TNF-α converting enzyme-mediated ErbB4 transactivation by TNF promotes colonic epithelial cell survival - PubMed (original) (raw)

TNF-α converting enzyme-mediated ErbB4 transactivation by TNF promotes colonic epithelial cell survival

Valda C Hilliard et al. Am J Physiol Gastrointest Liver Physiol. 2011 Aug.

Abstract

Disruption of intestinal epithelial homeostasis, including enhanced apoptosis, is a hallmark of inflammatory bowel disease (IBD). We have recently shown that tumor necrosis factor (TNF) increases the kinase activity of ErbB4, a member of the epidermal growth factor receptor family that is elevated in mucosa of IBD patients and that promotes colon epithelial cell survival. In this study, we tested the hypothesis that TNF transactivates ErbB4 through TNF-α converting enzyme (TACE)-mediated ligand release and that this transactivation is necessary to protect colonic epithelial cells from cytokine-induced apoptosis. Using neutralizing antibodies, we show that heparin-binding EGF-like growth factor (HB-EGF) is required for ErbB4 phosphorylation in response to TNF. Pharmacological or genetic inhibition of the metalloprotease TACE, which mediates HB-EGF release from cells, blocked TNF-induced ErbB4 activation. MEK, but not Src or p38, was also required for transactivation. TACE activity and ligand binding were required for ErbB4-mediated antiapoptotic signaling; whereas mouse colon epithelial cells expressing ErbB4 were resistant to TNF-induced apoptosis, TACE inhibition or blockade of ErbB4 ligand binding reversed the survival advantage. We conclude that TNF transactivates ErbB4 through TACE-dependent HB-EGF release, thus protecting colon epithelial cells from cytokine-induced apoptosis. These findings have important implications for understanding how ErbB4 protects the colon from apoptosis-induced tissue injury in inflammatory conditions such as IBD.

PubMed Disclaimer

Figures

Fig. 1.

TNF transactivates ErbB4 in colonic epithelial cells. A: ErbB4 expression in young adult mouse colon (YAMC) cells stably overexpressing ErbB4 (YAMC-ErbB4) or vector (YAMC-vector) was assessed by Western blot with antibodies specific for ErbB4 or actin (loading control). B: YAMC-ErbB4 cells were treated with TNF for 30 min at the indicated concentrations or with 10 ng/ml heregulin (HRG) for 10 min. C: YAMC-ErbB4 cells were treated with TNF (100 ng/ml) for the indicated time points or with HRG (10 ng/ml) for 10 min, as a positive control for activation. D: Immorto-Min colon epithelial (IMCE) cells were treated with 100 ng/ml TNF for the indicated times or with 10 ng/ml HRG for 10 min. E: Madin-Darby canine kidney (MDCK) cells were treated with TNF (100 ng/ml) for the indicated time points or with HRG (10 ng/ml) for 10 min. Cell lysates were analyzed by Western blot with antibodies specific for phosphorylated (PY-1284) ErbB4, total ErbB4, or actin (loading control). All blots are representative of at least 3 experiments.

Fig. 2.

Heparin-binding EGF-like growth factor (HB-EGF) ligand binding mediates ErbB4 transactivation by TNF. A: YAMC-ErbB4 cells were incubated with either control mouse IgG or an ErbB4 blocking antibody for 1 h before the addition of TNF (100 ng/ml) for 30 min or HRG (10 ng/ml) for 10 min. B: YAMC-ErbB4 cells were incubated with an ErbB4 blocking antibody for 1 h before the addition of 10 ng/ml HRG, 1 ng/ml betacellin (BTC), or 1 ng/ml EGF for 10 min. Phosphorylated ErbB4 (PY-1284), EGFR (PY-1068), ErbB2 (PY-1248), ErbB3 (1289), or total actin was assessed by Western blot. YAMC-ErbB4 cells were incubated for 1 h with HB-EGF neutralizing antibody (C), HRG neutralizing antibody (D), or BTC neutralizing antibody (E) before stimulation with 100 ng/ml TNF for 30 min and with 50 ng/ml HB-EGF, 10 ng/ml HRG, or 10 ng/ml BTC for 10 min. Treatment with mouse IgG served as the negative control. Cell lysates were analyzed by Western blot with antibodies specific for phosphorylated ErbB4 (PY-1284), total ErbB4, or actin. All blots are representative of at least 3 experiments.

Fig. 3.

TNF-α converting enzyme (TACE) mediates TNF transactivation of ErbB4 in colonic epithelial cells. YAMC-ErbB4 cells were incubated for 30 min with 50 μM of the broad spectrum metalloproteinase inhibitor GM6001 (A) or 10 μM of the TACE selective inhibitor TNF-α protease inhibitor (TAPI)-1 (B), then stimulated with TNF (100 ng/ml, 30 min) or HRG (1 ng/ml, 10 min). C: YAMC-ErbB4 cells were treated with 100 ng/ml TNF for various time points or with 20 ng/ml PMA for 1 min. Cell lysates were analyzed by Western blot with antibodies specific for phosphorylated TACE (PT-735), ERK, or total actin. D: TACE−/− colonic epithelial cells infected with ErbB4 and with either wild-type TACE or vector added back were treated with 100 ng/ml TNF for 30 min or 10 ng/ml HRG for 10 min. E: YAMC-ErbB4 cells were treated directly with 100 ng/ml TNF for 5 or 15 min or 10 ng/ml HRG for 10 min. TACE−/− colon epithelial cells expressing ErbB4 were treated with conditioned media (CM) from the corresponding YAMC cells for 10 min. Cell lysates were then analyzed by Western blot with antibodies specific for phosphorylated ErbB4 (PY-1284), total ErbB4, or actin. All blots are representative of at least 3 experiments.

