AJP - GI Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 292: G39-G52, 2007. First published August 3, 2006; doi:10.1152/ajpgi.00181.2006
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/G39    most recent
00181.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iizuka, M.
Right arrow Articles by Watanabe, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iizuka, M.
Right arrow Articles by Watanabe, S.

LIVER AND BILIARY TRACT

Morphogenic protein epimorphin protects intestinal epithelial cells from oxidative stress by the activation of EGF receptor and MEK/ERK, PI3 kinase/Akt signals

Masahiro Iizuka,1 Kenji Sasaki,1 Yohei Hirai,2 Kenichi Shindo,1 Shiho Konno,1 Hiroaki Itou,1 Shigetoshi Ohshima,1 Yasuo Horie,1 and Sumio Watanabe1

1Department of Internal Medicine, Akita University School of Medicine, Akita; 2Department of Morphoregulation, Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan

Submitted 30 April 2006 ; accepted in final form 16 July 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Epimorphin is a mesenchymal protein that regulates morphogenesis of epithelial cells. Our preliminary study suggested a novel function of epimorphin in enhancing survival of intestinal epithelial cells (IEC). Oxidative stress leads to cell injury and death and is suggested to be a key contributor to pathogenesis of inflammatory bowel disease. This study was conducted to determine whether epimorphin protects IEC from oxidative stress. Rat intestinal epithelial cell line IEC-6 was cultured with epimorphin (10 and 20 µg/ml), and the life span of IEC was assessed. The mean life span of IEC-6 cells was prolonged 1.9-fold (P < 0.0006) by treatment with epimorphin. We then examined the epimorphin signaling pathways. Epimorphin phosphorylated epidermal growth factor (EGF) receptor, activated the MEK/extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and phosphatidylinositol 3 (PI3) kinase/Akt pathways, phosphorylated Bad, and induced Bcl-XL and survivin. Hydrogen peroxide (1 mM) induced cell death in 92% of IEC-6 cells, but epimorphin dramatically diminished (88.7%) cell death induced by hydrogen peroxide (P < 0.0001). This protective effect of epimorphin was significantly attenuated by inhibitors of MEK and PI3 kinase (P < 0.0001) or EGF receptor-neutralizing antibody (P = 0.0007). In wound assays, the number of migrated cells in the wound area decreased (72.5%) by treatment with 30 µM hydrogen peroxide, but epimorphin increased the number of migrated cells 3.18-fold (P < 0.0001). These results support a novel function of epimorphin in protecting IEC from oxidative stress. This anti-oxidative function of epimorphin is dramatic and is likely mediated by the activation of EGF receptors and the MEK/extracellular signal-regulated kinase and PI3 kinase/Akt signaling pathways and through the induction of anti-apoptotic factors.

epimorphin; oxidative stress; intestinal epithelial cells; epidermal growth factor receptor; extracellular signal-regulated kinase

EPIMORPHIN WAS ORIGINALLY ISOLATED as a mesenchymal protein from mouse embryonic tissues that plays central role in epithelial-mesenchymal interactions (21). The epimorphin protein is composed of 289 amino acids with a hydrophobic stretch at the COOH terminus (21). Previous in vivo and in vitro studies have shown that epimorphin mediates morphogenesis in many tissues including skin, hair follicle, lung, mammary gland, pancreatic duct, and intestinal epithelium (17, 22–24, 29, 32, 40). However, the cognate epimorphin receptors and its signaling pathways have not been identified to date. A similar molecule was subsequently found as syntaxin-2, and members of the syntaxin protein family are involved in the docking and fusion of secretory vesicles with the plasma membrane (39).

It has been shown that oxidative stress leads to cell injury and death, and acceleration in aging and age-related diseases (16). The intestinal epithelium is exposed to various kinds of toxic agents and serves as an essential barrier. Excessive production of oxidants can induce inflammation, injury, damage to the microtubule cytoskeleton, and death in intestinal epithelial cells (IEC), and lead to mucosal barrier dysfunction (5, 27, 36). Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a chronic and refractory enterocolitis. It has been suggested that oxidative stress might be a key contributor to the pathogenesis of IBD (36).

During our laboratory's clinical investigations of the pathogenesis of IBD, we found that the expression and distribution of epimorphin were altered in the colonic mucosa of IBD patients (50). In this context, it was of interest that the expression of epimorphin increases during villous repair in the isografted intestine (20). Increased expression of epimorphin was also found in hepatic stellate cells during liver regeneration (56). In addition, in preliminary experiments, we unexpectedly found an important phenomenon suggesting that epimorphin might increase the viability and prolong the life span of IEC. Based on these findings, we hypothesized that the morphogenic protein epimorphin is also involved in restoration, protection, and survival of IEC. Thus the present study was conducted to elucidate this hypothesis, focusing particularly on the condition of oxidative stress. Here, we demonstrate a novel function of epimorphin to protect IEC from severe oxidative stress and prolong the life span. In addition, we elucidate the intracellular signaling pathways of epimorphin for the first time.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Cell culture. IEC-6, a nontransformed rat intestinal epithelial cell line (43), was purchased from ATCC (Manassas, VA). IEC-6 cells were cultured in DMEM (Invitrogen, Carlsbad, CA) with 5% fetal bovine serum and 5 µg/ml insulin. Cells were used at the 17th to 22nd passage for all experiments. HT-29, a human colon cancer cell line, was also purchased from ATCC and cultured in GIT medium (Wako, Osaka, Japan) with 15% fetal bovine serum.

Assay for viability of IEC-6 cells. Recombinant epimorphin was produced in Escherichia coli BL21 and purified as described previously (24) with a slight modification. The recombinant protein in bacterial cells was dissolved in PBS containing 4 M urea, applied onto Ni-NTA-agarose gel (Qiagen, Valencia, CA), washed with PBS several times, eluted with 200 mM imidazole, and dialyzed against PBS. The purity of thus-obtained recombinant protein was >90% as judged by electrophoresis. We initiated the culture of IEC-6 cells with 500 µl of the medium containing ~105 IEC-6 cells in the absence or presence of recombinant epimorphin (10 or 20 µg/ml) in 24-well dishes. The cultured cells were observed every 1–2 days, and the viability of IEC-6 cells in each medium was carefully assessed. In this study, we defined the life span of the IEC as the interval between the day of cell passage and the day when >90% of IEC-6 cells were dead. Data are expressed as means ± SD.

Western blot. Confluent IEC-6 monolayers grown on six-well dishes maintained for 24 h in serum-deprived DMEM were treated for 5, 15, 30, and 60 min, or 30 min, 60 min, 8 h, and 24 h with 20 µg/ml of recombinant epimorphin. Cells were rinsed in cold phosphate-free medium (Invitrogen) and placed on ice with 300 µl of lysis buffer per well [1% Triton X-100, 150 mM NaCl, 20 mM Tris·HCl, pH 7.5, 2 mM EDTA, containing 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktail, Complete Mini (Roche, Penzberg, Germany)]. Lysates were centrifuged (12,000 g, 10 min at 4°C), and the protein concentration in each supernatant was determined by colorimetric Bradford protein assay (Bio-Rad, Hercules, CA). Proteins from the resulting supernatant (15 µg per lane) were heated (90°C, 3 min), subjected to SDS-PAGE, and then transferred onto a polyvinylidene difluoride membrane (Millipore, Yonezawa, Japan), followed by immunoblotting with polyclonal antibodies specific to p44/42 [extracellular signal-regulated kinase (ERK) 1/2] mitogen-activated protein kinase (MAPK), phosphorylated p44/42 MAPK (Thr202/Tyr204), MEK1/2, phosphorylated MEK1/2 (Ser217/221), phosphatidylinositol 3 (PI3) kinase, Akt, phosphorylated Akt (Ser473), epidermal growth factor (EGF) receptor, phosphorylated EGF receptor (Tyr845, Tyr992), non-phosphorylated Src (Tyr416, Tyr527), phosphorylated Src (Tyr416, Tyr527), phosphorylated p38 MAPK (Thr180/Tyr182), phosphorylated SAPK/JNK (Thr183/Tyr185), phosphorylated 90-kDa ribosomal S6 kinase (90RSK) (Thy359/Ser363), Bad, phosphorylated Bad (Ser112), Bcl-XL (Cell Signaling, Beverly, MA), survivin (Santa Cruz Biotechnology, Santa Cruz, CA), actin (Sigma, St. Louis, MO), EGF (Santa Cruz Biotechnology), and transforming growth factor (TGF)-{alpha} (Santa Cruz Biotechnology). These antibodies were diluted 1:1,000 and used as the first antibody. Positive bands were visualized with the secondary horseradish peroxidase-conjugated antibody (Dako, Copenhagen, Denmark) (1:2,000 dilution) with the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Preparation of hydrogen peroxide. Cell death was induced in IEC by treatment with hydrogen peroxide. Based on the results of preliminary experiments, we determined the concentration of hydrogen peroxide that would induce the maximum degree of cell death in IEC-6 or HT-29 cells. Specifically, we used 1 mM of hydrogen peroxide for IEC-6 cells. However, we used a higher concentration of hydrogen peroxide (100 mM) for HT-29 cells because 1–10 mM of hydrogen peroxide was not sufficient to induce the maximum cell death in these cells. In wound assays, we used 30 µM of hydrogen peroxide since we confirmed in our preliminary experiments that ≥100 µM of hydrogen peroxide was too destructive for cellular wound healing and that 20–30 µM of hydrogen peroxide was an appropriate concentration for suppressing wound repair in IEC-6 cells (46).

