Departments of 1 Surgery and 2 Pathology, University of Maryland School of Medicine and 3 Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201
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ABSTRACT |
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Polyamines
are essential for early mucosal restitution that occurs by epithelial
cell migration to reseal superficial wounds after injury. Normal
intestinal epithelial cells are tightly bound in sheets, but they need
to be rapidly disassembled during restitution. -Catenin is involved
in cell-cell adhesion, and its tyrosine phosphorylation causes
disassembly of adhesion junctions, enhancing the spreading of cells.
The current study determined whether polyamines are required for the
stimulation of epithelial cell migration by altering
-catenin
tyrosine phosphorylation. Migration of intestinal epithelial cells
(IEC-6 line) after wounding was associated with an increase in
-catenin tyrosine phosphorylation, which decreased the binding
activity of
-catenin to
-catenin. Polyamine depletion by
-difluoromethylornithine reduced cytoplasmic free Ca2+
concentration ([Ca2+]cyt), prevented
induction of
-catenin phosphorylation, and decreased cell migration.
Elevation of [Ca2+]cyt induced by the
Ca2+ ionophore ionomycin restored
-catenin
phosphorylation and promoted migration in polyamine-deficient cells.
Decreased
-catenin phosphorylation through the tyrosine kinase
inhibitor herbimycin-A or genistein blocked cell migration, which was
accompanied by reorganization of cytoskeletal proteins. These results
indicate that
-catenin tyrosine phosphorylation plays a critical
role in polyamine-dependent cell migration and that polyamines induce
-catenin tyrosine phosphorylation at least partially through
[Ca2+]cyt.
ornithine decarboxylase; cell adhesion; tyrosine kinase; -catenin; intracellular calcium; restitution; intestinal epithelial
cells
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INTRODUCTION |
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-CATENIN
is a versatile intracellular protein that mediates cell-cell adhesion
and also actively participates in intracellular signal transduction
(5, 52). On one hand,
-catenin directly associates with
the highly conserved cytoplasmic domain of the cell adhesion protein
E-cadherin, the member of a family of transmembrane receptors that are
primarily found at the adherens junctions of adjacent cells and mediate
cell-cell contacts (14). The cadherin-catenin complex is
linked through
-catenin either directly (37) or indirectly to the actin cytoskeleton through the actin-binding protein
-actinin or vinculin (51). This association of the cadherin-catenin complex with the actin filament network is necessary for tight cell-cell interactions. Tyrosine phosphorylation of
-catenin in this complex is regulated by tyrosine kinases (TKs) and
protein-tyrosine phosphatases (PTPs), which increases the free pool of
phosphorylated
-catenin (PY-
-catenin) and consequently decreases
cell-cell contacts (14, 28, 39). On the other hand,
-catenin is able to translocate into the nucleus and plays an
additional function independent of cadherin-mediated cell adhesion (52). Through its interaction with members of the T-cell
factor/lymphoid enhancer factor (TCF/LEF) of DNA-binding proteins,
-catenin serves as a transcriptional regulator and induces the
expression of target genes (5, 27).
Intestinal mucosal restitution refers to the resealing of superficial
wounds after injury and occurs as a consequence of epithelial cell
migration into the defect, a process that is independent of cell
proliferation (30, 41). This early mucosal
reepithelialization following superficial wounding is a complex process
that includes the disassociation, dispersion, migration, and
reorganization of intestinal epithelial cells. Normal intestinal
epithelial cells are tightly bound together into sheets within
functional mucosa, but they need to be rapidly disassembled and
reassembled during epithelial restitution. The
cadherin/catenin-mediated cell-cell contacts are highly dynamic in the
intestinal mucosa, and this association has to be transiently disrupted
during intestinal epithelial cell migration after wounding. Although
the potential role for tyrosine phosphorylation of -catenin in the
regulation of intestinal epithelial restitution remains unclear, it has
been shown that increased PY-
-catenin in the cadherin-catenin
complex by transfection of the cytoplasmic TK such as v-src
gene (17, 24) or treatment with growth factors such as
epidermal growth factor (EGF) (19, 28, 42) causes unstable
cell-cell adhesion and enhances the spreading of cells. In contrast,
decreased PY-
-catenin through ectopic expression of the
PTP-LAR gene inhibits cell migration (28).
