Volume 116, Issue 1, Pages 78-89 (January 1999) Gastrin induces heparin-binding epidermal growth factor–like growth factor in rat gastric epithelial cells transfected with gastrin receptor  Yoshiji Miyazaki, Yasuhisa Shinomura, Shusaku Tsutsui, Shinichiro Zushi, Yoshifumi Higashimoto, Shuji Kanayama, Shigeki Higashiyama, Naoyuki Taniguchi, Yuji Matsuzawa  Gastroenterology  Volume 116, Issue 1, Pages 78-89 (January 1999) DOI: 10.1016/S0016-5085(99)70231-3 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Expression of gastrin/CCK-B–receptor mRNA by transfected RGM1 cells. Total RNAs isolated from parental RGM1 (lane 1), RGaR9 (lane 2), and RGaR19 cells (lane 3) were loaded and hybridized with a cDNA probe for human gastrin/CCK-B receptor. A GAPDH probe was used to control for RNA loading. RGaR9 and RGaR19 cells express the 2.7-kb gastrin/CCK-B–receptor mRNA. (B) Scatchard plot analysis of 125I-labeled gastrin 17 saturation binding studies. RGaR9 cells were incubated with 0.1–2.0 nmol/L 125I-labeled gastrin 17 in the presence or absence of 10 μmol/L unlabeled gastrin 17. The specific binding activity was calculated by subtraction of the radioactivity of the sample containing 10 μmol/L unlabeled gastrin 17 from the total activity. Each point represents the mean of results from 3 separate experiments. The Kd value is 0.39 nmol/L, and the average number of binding sites per cell is 2.72 × 103. Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Induction of HB-EGF and AR mRNAs by gastrin 17. Quiescent RGaR9 cells were incubated in serum-free medium with 10−8 mol/L gastrin 17 for the indicated times. Total RNA isolated from the cells was loaded and hybridized with cDNA probes for rat HB-EGF and AR. Blots were rehybridized with a GAPDH cDNA probe. Autoradiographs are representative of 4 experiments. (B) Quantitation of HB-EGF (•) and AR (○) mRNAs in A. Error bars indicate mean ± SE (n = 4). (C) Inhibition of gastrin-induced expression of HB-EGF and AR mRNAs by the gastrin/CCK-B–receptor antagonists. Quiescent RGaR9 cells were incubated in serum-free medium with 10−8 mol/L gastrin 17/CCK-8 for 2 hours under the following conditions: lane 1, untreated; lane 2, gastrin 17; lane 3, CCK-8; lane 4, gastrin 17 with L-740,093 (10 nmol/L); lane 5, gastrin with L-365,260 (100 nmol/L). Autoradiographs are representative of 4 experiments. Normalized hybridization signals of HB-EGF mRNA are expressed in relationship to values in gastrin-treated cells (lower panel). Error bars indicate means ± SE (n = 4). Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 (A) Induction of HB-EGF and AR mRNAs by gastrin 17. Quiescent RGaR9 cells were incubated in serum-free medium with 10−8 mol/L gastrin 17 for the indicated times. Total RNA isolated from the cells was loaded and hybridized with cDNA probes for rat HB-EGF and AR. Blots were rehybridized with a GAPDH cDNA probe. Autoradiographs are representative of 4 experiments. (B) Quantitation of HB-EGF (•) and AR (○) mRNAs in A. Error bars indicate mean ± SE (n = 4). (C) Inhibition of gastrin-induced expression of HB-EGF and AR mRNAs by the gastrin/CCK-B–receptor antagonists. Quiescent RGaR9 cells were incubated in serum-free medium with 10−8 mol/L gastrin 17/CCK-8 for 2 hours under the following conditions: lane 1, untreated; lane 2, gastrin 17; lane 3, CCK-8; lane 4, gastrin 17 with L-740,093 (10 nmol/L); lane 5, gastrin with L-365,260 (100 nmol/L). Autoradiographs are representative of 4 experiments. Normalized hybridization signals of HB-EGF mRNA are expressed in relationship to values in gastrin-treated cells (lower panel). Error bars indicate means ± SE (n = 4). Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Expression of (A) TGF-α and (B) BTC mRNAs in RGaR9 cells. Quiescent RGaR9 cells were incubated in serum-free medium with 10−8 mol/L gastrin 17 for the times indicated. (A) Poly(A)+ mRNA or (B) total RNA isolated from the cells was loaded and hybridized with a TGF-α or BTC cDNA probe, respectively. Blots were rehybridized with a GAPDH cDNA probe. Autoradiographs are representative of 4 experiments. (C) Quantitation of TGF-α (•) and BTC (○) mRNAs in A and B. Error bars indicate means ± SE (n = 4). Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Effects of cycloheximide and actinomycin D on gastrin-induced expression of HB-EGF mRNA. Quiescent RGaR9 cells were incubated in serum-free medium with 10−8 mol/L gastrin 17 for 2 hours under the following conditions: lane 1, untreated; lane 2, gastrin 17; lane 3, gastrin 17 with cycloheximide (5 μg/mL); lane 4, cycloheximide; and lane 5, gastrin 17 with actinomycin D (3 μg/mL). Total RNA isolated from the cells was loaded and hybridized with a cDNA probe for rat HB-EGF. Blots were rehybridized with a GAPDH cDNA probe. Autoradiographs are representative of 4 experiments. Normalized hybridization signals are expressed in relationship to values in gastrin-treated cells (lower panel). Error bars indicate means ± SE (n = 4). Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Involvement of (A) PKC and (B) MEK in gastrin-induced expression of HB-EGF and AR mRNAs. Quiescent RGaR9 cells were incubated in serum-free medium with or without 10−8 mol/L gastrin 17 for 2 hours. (A) To inhibit PKC activity, the cells were pretreated with 50–100 nmol/L staurosporine for 15 minutes (lanes 3 and 4) or with 250–500 nmol/L 12-o-tetradecanoyl phorbol 13-acetate (TPA) for 24 hours (lanes 5 and 6) before gastrin stimulation. (B) To inhibit MEK activity, the cells were treated with 50 nmol/L PD 98059 for 15 minutes before gastrin stimulation. Total RNA isolated from the cells was loaded and hybridized with cDNA probes for rat HB-EGF and AR. Blots were rehybridized with a GAPDH cDNA probe. Autoradiographs are representative of 4 experiments. Normalized hybridization signals of HB-EGF mRNA are expressed in relationship to values in gastrin-treated cells (lower panels). Error bars indicate means ± SE (n = 4). Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Effect of gastrin on HB-EGF mRNA expression in isolated parietal cells. Isolated guinea pig parietal cells were prepared as described in Materials and Methods. Cells were incubated in RPMI medium with or without 10−8 mol/L gastrin 17 for 2 hours. Total RNA isolated from the cells was loaded and hybridized with a cDNA probe for rat HB-EGF. Blots were rehybridized with cDNA probes for GAPDH and H+,K+-ATPase. Autoradiographs are representative of 4 experiments. (B) Quantitated hybridization signals are expressed in relationship to the control values. Error bars indicate means ± SE (n = 4). Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Kinetics of cell surface proHB-EGF processing by gastrin. RGaR9 cells were incubated with 10−8 mol/L gastrin 17 for the indicated periods, and cell surface proHB-EGF levels were measured by flow cytometry. Each histogram in A represents 2 × 104 cells. The closed area indicates cells treated with 10−8 mol/L gastrin 17; open areas indicate untreated control cells. (B) The absolute fluorescence intensities of the various time points shown in A were converted to relative fluorescence intensity as described in Materials and Methods. ■, Cells treated with 10−8 mol/L gastrin 17; 2, cells treated with gastrin and 100 nmol/L staurosporine. Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 Gastrin-induced cellular secretion of HB-EGF. Confluent RGaR9 cells were incubated in serum-free medium with or without 10−8 mol/L gastrin 17 for the indicated periods. 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–HB-EGF serum (antiserum 2998), and the results were visualized with an enhanced chemiluminescence detection system (Amersham). Western blots are representative of 4 experiments. Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 9 Gastrin-induced tyrosine phosphorylation of the EGF receptor. (A) RGaR9 cells were stimulated with 10−8 mol/L gastrin 17 (lanes 2–4), 10 ng/mL TGF-α (lane 5), or 10 ng/mL TGF-α with 250 nmol/L AG1478 (lane 6) for the times indicated. (B) The cells were stimulated with 10−8 mol/L gastrin 17 (lane 2), 10−8 mol/L gastrin 17 with antiserum to HB-EGF (antiserum 19, 1/100; lane 3), 10−8 mol/L gastrin 17 with 250 nmol/L AG1478 (lane 4), or 10 ng/mL TGF-α (lane 5) for 15 minutes. The treated cells were lysed, and immunoprecipitation was performed with anti-EGF receptor antibody (αEGFR). The precipitates were separated on SDS-PAGE and transferred to membranes. Immunoblots were probed with antiphosphotyrosine antibody (αPY), and the results were visualized with an Amersham enhanced chemiluminescence detection system. Filters were subsequently stripped of antibody and reprobed with αEGFR. Western blots are representative of 4 experiments. Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 10 Growth-stimulatory effects of (A and B) gastrin 17/CCK-8 and (C) EGF-related polypeptides on RGaR9 cells. Confluent RGaR9 cells in 96-well plates were placed in serum-free medium for 48 hours. The cells were subsequently incubated for 18 hours in serum-free medium with the indicated concentration of gastrin 17, CCK-8, TGF-α, HB-EGF, or AR; they were then labeled with 1 μCi/mL [3H]thymidine 18–22 hours later. The [3H]thymidine incorporated was measured by a Betaplate System (Amersham). Data represent the mean ± SE from 4–6 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. Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 11 Effects of (A) goat anti-rat HB-EGF serum and (B) tyrphostin AG1478 on gastrin-induced [3H]thymidine incorporation in RGaR9 cells. Quiescent RGaR9 cells were incubated in serum-free medium with 10−8 mol/L gastrin 17 for 18 hours in the presence or absence of goat anti-rat HB-EGF serum (antiserum 19) or 250 nmol/L AG1478. Thereafter, the cells were pulse labeled with [3H]thymidine for 4 hours, and the [3H]thymidine incorporated was measured by a Betaplate System (Amersham). Data represent the 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 gastrin-treated controls. Gastroenterology 1999 116, 78-89DOI: (10.1016/S0016-5085(99)70231-3) Copyright © 1999 American Gastroenterological Association Terms and Conditions