Detection of dead cells, apoptotic cells, and necrotic cells. We induced severe cell death in IEC-6 cells by treatment with 1 mM of hydrogen peroxide. The extent of cell death was assessed by measuring the levels of lactate dehydrogenase (LDH) in the culture medium. LDH level in culture medium was measured by colorimetric assay (SRL, Tokyo, Japan) and was expressed as the means ± SD. Dead cells were directly detected by staining with 4',6-diamino-2-phenylindole (DAPI) (Roche) using fluorescence microscopy. Furthermore, dead cells were distinguished as either apoptotic or necrotic cells by staining cells with annexin-V-fluorescein and propidium iodide (Roche), respectively. Namely, annexin-V-fluorescein-positive apoptotic cells were stained green only, and annexin-V-fluorescein and propidium iodide double-positive necrotic cells were stained orange or yellow/green. Dead cells were directly observed using fluorescence microscopy. The number of dead cells found in a standardized area (0.378 mm2) in IEC-6 cells 24 h after treatment with no reagent (control), 1 mM of hydrogen peroxide, 1 mM of hydrogen peroxide + 20 µg/ml of epimorphin, 1 mM of hydrogen peroxide + 20 µg/ml of epimorphin with the recommended concentration (10 µM) of the MEK inhibitor U0126 (Cell Signaling) or PI3 kinase inhibitor LY294002 (50 µM) (Cell Signaling), 1 mM of hydrogen peroxide + 20 µg/ml of epimorphin + 10 µM of MEK inhibitor + 50 µM of PI3 kinase inhibitor, or no reagent was counted, and the percentage of dead cells was calculated. The number of apoptotic cells or necrotic cells in a standardized area after treatment with no reagent (control), 1 mM of hydrogen peroxide, and 1 mM of hydrogen peroxide + 20 µg/ml of epimorphin was counted separately. Data are expressed as means + SD.

To elucidate the link between activation of the EGF receptor and the anti-cell death function of epimorphin, we pretreated HT-29 cells with the anti-human EGF receptor-neutralizing antibody (20 µg/ml) (Clone LA1, Upstate, Lake Placid, NY) for 24 h before exposure to hydrogen peroxide. In this experiment, we used HT-29 cells since the anti-human EGF receptor-neutralizing antibody recognizes only human cells and does not cross-react with rat intestinal cell line IEC-6 cells. We determined the concentration of the anti-human EGF receptor-neutralizing antibody based on the manufacturer's instruction. Preliminary experiments showed that the viability of HT-29 cells did not decrease after 24 h of culture with the antibody at this concentration. We used higher concentration of hydrogen peroxide (100 mM) to induce severe cell death in HT-29 cells. Induction of cell death after treatment with no reagent (control), 100 mM hydrogen peroxide, 100 mM of hydrogen peroxide + 20 µg/ml of epimorphin, and 100 mM of hydrogen peroxide + 20 µg/ml of epimorphin + 20 µg/ml of the anti-human EGF receptor-neutralizing antibody was assessed microscopically by staining with DAPI, and the extent of cell death was also assessed by measuring LDH level in the culture medium. We also assessed the effects of the EGF receptor-neutralizing antibody on the activation of the ERK1/2 MAPK and Akt signaling pathways and the induction of anti-apoptotic proteins by Western blot. Namely, we assessed the activations of the EGF receptor, MEK, ERK, PI3 kinase, Akt, 90RSK, and Bad and the induction of anti-apoptotic proteins (Bcl-XL and survivin) by 20 µg/ml of epimorphin 24 h after pretreatment with 20 µg/ml of the anti-human EGF receptor-neutralizing antibody.

Then, we assessed the effects of Src on anti-oxidative stress function of epimorphin using Src SH2 domain inhibitor (sc-3125, Santa Cruz Biotechnology). First, we assessed the effects of Src SH2 domain inhibitor on the activation of the EGF receptor by epimorphin using Western blot. Next, we assessed the effects of Src SH2 domain inhibitor on anti-cell death function of epimorphin under oxidative stress condition. Namely, the number of dead cells found in a standardized area in IEC-6 cells 24 h after treatment with no reagent (control), 1 mM of hydrogen peroxide, 1 mM of hydrogen peroxide + 20 µg/ml of epimorphin, 1 mM of hydrogen peroxide + 20 µg/ml of epimorphin + the recommended concentration (10–6 or 10–5 M) (18) of Src SH2 domain inhibitor was counted after DAPI-staining, and the percentage of dead cells was calculated.

Calcium ionophores, such as Br-A23187, can induce both necrotic cell death and apoptosis in many cell systems (41). We further used 5 µM Br-A23187 (Sigma) to induce cell death in IEC-6 cells. Induction and suppression of cell death in IEC-6 cells were assessed 50 min and 24 h after treatment with 5 µM Br-A23187 in the absence or presence of 20 µg/ml of epimorphin.

Wound assays. We performed wound assays with IEC-6 cells using a modification of the method described by Sato and Rifkin (11, 35, 48). Confluent monolayers of IEC-6 cells in 30-mm plastic dishes were wounded with a razor blade. Cells were washed with fresh serum-deprived medium, and the wounded monolayers were further cultured for 24 h in fresh serum-deprived medium in the presence of 30 µM H2O2, 30 µM H2O2 + various concentrations of epimorphin (2 µg/ml, 10 µg/ml, and 20 µg/ml), 30 µM H2O2 + 20 µg/ml of epimorphin + 10 µM of the MEK inhibitor U0126 or 50 µM of the PI3 kinase inhibitor LY294002, or no reagent (control). Migration of IEC-6 cells was assessed in a blinded fashion by determining of the number of migrating cells found across the wound border in a standardized wound area (944 µm) using an inverted microscope Olympus IX70 (Olympus, Tokyo, Japan) and DP controller (Olympus). Data are expressed as means + SD.

We also made artificial wounds with a uniform sized cell-free area (~2 mm) by mechanical cell denudation using a pencil-type mixer with a rotating silicon tip (57). Wound repair was monitored 24, 48, 72, and 100 h after treatment with no reagent (control), 30 µM H2O2, 30 µM H2O2 + 20 µg/ml epimorphin.

Bromodeoxyuridine staining. Proliferating cells in the wound areas were assessed 24 h after treatment with no reagent (control), 30 µM H2O2, or 30 µM H2O2 + 20 µg/ml epimorphin by indirect-immunohistochemical methods with anti-bromodeoxyuridine (BrdU) monoclonal antibody using the 5-bromo-2'-deoxy-uridine Labeling and Detection Kit II (Roche). BrdU-positive cells found across the wound border in a standardized wound area were counted, and the percentage of BrdU-positive cells was calculated. Data are expressed as means + SD.

