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INTRODUCTION |
Cell migration maintains the organization and integrity of the
mucosa of the gastrointestinal tract (1). New cells migrate from the
crypts of the small intestine onto the villi and differentiate as they
move toward the tips. Mature cells are sloughed into the lumen and
replaced from below. The damaged mucosa rapidly repairs itself through
two basic processes (2). The early phase consists of the loss of
damaged cells and the migration of remaining viable cells over the
denuded lamina propria. This process of early mucosal restitution
rapidly seals the wound and re-establishes the barrier to luminal
contents (3). The later phase of mucosal repair is the replacement of
lost cells by mitosis and does not take effect until 24 h or so
after injury. We have examined the early phase and the process of cell
migration using IEC-6 cells, a non-transformed, putative crypt cell
line derived from adult rat intestine and originally described by
Quaroni et al. (4). This model resembles the early phase of
mucosal healing in the gastrointestinal tract in that cell migration is
independent from DNA synthesis, has a complete dependence on actin
polymerization (5), and depends on polyamines (6).
Cell migration requires an intact and functioning cytoskeleton, which
for the most part consists of filamentous or F-actin, tubulin, and
intermediate fibers. F-actin is formed by the polymerization of 420-kDa
monomers termed G-actin. These filaments of actin with their associated
binding proteins make up the actin cortex, a dense network just inside
the inner surface of the plasma membrane (7). Long filaments of actin
traverse the cell as stress fibers, and short filaments extend into the
lamellipodia that are prominent during migration (8). Focal adhesions
provide the necessary attachments to the substrate and via the stress
fibers allow the cytoskeleton to exert force on the extracellular
matrix (9). Cells initiate migration in response to receptor signaling
via integrins and the extracellular matrix (10) or in response to soluble factors (11).
Soluble factor and integrin signaling is relayed to the cytoskeleton by
signal transduction pathways involving a subgroup of the Ras
superfamily of small GTP-binding proteins (12). The Rho (for
Ras homology) GTPases consist of three major
types of small (21 kDa) proteins that bind and hydrolyze GTP. Their
intrinsic GTPase activity is controlled by guanine nucleotide exchange
factors and GTPase-activating proteins, known as GAPs. Rho guanine
nucleotide dissociation inhibitor is an inhibitory guanine nucleotide
exchange factor that prevents the dissociation of guanosine diphosphate from Rho as well as from Rac and Cdc42, the two other members of this
family (13, 14). Rho and Rac regulate the polymerization of actin to
produce stress fibers and lamellipodia, respectively (15, 16). Cdc42
has been shown to be responsible for the formation of filopodia (12).
In NIH 3T3 fibroblasts, the Rho GTPases can be activated sequentially
in that activation of Cdc42 activates Rac, which in turn activates Rho
(12). Thus, there appears to be a mechanism for the coordinated
regulation of the cytoskeleton through activation of the Rho GTPases.
Our laboratory has been interested in the cellular actions of
polyamines, which are involved in many aspects of membrane function including stability, Ca2+ homeostasis, and ion transport
(17). Polyamines are also involved in the organization of the
cytoskeleton and cell migration. Polyamine-deficient Chinese hamster
ovary cells lack actin filaments and microtubules unless polyamines are
supplied exogenously (18). Inhibitors of polyamine synthesis prevent
concanavalin A-induced expression of
-tubulin and
-actin
mRNAs in mouse splenocytes (19). In vitro, polyamines
stimulate the rapid polymerization of G-actin and formation of bundles
from F-actin, indicating a possible direct effect of polyamines on
cytoskeletal organization (20). We have shown that the early phase of
mucosal healing, which is due to cell migration, requires polyamines
(21, 22) and that polyamine depletion inhibits migration in the IEC-6
cell model and leads to numerous alterations in the cytoskeleton. When
cells were depleted of polyamines with
-difluoromethylornithine
(DFMO),1 which inhibits ornithine
decarboxylase, the first rate-limiting enzyme in polyamine synthesis,
there was a significant decrease in actin stress fibers and a
corresponding increase in the density of the actin cortex (23). There
was also a redistribution of tropomyosin from stress fibers to the
actin cortex. Additional changes in response to polyamine depletion
included a marked reduction in the formation of lamellipodia and a
dissociation of actin from nonmuscle myosin II (24). Although no
changes occurred in the absolute amounts of G- and F-actin, the
association of actin with the sequestering protein thymosin
4 was
inhibited (25). All of the alterations in the cytoskeleton as well as
the inhibition of migration caused by DFMO were prevented if exogenous
polyamines were supplied in the presence of DFMO.