Studies from our laboratory (45-47) and others
(3, 23, 26) have demonstrated that the cellular polyamines
spermidine and spermine and their precursor putrescine are necessary
for the stimulation of cell migration after wounding and play an
important role in the maintenance of gastrointestinal mucosal
integrity. In an in vitro model mimicking the early cell
division-independent stage of intestinal epithelial restitution, cell
migration is associated with a dramatic increase in polyamine synthesis
(34, 47, 48). Depletion of cellular polyamines by
inhibiting ornithine decarboxylase (ODC), the first rate-limiting
enzyme in polyamine biosynthesis, with
D,L--difluoromethylornithine (DFMO) inhibits cell
migration, which is completely prevented by exogenous polyamines. Although exact roles of polyamines at cellular and molecular levels are
still unclear, increasing evidence indicates that polyamines modulate
cell migration after wounding through multiple signaling pathways
(22, 25, 54). In intestinal epithelial cells that do not
express voltage-dependent Ca2+ channels (VDCC), we have
recently reported (35, 49) that polyamines stimulate the
expression of K+ channel genes, resulting in membrane
hyperpolarization, and increase intracellular free Ca2+
concentration ([Ca2+]cyt). However, little is
known about the effects of this elevated [Ca2+]cyt following activation of
K+ channels except that it involves the activation of RhoA
signaling (33).
The current study was designed to test the hypothesis that polyamines
enhance cell migration during restitution by altering -catenin
tyrosine phosphorylation via a Ca2+-dependent process in
intestinal epithelial cells (IEC-6 line). First, we examined the
effects of cellular polyamines on the tyrosine phosphorylation of
-catenin during IEC-6 cell migration. Second, we determined whether
manipulating [Ca2+]cyt, either by increase or
decrease, would alter levels of
-catenin tyrosine phosphorylation in
the presence or absence of cellular polyamines. Third, we determined
whether inhibition of
-catenin tyrosine phosphorylation by treatment
with the specific TK inhibitors genistein and herbimycin-A would
decrease cell migration and alter cellular distribution of actin
filaments after wounding. Some of these data have been published in
abstract form (15).
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MATERIALS AND METHODS |
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Chemicals and supplies.
Disposable culture ware was purchased from Corning Glass Works
(Corning, NY). Tissue culture media and dialyzed fetal bovine serum
(dFBS) were obtained from GIBCO-BRL (Gaithersburg, MD), and
biochemicals were from Sigma (St. Louis, MO). The monoclonal antibodies
against phosphotyrosine (PY-20), -catenin,
-catenin, and
E-cadherin were purchased from Transduction Laboratories (Lexington, KY). DFMO was purchased from Ilex Oncology (San Antonio, TX). Matrigel
was from Collaborative Research (Bedford, MA). Rhodamine phalloidin and
unlabeled phalloidin were obtained from Molecular Probes (Eugene, OR).
Cell cultures and general experimental protocols. The IEC-6 cell line was purchased from the American Type Culture Collection (ATCC) at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (32). IEC-6 cells originated from intestinal crypt cells, as judged by morphological and immunological criteria. They are nontumorigenic and retain the undifferentiated character of epithelial stem cells.
Stock cells were maintained in T-150 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were subcultured once a week at 1:20; medium was changed three times weekly. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative, and passages 15-20 were used in the experiments. There were no significant changes of biological function and characterization from passages 15-20. The general protocol of the experiments and the methods used were similar to those described previously (33, 35). Briefly, IEC-6 cells were plated at 6 × 104/cm2 in DMEM plus 5% dFBS, 10 µg/ml insulin, and 50 µg/ml gentamicin. Cells were incubated in a humidified atmosphere at 37°C in a humidified atmosphere of 90% air-10% CO2 (vol/vol) for 4 days, followed by a period of different experimental treatments. In the first series of studies, we examined changes in tyrosine phosphorylation ofCell lysis and immunoprecipitation.
IEC-6 cell lysis and -catenin immunoprecipitation from whole cell
lysates were carried out according to the method of Müller et al.
(28) with minor changes. Briefly, 90 min before cell lysis, sodium orthovanadate, a specific inhibitor of PTP, was added to
cultures to inhibit PTP activity. Cells were washed with ice-cold D-PBS
three times and then lysed in ice-cold lysis buffer (50 mM HEPES, pH
7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 20 mM pyrophosphate, 1%
Triton X-100, 100 mM NaF, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 0.7 µg/ml pepstatin, 0.2 mM
ammonium molybdate, and 2 mM sodium orthovanadate). Cells were
sonicated for 20 s, whole cell lysates were centrifuged at 4°C,
and the supernatant was stored at
80°C until used. The protein concentration was measured by the Bradford method (7).
