EDITORIAL FOCUS
Epidermal and hepatocyte growth factors stimulate chemotaxis in an intestinal epithelial cell line

D. Brent Polk and Wei Tong

Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, Vanderbilt University, Nashville, Tennessee 37232-2576


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The migration of intestinal cells is important in the development and maintenance of normal epithelium, in a process that may be regulated by growth factors and cytokines. Although a number of growth factor receptors are expressed by intestinal cells, little progress has been made toward assignment of functional roles for these ligand-receptor systems. This study compares several growth factors and cytokines for their chemoattraction of the mouse small intestinal epithelial cell line. Epidermal and hepatocyte growth factors stimulated a rapid 30-fold chemotaxis of cells with delayed threefold migration toward transforming growth factor-beta 1. Despite stimulating proliferation, keratinocyte, fibroblast, or insulin-like growth factors did not stimulate directed migration. Chemotaxis required tyrosine kinase and phosphatidylinositol phospholipase C activities but not protein kinase C or mitogen-activated protein kinase activity. These findings suggest that the repertoire of growth factors capable of regulating directed intestinal epithelial cell migration is limited and that a divergence exists in the signal transduction pathways for directed vs. nondirected migration.

cellular migration; tyrosine kinase; proliferation; extracellular matrix


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS NOW APPARENT that intestinal epithelial cells express receptors for a number of peptide growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGF-I and -II), transforming growth factor (TGF)-beta , keratinocyte growth factor (KGF), and other fibroblast growth factors (FGFs) (2, 19, 24, 36, 42, 52). The ligation of these receptors and initiation of intracellular signal transduction have been demonstrated in vivo and in intestinal cell culture models (15, 18, 41, 57, 70, 75). Historically the predominant focus has been on the ability of such factors to alter cellular proliferation. However, the assignment of functional roles for these receptor-ligand systems has made little advance. For instance, the importance of TGF-alpha as an endogenous EGF receptor ligand was recently shown using a model of colitis, in which mice lacking expression of TGF-alpha developed a threefold more severe epithelial injury compared with genetic background controls with a 70% reduction in the severity of the illness by administration of exogenous TGF-alpha to the deficient animals (20). The TGF-alpha -null mouse, however, has not been reported to spontaneously develop either mucosal ulceration or inflammatory bowel disease (45, 46). Interestingly, in an EGF receptor-null mouse, the phenotype revealed impaired intestinal development with shortened villi, a pseudostratified epithelium, and death associated with a necrotizing enterocolitis-like disorder (47). This suggests that an individual ligand may not be indispensable for normal intestinal function; however, the signaling system of the EGF receptor may be required to maintain a healthy mucosal barrier.

Migration of the intestinal epithelial cell is important during intestinal development, during normal epithelial cell turnover, and during disease states involving ulceration of the single cell layer lining the gastrointestinal tract. Mucosal ulcer healing involves complex interactions between epithelial cells, the underlying matrix, and inflammatory and neural cells and may be aided by villus contraction. However, the initial epithelial cell response is the rapid wound closure (restitution) of the monolayer by migration of cells at the wound margins (22, 49, 50, 64). After in vivo injury, restitution of the protective monolayer is rapid, with enterocyte migration from the wound margins inward over the denuded supporting matrix, reestablishing continuity of the epithelium within minutes (8). Although it has been demonstrated in several models that many of these growth factors stimulate epithelial restitution, the mechanism of intestinal epithelial cellular migration is poorly understood (5, 15, 18).

Cellular migration is equally important in the movement of cells along the crypt-villus axis in the normal turnover of the epithelial lining. The rate of enterocyte migration along the length of the crypt-villus axis increases up to eightfold in the rat and threefold in humans during early postnatal development (33, 39). Interestingly, enterocytes arrested in the process of migration along this axis fail to fully differentiate (35). Perturbation of the initiation of cell migration out of the crypt onto the villus alters cell death programs and may promote chronic mucosal inflammation and adenoma formation (34). Clearly, a complete understanding of the mechanisms of intestinal cellular migration has significant implications for many areas of intestinal epithelial cell biology.

Another form of cellular migration is directed, chemotactic movement toward a growth factor or cytokine. Chemotaxis involves the sensing of a concentration gradient of chemoattractant, growth factor, or cytokine, reorganization of the actin cytoskeleton, and subsequent cellular movement toward the chemoattractant (7, 40). Blay and Brown (7) demonstrated chemotaxis of a rat intestinal epithelial cell toward EGF; however, surprisingly little is otherwise known about intestinal epithelial cell chemotaxis. There is a potential role for chemotaxis of intestinal epithelial cells in the complex process of crypt-villus migration if increased gradients of chemoattractants exist along the crypt-villus axis. Such a gradient has been described for TGF-alpha with the highest mRNA expression for this EGF receptor ligand at the villus tip (3). Two observations in transgenic mice make EGF receptor-regulated chemotaxis an attractive mechanism for modulating crypt-villus transit. First, the intestinal phenotype of the EGF receptor-null mouse shows impaired development, with shortened villi, a pseudostratified epithelium, and death associated with a necrotizing enterocolitis-like disorder (47). Second, a mouse lacking expression of the converting enzyme necessary for the final processing of TGF-alpha also has a similar phenotype, with an immature intestinal epithelium, blunted villi, and reduced cellular polarity (55).

We hypothesized that among growth factor receptor systems described to regulate intestinal epithelial cell proliferation or restitution a limited number would stimulate chemotaxis. A modified Boyden chamber assay was used with the conditionally immortalized mouse small intestinal epithelial (MSIE) cell line to address this potentially important aspect of intestinal cell migration. We studied various growth factors and cytokines and report that EGF and HGF receptor ligands, but not IGF-I, KGF, FGFs, or TGF-beta 1, stimulate rapid migration of intestinal cells toward the respective growth factor.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Human recombinant EGF was provided by Carlos George-Nascimento (Chiron, Emeryville, CA), porcine TGF-beta 1 was purchased from R&amp;D Systems (Minneapolis, MN), human recombinant KGF was generously provided by Amgen (Thousand Oaks, CA), and IGF-I was provided by Genentech (South San Francisco, CA). Human recombinant HGF, bovine FGF, rat tail collagen type I, mouse collagen type IV, mouse laminin, and ITS+ culture medium supplement were purchased from Collaborative Biomedical Products (Bedford, MA). Tyrphostin, AG-1478, LY-294002, calphostin C, PD-153035, and PD-98059 were purchased from Calbiochem (San Diego, CA), whereas U-73122, U-73343, D-609, and L-108 were purchased from Biomol Research Labs (Plymouth Meeting, PA). U-73122 and U-73343 were prepared as previously described (56). Genistein and fetal bovine serum (FBS) were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-active mitogen-activated protein (MAP) kinase rabbit polyclonal antibody was purchased from Promega (Madison, WI), whereas the anti-ERK1/ERK2 mouse monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY). Anti-phosphospecific Akt (Ser-473) and anti-Akt were purchased from New England Biolabs (Beverly, MA). Anti-protein kinase C (PKC)-alpha was from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant murine interferon-gamma (IFN-gamma ) and murine fibronectin were purchased from Life Technologies (Gaithersburg, MD). Other medium additives were purchased from Mediatech (Herndon, VA). Materials used for SDS-PAGE and Western blotting were from Bio-Rad Laboratories (Richmond, CA). The Boyden chamber was purchased from Neuroprobe (Cabin John, MD). Poretics polycarbonate membranes were purchased from Osmonics (Livermore, CA) and Aqua Polymount solution was purchased from Polysciences (Warrington, PA). RPMI-1640 medium, wortmannin, phorbol 12-myristate 13-acetate (PMA), and all other materials were purchased from Sigma Chemical (St. Louis, MO). All pharmacological inhibitors and agonists were dissolved in DMSO and added to the medium with the final concentration of DMSO <0.1%.

