DOP005. C-Jun N-terminal kinase 2 in non-hematopoietic cells protects against stress-induced apoptosis and improves gastrointestinal barrier functions
A.D. Mandic, E. Bennek, C. Liedtke, F.J. Cubero, C. Trautwein, G. Sellge, University Hospital Aachen, Department of Medicine III, Aachen, Germany
c-Jun N-terminal kinases (JNK), members of the mitogen-activated protein kinase family, regulate several cellular functions such as proliferation, differentiation, apoptosis as well as the production of cytokines and antimicrobial peptides. In this study we analyzed the role of JNK 1 and 2 isoforms in the regulation of the gastrointestinal barrier.
JNK1flox mice were crossed with Villin-Cre mice to create an intestinal epithelial-cell specific knockout (KO) of JNK1 (JNK1 ΔIEC). Furthermore, constitutive JNK2 knockout (JNK2−/−) and JNK1 ΔIEC/JNK2−/− double-KO mice were used. Bone marrow transplantation was performed to create chimeric mice with isolated JNK2-deletions in the hematopoietic or non-hematopoietic compartment. Mice were investigated under normal conditions and after induced colitis with 3% dextran sodium sulfate (DSS) for 7 days. Disease activity was monitored by the disease activity index (DAI) for DSS-induced colitis (based on weight loss, stool consistency, and bleeding), endoscopy, histology and functional testing of the intestinal barrier. Molecular analyses were performed by immmuhistochemistry, PCR, western blot and enzymatic assays.
JNK1 and JNK2 were equally expressed in the intestinal epithelium and phosphorylation was induced in both isoforms upon DSS colitis. Non-treated JNK1 ΔIEC, JNK2−/−, and JNK1 ΔIEC/JNK2−/− developed normally, did not show any phenotype up to 45 weeks. Upon induction of DSS colitis, disease activity was unchanged in JNK1 ΔIEC compared to WT animals, whereas JNK2−/− and JNK1 ΔIEC/JNK2−/− mice showed significantly higher disease activity with enhanced mucosa destruction, increased barrier dysfunction and higher inflammation as indicated by tissue myeloperoxidase levels, CD11b immunostaining, and mRNA expression of proinflammatory cytokine. The analysis of chimeric mice revealed that the loss of JNK2 in the non-hematopoietic compartment was mainly responsible for the enhanced disease phenotype. JNK2-deficient mice showed augmented caspase 3-dependent epithelial cell apoptosis and increased expression of genes related to endoplasmic reticulum stress. Cell culture experiments with the colon carcinoma cell line HT-29 showed enhanced apoptosis induction upon oxidative stress after treatment with the JNK inhibitor SP600125.
Our data indicate that JNK2, but not JNK1, protects against stress induced apoptosis in intestinal epithelial cells and therefore improves barrier function and secondary inflammation.