Statistics. Statistical analysis was performed with unpaired Student's t-test and Mann-Whitney U-test [1) the life span of IEC-6 cells, 2) LDH level in the culture medium, 3) the percentage of dead cells, 4) the number of apoptotic or necrotic cells, 5) the number of migrating cells (control vs. H2O2, H2O2 + 20 µg/ml epimorphin vs. H2O2 + 20 µg/ml epimorphin + MEK inhibitor or PI3 kinase inhibitor), 6) the ratio of BrdU-positive cells)] or unpaired Kruskal-Wallis test [the number of migrating cells (H2O2, H2O2+ 2 µg/ml, or 10 µg/ml, or 20 µg/ml epimorphin)], and probability of 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANT
 REFERENCES
 
Epimorphin prolongs the life span of IEC. We first attempted to elucidate whether epimorphin prolongs the life span of IEC using a nontransformed rat intestinal epithelial cell line, IEC-6. As shown in Fig. 1A, the mean life span of IEC-6 cells with no treatment (control) was 49.8 ± 10 days (33–66 days). In contrast, the mean life span of IEC-6 cells treated with 10 or 20 µg/ml of epimorphin was 93.7 ± 11.7 days (73–108 days) or 94.3 ± 11.9 days (78–114 days), respectively. Pictures of typical IEC-6 cells after 40, 52, and 104 days of culture with or without epimorphin are shown in Fig. 1B. Thus we confirmed that the mean life span of IEC-6 cells was significantly prolonged (~1.9-fold) (P < 0.0006) by treatment with epimorphin compared with those without the treatment.


Figure 1
View larger version (70K):
[in this window]
[in a new window]

  		Fig. 1. Epimorphin prolongs the life span of intestinal epithelial cells (IEC)-6. IEC-6 cells were cultured in 24-well dishes with DMEM-containing 5% fetal bovine serum and 5 µg/ml insulin. A: mean life span of IEC-6 cells (means ± SD) by treatment with no reagent (control) and 10 or 20 µg/ml of epimorphin (EPM). B: microscopic pictures of IEC-6 cells (x200) after 40, 52, and 104 days of treatment with 20 µg/ml of EPM (+) or without EPM (–).

 
Epimorphin activates EGF receptor, MEK/ERK1/2 MAPK, and PI3 kinase/Akt signaling pathways, and induces anti-apoptotic proteins in IEC. From these surprising effects of epimorphin on the life span of IEC, we next attempted to examine the signaling pathway of epimorphin in IEC-6 cells by Western blot. Namely, we investigated the activation of signaling proteins that belong to the MAPK and PI3 kinase-Akt pathways, since these signaling pathways have been shown to be closely involved in cell survival (4, 12, 34, 45, 53, 55). As shown in Fig. 2A, ERK1/2 MAPK was clearly activated by treatment with 20 µg/ml of epimorphin (Fig. 2A). However, we failed to find activations of other MAPK such as p38 MAPK and JNK by treatment with epimorphin (data not shown in Fig. 2). We investigated signaling proteins upstream or downstream of ERK1/2 MAPK and found that MEK1/2 and 90 RSK were also activated by treatment with epimorphin, respectively (Fig. 2A). Next, we found that both PI3 kinase and Akt were phosphorylated by treatment with epimorphin (Fig. 2B). Bad, which is downstream of ERK and Akt, was also phosphorylated (Fig. 2B). Then, we finally found that epimorphin phosphorylated EGF receptor on tyrosine residues 845 and 992 (Fig. 2C). Activation of Src was shown to be involved in the activation of EGF receptor at tyrosine 845 (9, 47). However, significant activation of Src (Tyr416) was not detected by treatment with epimorphin (Fig. 2C). On the other hand, it has been shown that the protein tyrosine kinase activity of the cellular Src protein is negatively regulated by phosphorylation at tyrosine residue 527 (Tyr527) (54). As shown in Fig. 2C, phosphorylation of Src (Tyr527) was observed both before and after treatment with epimorphin. Regarding survival proteins downstream of ERK1/2 MAPK and Akt, epimorphin increased the expression of Bcl-XL and survivin (Fig. 2D). In contrast, we could not clearly detect expression of EGF protein and TGF-{alpha} protein in IEC-6 cells before and after treatment with epimorphin by Western blot (not shown in Fig. 2).


Figure 2
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 2. Epimorphin activates epidermal growth factor (EGF) receptor, MEK-ERK1/2 MAPK, and PI3 kinase-Akt signaling pathways and induces anti-apoptotic proteins in IEC. A: results of Western blot of IEC-6 cells after treatment with 20 µg/ml EPM for the MEK-ERK signaling pathway using polyclonal antibodies to ERK1/2 MAPK, phospho ERK1/2 MAPK, MEK1/2, phospho MEK1/2, and phospho 90RSK. B: results of Western blot for the PI3 kinase-Akt pathway using polyclonal antibodies to PI3 kinase, Akt, phospho Akt, Bad, and phospho Bad. C: results of Western blot using polyclonal antibodies to EGF receptor, phospho (Tyr845 or Tyr992) EGF receptor, phospho and non-phospho Src (Tyr416 or Tyr527). D: results of Western blot for the survival proteins using polyclonal antibodies to Bcl-XL, survivin, and actin protein.

 
Epimorphin protects IEC from cell death induced by hydrogen peroxide, but inhibitors of MEK and PI3 kinase suppress the function of epimorphin. We investigated the protective function of epimorphin from injury or death of IEC induced by toxic agent. We used a high concentration of hydrogen peroxide to induce severe cell injury and cell death in IEC, since oxidative stress has been shown to be closely associated with the pathogenesis of IBD (36). As shown in Fig. 3A, LDH level in culture medium increased fourfold following exposure to 1 mM hydrogen peroxide (P < 0.01), which was significantly (64%) attenuated by epimorphin (P < 0.01). As shown in Fig. 3B, treatment with 1 mM hydrogen peroxide induced severe cell death in IEC-6 cells. However, the induction of cell death by hydrogen peroxide was dramatically suppressed by treatment with 20 µg/ml of epimorphin. Actually, as shown in Fig. 3, C and D, 1 mM hydrogen peroxide induced severe cell death in IEC-6 cells (percentage of dead cells: control 2.9%, 1 mM hydrogen peroxide 92.3%) (P < 0.0001), but the percentage of dead cells was significantly diminished by treatment with 20 µg/ml of epimorphin (from 92.3 to 10.4%) (P < 0.0001). We also found that this anti-cell death function of epimorphin was significantly suppressed by treatment with the MEK inhibitor U0126 and the PI3 kinase inhibitor LY294002 (Fig. 3, C and D). Actually, the percentage of dead cells increased 2.1-fold (P = 0.0007), 3.1-fold (P < 0.0001), or 8.16-fold (P < 0.0001) by treatment with both 10 µM of the MEK inhibitor, 50 µM of the PI3 kinase inhibitor, or 10 µM of the MEK inhibitor + 50 µM of the PI3 kinase inhibitor, respectively (Fig. 3C).


Figure 3
Figure 3
View larger version (178K):
[in this window]
[in a new window]

  		Fig. 3. Epimorphin protects IEC from cell death induced by hydrogen peroxide, but inhibitors of MEK and PI3 kinase suppress the function of epimorphin. A: LDH level (means ± SD) in culture medium 24 h after treatment of IEC-6 cells with no reagent (control), 1 mM H2O2, and 1 mM H2O2 + 20 µg/ml of EPM. B: IEC-6 cells 24 h after treatment with no reagent (control), 1 mM H2O2, and 1 mM H2O2 + 20 µg/ml EPM (x200) (top). Pictures at bottom are 4',6-diamino-2-phenylindole (DAPI)-stained IEC-6 cells 24 h after treatment with no reagent (control), 1 mM H2O2, and 1 mM H2O2 + 20 µg/ml EPM. C: percentage of dead cells (means + SD) in IEC-6 cells 24 h after treatment with no reagent (control) (open column), 1 mM of H2O2 with (light shaded column) or without (open column) 20 µg/ml EPM, 1 mM H2O2 + 20 µg/ml EPM + 10 µM of the MEK inhibitor (dark shaded column), 1 mM of H2O2 + 20 µg/ml EPM + 50 µM of the PI3 kinase inhibitor (dark shaded column), and 1 mM of H2O2 + 20 µg/ml EPM + 10 µM of the MEK inhibitor + 50 µM of the PI3 kinase inhibitor (filled column). D: DAPI-stained pictures of IEC-6 cells 24 h after treatment with no reagent (control), 1 mM H2O2 with or without 20 µg/ml EPM, 1 mM of H2O2 + 20 µg/ml EPM + 10 µM of the MEK inhibitor, 1 mM H2O2 + 20 µg/ml EPM + 50 µM of the PI3 kinase inhibitor, and 1 mM H2O2 + 20 µg/ml EPM + 10 µM of the MEK inhibitor + 50 µM of the PI3 kinase inhibitor.

