of integrin containing cell-matrix contacts. On the converse, PAFR deficient mice display delayed wound closure. In addition, intraperitoneal administration of a neutralizing TNF-α antibody inhibited intestinal mucosal wound repair. Our findings highlight a cross-talk between PAFR and TNF-α in orchestrating intestinal epithelial repair.

Pathobiology of several chronic inflammatory disorders, including ulcerative colitis and Crohn's disease is related to intermittent, spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). In this report, we show that TNFα promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor, Src and Rac1 signaling to promote wound closure. Consistent with these findings, delayed colonic mucosal repair was observed after administration of a neutralizing TNFα antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa, the pro-inflammatory milieu containing TNFα and PAF sets the stage for reparative events mediated by PAFR signaling.

The epithelial barrier plays a pivotal role in controlling mucosal homeostasis. In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces. Inflammatory mediators released at sites of mucosal injury by infiltrating immune cells and the epithelium have been proposed to influence wound repair mechanisms. We observed upregulation of pro‐inflammatory mediators Platelet Activating Factor (PAF) and Tumor Necrosis Factor alpha (TNF‐α) in healing colonic mucosal wounds. PAF signaling in epithelial cells is mediated by a G‐protein coupled receptor, platelet activating factor receptor (PAFR). In vitro studies demonstrated that PAFR signaling in response to PAF promotes intestinal epithelial cell migration and wound repair by the generation of reactive oxygen species and phosphorylation of focal adhesion kinase (FAK) at sites of cell‐matrix contacts. Epithelial pro‐repair properties of PAF were markedly augmented by exposure to TNF‐α and IFN‐γ and mechanistically linked to cytokine‐enhanced expression of epithelial PAFR. Complementary in vivo analyses of biopsy induced colonic mucosal wound closure revealed delayed mucosal wound healing in PAFR −/− mice compared to wildtype mice. These results support a pro‐repair effect of PAFR signaling in intestinal mucosal wounds that is potentiated by inflammatory cytokine TNF‐α and IFN‐γ signaling. Our findings delineate a novel cross‐talk between PAFR and pro‐inflammatory cytokines at sites of mucosal wounds that serves to coordinate epithelial repair and restore the mucosal barrier integrity. Support or Funding Information R01‐DK089763‐05A1: Formyl peptide receptors as mediators of intestinal mucosal homeostasis, National Institutes of Health; SI2282/1‐1, German Research Foundation

Chronisch entzündliche Darmerkrankungen führen zu Ulzerationen und Zerstörung der intestinalen epithelialen Barriere. Das klinische Ausmaß hängt mit der pathologischen Freisetzung pro-inflammatorischer Mediatoren wie Platelet Activating Factor (PAF) und Tumor Necrosis Factor alpha (TNF-a) zusammen. TNF-a neutralisierende Antikörper werden zur erfolgreichen Remissionsinduktion angewendet, wobei es jedoch bei einer signifikanten Patientenzahl zum Therapieversagen kommt, potentiell durch Inhibition pro-reparativer Funktionen von TNF-a und PAF.

The single pass, transmembrane proteins of the cadherin family have been appreciated as important proteins that regulate intercellular adhesion. In addition to this critical function, cadherins contribute to important signalling events that control cellular homeostasis. Many examples exist of classical, desmosomal and atypical cadherins participating in the regulation of signalling events that control homeostatic functions in cells. Much of the work on cadherin mediated signalling focuses on classical cadherins or on specific disease states such as pemphigus vulgaris. Cadherin mediated signalling has been shown to play critical roles during development, in proliferation, apoptosis, disease pathobiology and beyond. It is becoming increasingly clear that cadherins operate through a range of molecular mechanisms. The diversity of pathways and cellular functions regulated by cadherins suggests that we have only scratched the surface in terms of the roles that these versatile proteins play in signalling and cellular function.