Santos et al. (26) demonstrate that Rho was required for the
actual migration of IEC-6 cells after wounding or in response to growth
factor stimulation. Migration was inhibited after microinjection of Rho
guanine nucleotide dissociation inhibitor Clostridium
botulinum C3 ADP-ribosyltransferase toxin or Rho T19N,
a dominant negative form of RhoA. Inactivation of Rho by these means
not only inhibited migration but also altered the cytoskeleton in ways
that were identical to those produced by polyamine depletion. With this in mind we recently examined the effects of polyamine depletion on RhoA
(27). We found that DFMO caused a significant decrease in RhoA levels
in the cytoplasm and membranes of IEC-6 cells. This decrease was due to
an approximate 50% inhibition in RhoA synthesis. Neither the half-life
of RhoA nor the level of RhoA mRNA was affected. Constitutively
active HA-V14 RhoA cells migrated much more rapidly than
vector-transfected cells, and dominant negative HA-N19-RhoA cells
exhibited almost no motility. Surprisingly, the depletion of polyamines
almost totally inhibited the migration of the cells expressing
constitutively active Rho. Polyamine depletion did not affect the
activity of RhoA in the HA-V14-RhoA cells but inhibited it dramatically
in the vector-transfected cells (27). After the loss of polyamines,
constitutively active HA-V14-RhoA cells had fewer stress fibers and
took on the appearance of the HA-N19-RhoA cells and the
polyamine-depleted wild type cells. Thus, although RhoA activity is
essential for the migration of intestinal epithelial cells, it is not
sufficient. And although polyamines are necessary for RhoA synthesis
and activity, they are also required for an additional step either
upstream or downstream from RhoA that results in cell migration
(27).
Both Rac1 and Cdc42 are excellent candidates for additional steps in
the process of cell motility to be regulated by polyamines. There is
considerable signaling cross-talk between Rho, Rac, and Cdc42, and each
of these molecules regulates some aspect of cytoskeletal transformation
(12, 16). In fact Rac1 has also been shown to be involved in stress
fiber formation (16). In the current paper, we show that the activities
of Rac and Cdc42, like that of RhoA, are
polyamine-dependent. We demonstrate that the rates of
migration of IEC-6 cells transfected with constitutively active Rac or
Cdc42 are significantly more rapid than those of the corresponding wild
type cells or those transfected with empty vectors. We also demonstrate
that Rac1 activation leads to RhoA and Cdc42 activation independent of
polyamines, which explains the restoration of the cytoskeleton and
migration rate in constitutively active Rac1 cells depleted of polyamines.
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EXPERIMENTAL PROCEDURES |
Materials--
Disposable culture ware was purchased from
Corning Glass Works (Corning, NY). Media and other cell culture
reagents were obtained from Invitrogen. Dialyzed fetal bovine serum
(FBS) and other biochemicals were purchased from Sigma. Plasmids,
pMX-IRES-GFP-V12-Rac1, pMX-IRES-GFP-N17-Rac1, pMX-IRES-GFP-F28L-Cdc42, pMX-IRES-GFP-N17-Cdc42, and pMX-IRES-GFP (vector) were used for the transfection experiments. FuGENETM 6 transfection reagent was a gift from Roche Diagnostics. The primary antibodies, affinity-purified mouse monoclonal antibodies against RhoA
and affinity-purified rabbit polyclonal antibodies against Rac1 and
Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). Matrigel was obtained from Collaborative Research Inc. (Bedford,
MA). DFMO was the kind gift of Ilex Oncology Inc. (San Antonio, TX).