 
Epimorphin attenuates the induction of apoptosis and necrosis by hydrogen peroxide. As shown in Fig. 4, A and B, 1 mM hydrogen peroxide significantly increased both apoptotic cells (3.53-fold) and necrotic IEC-6 cells (19.6-fold) compared with control (P < 0.0001). Dead cells induced by 1 mM hydrogen peroxide in IEC-6 cells were comprised of 22% apoptotic cells (stained by annexin-V fluos as green) and 78% necrotic cells (stained by both annexin-V fluos and propidium iodine as orange or yellow/green). On the other hand, epimorphin significantly suppressed the induction of both apoptosis and necrosis by hydrogen peroxide (P < 0.0001). Actually, epimorphin decreased 67.3% of apoptotic cells or 79.3% of necrotic cells induced by hydrogen peroxide, respectively.


Figure 4
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 4. Epimorphin attenuates the induction of apoptosis and necrosis by hydrogen peroxide. A: the number of apoptotic cells (shaded column) (mean + SD/0.04 mm2) or necrotic cells (filled column) (mean + SD) in IEC-6 cells 24 h after treatment with no reagent (control), 1 mM H2O2, and 1 mM H2O2 + 20 µg/ml EPM. B: pictures of IEC-6 cells immunofluorescently stained with annexin-V-fluorescein and propidium iodide 24 h after treatment with no reagent (control), 1 mM H2O2, and 1 mM H2O2 + 20 µg/ml EPM (x200).

 
The EGF receptor-neutralizing antibody inhibits the anti-cell death function of epimorphin. To elucidate the link between the activation of EGF receptor and the anti-cell death function of epimorphin, we pretreated human colon cancer cell line HT-29 cells for 24 h with 20 µg/ml of the anti-human EGF receptor-neutralizing antibody (clone LA1, Upstate) and examined the anti-cell death function of epimorphin in the presence of hydrogen peroxide. In this experiment, we used HT-29 cells since the anti-human EGF receptor-neutralizing antibody recognizes only human cells and does not cross-react with rat intestinal cell line IEC-6 cells. As shown in Fig. 5A, 100 mM hydrogen peroxide induced severe cell death in HT-29 cells, but epimorphin dramatically suppressed the induction of cell death by hydrogen peroxide. However, pretreatment with the anti-human EGF receptor-neutralizing antibody abrogated the protective effect of epimorphin against cell death. Actually, more than 50% of epithelial cells were detached from the dishes by treatment with 100 mM hydrogen peroxide or 100 mM hydrogen peroxide + epimorphin following pretreatment with the anti-human EGF receptor-neutralizing antibody (Fig. 5A). The percentage of dead cells (mean ± SD) to total cells adhered to dishes after treatment with no reagent (control), 100 mM hydrogen peroxide, 100 mM hydrogen peroxide + epimorphin, or 100 mM hydrogen peroxide + epimorphin following pretreatment with the anti-human EGF receptor-neutralizing antibody was 2.0 ± 0.34, 83.3 ± 3.9, 13.4 ± 3.6, or 53.3 ± 8.5, respectively (Fig. 5B). Specifically, the anti-human EGF receptor-neutralizing antibody clearly suppressed the anti-cell death function of epimorphin (P = 0.0007). Similarly, as shown in Fig. 5C, LDH level in culture medium of HT-29 cells also increased dramatically (17.9-fold) by treatment with 100 mM hydrogen peroxide, but this increase was significantly suppressed (–86.3%) by epimorphin. However, pretreatment with the anti-human EGF receptor-neutralizing antibody restored the increase in LDH (5.0-fold) (Fig. 5C). Results of Western blot regarding the activation of EGF receptor, PI3 kinase-Akt and MEK-ERK signaling pathways, and the induction of the anti-apoptotic proteins by epimorphin after pretreatment with the EGF receptor-neutralizing antibody are shown in Fig. 5D. As shown in Fig. 5D, the activations of the signaling pathways including EGF receptor (Tyr845 and Tyr992), PI3 kinase, Akt, MEK, ERK1/2 MAPK, 90RSK, and Bad were significantly, but not completely, suppressed by treatment with the EGF receptor-neutralizing antibody. The induction of apoptotic proteins (Bcl-XL and survivin) was also significantly suppressed. These results suggest that epimorphin protects IEC from cell death caused by oxidative stress, probably through the activation of EGF receptor.


Figure 5
Figure 5
View larger version (85K):
[in this window]
[in a new window]

  		Fig. 5. The EGF receptor-neutralizing antibody inhibits the anti-cell death function of epimorphin. A: HT-29 cells 24 h after treatment with no reagent (control), 100 mM H2O2, 100 mM H2O2 + 20 µg/ml EPM, 100 mM H2O2 + 20 µg/ml EPM following for 24 h pretreatment with 20 µg/ml of the anti-human EGF receptor-neutralizing antibody (EGFR-Ab) (x200) (top). Bottom: DAPI-stained HT-29 cells 24 h after treatment with no reagent (control), 100 mM H2O2, 100 mM H2O2 + 20 µg/ml EPM, 100 mM H2O2 + 20 µg/ml EPM following for 24 h pretreatment with 20 µg/ml of the anti-human EGF receptor neutralizing antibody. B: percentage of dead cells to adhered epithelial cells (means + SD) 24 h after treatment of HT-29 cells with no reagent (control) (open column), 100 mM H2O2 (open column), 100 mM H2O2 + 20 µg/ml EPM (shaded column), 100 mM H2O2 + 20 µg/ml EPM following for 24 h pretreatment with 20 µg/ml of the anti-human EGF receptor-neutralizing antibody (EGFR Ab) (dark shaded column). C: LDH level (mean ± SD) in culture medium 24 h after treatment of HT-29 cells with no reagent (control), 100 mM H2O2, 100 mM of H2O2 + 20 µg/ml EPM, and 100 mM H2O2 + 20 µg/ml EPM followed by a 24-h pretreatment with 20 µg/ml of the anti-human EGF receptor-neutralizing antibody (EGFR-Ab). D: results of Western blot of HT-29 cells pretreated for 24 h with 20 µg/ml of the anti-human EGF receptor-neutralizing antibody to examine the activation of the EGF receptor-Akt/-ERK signaling pathways and the induction of the anti-apoptotic proteins by treatment with 20 µg/ml EPM. Western bolt was performed with polyclonal antibodies to EGF receptor, phospsho (Tyr845 or Tyr992) EGF receptor, PI3 kinase, Akt, phospho Akt, phospho MEK1/2, ERK1/2 MAPK, phospho ERK1/2 MAPK, phospho 90RSK, Bad, phospho Bad, Bcl-XL, survivin, and actin protein.

 
The Src SH2 domain inhibitor does not attenuate the anti-oxidative stress function of epimorphin. Results of Western blot regarding the activation of EGF receptor by treatment with epimorphin and Src SH2 domain inhibitor are shown in Fig. 6A. As shown in Fig. 6A, phosphorylation of both Tyr845 and Tyr992 sites of EGF receptor by epimorphin was not clearly suppressed by cotreatment with Src SH2 domain inhibitor. The percentage of dead cells (mean ± SD) 24 h after treatment with no reagent (control), 1 mM hydrogen peroxide, 1 mM hydrogen peroxide + 20 µg/ml of epimorphin, 1 mM hydrogen peroxide + 20 µg/ml of epimorphin + 10–6 M Src SH2 domain inhibitor, or 1 mM hydrogen peroxide + 20 µg/ml of epimorphin + 10–5 M Src SH2 domain inhibitor was 2.78 ± 0.82, 93.4 ± 4.13, 12.6 ± 2.54, 12.4 ± 3.19, or 13.2 ± 1.65, respectively (Fig. 6, B and C). Namely, the Src SH2 domain inhibitor did not attenuate the anti-cell death function of epimorphin under oxidative stress condition.


