Growth hormone reduces chloride secretion in human colonic epithelial cells via EGF receptor and extracellular regulated kinase1   Jimmy Y.C Chow, Katie Carlstrom, Kim E Barrett  Gastroenterology  Volume 125, Issue 4, Pages 1114-1124 (October 2003) DOI: 10.1016/S0016-5085(03)01211-3

Figure 1 Effect of GH on CCh-induced chloride secretion across T84 cells. (A) T84 cell monolayers were pretreated basolaterally with GH (10 nmol/L) for different times as shown, followed by addition of CCh. Chloride secretion was measured as changes in short-circuit current (▵Isc) and data are expressed as means ± SEM for 7–12 experiments. (B) T84 cell monolayers mounted in Ussing chambers were pretreated basolaterally with GH (0.1–1000 nmol/L) for 15 minutes before the basolateral addition of CCh (100 μmol/L). Data are expressed as mean ± SEM for 6–13 experiments. (A, B) Asterisks denote responses significantly different from those in the absence of GH. ∗P < 0.05; ∗∗P < 0.01 by one-way ANOVA with the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 2 Effect of GH on JAK2 phosphorylation and EGFr phosphorylation in T84 cells. (A) T84 cell monolayers were stimulated with GH on the basolateral side for various times as shown. Cell lysates were immunoprecipitated with an anti-JAK2 antibody, and Western blots were probed with a monoclonal antibody against phosphotyrosine. The panel depicts a single representative experiment. (B) Similar data were quantitated by image analysis and are means ± SEM for 3 experiments. (C, D) Same as panels A and B except that EGFr rather than JAK2 phosphorylation was assessed. Data are means ± SEM for 3 similar experiments. In all panels, asterisks denote responses significantly different from those in the absence of GH. ∗P < 0.05; ∗∗∗P < 0.001 by one-way ANOVA with the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 3 Effect of GH at different concentrations on EGFr phosphorylation in T84 cells. (A) T84 cell monolayers were stimulated with various concentrations of GH on the basolateral side. EGFr activation was assessed as in Figure 2. The upper depicts a single representative experiment. (B) Data were quantitated by image analysis and are means ± SEM for 7 similar experiments. Asterisks denote responses significantly different from those in the absence of GH. ∗P < 0.05, ∗∗P < 0.01 by one-way ANOVA with the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 4 Effect of AG490 on activation of JAK2 by GH. Cell monolayers were pretreated bilaterally with AG490 (50 μmol/L) for 30 minutes before the basolateral addition of GH (10 nmol/L), or were left untreated as controls. The cells then were lysed 1 minute after GH addition. JAK2 activation was assessed as in Figure 2. (A) A single representative experiment. (B) Data were quantitated by image analysis and are means ± SEM for 4 similar experiments. The asterisk denotes a response that differs significantly different from control. ∗P < 0.05 by one-way ANOVA with the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 5 Effect of AG490 on the ability of GH to cause EGFr phosphorylation and the inhibitory effect of GH on chloride secretion in T84 cells. (A, B) Monolayers were pretreated bilaterally with AG490 (20 μmol/L) for 30 minutes before the basolateral addition of GH (10 nmol/L). Other cells received buffer alone (control), AG490 alone, or GH alone. The cells were lysed 1 minute after GH addition. EGFr activation was assessed as in Figure 2. (A) A single representative experiment. (B) Data were quantitated by image analysis and are means ± SEM for 4 similar experiments, expressed as a percent of control phosphorylation. (C) Monolayers on inserts were mounted in Ussing chambers and pretreated bilaterally with AG490 (20 μmol/L) for 30 minutes before the basolateral addition of GH (10 nmol/L). This was followed 15 minutes later by basolateral addition of CCh (100 μmol/L). Other cells received CCh alone (control), CCh plus GH, or CCh plus AG490. Data are means ± SEM for 7 experiments and are expressed as peak increases in Isc (▵Isc) induced by CCh. (B, C) Asterisks denote responses that are significantly different from control. ∗∗P < 0.01; ∗∗∗P < 0.001; and +++ indicates responses in the presence of AG490 that were significantly different from GH alone. P < 0.001 by one-way ANOVA with the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 6 Effect of AG1478 on the ability of GH to cause EGFr phosphorylation and the inhibitory effect of GH on chloride secretion in T84 cells. (A, B) Monolayers were pretreated bilaterally with AG1478 (1 μmol/L) for 20 minutes before the basolateral addition of GH (10 nmol/L). Other cells received buffer alone (control), AG1478 alone, or GH alone. EGFr activation was assessed as in Figure 2. (A) A single representative experiment. (B) Data were quantitated by image analysis and are means ± SEM for 3 similar experiments. (C) Monolayers on inserts were mounted in Ussing chambers and pretreated bilaterally with AG1478 (1 μmol/L) for 20 minutes before the basolateral addition of GH (10 nmol/L). This was followed 15 minutes later by basolateral addition of CCh (100 μmol/L). Other cells received CCh alone (control), CCh plus GH, or CCh plus AG1478. Data are means ± SEM for 5 experiments and are expressed as peak increases in Isc (▵Isc) induced by CCh. (B, C) Asterisks denote responses that were significantly different from control, P < 0.05, and + indicates responses in the presence of AG1478 that were significantly different from GH alone. P < 0.05 by one-way ANOVA with the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 7 GH stimulates the activity of p38 MAPK in T84 cells. Monolayers were pretreated basolaterally with GH (10 nmol/L) for various times as shown. Lysates were blotted with a monoclonal antibody against the phosphorylated form of the ATF-2 transcription factor, which is a substrate of p38. (A) A single representative experiment. (B) Data were quantitated by image analysis, are expressed as arbitrary units, and are the means ± SEM for 5 experiments. Asterisks denote values that