Cell Culture--
The IEC-6 cell line was purchased from the
American Type Culture Collection at passage 13. 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 cell cultures were maintained in T-150 flasks in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 5% FBS, 10 µg
insulin, and 0.05 mg of gentamicin sulfate/ml. The flasks were
incubated at 37 °C in a humidified atmosphere of 90% air, 10%
CO2. Stock cells were passaged once a week at 1:20; medium was changed 3 times weekly. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative.
General Experimental Protocols--
The general protocol for the
experiments and the methods used were similar to those described
previously (27). In brief, IEC-6 cells were plated at 6.25 × 104 cells/cm2 in DMEM supplemented with 5%
dialyzed FBS, 10 µg of insulin, and 50 µg of gentamicin sulfate/ml
(DMEM/dialyzed FBS, control) or in DMEM/dialyzed FBS containing 5 mM DFMO or DFMO plus 10 µM putrescine. Cells
were grown at 37 °C in a humidified atmosphere of 90% air, 10%
CO2. They were fed every other day and serum-starved during
the 24 h before harvesting or cell migration assay. Previously we
have shown that maximal depletion of polyamines occurs after 4 day of
DFMO treatment (6, 28), and others also report an identical pattern of
polyamine depletion (29). In IEC-6 cells putrescine was completely
absent after 6 h of exposure to DFMO and after 24 h to
spermidine, and 40% of the spermine remained after 4 days. DFMO
inhibits ornithine decarboxylase, preventing the synthesis of
putrescine; therefore, exogenous addition of putrescine along with DFMO
restores endogenous spermidine and spermine. Thus, cells grown in DFMO
plus putrescine are an important control showing that effects are due
to the depletion of polyamines and not to pharmacological effects of
DFMO.
Cell Migration Assay--
Cells were grown in control medium for
4 days or in medium to which DFMO was added at the time of plating for
4 days or on days 1, 2, and 3 during feeding for 3-, 2-, and 1-day
treatments, respectively. Another group of cells was grown in DFMO plus
putrescine for 4 days. Cells were serum-starved for 24 h before
the experiment (during day 4). For the cell migration assay, plates
containing a confluent monolayer of cells were marked in the center by
drawing a line along the diameter of the plate with a black marker.
Wounding of the monolayer was carried out perpendicular to the marked
line using a gel-loading micro-tip. Plates were washed, and the area of
migration was photographed with a video camera system using NIH Image
software (Version 1.58*) at the intersection of the marked line and
wound edge at 0 h (WW0) and at desired time intervals (WWT). Cell migration was calculated as wound width covered at
time t (WW0
WWT) and expressed as % control. Each experiment was carried out three times in duplicate, and
each plate was wounded twice. Therefore, n was considered to
be 6 even though results are the means of 12 observations.
RhoA, Rac1, and Cdc42 Activation Assay--
Biological
activities of RhoA, Rac1, and Cdc42 proteins were assayed by pull-down
assays following the method of Kranenburg et al. (30).