Figure 6
View larger version (51K):
[in this window]
[in a new window]

  		Fig. 6. The Src SH2 domain inhibitor does not attenuate the anti-oxidative stress function of EPM. A: results of Western blot of IEC-6 cells after treatment with 20 µg/ml EPM + 10–5 M of Src SH2 domain inhibitor using polyclonal antibodies to EGF receptor and phospsho (Tyr845 or Tyr992) EGF receptor. B: percentage of dead cells (means + SD) 24 h after treatment with no reagent (control), 1 mM hydrogen peroxide, 1 mM hydrogen peroxide + 20 µg/ml EPM, 1 mM hydrogen peroxide + 20 µg/ml EPM + 10–6 M of Src SH2 domain inhibitor, or 1 mM hydrogen peroxide + 20 µg/ml EPM + 10–5 M of Src SH2 domain inhibitor. C: DAPI-stained pictures of IEC-6 cells 24 h after treatment with no reagent (control), 1 mM hydrogen peroxide, 1 mM hydrogen peroxide + 20 µg/ml EPM, 1 mM of hydrogen peroxide + 20 µg/ml of EPM + 10–5 M of Src SH2 domain inhibitor.

 
Epimorphin protects IEC-6 cells from cell death induced by Br-A23187. Calcium ionophores such as Br-A23187 induce both necrotic cell death and apoptosis in many cell systems (41). In this study, we also used Br-A23187, and cell death was induced in IEC-6 cells. As shown in Fig. 7, epimorphin clearly protected IEC-6 cells from cell death induced 50 min after treatment with 5 µM Br-A23187. Actually, cell death was induced in 64.4% of IEC-6 cells 50 min after treatment with 5 µM Br-A23187, but the percentage of cell death was reduced to 22.1% by co-culture with 20 µg/ml epimorphin (P < 0.001). However, epimorphin did not significantly protect IEC-6 cells from cell death after 24 h of treatment with Br-A23187 (data not shown).


Figure 7
View larger version (146K):
[in this window]
[in a new window]

  		Fig. 7. Epimorphin protects IEC-6 cells from cell death induced by Br-A23187. Top: IEC-6 cells 50 min after treatment with 5 µM Br-A23187 and 5 µM Br-A23187 + 20 µg/ml EPM (x200). Bottom: same IEC-6 cells as in top stained with DAPI.

 
Epimorphin prevents the delay of wound repair in IEC-6 cells caused by hydrogen peroxide. It has been shown that oxidative stress also suppresses wound repair (5, 46, 57). We finally focused on the effects of epimorphin on wound repair of IEC exposed to oxidative stress. We performed wound assays with IEC-6 cells using a modification of the method described by Sato and Rifkin (11, 35, 48). In wound assays, we used 30 µM of hydrogen peroxide since we confirmed in our preliminary experiments that high concentration (>100 µM) of hydrogen peroxide was too destructive for cellular wound healing and 20–30 µM hydrogen peroxide was an appropriate concentration for suppressing wound repair in IEC-6 cells (46).

As shown in Fig. 8, A and B, the number of epithelial cells migrating across the wound border decreased significantly (72.5%) after 24 h of exposure to 30 µM hydrogen peroxide compared with control (P < 0.0001). However, epimorphin prevented the decrease in the number of migrating epithelial cells caused by hydrogen peroxide in a concentration-dependent manner (P < 0.0001). Actually, the number of migrating epithelial cells increased 3.18-fold after treatment with 20 µg/ml epimorphin compared with those treated with only 30 µM hydrogen peroxide. On the other hand, co-culture with 10 µM of the MEK inhibitor or 50 µM of the PI3 kinase inhibitor decreased the number of migrating epithelial cells 62.2 or 65.7% compared with that with epimorphin (P < 0.0001). Thus each of these inhibitors suppressed the function of epimorphin (Fig. 8, A and B). We also used a model of artificial wounds by mechanical cell denudation using a rotating silicon tip (Fig. 8C), as described before (57). In control, the size of the cell-free area decreased gradually and completely closed 72 h after wound formation. On the other hand, wound repair was significantly disturbed by exposure to 30 µM hydrogen peroxide, and the cell-free area was not closed even 100 h after wound formation. However, the delay in wound repair caused by hydrogen peroxide was clearly prevented by treatment with 20 µg/ml epimorphin, and the cell-free area was almost closed 100 h after wound formation. Actually, 44.3, 15.0, or 30.3% of the cell-free area was repaired with epithelial cells 24 h after treatment with no reagent (control), 30 µM hydrogen peroxide, or 30 µM hydrogen peroxide + 20 µg/ml epimorphin, and 100, 58.3, or 95.4% of the cell-free area was repaired 72 h after treatment with no reagent (control), 30 µM hydrogen peroxide, or 30 µM hydrogen peroxide + 20 µg/ml epimorphin, respectively. Morphologically, most IEC near the wound margin were swollen, and cell integrity appeared to be loosened 72 h after exposure to hydrogen peroxide (Fig. 8D, left). In contrast, epimorphin clearly suppressed these morphological changes of IEC caused by hydrogen peroxide (Fig. 8D, right). The percentage of BrdU-positive proliferating epithelial cells in the wound area decreased following exposure to 30 µM hydrogen peroxide, which was abrogated by epimorphin (Fig. 8E). These results indicate that epimorphin prevents the delay of wound repair caused by hydrogen peroxide by enhancing both restitution and proliferation of IEC, probably through the activation of MEK-ERK and PI3 kinase-Akt signaling pathways.


Figure 8
Figure 8
View larger version (112K):
[in this window]
[in a new window]

  		Fig. 8. EPM prevents the delay of wound repair in IEC-6 cells caused by hydrogen peroxide. A: number of migrating cells (means + SD) across the wound border 24 h after treatment with no reagent (open column), 30 µM H2O2 (open column), 30 µM H2O2 + 2, 10, or 20 µg/ml EPM (light shaded columns), 30 µM H2O2 + 20 µg/ml EPM + 10 µM of the MEK inhibitor (dark shaded column), and 30 µM H2O2 + 20 µg/ml EPM + 50 µM of the PI3 kinase inhibitor (filled column). B: pictures of wounded IEC-6 cells 24 h after treatment with no reagent (control), 30 µM H2O2, 30 µM H2O2 + 20 µg/ml EPM, 30 µM H2O2 + 20 µg/ml EPM + 10 µM of the MEK inhibitor, and 30 µM H2O2 + 20 µg/ml EPM + 50 µM of the PI3 kinase inhibitor (x100). Wound lines are shown with arrows. C: pictures of IEC-6 cells wounded by mechanical cell denudation, 0, 24, 48, 72, and 100 h after treatment with no reagent (control) (top), 30 µM H2O2 (middle), 30 µM of H2O2 + 20 µg/ml of EPM (bottom) (x40). D: pictures of the wound margin of IEC-6 cells 72 h after treatment with 30 µM H2O2 (left) and 30 µM H2O2 + 20 µg/ml EPM (right) (x200). E: proliferating cells in wound areas were assessed by immunohistochemically staining with anti-bromodeoxyuridine (BrdU) monoclonal antibody. The percentage of BrdU-positive cells to all migrated cells in the wound area (means + SD) 24 h after treatment with no reagent (control) (open column), 30 µM H2O2 (open column), and 30 µM H2O2 + 20 µg/ml EPM (shaded column) is shown.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANT
 REFERENCES
 
