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TNF and epidermal growth factor (EGF) are well-known stimuli of cyclooxygenase (COX)-2 expression, and TNF stimulates transactivation of EGF receptor (EGFR) signaling to promote survival in colon epithelial cells. We hypothesized that COX-2 induction and cell survival signaling downstream of TNF are mediated by EGFR transactivation. TNF treatment was more cytotoxic to COX-2(-/-) mouse colon epithelial (MCE) cells than wild-type (WT) young adult mouse colon (YAMC) epithelial cells or COX-1(-/-) cells. TNF also induced COX-2 protein and mRNA expression in YAMC cells, but blockade of EGFR kinase activity or expression inhibited COX-2 upregulation. TNF-induced COX-2 expression was reduced and absent in EGFR(-/-) and TNF receptor-1 (TNFR1) knockout MCE cells, respectively, but was restored upon expression of the WT receptors. Inhibition of mediators of EGFR transactivation, Src family kinases and p38 MAPK, blocked TNF-induced COX-2 protein and mRNA expression. Finally, TNF injection increased COX-2 expression in colon epithelium of WT, but not kinase-defective EGFR(wa2) and EGFR(wa5), mice. These data indicate that TNFR1-dependent transactivation of EGFR through a p38- and/or an Src-dependent mechanism stimulates COX-2 expression to promote cell survival. This highlights an EGFR-dependent cell signaling pathway and response that may be significant in colitis-associated carcinoma.

Background & Aims: The ErbB4 receptor tyrosine kinase regulates cell growth, survival, and differentiation in several tissues, but its role in the gastrointestinal tract has not been reported. We tested the hypothesis that ErbB4 promotes intestinal cell survival and restitution following injury or inflammation., Methods: ErbB4 expression in human inflammatory bowel disease was determined by immunohistochemistry. Mice were subjected to dextran sulfate sodium (DSS, 3%) colitis or injected with tumor necrosis factor (TNF), and ErbB4 expression was quantified by immunohistochemistry and Western blot. Cultured young adult mouse colon (YAMC) cells were exposed to TNF, and ErbB4 messenger RNA, protein, and phosphorylation levels were measured. Cells transfected with ErbB4 small interfering RNA (siRNA), or over expressing ErbB4, were subjected to wound healing and apoptosis assays., Results: ErbB4 levels increased in Crohn's colitis and the colon epithelium of mice with DSS colitis or injected with TNF. In YAMC cells, TNF induced ErbB4 messenger RNA, protein, and phosphorylation; nuclear factor kappaB activation also stimulated ErbB4 accumulation. ErbB4 siRNA sensitized cells to TNF-stimulated apoptosis, while over expression blocked apoptosis induced by TNF plus cycloheximide. Additionally, ErbB4 siRNA decreased YAMC cell wound healing. ErbB4 knockdown attenuated, while over expression elevated, phosphorylation of Akt in response to TNF. Inhibition of the phosphatidylinositol 3-kinase/Akt signaling cascade reversed the ability of ErbB4 over expression to protect from cytokine-induced apoptosis., Conclusions: ErbB4 expression and signaling are key elements for TNF responses in vivo and in cell culture, protecting intestinal epithelial cells from apoptosis in the inflammatory environment, possibly through Akt activation.

Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2(-/-), but not TNFR1(-/-), mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1(-/-) MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases.

Internalization and proteolytic degradation of epidermal growth factor (EGF) receptor (R) following ligand binding is an important mechanism for regulating EGF-stimulated signals. Using pharmacological and RNA interference inhibition of p38 mitogen-activated protein kinase, we show that p38 is required for efficient EGF-induced EGFR destruction but not internalization. In the absence of p38 activity, EGF fails to stimulate the ubiquitin ligase Cbl or ubiquitinylation of EGFR, and internalized EGFR accumulates in intracellular vesicles containing caveolin-1. These effects are accompanied by loss of EGFR phosphorylation on Y1045, a phosphorylation site required for Cbl activation. Furthermore, similar to cells treated with p38 inhibitors, intestinal epithelial cells expressing Y1045F EGFR mutants show increased proliferation but not migration in response to EGF, thus uncoupling these biological responses. Together these data position p38 as a modulator of ligand-stimulated EGFR processing and demonstrate that this processing has a profound impact on the cellular outcome of EGFR signaling.