GST-Rho-kinase, GST-mDia, GST-PKN, and GST-PAK fusion proteins
were prepared by lysing the bacteria (Escherichia coli
BL21-DE-3pLysE strain transformed with GST fusion protein construct) in
a buffer containing 1% Nonidet P-40, 50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM MgCl2, and 10%
glycerol supplemented with protease inhibitors. The lysates were then
sonicated and cleared by centrifugation at 10,000 × g
for 15 min. The fusion proteins were recovered by the addition of
glutathione beads to the supernatants. The beads were washed three
times in cell lysis buffer and resuspended before the addition of the
cell lysates (200 µg of protein for RhoA and Cdc42 and 100 µg of
protein for Rac1). After 1 h of tumbling at 4 °C, beads were
washed with lysis buffer, and the amounts of RhoA, Rac1, and Cdc42
proteins bound to GST fusion proteins were analyzed by SDS-PAGE and
Western blot using RhoA-, Rac1-, and Cdc42-specific antibodies. Protein
(20 µg) from each sample was resolved by SDS-PAGE to determine the levels of RhoA, Rac1, and Cdc42 proteins.
Western Blot Analysis--
Protein was separated on 15%
SDS-PAGE and transferred to polyvinylidene difluoride membranes by
electroblotting. The membranes were then probed with an antibody
directed against one of the proteins (RhoA, Rac1, Cdc42, or actin).
Immunocomplexes were visualized by the enhanced chemiluminescence
detection system and quantitated by densitometric scanning.
Transfection--
IEC-6 cells were transfected with
constitutively active and dominant negative Rac1 and Cdc42 as well as
vector. DNA was prepared by using a Qiagen (endotoxin-free) plasmid
preparation kit. Cells were grown as mentioned earlier to 70-80%
confluence in 60-mm culture dishes. FuGENE 6 reagent was mixed with
constitutively active (pMX-IRES-GFP-V12-Rac1), dominant negative
(pMX-IRES-GFP-N17-Rac1), constitutively active
(pMX-IRES-GFP-F28L-Cdc42), dominant negative (pMX-IRES-GFP-N17-Cdc42),
or vector (pMX-IRES-GFP) DNA (3 µl: 2 µg) in serum-free medium to a
total volume of 100 µl and incubated 15 min at room temperature. The
reaction mixture was added dropwise onto monolayers of cells containing
4.0 ml of serum-free medium and further incubated for 12 h at
37 °C. Cells were fed after 12 h with fresh medium. The
limiting dilution technique was used to select stable clones. Selected
clones were grown to confluence, and the characterization of clones was
carried out by measuring cell migration and the direct visualization of
green fluorescence protein (GFP).
Immunohistochemical Localization of Endogenous and Recombinant
Rac1--
Transfected cells were grown for 4 days, trypsinized,
replated in 35-mm dishes (each containing a matrigel-coated glass
coverslip), and allowed to attach and spread. Cells were fixed with
4.0% formaldehyde, washed with DPBS, permeabilized with 0.2% Triton
X-100 for 5 min, and blocked with 3% BSA for 20 min. Cell monolayers
were stained with Rac1 antibody for 1 h followed by goat
anti-rabbit IgG-Texas Red (Chemicon Intl., Temecula, CA) at a 1:60
dilution for 1 h. Images were captured by digital confocal
microscopy and processed with NIH image.
Immunocytochemistry--
Control, DFMO, and DFMO plus
putrescine-treated (4 day) V12- Rac1, F28L-Cdc42, and
vector-transfected cells were plated in 35-mm dishes (each containing a
matrigel-coated glass coverslip) and allowed to attach and spread for
24 h. Cells were fixed with 4.0% formaldehyde, washed with DPBS,
permeabilized with 0.2% Triton X-100 for 5 min, and blocked with 3%
BSA for 20 min. Cell monolayers were stained with Texas-red conjugated
phalloidin for 1 h. Images were captured by digital confocal
microscopy and processed with NIH image.
Statistics--
All data are expressed as means ± S.E.
from six dishes. Autoradiographic results were repeated three times.
