Volume 120, Issue 1, Pages 108-116 (January 2001) Epidermal growth factor receptor mediates stress-induced expression of its ligands in rat gastric epithelial cells  Yoshiji Miyazaki, Shintaro Hiraoka, Syusaku Tsutsui, Shinji Kitamura, Yasuhisa Shinomura, Yuji Matsuzawa  Gastroenterology  Volume 120, Issue 1, Pages 108-116 (January 2001) DOI: 10.1053/gast.2001.20950 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of the mutant human EGFR in RGMHERCD cells. RGM1 cells and RGMHERCD-10 cells were incubated with a monoclonal antibody specific for the cell surface epitope of the human EGFR and then with fluorescein isothiocyanate–conjugated goat anti-mouse IgG. Fluorescence intensity of the cell surface was analyzed using a FACScan. Gastroenterology 2001 120, 108-116DOI: (10.1053/gast.2001.20950) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Mitogenic effects of EGF on RGM1 (○), RGM-neo (●), RGMHERCD-4 (▴), and RGMHERCD-10 (■) cells. Confluent cells in 96-well plates were placed in serum-free medium for 48 hours. The cells were subsequently incubated in serum-free medium with 0, 2.5, 5, or 10 ng/mL EGF for 18 hours; then they were labeled with 1 μCi/mL [3H]thymidine 18–22 hours later. The [3H]thymidine incorporated was measured by a Betaplate System (Amersham). Data represent mean ± SE from 6 identical experiments and are expressed as a percentage of the values seen in nonstimulated control cells. *P < 0.05 compared with values in control cells. (B) Growth curve of RGM1 (○), RGMHERCD-4 (▴), and RGMHERCD-10 (■) cells under serum-free conditions. Cells were seeded at 2 × 104 per 60-mm dish in 5% serum-containing medium and incubated for 24 hours. Next, serum was deprived and cells were cultured in serum-free medium. After 0, 1, 2, 4, 6, and 8 days, the cells were harvested by trypsinization, washed with PBS, and counted using a hemacytometer. Data represent mean ± SE from 6 identical experiments. *P < 0.05 compared with values in RGM1 cells. Gastroenterology 2001 120, 108-116DOI: (10.1053/gast.2001.20950) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Stress-induced tyrosine phosphorylation of EGFR. RGM1 cells were stimulated with (A) 450 μmol/L hydrogen peroxide or with (B) 600 mmol/L sorbitol for 0, 2, or 5 minutes. (C) RGM1 cells were treated or not treated with 450 μmol/L hydrogen peroxide for 5 minutes under the following conditions: lane 1, untreated; lane 2, hydrogen peroxide; lane 3, hydrogen peroxide with AG1478 (250 nmol/L); lane 4, hydrogen peroxide with NAC (10 mmol/L); lane 5, hydrogen peroxide with TAPI (0.1 μmol/L); lane 6, EGF (10 nmol/L). (D) Inhibition of tyrosine phosphorylation of the EGFR by a dominant-negative EGF receptor. RGM-neo (lanes 1–3), RGMHERCD-4 (lane 4), and RGMHERCD-10 (lane 5) cells were treated or not treated with 450 μmol/L hydrogen peroxide for 15 minutes under the following conditions: lane 1, untreated; lanes 2, 4, and 5, hydrogen peroxide; lane 3, hydrogen peroxide with AG1478 (250 nmol/L). The treated cells were lysed, and immunoprecipitation was performed with an anti-EGFR antibody (αEGFR). Tyrosine phosphorylation was determined by Western blot analysis using antiphosphotyrosine antibody (αPY). The results were visualized with an enhanced chemiluminescence detection system (ECL, Amersham). Filters were subsequently stripped of antibody and reprobed with anti-EGFR antibody (αEGFR). Western blots are representative of 4 experiments. Gastroenterology 2001 120, 108-116DOI: (10.1053/gast.2001.20950) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Stress-induced tyrosine phosphorylation of (A) ErbB-2, (B) ErbB-3, and (C) c-Met. RGM1 cells were treated or not treated with 450 μmol/L hydrogen peroxide for 5 minutes under the following conditions: lane 1, untreated; lane 2, hydrogen peroxide. As a positive control for c-Met phosphorylation, cells were treated with hepatocyte growth factor for 5 minutes (C, lane 3). The treated cells were lysed, and immunoprecipitation was performed with anti–ErbB-2 antibody (αErbB-2), anti–ErbB-3 antibody (αErbB-3), or