Cell culture. The conditionally immortalized MSIE cell line was generously provided by Robert Whitehead (Melbourne, Australia). The line was isolated from the H-2kb-tsA58 mouse expressing a heat-labile simian virus 40 large T antigen with an IFN-gamma -inducible promoter. These cells are identified as epithelial by anti-cytokeratin antibody staining and as intestinal by expression of dipeptidyl peptidase, lactase, and sucrase activities (69). In a related cell line, young adult mouse colon (YAMC), we demonstrated loss of the transformed phenotype (proliferation in suspension) when cells were cultured at 37°C for 24 h (37). Both MSIE and YAMC cells continue to proliferate until confluent and retain growth factor responsiveness when cultured as a monolayer at 37°C (56). Cells were grown on culture dishes coated with rat tail collagen type I (5 µg/cm2) in RPMI 1640 (pH 7.4) supplemented with 5% FBS, 3 g/l NaHCO3, 1% ITS+, 100,000 IU/l penicillin, 100 mg/l streptomycin, and 5 U/ml murine IFN-gamma . Cells were cultured under transforming (permissive) conditions at 33°C in the presence of IFN-gamma until confluent, and then the medium was removed. The cells were immediately washed twice with PBS and cultured under nontransforming (nonpermissive) conditions, 37°C in the absence of IFN-gamma until ready for use.

Cellular migration assays. The migration of cells was performed using a modified 48-well Boyden chamber apparatus. The polycarbonate membrane was coated with 5 ml of 5 µg/ml mouse fibronectin in carbonate buffer (pH 9.4) overnight before use. In some experiments, mouse fibronectin, mouse laminin, mouse collagen type IV, or rat tail collagen type I was used at 1 pM/cm2 to coat the membrane. Growth factors were diluted in RPMI 1640 and loaded into the lower wells of the chamber in triplicate. The wells were then covered with the matrix-coated polycarbonate membrane with 8-µm pores. Confluent cells, under nonpermissive conditions, were trypsinized and loaded into the top chamber at 50,000 cells/well. The chamber was incubated for various periods of time at 37°C in an atmosphere of 95% air and 5% CO2. At the end of the incubation time, the chamber was disassembled and the cells were scraped off the top of the membrane. The remaining cells were fixed in 100% methanol (10 min), washed in water, stained with hematoxylin (10 min), and then rinsed with water. The membrane was placed on a slide and covered with mounting medium and a coverslip. After slides were allowed to dry, three (×40) images were obtained of each well, recorded using an Optronix RGB camera (Goleta, CA) and quantitated by manual counting. The chemotaxis was calculated as the difference between the number of migrated cells in the presence of a gradient and in the absence (growth factor added to both the top and bottom wells) of a gradient.

Cellular proliferation assays. Cells were cultured under permissive conditions until confluent and then moved to nonpermissive conditions at 37°C (0.5% FBS) for 24 h. Growth factors were added for a 24-h incubation before cell number was determined by trypsinization and hemocytometry as previously described (37). The change in cell number over the 24-h growth factor treatment was compared with untreated controls, set at 100%.

Experimental protocols. All studies were performed following 24 h of culture under nonpermissive conditions. Cells were detached with trypsin-EDTA for 2 min, neutralized with 10% FBS-RPMI 1640 for 10 min, washed twice with RPMI 1640 without FBS, and then and pretreated with inhibitors for 45 min at 37°C. Inhibitors were dissolved in DMSO and diluted with RPMI 1640 before cell were loaded into the Boyden chamber at the final concentrations noted. For determination of MAP kinase or Akt phosphorylation states, cells were serum starved (0.5% FBS) overnight under nonpermissive conditions and then incubated in serum-free RPMI 1640 with or without PD-98059, wortmannin, or LY-294002. EGF, HGF, or TGF-beta 1 was added, and cells were incubated for various times at 37°C. Cells were washed twice with ice-cold PBS and scraped into buffer [50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 50 µM sodium molybdate (pH 7.5), 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 17.5 µg/ml phenylmethylsulfonyl fluoride]. Lysates were incubated in an ice bath for 30 min with frequent vortexing and clarified by centrifugation (16,000 g, 5 min) for determination of the supernatant protein content using the Bio-Rad DC protein assay.

Preparation of detergent soluble membrane fractions. Extracts were prepared essentially according to Black and colleagues (27). Cells were washed twice with ice-cold PBS and scraped into digitonin buffer (20 mM Tris, pH 7.5, 2 mM EGTA, 2 mM EDTA, 0.5 mg/ml digitonin, 10 mM NaF, 1 mM Na3VO4, 50 mM beta -glycerol phosphate, 10 mM Na4P2O7, 50 µM Na2MoO4, 0.5 mM benzamidine, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 17.5 µg/ml phenylmethylsulfonyl fluoride). Digitonin-insoluble fractions were prepared by ultracentrifugation (275,000 g, 12 min) at 4°C. The supernatant was discarded, and the pellet was resuspended in 150 µl of digitonin buffer with 1% Triton X-100 and incubated on ice for 30 min with frequent vortexing. The sample was then cleared by centrifugation (16,000 g, 30 min) at 4°C, and the supernatant was labeled detergent-soluble membrane fraction. A 30-µl aliquot of the supernatant was removed for protein determination, and the remainder was mixed with 5× Laemmli sample buffer (43) and incubated at 95°C for 5 min before separation by SDS-PAGE for Western blot analysis.

