gms | German Medical Science

Introduction: Intestinal epithelial intercellular junctions regulate barrier properties and have been linked to epithelial differentiation and programmed cell death (apoptosis). The survival of most normal intestinal epithelial cells (IECs) requires cell-cell and cell matrix adhesion, and loss of such cell adhesion induces epithelial cell death. Pathologically, pro-inflammatory cytokines such as Interferon gamma (IFN-γ) promote apoptosis in the inflamed epithelium in IBD. However mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions and are punctate structures comprised of transmembrane desmosomal cadherins termed Desmoglein-2 (Dsg2) and Desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia.

Material and methods: Cytokine incubation: TNF-α (10 ng/ml) and IFN-γ (100 U/ml) were added basolaterally to cultured T84 cells for 72h. Generation of antibodies to lipid raft-enriched epithelial junction proteins: To generate antibodies Balbmice were immunized intraperitoneally with lipid raft-enriched preparations. Splenocytes of mice with high titers of anti-epithelial antibodies fused with PU31 myeloma cells according to standard procedures. Immunofluorescence: Confluent monolayers and human colonic mucosa were stained with specific primary and secondary antibodies and visualized on a confocal microscope. SDS-PAGE/Western blotting: Pprotein concentrations were immunoblotted with specific antibodies. Immunoprecipitation: The antigen recognized by AH12.2 was immunoprecipitated using antibody coupled to sepharose beads. Mass spectrometry and identification of AH12.2 antigen: T84 cells were used to perform large-scale immunoprecipitations of the antigen recognized by AH12.2 antibody. siRNA experiments: siGENOME SMARTpool siRNA for Human Dsg2, Dsc2, LaminA/C, and a scramble control were purchased from Dharmacon. Expression of Dsg2 constructs: The DNA fragments encoding the Desmoglein cytoplasmic tail tagged with the flag epitope were generated by PCR using specific primers. The full length c-myc epitope tagged to human Dsg2 and the C-terminal flag tagged to human Dsg2 were transfected into SK-CO15 cells. Induction of Apoptosis and apoptosis assay: Beside of incubation with IFN-γ apoptosis was induced in confluent monolayers cultured in 6-well plates by adding 2 μg/ml Camptothecin (Camp). For inhibitor studies, the cells were pre-incubated for 60 min with Z-VAD(OMe)-FMK (50 μM, R&D systems, MN) and/or Calpeptin (52 nM, Calbiochem, CA) before adding camptothecin

Results: In the present study,we generated an antibody, AH12.2 that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2 is cleaved by cysteine proteases during the onset of intestinal epithelial cell apoptosis. SiRNA-mediated down regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a Cterminalfragment of Dsg2 regulates apoptosis and Dsg2 protein levels.

Conclusion: In conclusion, these findings suggest that apoptosis of IECs is triggered by molecular events that induce cell detachment through activation of cysteine proteases targeting Dsg2. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis during physiologic differentiation and inflammation. We propose that desmosome proteins play an important role in the induction of apoptosis during remodeling of epithelial cells.