Epimorphin was first identified by our co-author Dr. Hirai, as a mesenchymal protein that regulates morphogenesis of epithelial cells (20). Thereafter, the successive investigations have shown that this protein is involved in the epithelial morphogenesis of various organs (17, 22–24, 29, 32, 40). Although it has been shown that 19-amino acid motif (NL-peptide) within the cell-binding domain of epimorphin mediates binding of cells to epimorphin (29), the cognate epimorphin receptors and its signaling pathways have not been identified to date. In the present study, we demonstrated a new function of epimorphin protecting IEC from oxidative stress and prolonging the life span of IEC. In addition, we first elucidate the hypothetical intracellular signaling pathways of epimorphin. Specifically, epimorphin phosphorylates EGF receptor, activates both the MEK-ERK1/2 MAPK and PI3 kinase-Akt pathways, phosphorylates 90RSK and Bad, and finally induces cell survival proteins such as Bcl-XL and survivin (Fig. 9). It has been shown that EGF receptor regulates activations of MEK and PI3 kinase (26) and that activations of ERK and Akt can induce phosphorylation of Bad and disassociation of Bad from Bcl-XL (4, 12, 13, 45, 49) and upregulate survivin expression (3, 25). In our study, we also found that the EGF receptor-neutralizing antibody significantly attenuated the activations of the PI3 kinase-Akt and MEK-ERK signaling pathways and the induction of Bcl-XL and survivin. Thus we suggest that the activation of EGF receptor by epimorphin directly links the activations of PI3 kinase-Akt and MEK-ERK pathways and the induction of anti-apoptotic proteins. In these signaling pathways of epimorphin, it is critical but unclear whether epimorphin directly or indirectly phosphorylates EGF receptor. Previous in vitro and in vivo studies showed that Src phosphorylates the EGF receptor on Tyr 845 (9, 47). Another possibility was also suggested that epimorphin might induce ligands of EGF receptor, EGF or TGF-{alpha}, and indirectly phosphorylate EGF receptor. However, we failed to demonstrate clear activation of Src (Tyr416) and induction of EGF or TGF-{alpha} in IEC-6 cells by treatment with epimorphin. In contrast, phosphorylation at Src (Tyr 527), which negatively regulates activity of the cellular Src protein (18), was constantly observed before and after the treatment with epimorphin. Furthermore, the Src SH2 domain inhibitor did not suppress the activation of EGF receptor by epimorphin and the anti-oxidative stress function of epimorphin. On the other hand, we confirmed that epimorphin phosphorylates not only EGF receptor on Tyr 845 but also on Tyr 992. Based on these results, we suggest that Src was not activated by epimorphin and was not involved in the phosphorylation of EGF receptor and the anti-oxidative stress function of epimorphin. Although we did not demonstrate conclusively that epimorphin binds EGF receptor directly, these findings suggest the possibility that epimorphin might directly phosphorylate EGF receptor.


Figure 9
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 9. Schema of the hypothetic signaling pathways of epimorphin and mechanism of the anti-oxidative stress function in IEC.

 
It has been shown that oxidative stress leads to cell injury and cell death (16). Oxidative stress can inhibit calcium pumps in the endoplasmic reticulum and activate calcium release pathways with the consequence that calcium dumps into the cytosol from the endoplasmic reticulum to be taken up by mitochondria (33, 37, 52). Such uptake may induce a calcium-dependent mitochondrial permeability transition and cell death (33). The MEK-ERK and PI3 kinase-Akt pathways have been shown to be involved in cell survival under various conditions, including oxidative stress (4, 8, 12, 16, 44, 45, 53, 55), although conflicting findings have also been reported (6, 31, 51). Concerning the mechanisms of cell survival through these signaling pathways, the following findings have been shown. Activation of ERK induces phosphorylation of the proapoptotic protein Bad through the activation of RSK (4, 45). Activation of Akt also directly induces phosphorylation of Bad (12, 49). Phosphorylation of Bad may alter the affinity of Bad for Bcl-XL and promote dissociation of Bad from Bcl-XL, allowing for protection of cells from cell death (26). Recent studies have shown that Bcl-XL is involved in the inhibition of loss of mitochondrial membrane potential and mitochodrial membrane permeabilization (28). On the other hand, it has been shown that both activations of ERK and PI3/Akt signaling pathways upregulate the anti-apoptotic protein survivin (13, 25). Survivin contains a single baculovirus inhibitor of apoptotic proteins genes (2) and plays a role in antagonizing mitochondria-dependent apoptosis (1). In this study, we demonstrated that a high concentration of hydrogen peroxide induced severe cell death in IEC and that epimorphin dramatically suppressed cell death, including apoptosis and necrosis, induced by hydrogen peroxide. However, this anti-cell death function of epimorphin was clearly suppressed by treatment with the MEK inhibitor, the PI3 kinase inhibitor, and the EGF receptor-neutralizing antibody. Br-A23187 is a calcium ionophore that has been shown to increase mitochondrial free calcium and collapse mitochondrial membrane potential, and thus induces mitochondrial permeability transition and cell death (41). Our study also showed that epimorphin temporarily suppressed cell death of IEC induced by Br-A23187. These results suggest that activations of the EGF receptor-MEK/ERK and -PI3 kinase/Akt signaling pathways and the subsequent induction of survival factors are closely involved in the function of epimorphin to protect IEC from cell death induced by oxidative stress or mitochondria-dependent cell death (Fig. 8).

Excessive production of oxidants can induce damage of the microtubule cytoskeleton in IEC and leads to mucosal barrier dysfunction (5, 27, 36). Microtubules regulate cell morphology, cell migration, and polarity, and disruption of the microtubules by oxidative stress can limit cell function and inhibit cell restoration after wounding (5, 57). In our laboratory's recent (46) and current investigations, we demonstrated that oxidative stress induced by hydrogen peroxide strongly suppresses wound repair in IEC-6 cells. In contrast, our present study clearly demonstrated for the first time that epimorphin significantly prevents the delay of wound repair induced by hydrogen peroxide. Epimorphin increases not only the number of migrating cells but also the ratio of BrdU-positive proliferating cells in wound areas, both of which are significantly decreased by exposure to hydrogen peroxide. In addition, epimorphin prevents the morphological changes of IEC-6 cells induced by hydrogen peroxide. These results suggest that hydrogen peroxide diminishes both restitution and proliferation of IEC, but epimorphin recovers both functions of IEC. Based on the results of the wound assays and the proliferation assays, we suggest that enhancement of cell crawling is more closely involved in the function of epimorphin for wound healing than an enhancement of cell proliferation. It has been shown that both the MEK/ERK and PI3 kinase/Akt pathways are involved in the cell proliferation and migration of various types of cells (14, 15, 19, 42). Basuroy et al. (7) showed that EGF-activated ERK MAPK activity interrupts the mechanism involved in hydrogen peroxide-induced disruption of the actin cytoskeleton and the loss of interaction between tight junction proteins and the actin cytoskeleton in IEC. In this study, we demonstrated that treatment with each of the inhibitors of MEK or PI3 kinase clearly suppressed the function of epimorphin to prevent the delay of wound repair in IEC. These results suggest that the activation of each of the MEK-ERK or PI3 kinase-Akt signaling pathways is also involved in the function of epimorphin to prevent the delay of wound repair induced by hydrogen peroxide.

With regard to the function of epimorphin to prolong the life span of IEC, it has been shown that oxidants are important in the development of the senescent phenotype (16). In addition, previous studies with human diploid fibroblasts revealed that cells grown in low oxygen tension exhibit a prolonged life span (16, 38). Thus we suggest that an anti-oxidative stress mechanism is also involved in the function of epimorphin to prolong the life span of IEC-6 cells.

In conclusion, our study indicates that the morphogenic protein epimorphin also exhibits an anti-oxidative stress function and protects IEC from severe oxidative stress, suggesting the importance of mesenchymal-epithelial interactions for protection of IEC. The anti-oxidative stress function of epimorphin is dramatic and strong. In addition, it is of note that the signaling pathways of epimorphin were demonstrated for the first time in this study. Recent studies suggested that the EGF receptor-MAPK and PI3 kinase-Akt pathways are involved in morphogenesis (10, 30). Thus it is an intriguing question as to whether these signaling pathways are also involved in the morphogenic function of epimorphin. It has been suggested that oxidative stress is a key contributor to the pathogenesis of IBD (5, 27, 36). Thus we believe that epimorphin might shed light on a potential therapeutic strategy for oxidative stress-induced enterocolitis, such as IBD, in the near future.