Densitometry of Western blots was done by NIH image analysis. The
significance of the differences was determined using Dunnett's
multiple-range test (R), and values of p < 0.05 were considered significant.
 |
RESULTS |
Polyamine Depletion Inhibits the Activities of RhoA, Rac1, and
Cdc42--
To study the effect of polyamine depletion on activities of
Rho family GTPases, it is important to determine the relative amounts
of activated RhoA, Rac1, and Cdc42 in normal IEC-6 cells. Whole cell
extracts equivalent to 25-200 µg of protein were subjected to
pull-down assay using either GST-PKN and GST-mDia (for RhoA) or GST-PAK
(for Rac1 and Cdc42) protein. Pull-down samples along with 25 µg of
whole cell extract was resolved by SDS-PAGE and analyzed by Western
blot analysis. Confluent IEC-6 cells grown in control medium showed
high levels of active Rac1 protein in as low as 25 µg of total
protein (Fig. 1A). In contrast, a
significant amount of active RhoA protein was detected only in 200 µg
of total protein (Fig. 1B). Although active Cdc42 protein
was evident in as low as 50 µg of total protein, the total amount of
activated Cdc42 was lower than amounts of active Rac1 and RhoA (Fig.
1C). Total protein levels of RhoA and Rac1 were comparable
and significantly higher than that of Cdc42. Thus, in subsequent
experiments we used 100 µg of total protein for Rac1 and 200 µg of
total protein for the determination of RhoA and Cdc42 activity.
Polyamine depletion by treatment of cells with DFMO for 4 days
significantly decreased the activities of all three Rho GTPases (Fig.
2). Active RhoA decreased to 35 or 20% of
control levels depending on whether binding to mDia or to PKN was used
in the pull-down assay, respectively. There was also a 40% decrease in
the level of RhoA protein when cells were grown for 4 days in the
presence of DFMO. PAK1 was used to pull down both active Rac1 and
active Cdc42. Rac1 activity was reduced to 40% and Cdc42 activity was
reduced to 23% that of control levels. There was no change in the
level of Rac1 protein, but Cdc42 levels decreased to 45% of normal. In
each case supplying putrescine along with DFMO during growth prevented
the decrease (Fig. 2).

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Fig. 1.
Relative amounts of total and active
(GTP-bound) Rac1, RhoA, and Cdc42 proteins in confluent IEC-6 cells
grown for 4 days in control medium (DMEM-containing serum). Cell
extracts equivalent to 25-200 µg of total protein were subjected to
pull-down assays using GST-PAK or GST-PKN. Samples from the pull down
assays were resolved by SDS-PAGE along with 25 µg of total protein
from the same cell extracts. Western blot analysis was performed using
RhoA-, Rac1-, and Cdc42-specific antibodies to determine the levels of
activated and total Rac1, RhoA, and Cdc42 proteins. Representative
Western blots from three observations are shown.
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Fig. 2.
Polyamine depletion inhibits activation of
RhoA, Rac1, and Cdc42 in IEC-6 cells. Cells were grown in the
presence of 5 mM DFMO or DFMO plus 10 µM
putrescine for 4 days. Equal amounts of protein (200 µg for RhoA and
Cdc42 and 100 µg for Rac1) were used for the pull-down assays as
described under "Experimental Procedures." 25 µg of protein was
used to determine the levels of total RhoA, Rac1, and Cdc42 proteins.
Western blot (WB) analysis using RhoA-, Rac1-, and
Cdc42-specific antibodies was carried out. A, active RhoA
protein bound to GST-mDia. B, active RhoA protein bound to
GST-PKN. C, RhoA protein levels in whole cell extract.
D, active Rac1 protein bound to GST-PAK. E, Rac1
protein levels in whole cell extract. F, active Cdc42
protein bound to GST-PAK. G, Cdc42 protein levels in whole
cell extract. H, actin levels in whole cell extract.
Representative Western blots and densitometry readings from three
observations are shown.