Fig. 4.

ErbB4 transactivation by TNF requires MEK, but not p38 or Src kinases. A: YAMC-ErbB4 cells were incubated with 10 μM U0126 (MEK inhibitor) for 1 h before the addition of TNF (100 ng/ml) for either 15 or 30 min. B: YAMC-ErbB cells were pretreated with U0126 before 5-min TNF treatment. C: YAMC-ErbB4 cells were incubated with 2 μM of the Src inhibitor CGP77675 for 1 h before the addition of TNF for the indicated time points. D: YAMC-ErbB4 cells were incubated with 10 μM SB20190 (p38 inhibitor) for 1 h before TNF treatment for 15 or 30 min. Phosphorylation of ErbB4 (PY-1284), TACE (PT-735), ERK1/2, MSK1 (positive control for p38 inhibition), or FAK (PY-925, positive control for Src inhibition) or total ErbB4, TACE, ERK1/2, FAK, or actin was determined by Western blot analysis. All blots are representative of at least 3 experiments.

Fig. 5.

ErbB4 transactivation by TNF protects colonic epithelial cells from cytokine-induced apoptosis. YAMC cells expressing either vector (YAMC-vector) or ErbB4 (YAMC-ErbB4) were incubated with 10 μM TAPI-1 for 30 min (A) or ErbB4 blocking antibody for 1 h (B) before treatment with 1 μg/ml cycloheximide (CHX) and 100 ng/ml TNF for 3 h. Caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage were assessed by Western blot analysis of whole cell lysates. Actin was used as a loading control. C: vector and ErbB4-expressing YAMC cells were incubated with 10 μM TAPI-1 for 30 min or with an ErbB4 blocking antibody for 1 h before treatment with 1 μg/ml cycloheximide and 100 ng/ml TNF for 5 h. Subsequently, cells were subjected to terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling kit (TUNEL) apoptosis assay. Labeled nuclei from at least 5 random fields per condition were counted in each of 3 experiments.

Fig. 6.

ErbB4 transactivation by TNF promotes increased Akt activation. YAMC-vector and YAMC-ErbB4 cells were incubated with 10 μM TAPI-1 for 30 min (A) or with ErbB4 blocking antibody for 1 h (B) before treatment with 100 ng/ml TNF for 15 min. Cell lysates were then analyzed by Western blot with antibodies against phosphorylated Akt (PS-473), total Akt, ErbB4, or actin. All blots are representative of at least 3 experiments.

Fig. 7.

Model of ErbB4 transactivation by TNF in colonic epithelial cells TNF stimulation of colonic epithelial cells leads to activation of ERK1/2, which stimulates TACE activity. TACE cleaves membrane-bound HB-EGF, which then binds to ErbB4 and stimulates phosphorylation and activation. ErbB4 transactivation by TNF then mediates colonic epithelial cell survival through increased Akt signaling.

References

1. Brynskov J, Foegh P, Pedersen G, Ellervik C, Kirkegaard T, Bingham A, Saermark T. Tumour necrosis factor alpha converting enzyme (TACE) activity in the colonic mucosa of patients with inflammatory bowel disease. Gut 51: 37–43, 2002 - PMC - PubMed
1. Chalaris A, Adam N, Sina C, Rosenstiel P, Lehmann-Koch J, Schirmacher P, Hartmann D, Cichy J, Gavrilova O, Schreiber S, Jostock T, Matthews V, Hasler R, Becker C, Neurath MF, Reiss K, Saftig P, Scheller J, Rose-John S. Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice. J Exp Med 207: 1617–1624 - PMC - PubMed
1. Colon AL, Menchen LA, Hurtado O, De Cristobal J, Lizasoain I, Leza JC, Lorenzo P, Moro MA. Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis. Cytokine 16: 220–226, 2001 - PubMed
1. Danielsen AJ, Maihle NJ. The EGF/ErbB receptor family and apoptosis. Growth Factors 20: 1–15, 2002 - PubMed
1. Diaz-Rodriguez E, Montero JC, Esparis-Ogando A, Yuste L, Pandiella A. Extracellular signal-regulated kinase phosphorylates tumor necrosis factor alpha-converting enzyme at threonine 735: a potential role in regulated shedding. Mol Biol Cell 13: 2031–2044, 2002 - PMC - PubMed

TNF-α converting enzyme-mediated ErbB4 transactivation by TNF promotes colonic epithelial cell survival - PubMed (original) (raw)