Equal amounts of protein (300 µg) for each sample were incubated with the specific antibody against
-catenin (2 µg) for 3 h at
4°C; protein G-PLUS-agarose was added, and incubation continued
overnight at 4°C. The precipitates were washed five times with
ice-cold D-PBS, and the beads were resuspended in SDS sample buffer for subsequent Western blotting analysis.
Western blotting analysis.
The proteins from above the immunoprecipitation or the supernatant of
whole cell lysates were boiled for 5 min and then subjected to
electrophoresis on 7.5% acrylamide gel according to Laemmli (21). After the transfer of proteins onto nitrocellulose,
the filters were incubated for 1 h in 5% nonfat dry milk in 1×
TBST buffer (Tris-buffered saline containing Tween 20), except
the anti-phosphotyrosine immunoblot, which was incubated in 2% BSA in
TBST. Immunological evaluation was then performed for 1 h in 1%
BSA-TBST buffer containing the specific antibody against
phosphotyrosine, -catenin,
-catenin, or E-cadherin protein. The
filters were subsequently washed with 1× TBST and incubated for 1 h with the second antibody conjugated to peroxidase. After extensive
washing with 1× TBST, the immunocomplexes on the filters were reacted for 1 min with chemiluminescence reagent (NEL-100; NEN). Finally, the
filters were placed in a plastic sheet protector and exposed to
autoradiography film for 30 or 60 s.
Measurement of [Ca2+]cyt. Details of the digital imaging methods employed for measuring [Ca2+]cyt have been published (33). Briefly, IEC-6 cells were plated on 25-mm coverslips and incubated in culture medium containing 3.3 µM fura 2-AM for 30-40 min at room temperature (22-24°C). The fura 2-loaded cells were then superfused with standard bath solution for 20-30 min at 22-24°C to wash away extracellular dye and permit intracellular esterases to cleave cytosolic fura 2-AM into active fura 2. Fura 2 fluorescence (510-nm emission; 380- and 360-nm excitation) from the cells and background fluorescence were imaged by using a Nikon Diaphot microscope equipped for epifluorescence. Fluorescent images were obtained by using a microchannel plate image intensifier (Amperex XX1381; Opelco, Washington, DC) coupled by fiber optics to a Pulnix charge-coupled device video camera (Stanford Photonics, Stanford, CA).
Image acquisition and analysis were performed with a MetaMorph Imaging System (Universal Imaging). Video frames containing images of fura 2 fluorescence from cells and the corresponding background images (fluorescence from fields devoid of cells) were digitized at a resolution of 512 horizontal × 480 vertical pixels and eight bits by using a Matrix LC imaging board operating in an IBM-compatible computer. Images were acquired at a rate of one averaged image every 3 s when [Ca2+]cyt was changing and every 60 s when [Ca2+]cyt was relatively constant. [Ca2+]cyt was calculated from fura 2 fluorescent emission excited at 380 and 360 nm using the ratio method (31). In most experiments multiple cells (usually 10-15 cells) were imaged in a single field, and one arbitrarily chosen peripheral cytosolic area (4-6 × 4-6 pixels) from each cell was spatially averaged.Measurement of cell migration. The migration assays were carried out as described in our earlier publications (35, 47). Cells were plated at 6 × 104 cells/cm2 in DMEM/dFBS on 60-mm dishes thinly coated with Matrigel according to the manufacturer's instructions and were incubated as described for stock cultures. The cells were fed on day 2 and migration was tested on day 4. To initiate migration, the cell layer was scratched with a single-edged razor blade cut to ~27 mm in length. The scratch began at the diameter of the dish and extended over an area 7-10 mm wide. The migrating cells in six contiguous 0.1-mm squares were counted at ×100 magnification beginning at the scratch line and extending as far out as the cells had migrated. All experiments were carried out in triplicate, and the results were reported as the number of migrating cells per millimeter of scratch.
-Catenin and actin immunohistochemical staining.
