ABSTRACT
The tumor necrosis factor-receptor-associated factor 2 (TRAF2)- and Nck-interacting kinase (TNIK) is a ubiquitously expressed member of the germinal center kinase family. The TNIK functions in hematopoietic cells and the role of TNIK-TRAF interaction remain largely unknown. By functional proteomics we identified TNIK as interaction partner of the latent membrane protein 1 (LMP1) signalosome in primary human B-cells infected with the Epstein-Barr tumor virus (EBV). RNAi-mediated knockdown proved a critical role for TNIK in canonical NF-κB and c-Jun N-terminal kinase (JNK) activation by the major EBV oncoprotein LMP1 and its cellular counterpart, the B-cell co-stimulatory receptor CD40. Accordingly, TNIK is mandatory for proliferation and survival of EBV-transformed B-cells. TNIK forms an activation-induced complex with the critical signaling mediators TRAF6, TAK1/TAB2, and IKKß, and mediates signalosome formation at LMP1. TNIK directly binds TRAF6, which bridges TNIK's interaction with the C-terminus of LMP1. Separate TNIK domains are involved in NF-κB and JNK signaling, the N-terminal TNIK kinase domain being essential for IKKß/NF-κB and the C-terminus for JNK activation. We therefore suggest that TNIK orchestrates the bifurcation of both pathways at the level of the TRAF6-TAK1/TAB2-IKK complex. Our data establish TNIK as a novel key player in TRAF6-dependent JNK and NF-κB signaling and a transducer of activating and transforming signals in human B-cells.
Subject(s)
B-Lymphocytes/metabolism , CD40 Antigens/metabolism , MAP Kinase Signaling System , Protein Serine-Threonine Kinases/metabolism , Viral Matrix Proteins/metabolism , B-Lymphocytes/virology , CD40 Antigens/genetics , Cell Proliferation , Cell Transformation, Viral , Germinal Center Kinases , HEK293 Cells , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/metabolism , Herpesvirus 4, Human/pathogenicity , Humans , NF-kappa B/genetics , NF-kappa B/metabolism , Protein Binding , Protein Interaction Mapping/methods , Protein Serine-Threonine Kinases/genetics , Proteomics/methods , RNA Interference , Viral Matrix Proteins/geneticsABSTRACT
The interaction of nonpathogenic enteric bacteria with intestinal epithelial cells (IEC) in the absence of host-derived Interleukin 10 (IL-10) may contribute to the development of chronic inflammation. Functional proteome analysis of primary IEC from Enterococcus faecalis-monoassociated WT and IL-10-/- mice as well as IL-10 receptor reconstituted IEC revealed expression changes of proteins that are involved in endoplasmic reticulum stress, energy metabolism, and apoptosis, suggesting a protective role for IL-10 at the epithelial cell level.
Subject(s)
Enterococcus faecalis/metabolism , Epithelial Cells/metabolism , Inflammation/metabolism , Interleukin-10/metabolism , Receptors, Interleukin-10/metabolism , Animals , Apoptosis , Colitis/metabolism , Electrophoresis, Gel, Two-Dimensional , Endoplasmic Reticulum/metabolism , Galectin 3/metabolism , Interleukin-10/genetics , Interleukin-10/physiology , Intestinal Mucosa/metabolism , Mice , Mice, Transgenic , Proteomics/methodsABSTRACT
The loss of intestinal epithelial cell (IEC) function is a critical component in the initiation and perpetuation of chronic intestinal inflammation in the genetically susceptible host. We applied proteome analysis (PA) to characterize changes in the protein expression profile of primary IEC from patients with Crohn's disease (CD) and ulcerative colitis (UC). Surgical specimens from 18 patients with active CD (N = 6), UC (N = 6), and colonic cancer (N = 6) were used to purify primary IEC from ileal and colonic tissues. Changes in protein expression were identified using 2D-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS) as well as Western blot analysis. PA of primary IEC from inflamed ileal tissue of CD patients and colonic tissue of UC patients identified 21 protein spots with at least 2-fold changes in steady-state expression levels compared to the noninflamed tissue of control patients. Statistical significance was achieved for 9 proteins including the Rho-GDP dissociation inhibitor alpha that was up-regulated in CD and UC patients. Additionally, 40 proteins with significantly altered expression levels were identified in IEC from inflamed compared to noninflamed tissue regions of single UC (N = 2) patients. The most significant change was detected for programmed cell death protein 8 (7.4-fold increase) and annexin 2A (7.7-fold increase). PA in primary IEC from IBD patients revealed significant expression changes of proteins that are associated with signal transduction, stress response as well as energy metabolism. The induction of Rho GDI alpha expression may be associated with the destruction of IEC homeostasis under condition of chronic intestinal inflammation.
