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Desmoglein 2 regulates PI3‐kinase/AKT‐dependent expression of Claudin 2 in intestinal epithelial cells
Author(s) -
Burkard Natalie,
Meir Michael,
Flemming Sven,
Germer ChristophThomas,
Waschke Jens,
Schlegel Nicolas
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1049.14
Subject(s) - microbiology and biotechnology , protein kinase b , pi3k/akt/mtor pathway , tight junction , signal transduction , kinase , biology , chemistry , cancer research
Desmoglein 2 (Dsg2) is a desmosomal junctional protein expressed in all epithelia including the intestinal mucosa, and has been recently proposed to contribute in the pathogenesis of inflammatory bowel disease. In patients suffering from Crohn's disease (CD) it has been reported that reduced expression of intestinal Dsg2 is associated with impaired intestinal epithelial barrier function and accompanied with changes of tight junction (TJ) integrity. A previous study has provided that the application of a Dsg‐specific tandem peptide (TP) linking extracellular domains of adjacent desmogleins increases Dsg2‐mediated intercellular adhesion and strongly reduced TNFα‐induced expression of claudin2 (Cld2), which is referred to as “leaky” TJ protein. However the signaling pathways involved in the regulation of Cld2 by Dsg2 under inflammatory conditions are not elucidated yet. The incubation of differentiated CaCo2 cells with TNFα led to an impaired epithelial barrier function as revealed by decreased trans‐epithelial electric resistance (TER) and loss of intercellular adhesion in dispase‐based enterocyte dissociation assay. Simultaneously, Dsg2 was reduced at the cell borders and claudin2 expression was significantly augmented as confirmed in Western blot and RT‐PCR analyses. Interestingly, the application of TP restored all TNFα‐induced effects. Further analyses on signaling pathways demonstrated that both activation of the p38 mitogen‐activated protein kinase (p38MAPK) and phosphatidylinositol 3‐kinase (PI3K) with consecutive AKT phosphorylation are required for TNFα‐induced up‐regulation of Cld2 since the PI3‐kinase inhibitor LY2914002 and the p38MAPK inhibitor SB2021290 blocked up‐regulation of Cld2. Sequential experiments using these inhibitors determined that p38MAPK appears to be upstream of PI3K/AKT signaling. Interestingly, TP blocked both TNF‐induced activation of p38MAPK and PI3K/AKT signaling. By immunoprecipitation we found a direct interaction between Dsg2 and PI3‐kinase which was abrogated after TNFα incubation conforming to the observation in immunostaining that PI3‐kinase co‐localized with Dsg2 under steady state but dissociated from the cell borders to the cytoplasm under inflammatory conditions. These results were strengthened by knock‐down experiments using Dsg2‐siRNA where neither LY294002 nor TP were able to inhibit the PI3‐kinase signaling pathway and consecutive up‐regulation of Cld2. Our findings provide new insights into the contribution of Dsg2 in regulating TJ composition under inflammatory conditions. Based on our in vitro observations Dsg2 regulates Cld2 expression in a p38MAPK/PI3K/AKT‐dependent manner.