-Catenin staining was performed in IEC-6 cells according to the
method of Waterman-Storer et al. (50) with minor changes. Briefly, the cells were washed with D-PBS, fixed in 4%
paraformaldehyde, and then incubated with the specific anti-
-catenin
antibody used in Western blot analysis at 1:100 dilution overnight at
4°C. The cells were then washed three times and incubated with
secondary antibody conjugated with FITC (1:100) for 2 h at room
temperature. After the slides were rinsed three times, they were
mounted and viewed through a Zeiss confocal microscope (model LSM410).
Actin immunohistochemical staining was carried out as the method
described by Vielkind and Swierenga (43) with minor
changes. Briefly, cells were washed with D-PBS and then with D-PBS
without calcium and magnesium (D-PBS-Ca2+-Mg2+)
and fixed for 10 min at room temperature in one part 37% formaldehyde plus nine parts PEM buffer (10 mM PIPES, 5 mM EGTA, and 2 mM
MgCl2, pH 6.8, containing 0.2% Triton X-100) and
post-fixed with 95% ethanol at
20°C for 5 min. After being washed
two times with D-PBS-Ca2+-Mg2+, the cells were
covered with 0.2% Triton X-100 in PBS for 5 min. The fixed cells were
rehydrated in D-PBS-Ca2+-Mg2+ for 30 min at
room temperature and incubated for 30 min with 1% BSA in PBS (PBS/BSA)
to reduced nonspecific background staining. The cells were then
incubated with a 1:40 dilution of rhodamine-labeled phalloidin in
PBS/BSA for 45 min at room temperature. After three washes, slides were
mounted and examined in a confocal microscope.
Statistical analysis. All data are expressed as means ± SE from six dishes. Autoradiographic and immunofluorescence labeling results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined by using Dunnett's multiple range test (18).
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RESULTS |
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Effect of polyamines on total -catenin expression and
-catenin tyrosine phosphorylation.
To study the involvement of cellular polyamines in the regulation of
-catenin in normal intestinal epithelial cells, we examined total
-catenin protein expression, tyrosine phosphorylation of
-catenin, and cellular distribution of
-catenin after polyamine depletion in intact IEC-6 cells (nonwounding). Cells were grown for 4 and 6 days in the presence or absence of DFMO, the highly specific
inhibitor of ODC. Our previous studies have shown that exposure
of IEC-6 cells to DFMO for 4 and 6 days almost completely depletes
cellular polyamines. The levels of putrescine and spermidine were
undetectable at 4 and 6 days after DFMO treatment, whereas spermine was
decreased by >60% in DFMO-treated cells (35, 48).
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Effect of cellular polyamines on tyrosine phosphorylation of
-catenin during cell migration after wounding.
To determine changes in tyrosine phosphorylation of
-catenin during
intestinal epithelial cell migration, we collected whole cell lysates
at various times after wounding (removal of part of the monolayer), and
they were immunoprecipitated with the specific anti-
-catenin
antibody. Levels of PY-
-catenin in the precipitates were analyzed by
Western blotting with an anti-phosphotyrosine antibody. As shown in
Fig. 2A (top),
PY-
-catenin levels were significantly increased at 2 h and
peaked 6-8 h after wounding in control cells (without DFMO).
Maximum increases in PY-
-catenin levels were approximately three
times the prewounding control level [Fig. 2, A
(left) and Ba]. This induction of tyrosine
phosphorylation of
-catenin during intestinal epithelial cell
migration absolutely requires polyamines because depletion of cellular
polyamines by DFMO significantly inhibited the formation of
PY-
-catenin (Fig. 2A, middle). In DFMO-treated
cells, there were slight increases in PY-
-catenin after wounding.
Spermidine at a dose of 5 µM given together with DFMO prevented the
decreased PY-
-catenin in migrating IEC-6 cells. The levels of
PY-
-catenin in cells treated with DFMO plus spermidine were
indistinguishable from those of control cells after wounding (Fig.
2A, left vs. right). On the other
hand, there were no significant changes in levels of total
-catenin and E-cadherin proteins after wounding regardless of the presence or
absence of cellular polyamines [Fig. 2, A
(middle and bottom), Bb, and
Bc]. These findings indicate that intestinal epithelial cell migration after wounding is associated with a significant increase
in tyrosine phosphorylation of
-catenin, which is highly regulated
by cellular polyamines.
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Changes in PY--catenin binding activity in migrating cells.