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As shown in Fig. 3A the activities
of RhoA, Rac1, and Cdc42 decreased little when cells were treated with
the DFMO for 1, 2, and 3 days. There were dramatic decreases in the
activities of all 3 to below 50% that of control on the 4th day of
DFMO treatment. The levels of RhoA and Cdc42 proteins followed a
similar pattern, and there was no significant decrease in Rac1 protein
(Fig. 3A). Fig. 3B illustrates the means of the
quantified data of the activities of all three Rho GTPases. As in the
studies depicted in Fig. 2, the addition of putrescine to the
incubation medium containing DFMO prevented all of the effects of
ornithine decarboxylase inhibition.

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Fig. 3.
Time course of the effect of polyamine
depletion on the activation of RhoA, Rac1, and Cdc42 in IEC-6
cells. A control group of cells was grown for 4 days in DMEM/FBS
to confluence. 5 mM DFMO was added to other groups on days
0, 1, 2, or 3 during the 4-day growth period to deplete polyamines for
4, 3, 2, and 1 days respectively. Another group was exposed to DFMO
plus 10 µM putrescine (PUT) for 4 days.
A, 200 µg (for RhoA, Cdc42) and 100 µg (for Rac1) of
total protein from each sample was used for the pull-down assay using
GST-PKN or GST-PAK fusion protein. GST-PKN- or GST-PAK-bound proteins
were resolved by SDS-PAGE and subjected to Western blot analysis using
antibodies specific for RhoA, Rac1, and Cdc42. 25 µg of protein from
whole cell extracts was resolved by SDS-PAGE and subjected to Western
blot analysis for the detection of total RhoA, Rac1, and Cdc42 protein.
Representative Western blots from 6 observations are shown.
B, specific activities of RhoA, Rac1, and Cdc42 calculated
by the densitometric readings of the Western blots. Values are
means ± S.E. of six observations. *, significantly different from
control (p < 0.05).
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Rac1 and Cdc42 Protein Expression Alters IEC-6 Cell
Migration--
Clones of IEC-6 cells transfected with vector and with
dominant negative and constitutively active genes of Rac1 and Cdc42 were characterized by the direct visualization of green fluorescent protein and by determining rates of cell migration. Digital confocal micrographs of one clone of each of the five types are shown in Fig.
4. In each case, all cells expressed GFP,
indicating successful transfection and clonal selection. Transfection
with dominant negative constructs of both Rac1 and Cdc42 inhibited cell
migration compared with vector-transfected cells, whereas transfection
with constitutively active constructs significantly increased the rates of migration (Fig. 5A). As shown
in Fig. 5B, by 8 h vector-transfected cells had
migrated to cover ~40% of the original wound. This number was
reduced to 25% in cells carrying dominant negative Rac1 and to 10% in
cells with dominant negative Cdc42. On the other hand, cells
transfected with constitutively active Rac1 covered more than 85% of
the wound width and those with constitutively active Cdc42 had migrated
over 60% of the original wound.

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Fig. 4.
IEC-6 cells transfected with vector
(pMX-IRES-GFP), dominant negative (DN) Rac1, and Cdc42
(N17-Rac1, N17-Cdc42) and constitutively active (CA)
Rac1 and Cdc42 (V12-Rac1, F28L-Cdc42) were grown for 4 days as
described earlier and in 35-mm dishes (each containing a
matrigel-coated glass coverslip). Expression of green fluorescence
protein was detected by digital confocal microscopy, and the images
were processed with NIH image.
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Fig. 5.
Rac1 and Cdc42 protein expression influences
IEC-6 cell migration. A, cells transfected with vector
(pMX-IRES-GFP), constitutively active (CA) Rac1 and Cdc42,
and dominant negative (DN) Rac1 and Cdc42 were grown in
DMEM, 5% FBS for 4 days. Confluent monolayers were wounded with a
gel-loading tip in the center of plates marked to localize the wound
site. Plates were photographed immediately to record the wound width (0 h), washed, and incubated with fresh serum free medium. Plates were
photographed at the marked wound location after 10 h of
incubation. A plate containing vector cells at 0 and 10 h is shown
as a representative control. Representatives of three experiments are
shown. B, quantitative analysis of migration showing wound
width covered as compared with initial scratch size (0 h) using NIH
image analysis. Values are the mean ± S.E. of six observations.