Western blot analysis. Equivalent amounts of protein were mixed with Laemmli sample buffer (43), and total volumes were equilibrated with 1× Laemmli sample buffer. After incubation at 95°C for 5 min, the samples were separated by 10% SDS-PAGE. Gels were transferred to Hybond-P polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK) with a Bio-Rad semi-dry transfer cell and then blocked with 2% milk and 0.05% Tween 20 in PBS at 4°C overnight. At room temperature, the membranes were washed twice with PBS and then incubated with anti-active MAP kinase (1:10,000) or anti-phospho Akt (1:1,000), anti-Akt (1:1,000), or anti-PKC-alpha (1:250) for 2 h before washing with 1% milk and 0.05% Tween 20 in PBS for 15 min once and then for 5 min twice. The membranes were then incubated with horseradish peroxidase-recombinant-protein A (Zymed, San Francisco, CA) at 1:10,000 for 2 h. The membranes were washed three times again, as above, and proteins were detected by chemiluminescence (Renaissance kit, DuPont NEN Research Products, Boston, MA) and recorded on film (Kodak Scientific Imaging Film, Rochester, NY).

Statistical analysis. Values were compared using Student's t-test and Microsoft Excel version 5.0, with statistical significance set at the level of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EGF and HGF stimulate chemotactic migration of intestinal epithelial cells. To investigate the chemoattractant activities of various growth factors, we cultured MSIE cells in a modified Boyden chamber, with a fibronectin-coated polycarbonate membrane, at various concentrations of growth factors. As shown in Fig. 1A, after a 2-h incubation with various growth factors, only EGF and HGF stimulated directed migration. Cells also migrated toward 5% FBS. If cells were incubated for 18 h or longer, TGF-beta also stimulated chemotaxis. As shown in Fig 1B, continuous exposure for 18 h to TGF-beta 1, but not IGF-I, KGF, or FGF (data not shown), stimulated migration.


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Fig. 1.   Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) stimulate chemotactic migration of mouse small intestinal epithelial (MSIE) cells at 2 h (A), whereas transforming growth factor (TGF)-beta stimulates chemotaxis by 18 h (B). Cells were incubated under permissive conditions for 48 h and then serum-starved [0.5% fetal bovine serum (FBS)] for 24 h under nonpermissive conditions. Cells were then trypsinized and incubated in a modified Boyden chamber above fibronectin-coated polycarbonate membranes for 2 h (A) or 18 h (B) at 37°C in the presence or absence of the indicated growth factors at 10 ng/ml. Values are mean (fold) increase ± SD. * P < 0.001 compared with cells exposed to vehicle alone (control). KGF, keratinocyte growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor.

Growth factors regulate proliferation. To determine whether the cells expressed ligand-responsive growth factor receptors, we chose to utilize a proliferation assay. Cells were cultured under permissive conditions until confluent and then moved to 37°C for 24 h before the addition of EGF, HGF, TGF-beta 1, KGF, FGF, IGF-I, or FBS (5%) for 24 h before determining cell number by hemocytometry. As shown in Fig. 2, all growth factors studied increased cell proliferation except for TGF-beta 1, which, as expected, inhibited cellular proliferation.


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Fig. 2.   Growth factors regulate proliferation. MSIE cells were serum starved at 37°C, as in Fig. 1, before addition of various growth factors (10 ng/ml) for 24 h. Cells were removed by incubation with trypsin-EDTA for counting by hemocytometry. Change in cell number over the course of the growth factor treatment is shown relative to control, set at 100%. * P < 0.01 compared with control.

Directed migration of intestinal epithelial cells is matrix dependent. Intestinal epithelial cells in vivo migrate over various extracellular matrix components, including laminin, collagen IV, and fibronectin, among others, along the crypt-villus axis (6, 58). To determine whether matrix components altered the chemotactic migration of MSIE cells toward growth factors, the polycarbonate membrane separating the chambers was coated with various matrices and incubated for 2 h at 37°C. As shown in Fig. 3A, both EGF and HGF stimulated chemotaxis on collagen IV and laminin with the greatest migration detected on collagen IV and fibronectin. Interestingly, little chemotaxis occurred on rat collagen I, even though MSIE cells attach to this matrix within 30 min (Polk and Tong, unpublished observations). However, matrix components did not permit chemotaxis toward any of the other growth factors at 2 h (data not shown).



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Fig. 3.   Directed migration of intestinal epithelial cells is matrix dependent. MSIE cells (prepared as in Fig. 1A) were placed in the upper wells of a modified Boyden chamber above polycarbonate membranes coated with various matrix components (A) for 2 h at 37°C; noncoated membranes were also included for comparison. As indicated, either EGF (10 ng/ml; top) or HGF (50 ng/ml; bottom) was placed in the lower chamber of wells and compared with medium alone. Membranes were coated with rat collagen (r coll) I, mouse (m) collagen IV, laminin (Ln), or fibronectin (Fn) at 1 pM/cm2 (as described in MATERIALS AND METHODS). Migration in the presence or absence of matrix (B) was determined by coating with fibronectin either top of the membrane, where cells were initially loaded, or bottom of the membrane, where chemoattractant was added, or both. * P < 0.001 compared with control; @ P < 0.001 compared with top- or bottom-coated membranes.

We next asked whether there was a requirement for extracellular matrix in the in vitro chemotaxis of MSIE cells. Polycarbonate membranes were either coated on top, facing the upper chamber where cells were added to the Boyden chamber, or coated on bottom, facing the lower chamber, where chemoattractant was added or both sides of the membrane were coated or left uncoated. Cells were prepared and incubated for 2 h and then compared with controls, without added growth factor. As shown in Fig. 3B, directed migration toward EGF was significantly enhanced if the membrane was coated on both sides. Together, these data suggest that MSIE cells are behaving in a normal epithelial cell manner in the Boyden chamber assay, i.e., requiring adhesion to matrix before migration.

Directed migration requires tyrosine kinase activity. We and others have shown a requirement for tyrosine kinase activity in an intestinal epithelial wound-closure model (4, 56). In this series of experiments, we asked whether there was a similar requirement for tyrosine kinase activity in chemotactic migration. MSIE cells were prepared as above and studied for migration across fibronectin-coated membranes toward EGF or HGF at 2 h, or TGF-beta , at 18 h. As shown in Fig. 4A, genistein inhibited chemotaxis regardless of which growth factor was used as the chemoattractant.


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Fig. 4.   Tyrosine kinase activity is required for directed migration of intestinal cells. Migration across fibronectin-coated membranes toward either EGF or HGF at 2 h or TGF-beta 1 at 18 h was studied in the presence of nonspecific tyrosine kinase inhibitor genistein (40 µg/ml) (A) or the EGF receptor-specific tyrosine kinase inhibitors AG-1478 (150 nM) or PD-153035 (100 nM) (B). * P < 0.001 compared with control; @ P < 0.001 compared with respective growth factor treatment.