To examine the consequences of the
-catenin phosphorylation during
cell migration, we immunoprecipitated the cadherin-catenin complex at
various times after wounding by the specific antibody against
-catenin, and the binding activity of
-catenin was analyzed by
determining the levels of
-catenin or E-cadherin in this complex. Figure 3 clearly shows that increased
PY-
-catenin in the cadherin-catenin complex reduced its binding
activity to
-catenin but not to E-cadherin in migrating IEC-6 cells.
Although levels of total
-catenin protein (Fig. 3Ab) were
unaffected after wounding in IEC-6 cells, the binding activity of
-catenin to
-catenin decreased with the increase in
PY-
-catenin. Significant decrease in the binding activity to
-catenin occurred at 2 h, the time when the marked increase in
PY-
-catenin was observed after wounding. The maximum inhibition
occurred at 6 h after wounding, and the
-catenin binding activity was decreased by ~70% (Fig. 3B). We also
examined the binding activity of
-catenin to
-catenin during
migration in polyamine-deficient cells and demonstrated that,
consistent with the effect on tyrosine phosphorylation of
-catenin,
the binding activity of
-catenin in DFMO-treated cells remained
unchanged after wounding (data not shown).
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Effect of [Ca2+]cyt on
-catenin tyrosine phosphorylation during cell migration.
Our previous studies (35, 49) have demonstrated that
cellular polyamines regulate [Ca2+]cyt by
governing the driving force for Ca2+ influx via controlling
activity of voltage-gated K+ (Kv) channels in IEC-6 cells
that do not express VDCC. Depletion of cellular polyamines by DFMO
inhibited Kv channel expression and resulted in membrane depolarization
(data not shown), which was associated with a decrease in
[Ca2+]cyt (Fig.
4A).
[Ca2+]cyt in DFMO-treated cells was ~50%
of normal values (without DFMO), and addition of spermidine to the
cultures containing DFMO restored [Ca2+]cyt
to normal levels. In addition, exposure of both control and polyamine-deficient cells to the Ca2+ ionophore ionomycin
reversibly increased [Ca2+]cyt by promoting
Ca2+ influx (Fig. 4B).
[Ca2+]cyt in control cells was increased
after the addition of 1 µM ionomycin for 5 min and rapidly returned
to basal levels when ionomycin was washed out (Fig. 4Ba).
Exposure of polyamine-deficient cells to ionomycin also significantly
increased [Ca2+]cyt, but the peak of
ionomycin-induced Ca2+ influx was reduced compared with
that observed in control cells. This reduced response of DFMO-treated
cells to ionomycin resulted from a decrease in the
Ca2+-driving force for Ca2+ influx, because
polyamine depletion inhibited Kv channel expression and resulted in
membrane depolarization (33, 35).
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Association of observed changes in PY--catenin and rates of cell
migration.
Consistent with the inhibitory effect on tyrosine phosphorylation of
-catenin, polyamine depletion by DFMO also significantly inhibited
cell migration in IEC-6 cells, which was completely prevented by
spermidine given together with DFMO (Fig.
6A). Furthermore, removal of
extracellular Ca2+ from the cultural medium decreased cell
migration in controls (Fig. 6Ba) and DFMO-treated cells
(Fig. 6Bb), whereas [Ca2+]cyt
increased by ionomycin promoted cell migration. Rates of cell migration
were decreased by ~80% when the Ca2+-free medium was
given immediately after wounding in both two groups. Exposure of IEC-6
cells to the Ca2+-free medium for 6 h did not alter
cell attachment and cell viability (data not shown). In cells exposed
to ionomycin, the rate of cell migration was increased by ~20% in
controls and ~40% in DFMO-treated cells, respectively. These results
indicate that changes in
-catenin tyrosine phosphorylation are
accompanied by the rates of intestinal epithelial cell migration.
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Effects of inhibition of -catenin tyrosine phosphorylation by TK
inhibitors on cell migration.