   GRANT
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANT
REFERENCES
 
This work was supported in part by a grant from Grant-In-Aid for Scientific Research from Japan Society for the Promotion of Science.


   ACKNOWLEDGMENTS
 
We thank Takako Sasaki and Yuki Watanabe for technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Iizuka, Dept. of Internal Medicine, Akita Univ. School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan (e-mail: maiizuka{at}doc.med.akita-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 

  1. Altieri DC. Validating surviving as a cancer therapeutic target. Nat Rev Cancer 3: 46–54, 2003.[CrossRef][Web of Science][Medline]
  2. Ambrosini G, Adida C, and Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 3: 917–912 1997.[CrossRef][Web of Science][Medline]
  3. Asanuma H, Torigoe T, Kamiguchi K, Hirohashi Y, Ohmura T, Hirata K, Sato M, and Sato N. Survivin expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer cells. Cancer Res 65: 11018–11025, 2005.[Abstract/Free Full Text]
  4. Ballif BA and Blenis J. Molecular mechanisms mediating mammalian mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK cell survival signals. Cell Growth Differ 12: 397–408, 2001.[Free Full Text]
  5. Banan A, Choudhary S, Zhang Y, Fields JZ, and Keshavarzian A. Oxidant-induced intestinal barrier disruption and its prevention by growth factors in a human colonic cell line: role of the microtubule cytoskeleton. Free Radic Biol Med 28: 727–738, 2000.[CrossRef][Web of Science][Medline]
  6. Basu A and Tu H. Activation of ERK during DNA damage-induced apoptosis involves protein kinase Cdelta. Biochem Biophys Res Commun 334: 1068–1073, 2005.[CrossRef][Web of Science][Medline]
  7. Basuroy S, Seth A, Elias B, Naren A, and Rao R. MAP kinase interacts with occludin and mediates EGF-induced prevention of tight junction disruption by hydrogen peroxide. Biochem J 393: 69–77, 2006.[CrossRef][Web of Science][Medline]
  8. Bhattacharya S, Ray RM, and Johnson LR. Prevention of TNF-{alpha}-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2. Am J Physiol Gastrointest Liver Physiol 286: G479–G490, 2004.[Abstract/Free Full Text]
  9. Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, and Parsons SJ. cSrc-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr101 is associated with modulation of receptor function. J Biol Chem 274: 8335–8343, 1999.[Abstract/Free Full Text]
  10. Cabernard C and Affolter M. Distinct roles for two receptor tyrosine kinases in epithelial branching morphogenesis in Drosophila. Dive Cell 9: 831–842, 2005.
  11. Ciacci C, Lind SE, and Podolsky DK. Tranforming growth factor beta regulation of migration in wounded rat intestinal epithelial monolayers. Gastroenterology 105: 93–101, 1993.[Web of Science][Medline]
  12. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, and Greenberg ME. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91: 231–241, 1997.[CrossRef][Web of Science][Medline]
  13. Del Peso L, Gonzalez-Garcia M, Page C, Herrera R, and Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278: 687–689, 1997.[Abstract/Free Full Text]
  14. El-Assal ON and Besner GE. HB-EGF enhances restitution after intestinal ischemia/reperfusion via PI3K/AKT and MEK/ERK1/2 activation. Gastroenterology 129: 609–625, 2005.[CrossRef][Web of Science][Medline]
  15. Etscheid M, Beer N, and Dodt J. The hyaluronan-binding protease upregulates ERK1/2 and PI3K/Akt signaling pathways in fibroblasts and stimulates cell proliferation and migration. Cell Signal 17: 1486–1494, 2005.[CrossRef][Web of Science][Medline]
  16. Finkel T and Holbrook NJ. Oxidants, oxidative stress and the biology of aging. Nature 408: 239–247, 2000.[CrossRef][Medline]
  17. Fritsch C, Swietlicki EA, Lefebvre O, Kedinger M, Iordanov H, Levin MS, and Rubin DC. Epimorphin expression in intestinal myofibroblasts induces epithelial morphogenesis. J Clin Invest 110: 1629–1641, 2002.[CrossRef][Web of Science][Medline]
  18. Gilmer T, Rodriguez M, Jordan S, Crosby R, Alligood K, Green M, Kimery M, Wagner C, Kinder D, Charifson P, Hassell AM, Willard D, Luther M, Rusnak D, Sternbach DD, Mehrotra M, Peel M, Shampine L, Davis R, Robbins J, Patel IR, Kassel D, Burkhart W, Moyer M, Bradshaw T, and Berman J. Peptide inhibitors of src SH3-SH2-phosphoprotein interactions. J Biol Chem 269: 31711–31719, 1994.[Abstract/Free Full Text]
  19. Göke M, Kanai M, Lynch-Devaney K, and Podolsky DK. Rapid mitogen-activated protein kinase activation by transforming growth factor {alpha} in wounded rat intestinal epithelial cells. Gastroenterology 114: 697–705, 1998.[CrossRef][Web of Science][Medline]
  20. Goyal A, Singh R, Swietlicki EA, Levin MS, and Rubin DC. Characterization of rat epimorphin/syntaxin 2 expression suggests a role in crypt-villus morphogenesis. Am J Physiol Gastrointest Liver Physiol 275: G114–G124, 1998.[Abstract/Free Full Text]
  21. Hirai Y, Takabe K, Takashina M, Kobayashi S, and Takeichi M. Epimorphin: a mesenchymal protein essential for epithelial morphogenesis. Cell 69: 471–481, 1992.[CrossRef][Web of Science][Medline]
  22. Hirai Y, Nakagawa S, and Takeichi M. Reexamination of the properties of epimorphin and its possible roles. Cell 73: 426–427, 1993.[CrossRef][Web of Science][Medline]
  23. Hirai Y, Lochter A, Galosy S, Koshida S, Niwa S, and Bissell MJ. Epimorphin functions as a key morphoregulator for mammary epithelial cells. J Cell Biol 140: 159–169, 1998.[Abstract/Free Full Text]
  24. Hirai Y, Radisky D, Boudreau R, Simian M, Stevens ME, Oka Y, Takebe K, Niwa S, and Bissell MJ. Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta. J Cell Biol 153: 785–794, 2001.[Abstract/Free Full Text]
  25. Hosseinimehr SJ, Inanami O, Hamasu T, Takahashi M, Kashiwakura I, Asanuma T, and Kuwabara M. Activation of c-kit by stem cell factor induces radioresistance to apoptosis through ERK-dependent expression of survivin in HL60 cells. J Radiat Res (Tokyo) 45: 557–561, 2004.[Medline]
  26. Jorissen RN, Walker F, Pouliot N, Garrett TPJ, Ward CW, and Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signaling. Exp Cell Res 284: 31–53, 2003.[CrossRef][Web of Science][Medline]
  27. Keshavarzian A, Banan A, Farhadi A, Komanduri S, Mutlu E, Zhang Y, and Fields JZ. Increases in free radicals and cytoskeletal protein oxidation and nitration in the colon of patients with inflammatory bowel disease. Gut 52: 720–728, 2003.[Abstract/Free Full Text]
  28. Kim R. Unknotting the roles of Bcl-2 and Bcl-xL in cell death. Biochem Biophys Res Commun 333: 336–343, 2005.[CrossRef][Web of Science][Medline]
  29. Koshiba S and Hirai Y. Identification of cellular recognition sequence of epimorphin and critical role of cell/epimorphin interaction in lung branching morphogenesis. Biochem Biophys Res Commun 234: 522–525, 1997.[CrossRef][Web of Science][Medline]
  30. Kumar V, Zhang MX, Swank MW, Kunz J, and Wu GY. Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways. J Neurosci 25: 11288–11299, 2005.[Abstract/Free Full Text]
  31. Lee WC, Choi CH, Cha SH, Oh HL, and Kim YK. Role of ERK in hydrogen peroxide-induced cell death of human glioma cells. Neurochem Res 30: 263–270, 2005.[CrossRef][Web of Science][Medline]
  32. Lehnert L, Lerch MM, Hirai Y, Kruse ML, Schmiegel W, and Kalthoff H. Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro. J Cell Biol 152: 911–921, 2001.[Abstract/Free Full Text]
  33. Lemasters JJ. Dying a thousand deaths: redundant pathways from different organelles to apoptosis and necrosis. Gastroenterology 129: 351–360, 2005.[CrossRef][Web of Science]
  34. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, and Altieri DC. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396: 580–584, 1998.[CrossRef][Medline]
  35. MacCormack SA, Viar MJ, and Johnson LR. Migration of IEC-6 cells: a model for mucosal healing. Am J Physiol Gastrointest Liver Physiol 263: G426–G435, 1992.[Abstract/Free Full Text]
  36. McKenzie SJ, Baker MS, Buffinton GD, and Doe WF. Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease. J Clin Invest 98: 136–141, 1996.[Web of Science][Medline]
  37. Orrenius S, Zhivotovsky B, and Nicotera P. Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4: 552–565, 2003.[CrossRef][Web of Science][Medline]
  38. Packer L and Fuehr K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 267: 423–425, 1977.[CrossRef][Medline]
  39. Pelham HRB. Is epimorphin involved in vesicular transport? Cell 73: 425–426, 1993.[CrossRef][Web of Science][Medline]
  40. Plateroti M, Rubin DC, Duluc I, Singh R, Foltzer-Jourdainne C, Freund JN, and Kedinger M. Subepithelial fibroblast cell lines from different levels of gut axis display regional characteristics. Am J Physiol Gastrointest Liver Physiol 274: G945–G954, 1998.[Abstract/Free Full Text]
  41. Qian T, Herman B, and Lemasters JJ. The mitochondrial permeability transition mediates both necrotic and apoptotic death of hepatocytes exposed to Br-A23187. Toxicol Appl Pharmacol 154: 117–125, 1999.[CrossRef][Web of Science][Medline]
  42. Qian Y, Zhong X, Flynn DC, Zheng JZ, Qiao M, Wu C, Dedhar S, Shi X, and Jiang BH. ILK mediates actin filament rearrangements and cell migration and invasion through PI3K/Akt/Rac1 signaling. Oncogene 24: 3154–3165, 2005.[CrossRef][Web of Science][Medline]
  43. Quaroni A, Wands J, Trelstad RL, and Isselbacher KJ. Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell Biol 80: 248–265, 1979.[Abstract/Free Full Text]
  44. Rice PL, Beard KS, Driggers LJ, and Ahnen DJ. Inhibition of extracellular-signal regulated kinase 1/2 is required for apoptosis of human colon cancer cells in vitro by sulindac metabolites. Cancer Res 64: 8148–8151, 2004.[Abstract/Free Full Text]
  45. Rosseland CM, Wierod L, Oksvold MP, Werner H, Ostvold AC, Thoresen GH, Paulsen RE, Huitfeldt HS, and Skarpen E. Cytoplasmic retention of peroxide-activated ERK provides survival in primary cultures of rat hepatocytes. Hepatology 42: 200–207, 2005.[CrossRef][Web of Science][Medline]
  46. Sasaki K, Iizuka M, Konno S, Shindo K, Sato A, Horie Y, and Watanabe S. Ecabet sodium prevents the delay of wound repair in intestinal epithelial cells induced by hydrogen peroxide. J Gastroenterol 40: 474–482, 2005.[CrossRef][Web of Science][Medline]
  47. Sato K, Sato A, Aoto M, and Fukami Y. cSrc phosphorylates epidermal growth factor receptor on tyrosine 845. Biochem Biophys Res Commun 215: 1078–1087, 1995.[CrossRef][Web of Science][Medline]
  48. Sato Y and Rifkin DB. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta1-like molecule by plasmin during coculture. J Cell Biol 109: 309–315, 1989.[Abstract/Free Full Text]
  49. Sheid MP, Schubert KM, and Duronio V. Regulation of Bad phosphorylation and association with Bcl-xL by the MAPK/Erk kinase. J Biol Chem 274: 31108–31113, 1999.[Abstract/Free Full Text]
  50. Shirasaka T, Iizuka M, Yukawa M, Hirai Y, Horie Y, Itou H, Kon-no S, Fukushima T, and Watanabe S. Altered expression of epimorphin in ulcerative colitis. J Gastroenterol Hepatol 18: 570–577, 2003.[CrossRef][Web of Science][Medline]
  51. Song P, Wei J, and Wang HC. Distinct roles of the ERK pathways in modulating apoptosis of Ras-transformed and non-transformed cells induced by anticancer agent FR901228. FEBS Lett 579: 90–94, 2005.[CrossRef][Web of Science][Medline]
  52. Szabadkai G and Rizzuto R. Participation of endoplasmic reticulum and mitochodrial calcium handling in apoptosis: more than just neighborhood? FEBS Lett 567: 111–115, 2004.[CrossRef][Web of Science][Medline]
  53. Tapodi A, Debreceni B, Hanto K, Bognar Z, Wittmann I, Gallyas F Jr, Varbiro G, and Sumegi B. Pivotal role of Akt activation in mitochondrial protection and cell survival by poly (ADP-ribose) polymerase-1 inhibition in oxidative stress. J Biol Chem 280: 35767–35775, 2005.[Abstract/Free Full Text]
  54. Thomas JE, Soriano P, and Brugge JS. Phosphorylation of c-Src on tyrosine 527 by another protein tyrosine kinase. Science 254: 568–571, 1991.[Abstract/Free Full Text]
  55. Umeda J, Sano S, Kogawa K, Motoyama N, Yoshikawa K, Itami S, Kondoh G, Watanabe T, and Takeda J. In vivo cooperation between Bcl-xL and the phosphoinositide 3-kinase-Akt signaling pathway for the protection of epidermal keratinocytes from apoptosis. FASEB J 17: 610–620, 2003.[Abstract/Free Full Text]
  56. Watanabe S, Hirose M, Wang XE, Ikejima K, Oide H, Kitamura T, Takei Y, Miyazaki A, and Sato N. A novel hepatic stellate (Ito) cell-derived protein, epimorphin, plays a key role in the late stages of liver regeneration. Biochem Biophys Res Commun 250: 486–490, 1998.[CrossRef][Web of Science][Medline]
  57. Watanabe S, Wang XE, Hirose M, Osada T, Tanaka H, and Sato N. Rebamipide prevented delay of wound repair induced by hydrogen peroxide and suppressed apoptosis of gastric epithelial cells in vitro. Dig Dis Sci 43: 107–112, 1998.