Next, because a requirement for EGF receptor tyrosine kinase activity has been demonstrated for fibroblast migration toward fibronectin, we asked whether there was a similar requirement for intestinal cell migration (44). To determine the role of the EGF receptor tyrosine kinase activity in directed migration, cells were studied in the presence of either AG-1478 or PD-153035, tyrphostins specifically inhibiting EGF receptor tyrosine kinase activity. As shown in Fig. 4B, the tyrphostins blocked EGF- but not HGF-stimulated chemotaxis. Similar results were seen for TGF-beta 1 at 18 h, with no inhibition by either EGF receptor-specific tyrosine kinase inhibitor.

Mitogen activated protein kinase activity is not required for growth factor-stimulated chemotaxis. Because a number of functional responses to growth factors, including cytoskeletal reorganization (59), appear to be coordinated by MAP (ERK1/ERK2) kinase activities, we determined to study directed migration toward EGF, HGF, and TGF-beta 1 in the presence of the selective MAP kinase kinase (MEK1) inhibitor, PD-98059. Cells were prepared as above and incubated in the presence of PD-98059 and compared for migration toward EGF or HGF at 2 h or toward TGF-beta 1 at 18 h. As shown in Fig. 5A, chemotactic migration toward any of the growth factors was not attenuated by the inhibitor of MAP kinase activation.


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Fig. 5.   Mitogen-activated protein kinase activity is not required for directed intestinal cell migration. A: Chemotaxis toward EGF, HGF, or TGF-beta 1 was studied in the presence of the mitogen-activated protein kinase kinase inhibitor PD-98059. Cells were prepared for migration across fibronectin-coated membranes as before and incubated in the presence of PD-98059 (10 µM), and migration was determined at 2 h for EGF or 18 h for TGF-beta 1. Cells were incubated with PD-98059 for 60 min before treatment with either 10 ng/ml EGF, HGF, or TGF-beta 1. B: lysis and separation of 50 µg of protein by SDS-PAGE for Western blot analysis with anti-activated ERK1/ERK2 or anti-ERK1/ERK2 antibodies (Ab). * P < 0.001 compared with control.

To determine whether PD-98059 inhibited ERK1/ERK2 activation, cells were treated with EGF, HGF, or TGF-beta 1 in the presence of PD-98059. Cellular lysates were prepared as described in MATERIALS AND METHODS and separated by SDS-PAGE for Western blot analysis with anti-activated ERK1/ERK2 antibodies. As shown in Fig. 4B, EGF and HGF, but not TGF-beta 1, activated ERK1/ERK2, which was blocked by PD-98059.

Inhibitors of phosphatidylinositol phospholipase C activity attenuate directed cell migration. Our laboratory has recently reported the requirement of phosphatidylinositol phospholipase C (PLC) and PKC activities for intestinal epithelial cell migration in a wound closure assay (56). We asked whether PLC, PKC, or phosphoinositide (PI) 3-kinase activities were required for chemotaxis of MSIE cells toward EGF or HGF. As shown in Fig. 6, the active PLC inhibitors U-73122, D-609, and L-108 blocked either EGF or HGF stimulation of cell migration. The inactive U-73122 analog, U-73343, did not inhibit chemotaxis. Interestingly, as shown in Fig. 7A, neither PI 3-kinase inhibitors (wortmannin or LY-294002) nor PKC inhibitors (calphostin C or prolonged exposure to phorbol ester) (71, 74) altered chemoattraction toward either growth factor.


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Fig. 6.   Inhibitors of phospholipase C (PLC) attenuate chemotaxis toward either EGF or HGF. Cells were prepared as before and loaded in the upper chamber with U-73122 (1 µM), D-609 (100 µM), L-108 (10 µM), or the inactive analog U-73343 (1 mM). Either EGF (A) or HGF (B) were added to medium in the lower chamber of the apparatus at the indicated concentrations. Chemotaxis was measured at 2 h as in Fig. 1.




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Fig. 7.   Neither phosphoinositide (PI) 3-kinase nor protein kinase C (PKC) inhibitors affect directed cell migration toward either EGF or HGF. For inhibition of PI 3-kinase, cells were incubated with wortmannin (WT, 100 nM), LY-294002 (LY, 5 µM), or, for inhibition of PKC, with calphostin C (Cal C, 100 nM) or subjected to prolonged exposure to phorbol 12-myristate 13-acetate (PMA, 10 ng/ml for 16 h), as described in MATERIALS AND METHODS. Chemotaxis assays were performed as above in the presence of the various inhibitors, including genistein (gen, 40 µg/ml) as a positive control (A). EGF (top) or HGF (bottom) was added to the medium in lower chamber as in Fig. 6. * P < 0.001 compared with control; @ P < 0.001 compared with EGF or HGF, as indicated. B: to verify function of inhibitors in intestinal cells, cells were treated with EGF (10 ng/ml) or HGF (50 ng/ml) for 15 min in the presence of indicated inhibitors at concentrations given in A. Cells were lysed and 25 µg of protein were separated by SDS-PAGE for Western blot analysis with the indicated primary antibody.

To verify the effect of PI 3-kinase inhibitors, MSIE cells were incubated with wortmannin or LY-294002 or, as control, with the MEK1 inhibitor PD-98059. We used the recent recognition of PI 3-kinase activity as sufficient and necessary for growth factor-stimulated phosphorylation and activation of Akt (25, 26). Cells were treated with EGF or HGF and lysed, and equal amounts of Triton-soluble protein were separated by SDS-PAGE for Western blot analysis with anti-phospho Akt. As shown in Fig. 7B, top, both EGF and HGF stimulated Akt phosphorylation, which was inhibited by either wortmannin or LY-294002 but not PD-98059.

The effects of PKC inhibitors were studied by membrane translocation of PKC-alpha in response to phorbol ester (27). Cells were treated with phorbol ester for 5 min, and detergent-soluble membrane fractions were prepared. To determine the effect of PKC inhibitors, cells were either preincubated with PMA for 16 h to downregulate PKC expression (71, 74) or incubated with calphostin C. The MEK1 inhibitor PD-98059 was used as control. As shown in Fig. 7B, bottom, PMA stimulated PKC-alpha membrane translocation, which was blocked by long-term treatment with PMA or calphostin C but not PD-98059.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A fundamental question in the study of growth factor effects on intestinal epithelia regards the assignment of functional roles for these receptor-ligand systems. Our study shows that, among a number of growth factors and cytokines with reported functional effects on the intestinal epithelial cell, the regulation of directed cell migration is surprisingly limited. We also report that whether the chemotactic response is rapid, as toward EGF or HGF, or delayed, as with TGF-beta 1, there is a requirement for tyrosine kinase and PLC activities.