To study the role of induced
-catenin tyrosine phosphorylation in
the process of cell migration after wounding, we carried out two types
of experiments by using the potent and specific inhibitors of TKs,
genistein (1) and herbimycin-A (12). In the
first study, we examined whether inhibition of tyrosine kinase activity
by genistein or herbimycin-A altered the levels of PY-
-catenin and
cell migration in control cells (without DFMO). Figure
7 clearly shows that exposure to
genistein and herbimycin-A during cell migration decreased levels of
PY-
-catenin but had no effect on total
-catenin protein in IEC-6
cells. When various doses of genistein were tested,
-catenin
tyrosine phosphorylation was inhibited dose dependently, with
concentrations ranging from 25 to 100 µM. Maximum inhibition of
PY-
-catenin occurred at 100 µM, where the levels of PY-
-catenin
were decreased by ~80% (Fig. 7B, left). In
cells treated with herbimycin-A, PY-
-catenin levels were decreased
by ~45, ~60, and ~80% at doses of 0.5, 1, and 2 µg/ml,
respectively (Fig. 7B, right). Consistent with
the effect on
-catenin phosphorylation, exposure to genistein or
herbimycin-A also dose-dependently inhibited cell migration in control
cells (Fig. 8). When various doses of
genistein were given immediately after wounding, rates of cell
migration were decreased by ~25, ~40, and ~70% at 25, 50, and
100 µM, respectively. Similarly, cell migration was inhibited by
~30, ~40, and ~55% when herbimycin-A was given at doses of 0.5, 1, and 2 µg/ml during the period of cell migration. There was no
apparent loss of cell viability in cells treated with genistein or
herbimycin-A (Fig. 8B).
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Effects of [Ca2+]cyt
and PY--catenin on distribution of actin filaments.
In this study, we examined whether increasing or decreasing
PY-
-catenin levels altered cellular distribution of actin filaments after wounding. In control migrating cells, long stress fibers could be
seen traversing the cell, a network of cross-linked actin fibers of
actin cortex just inside the plasma membrane (Fig.
10Aa). Some very fine short
actin fibers outside the actin cortex extended into lamellipodia,
sometimes with a bright outer edge. Exposure of control cells to the
Ca2+-free medium during migration greatly reduced long
stress fibers, and in some cells they appeared to be absent (Fig.
10A, a vs. b). There was a pronounced
actin cortex around the entire cells that could be seen over the entire
monolayer. Decreased PY-
-catenin by genistein, herbimycin-A or
polyamine depletion with DFMO also affected cellular organization of
actin filaments (Fig. 10A, c, d, and
e). The features of distribution of actin stress fibers in
all these three groups were similar to those observed in cells exposed
to the Ca2+-free medium (Fig. 10A, b
vs. c, d, or e). The number of long
stress fibers was remarkably reduced, but the heavy actin cortex was observed around the whole cell in every experiment. Increased [Ca2+]cyt by exposure of polyamine-deficient
cells to ionomycin not only promoted tyrosine phosphorylation of
-catenin (Fig. 9, A and B) but also restored
the distribution of actin filaments to near normal (Fig.
10A, e vs. f). The organization of
actin stress fibers in cells treated with DFMO but exposed to ionomycin
after wounding was indistinguishable from that of control cells (Fig. 10A, a vs. f). On the other hand, the
quantity of actin protein in the different treatment groups, as
measured by Western blotting analysis, showed no significant difference
between them (Fig. 10B). These data indicate that
PY-
-catenin affects the regulation of actin distribution but has no
effect on actin protein formation.
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DISCUSSION |
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Epithelial cell migration is a primary process during rapid
mucosal restitution after superficial wounds in the gastrointestinal tract and requires precise control. Although cellular polyamines are
absolutely required for this process, few specific functions of
polyamines in epithelial cell migration have been defined. The dynamic
link between the cadherin-catenin complex and the actin filament
networks is essential for tight cell-cell contacts (2,
50), but the disruption of this association is crucial for the
process through which epithelial cells are rapidly disassembled during
restitution. Several studies have shown that disassembly of adhesion
junctions is frequently associated to augmented tyrosine phosphorylation of proteins presented in the complex (2, 4, 20,
39, 44). Among these proteins, the most relevant changes have
been detected in -catenin. The results presented here provide, to
our knowledge, the first demonstration that normal intestinal epithelial cell migration after wounding is accompanied by a
significant increase in tyrosine phosphorylation of
-catenin.
Cellular polyamines highly regulate this process by controlling
[Ca2+]cyt. These results suggest that
tyrosine phosphorylation of
-catenin is involved in rapid early
mucosal restitution and that polyamines are required for the
stimulation of epithelial cell migration after wounding through, at
least partially, alteration of cell-cell adhesion.
