HB-EGF Enhances Restitution After Intestinal Ischemia/Reperfusion via PI3K/Akt and MEK/ERK1/2 Activation  Osama N. El-Assal, Gail E. Besner  Gastroenterology  Volume 129, Issue 2, Pages 609-625 (August 2005) DOI: 10.1053/j.gastro.2005.05.054 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Intestinal I/R injury induces endogenous HB-EGF expression, and administration of exogenous HB-EGF enhances recovery from intestinal injury. (A) Reverse-transcription polymerase chain reaction showing that intestinal I/R injury induces endogenous HB-EGF mRNA expression. HB-EGF responsive receptors (ErbB-1, ErbB-4, and nardilysin C [NRDc]) were also induced at the mRNA level starting 3 hours after reperfusion. (B) Serum LDH levels were significantly increased by intestinal I/R compared with control levels (*P < .001). In vivo treatment with HB-EGF (closed bars) resulted in a significant reduction in LDH levels compared with I/R animals (open bars) by 6 hours after reperfusion (**P < .05). (C) The histologic injury score was evaluated in the last 5 cm of ileum. At the end of ischemia, there was no significant difference between untreated (open bars) and HB-EGF-treated (closed bars) animals. Histologic damage reached significantly lower levels in both HB-EGF-treated (**P < .001) and untreated (*P < .05) animals 3 hours after reperfusion compared with the corresponding levels at the end of ischemia. Animals treated with HB-EGF had significantly lower histologic injury scores compared with untreated animals after 3 hours of reperfusion (P < .05). Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 HB-EGF promotes intestinal healing by restitution rather than proliferation early after intestinal I/R injury. (A) Western blotting of intestinal PCNA expression in rats treated with or without HB-EGF. PCNA was expressed as percent of β-actin expression (right panel). PCNA expression was not increased but rather decreased within the first 2 hours after reperfusion in HB-EGF-treated (closed bars) and untreated animals (open bars). Data are presented as mean ± SE. (B) Evidence of early intestinal recovery by restitution in rat ileum stained with H&E (a1, b1, c1, d1, e1, f1, and g1) or with periodic acid-Schiff (a2, b2, c2, d2, e2, f2, and g2) to demonstrate goblet cells. (a1, a2) Normal ileum. At the end of ischemia, (b1) untreated animals and (c1) HB-EGF-treated animals showed the highest degree of injury. (b2 and c2) Some goblet cells remained attached to the villi despite loss of surrounding enterocytes. One hour after reperfusion, there was a reduction in the degree of injury in both (d) untreated and (e) HB-EGF-treated animals, with HB-EGF-treated animals showing better histologic recovery. Evidence of restitution included short, contracted villi; broad villous tips; and restoration of mucosal continuity with a single layer of multiple goblet cells (d2 and e2, arrowheads). Three hours after reperfusion, (g) HB-EGF-treated animals showed significant structural recovery with more complete restitution compared with (f) untreated animals. Goblet cells were reduced and replaced by (f2 and g2) enterocytes. Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 HB-EGF promotes restitution and preserves gut barrier function after intestinal I/R injury. (A) Quantification of restitution as determined by the number of structurally incompetent villi. The highest numbers of incompetent villi were detected at the end of the ischemic period, which gradually declined after reperfusion in both untreated (open bars) and HB-EGF-treated (closed bars) animals. The number of incompetent villi in HB-EGF-treated rats was significantly lower 1 and 3 hours after reperfusion (*P < .05) than at the end of ischemia. Three hours after reperfusion, untreated rats had a significantly lower number of communicating villi (**P < .05) than at the end of ischemia; however, they had a significantly lower recovery compared with HB-EGF-treated rats. (B) Intestinal mucosal permeability to FD-4 3 hours after reperfusion using everted ileal sacs incubated ex vivo. I/R resulted in significant impairment of gut barrier function as indicated by increased FD-4 clearance compared with control rats (*P < .05). Treatment with HB-EGF resulted in significant improvement in gut barrier function compared with I/R rats (**P < .05). Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 The effect of intestinal I/R and HB-EGF treatment on the activation of Akt and ERK1/2. (A) Western blotting for detection of Akt phosphorylation in rat intestine. Phospho-Akt was not detectable in control animals and only weakly detected in untreated animals subjected to ischemia alone. HB-EGF administration (closed bars) accelerated and enhanced Akt activation, which was significantly higher 30 minutes and 1 hour after reperfusion compared with untreated animals (open bars). (B) Western blotting for detection of ERK1/2 phosphorylation in rat intestine. ERK1/2 phosphorylation was not detectable in control animals or in untreated animals subjected to ischemia alone. Treatment with HB-EGF (closed bars) resulted in stronger activation of ERK1/2 at the end of the ischemic period, and at 30 minutes and 1 hour after reperfusion, compared with I/R injury alone (open bars). Activated Akt and ERK were expressed as percent of total protein (lower panel). Data are presented as mean ± SE (*P < .05). Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 HB-EGF activates PI3K/Akt and MEK/ERK1/2 via activation of ErbB-1 in RIE-1 cells. (A) Western blotting for detection of ErbB-1 phosphorylation. rHB-EGF induces activation of ErbB-1 in RIE-1 cells in vitro in a time-dependent manner. ErbB-1 activation was inhibited by AG1478 (100 nmol/L; a specific ErbB-1 inhibitor) but not by LY194002 (10 μmol/L; a specific PI3K/Akt inhibitor) or by U0126 (10 μmol/L; a specific MEK/ERK inhibitor). (B) Western blotting for detection of Akt phosphorylation. rHB-EGF induces Akt phosphorylation in RIE-1 cells in vitro in a time-dependent fashion. Pretreatment with AG1478 or LY294002 (10 μmol/L), but not LY 303511 (10 μmol/L; the chemically related nonactive inhibitor for PI3K/Akt), for 30 minutes completely blocked HB-EGF-induced Akt phosphorylation. (C) Western blotting for detection of ERK1/2 phosphorylation. rHB-EGF induces ERK1/2 phosphorylation in RIE-1 cells in vitro in a time-dependent fashion, which was inhibited by prior treatment with AG1478 (100 nmol/L) or U0126 (10 μmol/L) for 30 minutes. U0124 (10 μmol/L), the chemically related nonactive inhibitor for MEK/ERK, did not affect HB-EGF-induced ERK activation. Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 HB-EGF induces cell migration of RIE-1 cells after scrape wounding in a PI3K/Akt- and ERK1/2-dependent fashion. (A) Time course of RIE-1 wound healing with or without rHB-EGF treatment in vitro. rHB-EGF significantly enhanced the percent wound healing at 4, 8, and 18 hours after wounding. Data are presented as mean ± SE (*P < .05 vs control). (B) Inhibition of ErbB-1 activation using AG1478 significantly reduced both intrinsic and HB-EGF-induced restitution 18 hours after wounding. Pretreatment with LY294002, wortmannin, or U0126 resulted in significant reduction of intrinsic and HB-EGF-induced cell migration. PD98059 inhibited only HB-EGF-induced cell migration. Data are presented as mean ± SE (*P < .05 vs control; **P < .05 vs HB-EGF-treated rats). (C) Examples of the healing after scrape wounding. (1) Untreated cells immediately after wounding. All other panels represent cells 18 hours after wounding as follows: (2) untreated cells, (3) cells treated with HB-EGF (10 ng/mL), (4) cells treated with AG1478 (100 nmol/L), (5) cells treated with AG1478 for 30 minutes followed by rHB-EGF (10 ng/mL), (6) cells treated with LY294002 (10 μmol/L), (7) cells treated with LY294002 for 30 minutes followed by HB-EGF (10 ng/mL), (8) cells treated with wortmannin (100 nmol/L), (9) cells treated with wortmannin (100 nmol/L) followed by HB-EGF (10 ng/mL), (10) cells treated with U0126 (10 μmol/L), (11) cells treated with U0126 (10 μmol/L) for 30 minutes followed by HB-EGF (10 ng/mL), (12) cells treated with PD98059 (100 nmol/L), and (13) cells treated with PD98059 (100 nmol/L) followed by HB-EGF (10 ng/mL). (D) Quantitative cell migration assay. This assay quantitatively assessed cell migration based on the