Although EGF has been shown to have chemoattractant properties in the rat intestinal epithelial cell line, to our knowledge this is the first report demonstrating that HGF has similar chemoattractant properties (7). Both EGF and HGF have been demonstrated to increase intestinal cell proliferation (15, 60, 65) and alter cytoskeletal organization and intracellular communication (63, 66, 68, 77). Both factors have been shown to induce tyrosine phosphorylation of substrates, including PLC-gamma 1, which may be involved in cellular migration (53, 67).

One possible explanation for chemotaxis toward EGF and HGF, to the exclusion of the other factors studied, is that the MSIE cells produce ligands for the IGF-I, KGF, or FGF receptors in concentrations that preclude the development of a gradient across the Boyden chamber. However, we do not feel this adequately explains the differences, based on the following three observations. First, conditioned MSIE cell medium stimulates tyrosine phosphorylation of the EGF receptor, suggesting production of ligand by the cells. Second, a full concentration range from 0.1 to 100 ng/ml was studied for each growth factor, with no migration detected regardless of the concentration (due to space limitations only data for 10 ng/ml are shown). Third, we confirmed these observations in the nontransformed rat small intestinal cell (IEC-6) line, with EGF and HGF stimulating rapid chemotaxis and delayed migration toward TGF-beta 1 but not toward IGF-I, KGF, or FGF. It is also noteworthy that, in the KGF receptor system, the ligand is not expressed by epithelial cells (23).

The ultrastructural events during restitution have been observed in several laboratories, yet little information is known about cytoskeletal organization during the process of chemotaxis (77). Here, we demonstrate an apparent requirement for intestinal cell adhesion before effective chemotactic migration, suggesting some of the cytoskeletal changes between these two forms of migration are likely to overlap. Although it has been shown that the EGF receptor can directly bind actin (66), significant questions remain regarding the organization of the plasma membrane receptors sensing the chemoattractant and their potential interaction with the cytoskeleton.

Our findings suggest that PLC activity is necessary for signal transduction of effective chemotaxis regardless of the growth factor. We have shown the inhibitors used here block increased cellular PLC and in vitro PLC-gamma 1 activities in MSIE cells (56). Both tyrosine kinase and PLC activities were necessary for EGF receptor-stimulated intestinal cell migration in a wound closure assay. PI 3-kinase and PKC activities were also important, although not absolutely required, in this nondirected cellular migration. There may be cell-type specificity in the signal transduction pathways involved in growth factor-mediated chemotaxis. For instance, platelet-derived growth factor-stimulated chemotaxis of fibroblasts requires PI 3-kinase and PKC activities (12). We have used the PI 3-kinase regulation of Akt phosphorylation to demonstrate the inhibition of both EGF and HGF stimulation of PI 3-kinase signaling by wortmannin and LY-294002 (25, 38, 62, 72). To demonstrate the effect of PKC inhibitors, we have used the supraphysiological activation of PKC-alpha membrane translocation by phorbol esters (27). In addition, PKC inhibition by calphostin C and downregulation of expression by continuous phorbol ester exposure are well documented (31, 71). Thus it is unlikely that PI 3-kinase or PKC activities are required for MSIE chemotaxis toward EGF or HGF.

The observation that PI 3-kinase and PKC are not required for intestinal cell chemotaxis is at odds with our observations in a wound closure assay of migration, suggesting a potential divergence in signal transduction pathways for nondirected (wound closure) vs. directed (chemotactic) cell migration (56). The wound closure assay was performed using cells plated on collagen I for several days before injury (56), whereas the chemotaxis assay performed here used a variety of individual matrices in short-term culture. Presumably, the cells in the wound closure assay alter their extracellular matrix and integrin expression during the 7-day culture before and immediately after injury (29, 56). Recent reports indicate the existence of novel signal transduction mechanisms regulating cell migration between the EGF receptor and extracellular matrix via integrins (44, 51, 76).

Chemotaxis of intestinal epithelial cells does not appear to require enhanced MAP kinase (ERK1/ERK2) activity. This "noneffect" was seen whether EGF, HGF, or TGF-beta 1 was the growth factor studied. We did not detect an increase in MAP kinase activity from TGF-beta 1 stimulation, which is in contrast to other laboratories (30, 73). Whether or not MAP kinases are regulatory in cellular migration appears to be determined by cell type, extracellular matrix, and perhaps ligand. Fibroblasts transfected with mutant EGF receptors competent for activation of ERK1/ERK2 but not PLC-gamma 1 lost the ability to migrate in response to EGF (10). However, fibroblast migration toward soluble fibronectin required MAP kinase activity (1), whereas chemotaxis of Chinese hamster ovary cells appears to be inhibited by MAP kinase activity (59). In the IEC-6 cell line, MAP kinase is activated by wounding in a migration model by an EGF receptor ligand-dependent mechanism (28). It has also been suggested that other MAP kinase family members may be necessary for IEC-6 cell monolayer restitution (13).

Substantial evidence indicates a regulatory role for TGF-beta 1 in growth factor-mediated intestinal epithelial restitution; therefore, we were surprised at the lack of early chemotactic response to TGF-beta 1 (11, 14-18). One mechanism of TGF-beta 1 regulation of cellular migration may be in the alteration of expression of extracellular matrix and/or cellular integrins during restitution (29). Consequently, the effects of TGF-beta 1 in this assay may be to alter the cellular and extracellular environment in favor of migration. Unlike EGF and HGF, which initiate intracellular signaling via tyrosine kinase receptors, TGF-beta 1 initiates signaling via intrinsic serine/threonine kinase activity of its type I or II receptors (9, 32). Although the earliest migration detected toward TGF-beta 1 was at 18 h, as opposed to EGF or HGF, seen as early as 30 min (data not shown), migration toward all three factors was inhibited by the tyrosine kinase inhibitor genistein. This may be explained by inhibition of receptor tyrosine kinase for EGF or HGF but does not explain the inhibition of TGF-beta 1-stimulated migration. However, the principle effect of genistein on all three growth factors may be inhibition of focal adhesion complex assembly (48), a necessary regulatory step in the cytoskeletal reorganization during cellular migration (21). The clustering of integrin receptors at focal adhesions stimulates tyrosine phosphorylation of focal adhesion kinase and Src family tyrosine kinases, both of which are inhibited by genistein (reviewed in Ref. 61). In fact, it has recently been shown that focal adhesion kinase autophosphorylation is a critical regulatory step during chemotaxis (54). Therefore, if TGF-beta enhances migration by extracellular matrix and/or integrin expression, the earliest tyrosine phosphorylation necessary for focal adhesion complex formation regulating migration may be inhibited by genistein. A full mechanistic explanation of the requirement for tyrosine kinase activity in TGF-beta -mediated chemotaxis will require additional studies.

In summary, we have shown that, among several growth factors that are known to alter intestinal cell function, only EGF and HGF stimulate rapid chemotactic migration. A delayed, less pronounced response was seen toward TGF-beta 1. A requirement for tyrosine kinase and PLC activities was shown for directed migration; however, inhibitors of MAP kinase, PI 3-kinase and PKC, did not alter the response. These findings suggest that a limited repertoire of growth factors are capable of regulating directed migration and divergence in the signal transduction pathways for directed vs. nondirected cell migration. Determining the mechanistic basis for this apparent divergence in signal transduction pathways leading to enhanced intestinal epithelial cell migration will require further investigation.


    ACKNOWLEDGEMENTS

We thank Steve Hanks for critical comments, J. J. Owen for advice establishing the modified Boyden chamber assay, and Jennifer Black for advice regarding PKC isozyme detection.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02212 and a Crohn's and Colitis Foundation of America Research Grant.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. B. Polk, Dept. of Pediatrics, Division of Pediatric Gastroenterology & Nutrition, Nashville, TN 37232-2576 (E-mail: d-brent.polk{at}mcmail.vanderbilt.edu).

Received 28 July 1998; accepted in final form 20 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anand-Apte, B., B. R. Zetter, A. Viswanathan, R.-G. Qiu, J. Chen, R. Ruggieri, and M. Symons. Platelet-derived growth factor and fibronectin-stimulated migration are differentially regulated by the Rac and extracellular signal-regulated kinase pathways. J. Biol. Chem. 272: 30688-30692, 1997[Abstract/Free Full Text].

2.   Barnard, J. A., R. D. Beauchamp, R. J. Coffey, and H. L. Moses. Regulation of intestinal epithelial cell growth by transforming growth factor type beta . Proc. Natl. Acad. Sci. USA 86: 1578-1582, 1989[Abstract].

3.   Barnard, J. A., W. H. Polk, H. L. Moses, and R. J. Coffey. Production of transforming growth factor-alpha by normal rat small intestine. Am. J. Physiol. 261 (Cell Physiol. 30): C994-C1000, 1991[Abstract/Free Full Text].

4.   Basson, M. D., I. M. Modlin, S. D. Flynn, B. P. Jean, and J. A. Madri. Independent modulation of enterocyte migration and proliferation by growth factors, matrix proteins, and pharmacologic agents in an in vitro model of mucosal healing. Surgery 112: 299-308, 1992[Medline].

5.   Basson, M. D., I. M. Modlin, and J. A. Madri. Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. J. Clin. Invest. 90: 15-23, 1992[Medline].

6.   Beaulieu, J.-F., and P. H. Vachon. Reciprocal expression of laminin A-chain isoforms along the crypt-villus axis in the human small intestine. Gastroenterology 106: 829-839, 1994[Medline].

7.   Blay, J., and K. D. Brown. Epidermal growth factor promotes the chemotactic migration of cultured rat intestinal epithelial cells. J. Cell. Physiol. 124: 107-112, 1985[Medline].

8.   Buck, R. C. Ultrastructural features of rectal epithelium of the mouse during the early phases of migration to repair a defect. Virchows Arch. B Cell. Pathol. 51: 331-340, 1986[Medline].

9.   Carcamo, J., F. M. B. Weis, F. Ventura, R. Wieser, J. L. Wrana, L. Attisano, and J. Massague. Type I receptors specify growth-inhibitory and transcriptional responses to transforming growth factor beta  and activin. Mol. Cell. Biol. 14: 3810-3821, 1994[Abstract].

10.   Chen, P., H. Xie, M. C. Sekar, K. Gupta, and A. Wells. Epidermal growth factor receptor-mediated cell motility: phospholipase C activity is required, but mitogen-activated protein kinase activity is not sufficient for induced cell movement. J. Cell. Biol. 127: 847-857, 1994[Abstract].

11.   Ciacci, C., S. E. Lind, and D. K. Podolsky. Transforming growth factor beta  regulation of migration in wounded rat intestinal epithelial monolayers. Gastroenterology 105: 93-101, 1993[Medline].

12.   Derman, M. P., A. Toker, J. H. Hartwig, K. Spokes, J. R. Falck, C.-S. Chen, L. C. Cantley, and L. G. Cantley. The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C. J. Biol. Chem. 272: 6465-6470, 1997[Abstract/Free Full Text].

13.   Dieckgraefe, B. K., D. M. Weems, S. A. Santoro, and D. H. Alpers. ERK and p38 MAP kinase pathways are mediators of intestinal epithelial wound-induced signal transduction. Biochem. Biophys. Res. Commun. 233: 389-394, 1997[Medline].

14.   Dignass, A., K. Lynch-Devaney, H. Kindon, L. Thim, and D. K. Podolsky. Trefoil peptides promote epithelial migration through a transforming growth factor beta -independent pathway. J. Clin. Invest. 94: 376-383, 1994[Medline].

15.   Dignass, A. U., K. Lynch-Devaney, and D. K. Podolsky. Hepatocyte growth factor/scatter factor modulates intestinal epithelial cell proliferation and migration. Biochem. Biophys. Res. Commun. 202: 701-709, 1994[Medline].

16.   Dignass, A. U., and D. K. Podolsky. Cytokine modulation of intestinal epithelial cell restitution: central role of transforming growth factor beta . Gastroenterology 105: 1323-1332, 1993[Medline].

17.   Dignass, A. U., and D. K. Podolsky. Interleukin 2 modulates intestinal epithelial cell function in vitro. Exp. Cell. Res. 225: 422-429, 1996[Medline].

18.   Dignass, A. U., S. Tsunekawa, and D. K. Podolsky. Fibroblast growth factors modulate intestinal epithelial cell growth and migration. Gastroenterology 106: 1254-1262, 1994[Medline].

19.   DiRenzo, M. F., R. P. Narsimhan, M. Olivero, S. Bretti, S. Giordano, E. Medico, P. Gaglia, P. Zara, and P. M. Comoglio. Expression of the Met/HGF receptor in normal and neoplastic human tissues. Oncogene 6: 1997-2003, 1991[Medline].

20.   Egger, B., F. Procaccino, J. Lakshmanan, M. Reinshagen, P. Hoffmann, A. Patel, W. Reuben, S. Gnanakkan, L. Liu, L. Barajas, and V. E. Eysselein. Mice lacking transforming growth factor alpha  have an increased susceptibility to dextran sulfate-induced colitis. Gastroenterology 113: 825-832, 1997[Medline].

21.   Ezzell, R. M., W. H. Goldmann, N. Wang, N. Parasharama, and D. E. Ingber. Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton. Exp. Cell Res. 231: 14-26, 1997[Medline].

22.   Feil, W., E. R. Lacy, Y.-M. M. Wong, D. Burger, E. Wenzl, M. Starlinger, and R. Schiessel. Rapid epithelial restitution of human and rabbit colonic mucosa. Gastroenterology 97: 685-701, 1989[Medline].

23.   Finch, P. W., J. S. Rubin, T. Miki, D. Ron, and S. A. Aaronson. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 245: 752-755, 1989[Medline].

24.   Forgue-Lafitte, M.-E., M. Laburthe, M.-C. Chamblier, A. J. Moody, and G. Rosselin. Demonstration of specific receptors for EGF-urogastrone in isolated rat intestinal epithelial cells. FEBS Lett. 114: 243-246, 1980[Medline].

25.   Franke, T. F., D. R. Kaplan, L. C. Cantley, and A. Toker. Direct Regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-biphosphate. Science 275: 665-668, 1997[Abstract/Free Full Text].

26.   Franke, T. F., S.-I. Yang, T. O. Chan, K. Datta, A. Kazlauskas, D. K. Morrison, D. R. Kaplan, and P. N. Tsichlis. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81: 727-736, 1995[Medline].

27.   Frey, M. R., M. L. Saxon, X. Zhao, A. Rollins, S. S. Evans, and J. D. Black. Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21waf1/cip1 and p27kip1 and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells. J. Cell. Biol. 272: 9424-9435, 1997.

28.   Göke, M., M. Kanai, K. Lynch-Devaney, and D. K. Podolsky. Rapid mitogen-activated protein kinase activation by transforming growth factor alpha  in wounded rat intestinal epithelial cells. Gastroenterology 114: 697-705, 1998[Medline].

29.   Göke, M., A. Zuk, and D. K. Podolsky. Regulation and function of extracellular matrix in intestinal epithelial restitution in vitro. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G729-G740, 1996[Abstract/Free Full Text].

30.   Hartsough, M. T., and K. M. Mulder. Transforming growth factor beta  activation of p44mapk in proliferating cultures of epithelial cells. J. Biol. Chem. 270: 7117-7124, 1995[Abstract/Free Full Text].

31.   Hartzell, H. C., and A. Rinderknecht. Calphostin C, a widely used protein kinase C inhibitor, directly and potently blocks L-type Ca channels. Am. J. Physiol. 270 (Cell Physiol. 39): C1293-C1299, 1996[Abstract/Free Full Text].

32.   Henis, Y. I., A. Moustakas, H. Y. Lin, and H. F. Lodish. The types II and III transforming growth factor-beta receptors form homo-oligomers. J. Cell. Biol. 126: 139-154, 1994[Abstract].

33.   Herbst, J. J., P. Sunshine, and N. Kretchmer. Intestinal malabsorption in infancy and childhood. Adv. Pediatr. 16: 11-64, 1969[Medline].

34.   Hermiston, M. L., and J. I. Gordon. Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin. Science 270: 1203-1207, 1995[Abstract].

35.   Hermiston, M. L., W. H. Wong, and J. I. Gordon. Forced expression of E-cadherin in the mouse intestinal epithelium slows cell migration and provides evidence of nonautonomous regulation of cell fate in a self-renewing system. Genes Dev. 10: 985-996, 1996[Abstract].

36.   Housley, R. M., C. F. Morris, W. Boyle, B. Ring, R. Blitz, J. E. Tarpley, S. L. Aukerman, P. L. Devine, R. H. Whitehead, and G. F. Pierce. Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract. J. Clin. Invest. 94: 1767-1777, 1994.

37.   Kaiser, G. C., and D. B. Polk. Tumor necrosis factor alpha  regulates proliferation in a mouse intestinal cell line. Gastroenterology 112: 1231-1240, 1997[Medline].

38.   Karnam, P., M. L. Standaert, L. Galloway, and R. V. Farese. Activation and translocation of Rho (and ribosylation factor) by insulin in rat adipocytes. J. Biol. Chem. 272: 6136-6140, 1997[Abstract/Free Full Text].

39.   Koldovsky, O., P. Sunshine, and N. Kretchmer. Cellular migration of intestinal epithelia in suckling and weaned rats. Nature 212: 1389-1390, 1966[Medline].

40.   Kundra, V., J. A. Escobedo, A. Kazlauskas, H. K. Kim, S. G. Rhee, L. T. Williams, and B. R. Zetter. Regulation of chemotaxis by the platelet-derived growth factor receptor-beta . Nature 367: 474-476, 1994[Medline].

41.   Kurokowa, M., K. Lynch, and D. K. Podolsky. Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta  inhibits proliferation and stimulates differentiation. Biochem. Biophys. Res. Commun. 142: 775-782, 1987[Medline].

42.   Laburthe, M., C. Rouyer-Fessard, and S. Gammeltoft. Receptors for insulin-like growth factors I and II in rat gastrointestinal epithelium. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G457-G462, 1988[Abstract/Free Full Text].

43.   Laemmli, E. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

44.   Li, J., M.-L. Lin, G. J. Wiepz, A. G. Guadarrama, and P. J. Bertics. Integrin-mediated migration of murine B82L fibroblasts is dependent on the expression of an intact epidermal growth factor receptor. J. Biol. Chem. 274: 11209-11219, 1999[Abstract/Free Full Text].

45.   Luetteke, N. C., T. H. Qui, R. L. Peiffer, P. Oliver, O. Smithies, and D. C. Lee. TGF alpha  deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice. Cell 73: 263-278, 1993[Medline].

46.   Mann, G. B., K. J. Fowler, A. Gabriel, E. C. Nice, R. L. Williams, and A. R. Dunn. Mice with a null mutation of the TGF alpha  gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation. Cell 73: 249-261, 1993[Medline].

47.   Miettinen, P. J., J. E. Berger, J. Meneses, Y. Phung, R. A. Pedersen, Z. Werb, and R. Derynck. Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376: 337-341, 1995[Medline].

48.   Miyamoto, S., H. Teramoto, O. A. Coso, J. S. Gutkind, P. D. Burbelo, S. K. Akiyama, and K. M. Yamada. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J. Cell. Biol. 131: 791-805, 1995[Abstract].

49.   Moore, R., S. Carlson, and J. L. Madara. Villus contraction aids repair of intestinal epithelium after injury. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G274-G283, 1989[Abstract/Free Full Text].

50.   Moore, R., J. Madri, S. Carlson, and J. L. Madara. Collagens facilitate epithelial migration in restitution of native guinea pig intestinal epithelium. Gastroenterology 102: 119-130, 1992[Medline].

51.   Moro, L., M. Venturino, C. Bozzo, L. Silengo, F. Altruda, L. Beguinot, G. Tarone, and P. Defilippi. Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival. EMBO J. 17: 6622-6632, 1998[Abstract/Free Full Text].

52.   New, B. A., and L. C. Yeoman. Identification of basic fibroblast growth factor sensitivity and receptor and ligand expression in human colon tumor cell lines. J. Cell. Physiol. 150: 320-326, 1992[Medline].

53.   Okano, Y., K. Mizuno, S. Osada, T. Nakamura, and Y. Nozawa. Tyrosine phosphorylation of phospholipase Cgamma in c-met/HGF receptor-stimulated hepatocytes: comparison with HepG2 hepatocarcinoma cells. Biochem. Biophys. Res. Commun. 190: 842-848, 1993[Medline].

54.   Owen, J. D., P. J. Ruest, D. W. Fry, and S. K. Hanks. Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2. Mol. Cell. Biol. 19: 4806-4818, 1999[Abstract/Free Full Text].

55.   Peschon, J. J., J. L. Slack, P. Reddy, K. L. Stocking, S. W. Sunnarborg, D. C. Lee, W. E. Russell, B. J. Castner, R. S. Johnson, J. N. Fitzner, R. W. Boyce, N. Nelson, C. J. Kozlosky, M. F. Wolfson, C. T. Rauch, D. P. Cerretti, R. J. Paxton, C. J. March, and R. A. Black. An essential role for ectodomain shedding in mammalian development. Science 282: 1281-1284, 1998[Abstract/Free Full Text].

56.   Polk, D. B. Epidermal growth factor receptor-stimulated intestinal epithelial cell migration requires phospholipase C activity. Gastroenterology 117: 493-502, 1998.

57.   Polk, D. B. Ontogenic regulation of PLCgamma 1 activity and expression in the rat small intestine. Gastroenterology 107: 109-116, 1994[Medline].

58.   Potten, C. S. Epithelial cell growth and differentiation. II. Intestinal apoptosis. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G253-G257, 1997[Free Full Text].

59.   Reszka, A. A., J. C. Bulinski, E.G. Kreb, and E. H. Fischer. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions. Mol. Biol. Cell 8: 1219-1232, 1997[Abstract].

60.   Scheving, L. A., Y. C. Yeh, T. H. Tsai, and L. E. Scheving. Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the duodenum, jejunum, ileum, caecum, colon, and rectum of the adult male mouse. Endocrinology 106: 1498-1503, 1980[Abstract].

61.   Schlaepfer, D. D., and T. Hunter. Integrin signalling and tyrosine phosphorylation: just the FAKs? Trends Cell. Biol. 8: 151-157, 1998[Medline].

62.   Sizemore, N., S. Leung, and G. R. Stark. Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-kB p65/RelA subunit. Mol. Cell. Biol. 19: 4798-4805, 1999[Abstract/Free Full Text].

63.   Stoker, M., E. Gherardi, M. Perryman, and J. Gray. Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature 327: 239-242, 1987[Medline].

64.   Svanes, K., S. Ito, K. Takeuchi, and W. Silen. Restitution of the surface epithelium of the in vitro frog gastric mucosa after damage with hyperosmolar sodium chloride. Gastroenterology 82: 1409-1426, 1982[Medline].

65.   Ulshen, M. H., L. E. Lyn-Coo, and R. H. Raasch. Effects of intraluminal epidermal growth factor on mucosal proliferation in the small intestine of adult rats. Gastroenterology 91: 1134-1140, 1986[Medline].

66.   Van Bergen en Henegouwen, P. M. P., D. den Hartigh, P. Romeyn, A. J. Verkleij, and J. Boonstra. The epidermal growth factor receptor is associated with actin filaments. Exp. Cell. Res. 199: 90-97, 1992[Medline].

67.   Wahl, M. I., S. Nishibe, P.-G. Suh, S. G. Rhee, and G. Carpenter. Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracellular calcium. Proc. Natl. Acad. Sci. USA 86: 1568-1572, 1989[Abstract].

68.   Weidner, K. M., J. Behren, J. Vandekerckov, and W. Birchmeier. Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J. Cell. Biol. 111: 2097-2108, 1990[Abstract].

69.   Whitehead, R. H., P. E. VanEeden, M. D. Noble, P. Ataliotis, and P. S. Jat. Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. Proc. Natl. Acad. Sci. USA 90: 587-591, 1993[Abstract].

70.   Winesett, D. E., M. H. Ulshen, E. C. Hoyt, N. K. Mohapatra, C. R. Fuller, and P. K. Lund. Regulation and localization of the insulin-like growth factor system in small bowel during altered nutrient status. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G631-G640, 1995[Abstract/Free Full Text].

71.   Woodgett, J. R., and T. Hunter. Immunological evidence for two physiological forms of protein kinase C. Mol. Cell. Biol. 7: 85-96, 1987[Medline].

72.   Woscholski, R., T. Kodaki, M. McKinnon, M. D. Waterfield, and P. J. Parker. A comparison of demthoxyviridin and wortmannin as inhibitors of phosphatidylinositol 3-kinase. FEBS Lett. 342: 109-114, 1994[Medline].

73.   Yan, Z., S. Winawer, and E. Friedman. Two different signal transduction pathways can be activated by transforming growth factor beta 1 in epithelial cells. J. Biol. Chem. 269: 13231-13237, 1994[Abstract/Free Full Text].

74.   Yeo, E.-J., A. Kazlauskas, and J. H. Exton. Activation of phospholipase C-gamma is necessary for stimulation of phospholipase D by platelet-derived growth factor. J. Biol. Chem. 269: 27823-27826, 1994[Abstract/Free Full Text].

75.   Zeeh, J. M., F. Procaccino, P. Hoffman, S. L. Aukerman, J. A. McRoberts, S. Soltani, G. F. Pierce, J. Lakshmanan, D. Lacey, and V. E. Eysselein. Keratinocyte growth factor ameliorates mucosal injury in an experimental model of colitis in rats. Gastroenterology 110: 1077-1083, 1996[Medline].

76.   Zhao, M., A. Dick, J. V. Forrester, and C. D. McCaig. Electric field-directed cell motility involves up-regulated expression an asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin. Mol. Biol. Cell 10: 1259-1276, 1999[Abstract/Free Full Text].

77.   Zigmond, S. H. Signal transduction and actin filament organization. Current Opin. Cell. Biol. 8: 66-73, 1996[Medline].


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