The findings reported here indicate that tyrosine phosphorylation of
-catenin reduces its interaction with
-catenin during intestinal
epithelial cell migration. This specific change in the binding activity
of PY-
-catenin to
-catenin is cell type-dependent because
induction of PY-
-catenin in NBT-II cells has been shown to decrease
its binding activity to both
-catenin and E-cadherin (28). The integrity of the cadherin-catenin complex is
essential for strong cell-cell adhesion; therefore, this reduced
interaction may lead to an overall decrease in intercellular contacts,
thus promoting cell disassembly and consequent spreading during
restitution. E-cadherin is the predominant cadherin in epithelial
tissue and is responsible for the correct establishment and maintenance
of adherens junctions (28, 39, 44). The adhesive function
of E-cadherin requires the attachment to the actin cytoskeleton, an
association mediated by catenins.
-Catenin is considered to be a
tyrosine phosphorylation-sensitive component of the adhesion complexes,
and the activation of tyrosine phosphorylation of
-catenin disrupts
adherens junctions and dissociates E-cadherin from the cytoskeleton
(4, 28, 39, 44, 53). Although the exact mechanisms
involved are still unclear, different possibilities are proposed to
explain the adhesive changes of cadherin-catenin complex in response to
-catenin tyrosine phosphorylation (5, 39, 50). First,
alteration in affinity of PY-
-catenin to
-catenin leads to a
decreased stability of cadherin-catenin complex during epithelial cell
migration. Second, conformational changes in the cadherin-catenin
complex following tyrosine phosphorylation of
-catenin result in the
disruption of linkage within cadherin and the cytoskeleton. Finally,
recruitment of unknown proteins or factors to the cadherin-catenin
complex enhances the dissociation of the cadherin-catenin from the cytoskeleton.
Cellular polyamines regulate tyrosine phosphorylation of -catenin
through Ca2+ in intestinal epithelial cells. Although
depletion of cellular polyamines by DFMO did not affect basal level and
cellular distribution of
-catenin protein in unwounded intestinal
epithelial cells, it almost completely prevented the induction of
tyrosine phosphorylation of
-catenin during epithelial cell
migration after wounding (Fig. 2). This inhibitory effect of polyamine
depletion on
-catenin tyrosine phosphorylation is consistent with
data from others (36, 40) who have reported that
polyamine-deficient IEC-6 cells have a general decrease in tyrosine
phosphorylation of focal adhesion kinase (FAK), which is associated
with the inhibition of both FAK activity and cell attachment. The
results presented in this report, however, have further demonstrated
that polyamines regulate tyrosine phosphorylation through a process
dependent on [Ca2+]cyt in intestinal
epithelial cells. As noted in Fig. 5, reduction of
[Ca2+]cyt through polyamine depletion or the
removal of extracellular Ca2+ inhibited tyrosine
phosphorylation of
-catenin during cell migration, whereas elevation
of [Ca2+]cyt by the Ca2+
ionophore ionomycin not only increased PY-
-catenin levels in control
cells but also restored the tyrosine phosphorylation to near normal in
polyamine-deficient cells.
Our previous studies have demonstrated that elevated
[Ca2+]cyt is a major mediator for the
stimulation of intestinal epithelial cell migration following an
increase in cellular polyamines (33, 35, 49). Cytoplasmic
free Ca2+ is an important intracellular second messenger
that modulates a large number of physiological functions (6, 8,
9). [Ca2+]cyt is controlled by
Ca2+ influx through Ca2+-permeable channels in
the plasma membrane and Ca2+ release from internal
Ca2+ stores (8, 31). Ca2+ influx
depends on the Ca2+ driving force (i.e., the
electrochemical gradient across the plasma membrane), which is
predominantly regulated by membrane potential
(Em) while the Ca2+ concentration
gradient is constant (10, 11, 13). Polyamines regulate
[Ca2+]cyt concentration primarily by
governing Em through control of Kv channel
expression in intestinal epithelial cells. Polyamine depletion
decreases Kv channel expression and voltage-gated K+
currents, leading to membrane depolarization and decrease in [Ca2+]cyt through reduction of the driving
force for Ca2+ influx (35, 49). We have
recently demonstrated that small GTPase RhoA is a downstream target of
elevated [Ca2+]cyt following activation of
K+ channels by increased cellular polyamines and that
Ca2+-activated RhoA activity increases stress fiber
formation in migrating cells during restitution (33). The
current studies provide additional new information that tyrosine
phosphorylation of -catenin is also implicated in the signaling
pathway of Ca2+-mediated intestinal epithelial cell
migration following increased polyamines after wounding.