Boyden dual chamber principle, with 8-μm pore size membranes placed between the upper and lower chambers. RIE-1 cells were incubated in the upper chamber at a density of 5 × 105 cells/mL. The lower chamber was either supplemented with rHB-EGF (10 ng/mL) or an equivalent volume of 0.1% bovine serum albumin in phosphate-buffered saline. The upper chamber was supplemented with AG1478 (100 nmol/L), LY294002 (10 μmol/L), U0126 (10 μmol/L), or an equivalent volume of dimethyl sulfoxide. The number of migrating cells was evaluated fluorometrically and compared with standard curves generated using known numbers of RIE-1 cells, according to the recommendations of the manufacturer. rHB-EGF significantly increased the number of migrating cells after 24 hours compared with control cells. AG1478, LY294002, and U0126 reduced the migration of control cells and HB-EGF-treated cells. All parameters were tested in quadruplicate. Data are presented as mean ± SE (*P < .05 vs control; **P < .05 vs HB-EGF treatment). Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 HB-EGF induces cell migration of RIE-1 cells after scrape wounding in a PI3K/Akt- and ERK1/2-dependent fashion. (A) Time course of RIE-1 wound healing with or without rHB-EGF treatment in vitro. rHB-EGF significantly enhanced the percent wound healing at 4, 8, and 18 hours after wounding. Data are presented as mean ± SE (*P < .05 vs control). (B) Inhibition of ErbB-1 activation using AG1478 significantly reduced both intrinsic and HB-EGF-induced restitution 18 hours after wounding. Pretreatment with LY294002, wortmannin, or U0126 resulted in significant reduction of intrinsic and HB-EGF-induced cell migration. PD98059 inhibited only HB-EGF-induced cell migration. Data are presented as mean ± SE (*P < .05 vs control; **P < .05 vs HB-EGF-treated rats). (C) Examples of the healing after scrape wounding. (1) Untreated cells immediately after wounding. All other panels represent cells 18 hours after wounding as follows: (2) untreated cells, (3) cells treated with HB-EGF (10 ng/mL), (4) cells treated with AG1478 (100 nmol/L), (5) cells treated with AG1478 for 30 minutes followed by rHB-EGF (10 ng/mL), (6) cells treated with LY294002 (10 μmol/L), (7) cells treated with LY294002 for 30 minutes followed by HB-EGF (10 ng/mL), (8) cells treated with wortmannin (100 nmol/L), (9) cells treated with wortmannin (100 nmol/L) followed by HB-EGF (10 ng/mL), (10) cells treated with U0126 (10 μmol/L), (11) cells treated with U0126 (10 μmol/L) for 30 minutes followed by HB-EGF (10 ng/mL), (12) cells treated with PD98059 (100 nmol/L), and (13) cells treated with PD98059 (100 nmol/L) followed by HB-EGF (10 ng/mL). (D) Quantitative cell migration assay. This assay quantitatively assessed cell migration based on the Boyden dual chamber principle, with 8-μm pore size membranes placed between the upper and lower chambers. RIE-1 cells were incubated in the upper chamber at a density of 5 × 105 cells/mL. The lower chamber was either supplemented with rHB-EGF (10 ng/mL) or an equivalent volume of 0.1% bovine serum albumin in phosphate-buffered saline. The upper chamber was supplemented with AG1478 (100 nmol/L), LY294002 (10 μmol/L), U0126 (10 μmol/L), or an equivalent volume of dimethyl sulfoxide. The number of migrating cells was evaluated fluorometrically and compared with standard curves generated using known numbers of RIE-1 cells, according to the recommendations of the manufacturer. rHB-EGF significantly increased the number of migrating cells after 24 hours compared with control cells. AG1478, LY294002, and U0126 reduced the migration of control cells and HB-EGF-treated cells. All parameters were tested in quadruplicate. Data are presented as mean ± SE (*P < .05 vs control; **P < .05 vs HB-EGF treatment). Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 HB-EGF-induced F-actin assembly and lamellipodia formation require PI3K/Akt and MEK/ERK1/2 activation. (A) F-actin was stained with phalloidin-rhodamine 3 hours after scrape wounding and treatment of the cells. (1) Untreated control cells showed intrinsic reorganization of F-actin fibers and early migration beyond the wound margin (dotted line). (2) HB-EGF-treated cells showed significant features of cell polarization, elongated stress fibers, multiple filopodia and lamellipodia (arrows and arrowheads), and advanced migration in the direction of wound healing. LY294002 caused significant retardation of (3) intrinsic and (4) HB-EGF-induced cell polarization, F-actin reorganization, and cell motility. Treatment with U0126 also resulted in significant delay of (5) intrinsic and (6) HB-EGF-induced cell motility and F-actin reorganization. The long arrow in panel 1 indicates direction of wound healing. (B) The average number of lamellipodia per cell projecting into the wound space was quantified 3 hours after wounding in vitro. Treatment with rHB-EGF resulted in a significant increase in the number of lamellipodia per cell, while treatment with LY294002 or U0126 significantly reduced both intrinsic and HB-EGF-induced lamellipodia formation. Data are presented as mean ± SE (*P < .05 vs control; **P < .05 vs HB-EGF-treated rats). Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 HB-EGF induces independent activation of PI3K/Akt and MEK/ERK1/2. (A) Western blotting showing that blocking of PI3K using LY294002 (10 μmol/L) specifically inhibited HB-EGF-induced Akt activation but did not affect HB-EGF-induced ERK activation. Likewise, U0126 blocked HB-EGF-induced ERK1/2 activation but not Akt activation. (B) Simultaneous addition of LY294002 and U0126 resulted in a synergistic inhibitory effect on intrinsic cell migration compared with control or with addition of either inhibitor alone (*P < .01) and on HB-EGF-induced cell migration compared with addition of HB-EGF alone or with addition of HB-EGF plus either inhibitor alone (**P < .001). Inhibition of ErbB-1 activation with AG1478 resulted in a greater inhibitory effect on intrinsic or HB-EGF-induced cell migration compared with all other groups. Data are presented as mean ± SE. Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Endogenous HB-EGF is involved in scrape wound-induced ErbB-1 and ERK1/2 activation and in intrinsic restitution in CaCo-2 cells in vitro. (A) Western blotting of CaCo-2 cells. Scrape wounding of Caco-2 cells resulted in activation of ErbB-1 and ERK1/2 in a time-dependent fashion, which was inhibited by AG1478 (100 nmol/L). Blocking of endogenous HB-EGF using anti-HB-EGF neutralizing antibody (10 μg/mL) or CRM197 (10 μg/mL) significantly reduced wound-induced ErbB-1 and ERK1/2 activation. (B) Intestinal wound healing in CaCo-2 cells. rHB-EGF significantly increased intestinal wound healing in Caco-2 cells compared with untreated cells 18 hours after wounding. ErbB-1 inhibition or blocking of endogenous HB-EGF using either anti-HB-EGF neutralizing antibody (10 μg/mL) or CRM197 (10 μg/mL) resulted in a significant reduction in the rate of spontaneous intestinal wound healing. Data are presented as mean ± SE (*P < .05 compared with untreated cells). Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 10 Summary of the early events after intestinal I/R and the role of HB-EGF. I/R results in tissue damage, loss of gut barrier function, and subsequent development of remote complications, including multiple organ dysfunction syndrome (MODS). At the same time, reperfusion leads to activation of PI3K/Akt and MEK/ERK1/2, which are involved in restitution, leading to tissue recovery and restoration of barrier function. Akt and ERK1/2 may also be involved in activation of prosurvival pathways that enhance mucosal tolerance to reperfusion damage. Induction of endogenous HB-EGF occurs after I/R, which may be involved in activation of PI3K/Akt and MEK/ERK1/2 via activation of ErbB1, and may be sufficient to protect the intestine from subclinical levels of injury but is not sufficient to protect the intestine from more significant degrees of injury. However, administration of rHB-EGF may result in reduction in reperfusion damage, enhanced restitution, and functional recovery of the gut barrier via accelerated production and enhanced activation of Akt and ERK1/2. Gastroenterology 2005 129, 609-625DOI: (10.1053/j.gastro.2005.05.054) Copyright © 2005 American Gastroenterological Association Terms and Conditions