are significantly different from 0 minutes. ∗P < 0.05; ∗∗P < 0.01 by one-way ANOVA followed by the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 8 GH stimulates ERK phosphorylation in T84 cells. Monolayers were pretreated basolaterally with GH (10 nmol/L) for various times as shown. Lysates were Western blotted with a monoclonal antibody against phospho-ERK. (A) A single representative experiment. (B) Data were quantitated by image analysis, are expressed as arbitrary units, and are means ± SEM for 3 experiments. Asterisks denote values that are significantly different from 0 minutes. ∗P < 0.05; ∗∗P < 0.01 by one-way ANOVA followed by the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 9 Effect of AG490 on the ability of GH to cause p38 phosphorylation in T84 cells. Monolayers were pretreated bilaterally with AG490 (50 μmol/L) for 30 minutes before the basolateral addition of GH (10 nmol/L). Other cells received buffer alone (control), AG490 alone, or GH alone. p38 activation was assessed by blotting lysates with an antibody specific for the phosphorylated form of p38. (A) A single representative experiment. (B) Data were quantitated by image analysis and are the means ± SEM for 3 experiments. ∗Significantly different from control, P < 0.05 by one-way ANOVA with the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 10 Effect of AG490 on the ability of GH to increase ERK1/2 activity in T84 cells. Monolayers were pretreated bilaterally with AG490 (100 μmol/L) for 30 minutes before the basolateral addition of GH (10 nmol/L). Other cells received buffer alone (control), AG490 alone, or GH alone. ERK activation was assessed in lysates by Western blotting with an antibody specific for the phosphorylated forms of Elk, which is a substrate for ERK1/2. (A) A single representative experiment. (B) Data were quantitated by image analysis and are means ± SEM for 4 experiments. ∗∗∗Significantly different from control, P < 0.001 by one-way ANOVA followed by the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 11 Effect of AG1478 on the ability of GH to cause phosphorylation of ERK1/2 in T84 cells. Monolayers were pretreated bilaterally with AG1478 (1 μmol/L) for 20 minutes before the basolateral addition of GH (10 nmol/L). Other cells received buffer alone (control), AG1478 alone, or GH alone. ERK activation was assessed in lysates by Western blotting with an antibody specific for the phosphorylated forms. (A) A single representative experiment. (B) Data were quantitated by image analysis and are means ± SEM for 5 experiments. ∗∗∗Significantly different from control, P < 0.001 by one-way ANOVA with the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 12 Effect of AG1478 on the ability of GH to cause phosphorylation of p38 in T84 cells. Monolayers were pretreated bilaterally with AG1478 (1 μmol/L) for 20 minutes before the basolateral addition of GH (10 nmol/L). Other cells received buffer alone (control), AG1478 alone, or GH alone. p38 activation was assessed by Western blotting of lysates with an antibody specific for the phosphorylated form. (A) A single representative experiment. (B) Data were quantitated by image analysis and are means ± SEM for 4 experiments. ∗Significantly different from control, P < 0.05 by one-way ANOVA with the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 13 Effect of PD98059 on the inhibitory action of GH on CCh-induced chloride secretion in T84 cells. (A, B) Monolayers were pretreated bilaterally with PD98059 (20 μmol/L) for 15 minutes before the basolateral addition of GH (10 nmol/L). Other cells received buffer alone (control), PD98059 alone, or GH alone. The cells were lysed 1 minute after GH addition. Western blots were probed with an antibody against phospho-ERK1/2. (A) A single representative experiment. (B) Data were quantitated by image analysis and are means ± SEM for 3 similar experiments, expressed as a percent of control phosphorylation. ∗Significantly different from control, P < 0.05 by one-way ANOVA with the Student-Newman-Keul post hoc test. (C) Monolayers were mounted in Ussing chambers and pretreated bilaterally with PD98059 (20 μmol/L) for 15 minutes before the basolateral addition of GH (10 nmol/L). This was followed 15 minutes later by basolateral addition of CCh (100 μmol/L). Other cells received CCh alone (control), CCh plus GH, or CCh plus PD98059. Data are means ± SEM for 6 experiments and are expressed as the peak increases in Isc (▵Isc) induced by CCh. The asterisk denotes a response that differs significantly from that induced by CCh alone. ∗P < 0.05 by one-way ANOVA followed by the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)

Figure 14 Effect of SB203580 on the inhibitory action of GH on CCh-induced chloride secretion in T84 cells. (A, B) Monolayers were pretreated bilaterally with SB203580 (10 μmol/L) for 15 minutes before the basolateral addition of GH (10 nmol/L). Other cells received buffer alone (control), SB203580 alone, or GH alone. The cells were lysed 5 minutes after GH addition. Western blots were probed with an antibody against phospho-p38. (A) A single representative experiment. (B) Data were quantitated by image analysis and are means ± SEM for 3 similar experiments, expressed as a percent of control phosphorylation. ∗∗Significantly different from control, P < 0.01 by one-way ANOVA with the Student-Newman-Keul post hoc test. (C) Monolayers were mounted in Ussing chambers and pretreated bilaterally with SB203580 (10 μmol/L) for 15 minutes before the basolateral addition of GH (10 nmol/L). This was followed 15 minutes later by basolateral addition of CCh (100 μmol/L). Other cells received CCh alone (control), CCh plus GH, or CCh plus SB203580. Data are means ± SEM for 8 experiments and are expressed as the peak increases in Isc (▵Isc) induced by CCh. Asterisks denote a response that differs significantly from those induced by CCh alone. ∗P < 0.05; ∗∗∗P < 0.001 by one-way ANOVA followed by the Student-Newman-Keul post hoc test. Gastroenterology 2003 125, 1114-1124DOI: (10.1016/S0016-5085(03)01211-3)