Activation of -catenin tyrosine phosphorylation due to elevated
[Ca2+]cyt plays a critical role in
polyamine-dependent cell migration during early epithelial restitution.
Decreased PY-
-catenin levels caused by treatment with the specific
TK inhibitors genistein and herbimycin-A inhibited normal cell
migration (without DFMO) (Figs. 7 and 8). Consistent with our current
observations, it has been shown that induction of the tyrosine
phosphorylation of
-catenin by transfection of the cytoplasmic
tyrosine kinases such as v-src gene (17, 24) or
treatment with growth factors (19, 28, 42) causes unstable
cell-cell adhesion and promotes cell migration. In contrast, decreased
PY-
-catenin by transfection of either a dominant negative
src mutant or the PTP-LAR gene inhibits cell
migration (28, 38). An interesting and extended finding obtained in the current study is that decreased PY-
-catenin also prevents the restoration of cell migration by increasing
[Ca2+]cyt with ionomycin in
polyamine-deficient cells (Fig. 9). Taken together, the current results
and our previous findings (33, 35, 49) strongly support
the contention that polyamines stimulate intestinal epithelial cell
migration after wounding, at least partially, by altering cell-cell
adhesion through
-catenin tyrosine phosphorylation mediated by
[Ca2+]cyt.
Our results also show that tyrosine phosphorylation of -catenin is
involved in the regulation of cellular distribution of the cytoskeleton
during epithelial cell migration. Decreases in tyrosine phosphorylation
of
-catenin by genistein or herbimycin-A resulted in reorganization
of actin filaments. The numbers of long stress fibers were greatly
reduced, and the heavy actin cortex around the whole cell was observed
(Fig. 10). Although the exact mechanisms involved are obscure, it is
likely that the regulatory effect of
-catenin on cellular
distribution of actin filaments is independent of its role at adherens
junctions. It has been shown that
-catenin is an intracellular
mediator and interacts with different proteins and transcription
factors to perform various distinct signaling functions (5, 27,
52). Our previous studies demonstrated that elevation of
[Ca2+]cyt following increased cellular
polyamine levels activates RhoA activity during intestinal epithelial
cell migration (33). Activated Rho-A protein interacts
with cellular targets or effectors to regulate cytoskeletal
rearrangements and focal adhesions (16, 29). However, it
is not clear at present whether the involvement of
-catenin in the
regulation of actin filament networks is mediated by the interaction
between PY-
-catenin and RhoA during intestinal epithelial cell migration.
In summary, these results indicate that tyrosine phosphorylation of
-catenin is implicated in the signaling pathway of
polyamine-dependent intestinal epithelial cell migration after
wounding. Depletion of cellular polyamines reduces
[Ca2+]cyt and decreases tyrosine
phosphorylation of
-catenin, which accompany a decrease in
epithelial cell migration. An increase in
[Ca2+]cyt promotes
-catenin tyrosine
phosphorylation and stimulates epithelial cell migration regardless of
the presence or absence of cellular polyamines. Decreased
-catenin
phosphorylation by using the TK inhibitor either genistein or
herbimycin-A impairs epithelial cell migration after wounding. These
findings support a function of polyamines in the regulation of
-catenin tyrosine phosphorylation and the control of cell-cell
contacts during epithelial restitution. An increase in cellular
polyamines induces the levels of PY-
-catenin by altering
[Ca2+]cyt and decreases the association of
the cadherin-catenin complex with the cytoskeleton, thus promoting
epithelial cell disassembly and spreading after wounding.
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ACKNOWLEDGEMENTS |
---|
This work was supported by a Merit Review Grant from the Department of Veterans Affairs (to J.-Y. Wang), a Baltimore Research and Education Foundation Grant (to J. N. Rao), and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57819 (to J.-Y. Wang). J.-Y. Wang is a Research Career Scientist, Medical Research Service, Department of Veterans Affairs.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201 (E-mail: jwang{at}smail.umaryland.edu).
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.
April 24, 2002;10.1152/ajpcell.00054.2002
Received 4 February 2002; accepted in final form 18 April 2002.
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