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
J. Teerapornpuntakit, N. Dorkkam, K. Wongdee, N. Krishnamra, and N. Charoenphandhu
Endurance swimming stimulates transepithelial calcium transport and alters the expression of genes related to calcium absorption in the intestine of rats
Am J Physiol Endocrinol Metab, April 1, 2009; 296(4): E775 - E786.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M.-L. Wang, S. A. Keilbaugh, T. Cash-Mason, X. C. He, L. Li, and G. D. Wu
Immune-mediated signaling in intestinal goblet cells via PI3-kinase- and AKT-dependent pathways
Am J Physiol Gastrointest Liver Physiol, November 1, 2008; 295(5): G1122 - G1130.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
L. S. Chaturvedi, C. P. Gayer, H. M. Marsh, and M. D. Basson
Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells
Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1350 - C1361.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
Y. Hirai, M. J. Bissell, and D. C. Radisky
Extracellular localization of epimorphin/syntaxin-2
Blood, October 15, 2007; 110(8): 3082 - 3082.
[Full Text] [PDF]

Home page
 J. Cell Sci.Home page
Y. Hirai, C. M. Nelson, K. Yamazaki, K. Takebe, J. Przybylo, B. Madden, and D. C. Radisky
Non-classical export of epimorphin and its adhesion to {alpha}v-integrin in regulation of epithelial morphogenesis
J. Cell Sci., June 15, 2007; 120(12): 2032 - 2043.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/G39    most recent
00181.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iizuka, M.
Right arrow Articles by Watanabe, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iizuka, M.
Right arrow Articles by Watanabe, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.