Subject(s)
Gene Expression Regulation , Guanine Nucleotide Dissociation Inhibitors/analysis , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/chemistry , Proteins/analysis , Proteomics/methods , Apoptosis , Blotting, Western , Colitis, Ulcerative/pathology , Colonic Neoplasms/pathology , Crohn Disease/pathology , Electrophoresis, Gel, Two-Dimensional , Energy Metabolism , Guanine Nucleotide Dissociation Inhibitors/genetics , Humans , Signal Transduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Stress, Physiological , Up-Regulation , rho-Specific Guanine Nucleotide Dissociation InhibitorsABSTRACT
BACKGROUND & AIMS: The initiation of endoplasmic reticulum (ER)-mediated stress responses in intestinal epithelial cells (IEC) may contribute to the pathogenesis of chronic intestinal inflammation. The aim of the study was to use functional epithelial cell proteomics to characterize anti-inflammatory mechanisms of interleukin 10 (IL-10). METHODS: Primary IEC were isolated from Enterococcus faecalis-monoassociated IL-10-deficient (IL-10-/-) and wild-type mice to perform 2D-gel sodium-dodecyl-sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. In addition, IEC from 6 patients with active Crohn's disease, ulcerative colitis, and sigmoid diverticulitis as well as noninflamed controls were purified. Molecular protective mechanisms of IL-10 were characterized in tumor necrosis factor (TNF)-stimulated IL-10 receptor (IL-10R) reconstituted epithelial cells. RESULTS: Primary IEC from IL-10-/- mice as well as inflammatory bowel disease patients revealed increased expression levels of the glucose-regulated ER stress protein (grp)-78 under conditions of chronic inflammation. Consistent with the observation that TNF induced ER stress responses through grp-78 redistribution from the ER lumen to the cytoplasmic IkappaB kinase complex, grp-78 knockdown completely abolished TNF-induced nuclear factor-kappaB RelA phosphorylation in epithelial cell cultures. Interestingly, IL-10 inhibited grp-78 protein and messenger RNA expression in IL-10R reconstituted IEC. Chromatin immunoprecipitation analysis and immunofluorescence microscopy revealed that IL-10-mediated p38 signaling inhibited TNF-induced recruitment of the ER-derived activating transcription factor (ATF)-6 to the grp-78 promoter likely through the blockade of ATF-6 nuclear translocation. CONCLUSIONS: Primary IEC from inflamed IL-10-/- mice and inflammatory bowel disease patients revealed activated ER stress responses in the intestinal epithelium. IL-10 inhibits inflammation-induced ER stress response mechanisms by modulating ATF-6 nuclear recruitment to the grp-78 gene promoter.
Subject(s)
Endoplasmic Reticulum/metabolism , Inflammatory Bowel Diseases/immunology , Interleukin-10/immunology , Intestinal Mucosa/immunology , Activating Transcription Factor 6/metabolism , Animals , Cell Nucleus/metabolism , Cells, Cultured , Chronic Disease , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum Chaperone BiP , Enterococcus faecalis , Epithelial Cells/cytology , Epithelial Cells/immunology , Gram-Positive Bacterial Infections/immunology , Gram-Positive Bacterial Infections/metabolism , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/microbiology , Interleukin-10/genetics , Interleukin-10/metabolism , Intestinal Mucosa/cytology , Intestinal Mucosa/pathology , Mice , Mice, Inbred Strains , Mice, Mutant Strains , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Promoter Regions, Genetic/immunology , Proteomics , Signal Transduction/immunology , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in interleukin-10 geneeficient mice (IL-10(-/-)). Bacteria-specific effects on the epithelium are difficult to distinguish because of the complex nature of the gut microflora. We showed that IL-10(-/-) mice compared to wild-type mice fail to inhibit pro-inflammatory gene expression in native intestinal epithelial cells after the colonization with colitogenic Gram-positive Enterococcus faecalis. Of interest, pro-inflammatory gene expression was transient after 1 week of E. faecalis monoassociation in IECs from wild-type mice but persisted after 14 weeks of bacterial colonization in IL-10(-/-) mice. Accordingly, wild-type IECs expressed phosphorylated NF-kappaB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10(-/-) mice triggered persistent RelA but no Smad2 phosphorylation in IECs at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and pro-inflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice but persisted in IL-10(-/-) IECs. Of note, TGF-beta-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-kappa Bependent gene expression in E. faecalis-stimulated IEC lines. In conclusion, E. faecalis-monoassociated IL-10(-/-) but not wild-type mice lack protective TGF-beta/Smad signaling and fail to inhibit TLR2-mediated pro-inflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.
Subject(s)
Inflammation/genetics , Interleukin-10/deficiency , Interleukin-10/genetics , Intestinal Mucosa/physiopathology , Toll-Like Receptor 2/metabolism , Animals , Disease Models, Animal , Enterococcus faecalis/isolation & purification , Gene Expression Regulation , Inflammation/microbiology , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/microbiology , Intestinal Mucosa/pathology , Mice , Mice, Knockout , Transforming Growth Factor beta/physiologyABSTRACT
Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in IL-10 gene-deficient mice (IL-10(-/-)). Bacteria-specific effects on the epithelium are difficult to dissect due to the complex nature of the gut microflora. We showed that IL-10(-/-) mice compared with wild-type mice fail to inhibit proinflammatory gene expression in native intestinal epithelial cells (IEC) after the colonization with colitogenic Gram-positive Enterococcus faecalis. Interestingly, proinflammatory gene expression was transient after 1 wk of E. faecalis monoassociation in IEC from wild-type mice, but persisted after 14 wk of bacterial colonization in IL-10(-/-) mice. Accordingly, wild-type IEC expressed phosphorylated NF-kappaB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10(-/-) mice triggered persistent RelA, but no Smad2 phosphorylation in IEC at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and proinflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice, but persisted in IL-10(-/-) IEC. Of note, TGF-beta1-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-kappaB-dependent gene expression in IEC lines. In conclusion, E. faecalis-monoassociated IL-10(-/-), but not wild-type mice lack protective TGF-beta/Smad signaling and fail to inhibit TLR2-mediated proinflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.
