Volume 114, Issue 4, Pages 697-705 (April 1998) Rapid mitogen-activated protein kinase activation by transforming growth factor α in wounded rat intestinal epithelial cells  Michael Göke, Michiyuki Kanai, Kathryn Lynch–Devaney, Daniel K. Podolsky  Gastroenterology  Volume 114, Issue 4, Pages 697-705 (April 1998) DOI: 10.1016/S0016-5085(98)70583-9 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Time course for tyrosine phosphorylation in total cell lysates from wounded IEC-6 cells. Twenty-five micrograms of proteins from wounded (1, 5, 15, 30, 60, and 360 minutes after wounding; w1–w360) and intact confluent monolayers (con 0) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) analysis and Western blot transfer. Hybridization with an anti–PY-20 antibody, detection, and autoradiography were performed as described in Materials and Methods. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Tyrosine phosphorylation of ERK1 in wounded IEC-6 monolayers. Lysates containing equal amounts of protein (300 μg/tube) obtained from wounded (5, 15, 30, and 360 minutes after wounding; w5–w360) and intact confluent monolayers (con 0) were immunoprecipitated with anti-ERK1 antibody. Immune complexes were subjected to SDS-PAGE analysis and Western blot transfer. (A) Phosphorylation of ERK1 protein was assessed by hybridization with anti–PY-20 antibody. (B) For evaluation of ERK1 protein expression, the same blot was stripped and reprobed with anti-ERK1 antibody. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Assessment of ERK1, ERK2, and Raf-1 kinase activity in wounded intestinal epithelial cells. Lysates containing equal amounts of protein (300 μg/tube) from wounded (1, 5, and 60 minutes after wounding; w1–w60) and intact confluent monolayers (con 0) were immunoprecipitated with (A) anti-ERK1, (B) anti-ERK2, and (C) anti–Raf-1 antibodies, respectively. Immune complexes were washed and subjected to an in vitro kinase reaction for 20 minutes at 30°C using [γ-32P]ATP and MBP as a substrate, and analyzed by SDS-PAGE and autoradiography as described in Materials and Methods. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Assessment of ERK1, ERK2, and Raf-1 kinase activity in wounded intestinal epithelial cells. Lysates containing equal amounts of protein (300 μg/tube) from wounded (1, 5, and 60 minutes after wounding; w1–w60) and intact confluent monolayers (con 0) were immunoprecipitated with (A) anti-ERK1, (B) anti-ERK2, and (C) anti–Raf-1 antibodies, respectively. Immune complexes were washed and subjected to an in vitro kinase reaction for 20 minutes at 30°C using [γ-32P]ATP and MBP as a substrate, and analyzed by SDS-PAGE and autoradiography as described in Materials and Methods. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Assessment of ERK1, ERK2, and Raf-1 kinase activity in wounded intestinal epithelial cells. Lysates containing equal amounts of protein (300 μg/tube) from wounded (1, 5, and 60 minutes after wounding; w1–w60) and intact confluent monolayers (con 0) were immunoprecipitated with (A) anti-ERK1, (B) anti-ERK2, and (C) anti–Raf-1 antibodies, respectively. Immune complexes were washed and subjected to an in vitro kinase reaction for 20 minutes at 30°C using [γ-32P]ATP and MBP as a substrate, and analyzed by SDS-PAGE and autoradiography as described in Materials and Methods. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of wound-conditioned medium on Raf-1, ERK1, and ERK2 kinase activity of intestinal epithelial cells. Conditioned medium was collected from wounded IEC-6 cultures 5 minutes after wounding or from unwounded confluent monolayers and then added to serum-starved intact confluent IEC-6 monolayers. After culture of IEC-6 cells in the presence of either wound conditioned (w) or control conditioned medium (con) for 5 minutes, cell lysates were harvested. Equal amounts of protein (300 μg/tube) were immunoprecipitated with anti–Raf-1, anti-ERK1, and anti-ERK2 antibodies, respectively. Immune complexes were subjected to an in vitro kinase assay with MBP as a substrate, then analyzed by SDS-PAGE and autoradiography. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of neutralizing anti–TGF-α and anti–TGF-β antibodies on ERK1 and ERK2 activity of intestinal epithelial cells. Confluent serum-starved IEC-6 cells were cultured for 5 minutes with conditioned medium collected from unwounded confluent monolayers (con), untreated wounded monolayers (w), or wounded monolayers containing 15 μg/mL normal rabbit IgG (NR IgG), anti–TGF-α antibody, or anti–TGF-β antibody. Equal amounts of proteins in lysates were immunoprecipitated with (A) anti-ERK1 and (B) anti-ERK2 antibodies, respectively. ERK1 and ERK2 in vitro kinase activity was assessed using MBP as a substrate and analyzed as in Figures 3 and 4. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of wound-conditioned medium on IEC-6 cell proliferation. Confluent serum-starved monolayers were cultured for 20 hours in the presence of fresh serum-deprived medium (DMEM), conditioned medium from intact confluent control monolayers (con CM), or wound-conditioned medium in the absence or presence of 15 μg/mL neutralizing anti–TGF-α antibody (w CM and w CM + anti–TGF-α, respectively). Cells were fixed and cell proliferation assessed by measurement of [3H]thymidine incorporation (in cpm) as detailed in Materials and Methods. *P = 0.029 vs. con CM. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of TGF-α on ERK1, ERK2, and Raf-1 activity in intestinal epithelial cells. Lysates from confluent serum-deprived IEC-6 monolayers were harvested 5 minutes after stimulation with TGF-α (5 or 10 ng/mL) or BSA (dissolved in the same carrier as that for TGF-α). In parallel studies, serum-deprived IEC-6 monolayers were exposed to conditioned medium (CM) obtained from wounded or unwounded monolayers for 5 minutes. The conditioned medium was collected from dishes with or without change in medium (“wash” or “no wash”). (A) Raf-1 and (B) ERK1 and ERK2 were determined after immunoprecipitation with relevant antibody followed by in vitro kinase assay as described in the text. Activity of ERK1, ERK2, and Raf-1 proteins was determined by an in vitro kinase assay and analyzed as described in Materials and Methods. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of wounding on JNK1 activity in intestinal epithelial cells. (A) Lysates containing equal amounts of protein were harvested from wounded monolayers (1, 5, 15, 30, and 60 minutes after wounding; w1–w60), and confluent control (con 0) IEC-6 monolayers were immunoprecipitated with anti-JNK1 antibody. Immune complexes were subjected to an in vitro kinase assay using [γ-32P]ATP and c-Jun–glutathione-S-transferase fusion protein substrate as described in Materials and Methods. (B) Intact confluent serum-starved IEC-6 monolayers were cultured in the presence of either wound-conditioned (w CM) or control-conditioned (con CM) medium for 5 minutes and harvested. In parallel, a wounded monolayer 5 minutes after injury and confluent monolayer were harvested. JNK1 in vitro kinase activity was assessed as described above. Gastroenterology 1998 114, 697-705DOI: (10.1016/S0016-5085(98)70583-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions