Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, Vanderbilt University, Nashville, Tennessee 37232-2576
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ABSTRACT |
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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-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
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INTRODUCTION |
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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)-, 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-
as an
endogenous EGF receptor ligand was recently shown using a model of
colitis, in which mice lacking expression of TGF-
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-
to the deficient animals
(20). The TGF-
-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- 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-
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-1, stimulate rapid
migration of intestinal cells toward the respective growth factor.
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MATERIALS AND METHODS |
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Materials.
Human recombinant EGF was provided by Carlos George-Nascimento (Chiron,
Emeryville, CA), porcine TGF-1 was purchased from R&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)-
was from Santa Cruz
Biotechnology (Santa Cruz, CA). Recombinant murine interferon-
(IFN-
) 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--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-
. Cells were cultured under transforming (permissive) conditions
at 33°C in the presence of IFN-
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-
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-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 -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- (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.
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RESULTS |
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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- also stimulated
chemotaxis. As shown in Fig 1B, continuous exposure for 18 h to
TGF-
1, but not IGF-I, KGF, or FGF (data not shown), stimulated
migration.
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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-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-
1, which, as expected, inhibited cellular proliferation.
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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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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-, 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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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-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-
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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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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DISCUSSION |
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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-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-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-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-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-
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-1 was the growth factor studied. We
did not detect an increase in MAP kinase activity from TGF-
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-
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-1 in growth
factor-mediated intestinal epithelial restitution; therefore, we were
surprised at the lack of early chemotactic response to TGF-
1 (11,
14-18). One mechanism of TGF-
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-
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-
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-
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-
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-
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-
-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-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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