Transactivation of EGF receptor and ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis

Abstract

TNF is a pleiotropic cytokine that activates both anti- and proapoptotic signaling pathways, with cell fate determined by the balance between these two pathways. Activation of ErbB family members, including EGF receptor (EGFR/ErbB1), promotes cell survival and regulates several signals that overlap with those stimulated by TNF. This study was undertaken to determine the effects of TNF on EGFR and ErbB2 activation and intestinal epithelial cell survival. Mice, young adult mouse colon epithelial cells, and EGFR knockout mouse colon epithelial cells were treated with TNF. Activation of EGFR, ErbB2, Akt, Src, and apoptosis were determined in vivo and in vitro. TNF stimulated EGFR phosphorylation in young adult mouse colon epithelial cells, and loss of EGFR expression or inhibition of kinase activity increased TNF-induced apoptosis, which was prevented in WT but not by kinase-inactive EGFR expression. Similarly, TNF injection stimulated apoptosis in EGFR-kinase-defective mice (EGFR(wa2)) compared with WT mice. TNF also activated ErbB2, and loss of ErbB2 expression increased TNF-induced apoptosis. Furthermore, Src-kinase activity and the expression of both EGFR and ErbB2 were required for TNF-induced cell survival. Akt was shown to be a downstream target of TNF-activated EGFR and ErbB2. These findings demonstrate that EGFR and ErbB2 are critical mediators of TNF-regulated antiapoptotic signals in intestinal epithelial cells. Given evidence for TNF signaling in the development of colitis-associated carcinoma, this observation has significant implications for understanding the role of EGFR in maintaining intestinal epithelial cell homeostasis during cytokine-mediated inflammatory responses.

Fig. 1.

TNF stimulates EGFR and ErbB2 activation in intestinal epithelial cells. YAMC cells were treated with TNF at the indicated concentrations for various times. EGFR and ErbB2 phosphorylation was detected by Western blot analysis of cellular lysates with anti-EGFR-Y1068 and anti-ErbB2-Y1248 antibodies, respectively. Anti-actin antibody was used as a loading control. Data in this and subsequent figures are representative of at least three separate experiments.

Fig. 2.

TNF transactivation of EGFR and ErbB2 requires both intact EGFR and ErbB2 in intestinal epithelial cells. Cells were treated with TNF (100 ng/ml) for 2 h or EGF (10 ng/ml) for 5 min in the presence or absence of 1-h pretreatment with EGFR receptor tyrosine kinase inhibitor AG1478 (150 nM) or vehicle DMSO. AG1478 and DMSO were maintained during the entire course of cytokine treatment. EGFR and ErbB2 phosphorylation was detected as described in Fig. 1. Anti-EGFR and anti-ErbB2 antibodies were used to detect EGFR and ErbB2 expression levels, respectively.

Fig. 3.

EGFR-kinase activity is required for survival of intestinal epithelial cells exposed to TNF. Cells were treated with TNF (100 ng/ml), IL-1α (10 ng/ml), or IFN-γ (100 ng/ml) as indicated for 8 h in the presence or absence of 1-h pretreatment with EGFR receptor kinase inhibitor AG1478 (150 nM). (A) Cells were fixed for TUNEL assay, with apoptotic nuclei labeled with FITC (green) and nuclei labeled with DAPI (blue). FITC- and DAPI-labeled images were taken from the same field. Arrowheads indicate apoptotic nuclei. (B and C) Apoptosis was determined by counting at least 500 cells. The percentage of cells undergoing apoptosis is shown. *, P < 0.05 compared with control, the treatment with AG1478, or TNF. (D) Caspase activity in living cells was detected by using the sulforhodamine multicaspase activity kit with caspase-active cells stained as red. (E) The percentage of cells with active caspase.

Fig. 4.

Suppression of EGFR and ErbB2 expression promotes apoptosis in intestinal epithelial cells treated with TNF. YAMC cells transfected with EGFR (A and B) or ErbB2 siRNA or nontargeting siRNA (C and D) for 24 h were treated with TNF (100 ng/ml) for 8 h to detect apoptosis by using TUNEL assay (B and D), as described in Fig. 3. EGFR (A) or ErbB2 (C) protein levels were detected as described in Fig. 2.

Fig. 5.

Loss of EGFR kinase activity promotes colon epithelial cell apoptosis in mice injected with TNF. WT and EGFR^wa2 (EGFR-kinase-defective) mice were injected with TNF (10⁴ units) or PBS for 24 h. (A) Total colon mucosal lysates were collected for Western blot analysis with indicated antibodies. Paraffin-embedded colon tissues were double-stained with rabbit anti-EGFR-Tyr-1068 (green in B) and mouse anti-E-cadherin (red in B) or with anti-active-caspase-3 antibodies (red in E) or DAPI (blue in B and E). Images were taken from the same field by using fluorescence microcopy and overlaid in B and C (40× magnification). Arrowheads (B) indicate costaining of EGFR-Tyr-1068 and E-cadherin. Arrows (E) indicate activated caspase-3-stained cells. (C) Paraffin-embedded colon tissues were studied for apoptosis by using ISOL staining, and apoptotic nuclei labeled with peroxidase were visualized by using differential interference contrast microscopy. Arrowhead indicates ISOL-labeled apoptotic nuclei. (D) The number of apoptotic nuclei per 100 colonic glands is shown. Shown is one representative of seven mice in each treatment group.

Fig. 6.

Src mediates TNF transactivation of EGFR and ErbB2 and cell survival in intestinal epithelial cells. YAMC cells (A) and cells transfected with a siRNA mixture for Src family members (Src, Fyn, and Yes) siRNA or nontargeting siRNA for 24 h (B) were treated with TNF (100 ng/ml) for 2 h or as indicated in A or EGF (10 ng/ml) for 5 min. Cellular lysates were prepared to detect Src activation by Western blot analysis using anti-phospho-Tyr-416 (P) Src antibody, and EGFR and ErbB2 phosphorylation was detected as described in Fig. 1. Anti-Src, anti-Fyn, and anti-Yes antibodies were used to detect Src, Fyn, and Yes protein levels, respectively. YAMC cells were treated with TNF (100 ng/ml) for 8 h in the presence or absence of 1-h pretreatment with Src inhibitors, CGP (0.2 μM), PP1 (1 μM), or PP2 (1 μM). Apoptosis was detected by using TUNEL assay, as described in Fig. 3. (C) The percentage of cells undergoing apoptosis is shown.

Fig. 7.

EGFR and ErbB2 are required for Akt activation in intestinal epithelial cells treated with TNF. EGFR^−/−MCE cells stably expressing WT EGFR or vector only (A) and YAMC cells transfected with ErbB2 siRNA or nontargeting siRNA (B) were treated with TNF (100 ng/ml) for 2 h or with EGF (10 ng/ml) for 5 min. Akt and ERK1/2 activation were detected by Western blot analysis of cellular lysates with anti-phospho (P)-Ser 473 Akt and anti-P-ERK1/2 antibodies, with total expression levels detected by using anti-Akt and anti-ERK1/2 antibodies, respectively.