anti–c-Met antibody (αMet). Tyrosine phosphorylation was determined by Western blot analysis using anti-phosphotyrosine antibody (αPY). The results were visualized with an enhanced chemiluminescence detection system (ECL, Amersham). Filters were subsequently stripped of antibody and reprobed with αErbB-2, αErbB-3, or αMet. Western blots are representative of 4 experiments. Gastroenterology 2001 120, 108-116DOI: (10.1053/gast.2001.20950) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Stress-induced expression of HB-EGF, AR, and TGF-α transcripts. RGM1 cells were incubated in (A) serum-free medium with 450 μmol/L hydrogen peroxide or in (B) high-osmolarity medium (600 mmol/L sorbitol) for 15 minutes and then transferred to a normal osmolarity medium. After 0, 0.25, 0.5, 1, 3, 5, 7, or 24 hours, total RNA was extracted, loaded, and hybridized with cDNA probes for rat HB-EGF and AR. For detection of TGF-α transcripts, poly(A)+ mRNA was loaded and hybridized with a cDNA probe for rat TGF-α. Blots were rehybridized with a GAPDH cDNA probe. Gastroenterology 2001 120, 108-116DOI: (10.1053/gast.2001.20950) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of NAC and AG 1478 on the stress-induced expression of HB-EGF and AR mRNAs. RGM1 cells were incubated in (A) serum-free medium with 450 μmol/L hydrogen peroxide or in (B) high-osmolarity medium (600 mmol/L sorbitol) for 15 minutes and then transferred to a normal osmolarity medium. Lanes in A and B show the following conditions: lane 1, untreated; lane 2, (A) hydrogen peroxide or (B) sorbitol; lane 3, (A) hydrogen peroxide or (B) sorbitol with AG1478; lane 4, (A) hydrogen peroxide or (B) sorbitol with NAC. After 3 hours, total RNA was extracted, loaded, and hybridized with cDNA probes for rat HB-EGF and AR. Blots were rehybridized with a GAPDH cDNA probe. Gastroenterology 2001 120, 108-116DOI: (10.1053/gast.2001.20950) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Introduction of a dominant-negative EGFR inhibits the oxidative stress–induced expression of HB-EGF and AR transcripts. RGM-neo, RGMHERCD-4, and RGMHERCD-10 cells were incubated in serum-free medium with 450 μmol/L hydrogen peroxide. After 0, 2, 4, or 8 hours, total RNA was extracted, loaded, and hybridized with cDNA probes for rat HB-EGF and AR. Blots were rehybridized with a GAPDH cDNA probe. Quantitated hybridization signals were normalized to the control gene GAPDH and were expressed relative to control values (n = 4, means ± SE). Gastroenterology 2001 120, 108-116DOI: (10.1053/gast.2001.20950) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Activation of HB-EGF promoter by oxidative stress. The cells were transiently transfected with a reporter construct containing a 1.73-kb region of the murine HB-EGF gene encompassing sequences from positions −1884 to −155 upstream of the translation initiation codon in exon 1. After the cells were treated or not treated with 450 μmol/L hydrogen peroxide for 12 hours, the luciferase activity was measured. The enzyme activity was normalized for efficiency of transfection, and relative values were determined. Transfection experiments were carried out 5 times independently, and the average of the 5 results was calculated. Gastroenterology 2001 120, 108-116DOI: (10.1053/gast.2001.20950) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 9 Oxidative stress–induced cellular secretion of HB-EGF. Confluent cells were incubated in serum-free medium with or without 450 mmol/L hydrogen peroxide for 0, 15, 30, 45, 60, or 120 minutes. The conditioned media were pooled and applied to a 30-μL heparin-Sepharose CL-6B column. The proteins bound to heparin-Sepharose beads were fractionated by 15% SDS-PAGE and transferred to membranes. HB-EGF protein was detected by Western blot with anti-mouse HB-EGF antibody (Santa Cruz), and the results were visualized with an enhanced chemiluminescence detection system (ECL, Amersham). Gastroenterology 2001 120, 108-116DOI: (10.1053/gast.2001.20950) Copyright © 2001 American Gastroenterological Association Terms and Conditions