Tu1825 Protease-Activated Receptor 2 Exerts a Tonic Anti-Apoptotic Effect in HT-29 Colonic Epithelial Cells

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Backround: The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors, which has been strongly associated with intestinal repair and the development of several human cancers. Methods: The human colonic myofibroblast cell line 18Co was grown to confluence on 35x10mm cell culture dishes and was used from passages 10-14. 18Co cells were exposed to TNF-α (10 ng/ml) and EGF (5 ng/ ml) under varying conditions. EGFR protein expression, EGFR tyrosine phosphorylation at Y845 and Y1068, p42/44 MAPK phosphorylation, and COX-2 expression were assessed by Western blot analysis. Live cell imaging was performed using Quantum dot technology. Experiments were also performed using primary myofibroblasts harvested from human colon tissue. Results: Treatment of 18Co cells with TNF-α led to a time-dependent increase in cell surface EGFR expression, an effect that was completely prevented by inhibition of protein synthesis with cycloheximide. Stimulation of up-regulated EGFR with EGF or HBEGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a synergistic increase in COX-2 expression compared to 18Co cells treated with EGF andHB-EGF alone. Importantly, TNF-α also increased EGFR expression in primarymyofibroblasts isolated from human colon tissue. Conclusions: Our results imply that TNF-α induced a significant upregulation of EGFR in human colonic myofibroblasts that is associated with enhanced signaling and increased functional activity (ERK, COX-2). Induction of EGFR expression by TNF-α provides a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes IBD, and which may contribute to a microenvironment that predisposes to colitis-associated cancer.