Desmosomal cadherins mediate intercellular adhesion and have also been shown to regulate homeostatic signaling in epithelial cells. We have previously reported that select pro-inflammatory cytokines induce Dsg2 ectodomain cleavage and shedding from intestinal epithelial cells (IECs). Dsg2 extracellular cleaved fragments (Dsg2 ECF) function to induce paracrine pro-proliferative signaling in epithelial cells. In this study, we show that exposure of IECs to pro-inflammatory cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) resulted in Dsg2 intracellular cleavage and generation of a ~55 kDa fragment (Dsg2 ICF). Dsg2 intracellular cleavage is mediated by caspase-8 and occurs prior to Dsg2 extracellular cleavage and the execution of apoptosis. Expression of exogenous Dsg2 ICF in model IECs resulted in increased sensitivity to apoptotic stimuli and apoptosis execution. Additionally, expression of the Dsg2 ICF repressed the anti-apoptotic Bcl-2 family member proteins Bcl-XL and Mcl1. Taken together, our findings identify a novel mechanism by which pro-inflammatory mediators induce modification of Dsg2 to activate apoptosis and eliminate damaged cells, while also promoting release of Dsg2 ECF that promotes proliferation of neighboring cells and epithelial barrier recovery.

Die einschichtige intestinale Epithelschicht stellt eine physiologische Trennschicht zwischen dem intestinalen Lumen und dem darunterliegenden Gewebe dar und ist somit ein Schlüsselfaktor in der Regulation der intestinalen Homöostase. Verantwortlich für die Aufrechterhaltung der Epitehlbarriere sind interzelluläre Junktionsproteine, die aus dem apikalen Junktionskomplex und den basalen Desmosomen bestehen. In einer Vielzahl von Erkrankungen, z.B. chronisch entzündlichen Darmerkrankungen, kommt es zu einer epithelialen Verletzung einhergehend mit einer gestörten Barrierefunktion. Daher ist eine intakte intestinale Wundheilung essenziell zur Re-Etablierung der mukosalen Homöostase und zur Verhinderung einer chronischen Inflammation.

The intestinal epithelial barrier is comprised of a monolayer of simple columnar intestinal epithelial cells (IECs) that serve to protect underlying tissues from antigens and pathogens. Under conditions of intestinal inflammation this barrier breaks down, allowing these antigens and pathogens to gain access to the underlying mucosa. The aim this project is to further our understanding of the mechanisms responsible for barrier breakdown during intestinal inflammation. Epithelial barrier properties are achieved by a series of intercellular junctions that include an Apical Junctional Complex and desmosomes. The intercellular contacts of desmosomes are established by transmembrane cadherin proteins, the desmogleins and desmocollins. Pro‐inflammatory cytokines (e.g. interferon gamma (IFN‐γ) and tumor necrosis factor alpha (TNF‐α)) induce cadherin cleavage and the resulting cadherin protein fragments influence epithelial homeostasis. Using model IECs plated on transwell permeable supports, we have observed that IFN‐γ and TNF‐α or TNF‐α related apoptosis inducing ligand (TRAIL) induce desmoglein‐2 (Dsg2) intracellular cleavage resulting in generation of an intracellular fragment (ICF). This Dsg2 intracellular cleavage is, in part, mediated by caspase activation and occurs prior to Dsg2 extracellular cleavage. Previously published data suggests that Dsg2 ICF generation is associated with redistribution of another desmosomal protein, plakoglobin (Pg). We show here that exogenous expression of Myc‐tagged Dsg2 ICF using adenoviral expression constructs results in downregulation of key Pg signaling targets (c‐Myc and Bcl‐X L ), increases apoptosis, and decreassd cell proliferation. Taken together, our findings suggest that IFN‐γ and TNF‐α induced Dsg2 ICF generation activates Pg signaling thereby promoting increased epithelial apoptosis and reduced proliferation. Support or Funding Information Intestinal Epithelial Tight Junction Structure‐Function: R01 DK059888‐15