*, Significantly different from vector (p < 0.05).
DN, dominant negative; CA, constitutively
active.
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Expression of Activated Recombinant Rac1 and Cdc42 Influences the
Migration of Polyamine-depleted IEC-6 Cells--
Polyamine depletion
inhibited the migration rate of vector-transfected cells by ~50%
(Fig. 6, A and B).
Cells transfected with constitutively active Rac1 had migrated to close
~85% of the initial wound width by 8 h, and polyamine depletion
had no significant effect in these cells (Fig. 6, A and
B). Polyamine depletion inhibited migration of cells
expressing constitutively active Cdc42 by ~30%. Supplying putrescine
to cells incubated with DFMO prevented its effects on the migration of
cells expressing only vector and those expressing active Cdc42. In
summary, polyamine depletion had no effect on the migration of cells
expressing Rac1. It did inhibit the migration of cells expressing Cdc42
but to a lesser extent compared with cells transfected with vector.

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Fig. 6.
Activated recombinant Rac1 and Cdc42
expression influences the migration of polyamine-depleted IEC-6
cells. A, vector (pMX-IRES-GFP) and constitutively
active V12-Rac1- and F28L-Cdc42-transfected cells were grown in control
media, control media plus 5 mM DFMO, and control media plus
DFMO plus 10 µM putrescine for 4 days. After serum
starvation, monolayers were wounded as described in Fig. 5. 0- and 8-h
images of wounds are shown for control, DFMO and DFMO + putrescine
(Put) group from the same plate. Panels are representative
of six observations. B, quantitative analysis of migration
showing wound width covered (%) compared with initial scratch size
(0 h) using NIH image analysis. Values are the mean ± S.E. of six
observations. *, Significantly different from control
(p < 0.05).
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Polyamine Depletion Does Not Alter Rac1 or Cdc42 Activity in Cells
Expressing Those Proteins--
The expression of active Rac1 or active
Cdc42 prevented the inhibition of the activities of those respective
proteins that occurred in normal wild type IEC-6 cells after polyamine
depletion (Fig. 7). As one would expect,
incubation with DFMO inhibited Rac1 activity in vector-transfected
cells to the same extent as it did in wild type cells. Fig. 7 also
shows that polyamine depletion had no effect once vector-transfected
cell extracts had been activated with GTP
S. These data suggest that,
irrespective of total protein levels, activated Rac1 and Cdc42 protein
levels were significantly higher in DFMO-treated cells expressing
constitutively active proteins, and Rac1 activation in the absence of
polyamines is not limited by guanine nucleotide exchange factors.

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Fig. 7.
Polyamine depletion does not alter Rac1 and
Cdc42 activation in constitutively active (V12) Rac and (F28L) Cdc42
cells but inhibits Rac1 activation in vector (pMX-IRES-GFP)-transfected
IEC-6 cells. Extracts of control cells (C) and those
grown in DFMO (D) or DFMO plus putrescine (DP)
for 4 days were subjected to the GST-PAK pull down assay described
"Experimental Procedures." Extracts of vector-transfected cells
were also preincubated with GTP S for 15 min before the addition of
GST-PAK for the pull-down assay. 25 µg of total protein was used to
determine total Rac1 and Cdc42 protein. Western blot analysis was
performed using Rac1- and Cdc42-specific antibodies to determine the
levels of activated and total Rac1 and Cdc42 proteins. Representative
Western blots and densitometry readings from three observations are
shown.
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Expression of Constitutively Active Rac1 Prevents Cytoskeletal
Changes Associated with Polyamine Depletion--
As pointed out in the
Introduction, after polyamine depletion wild type IEC-6 cells lose
lamellipodia and stress fibers and develop a heavy actin cortex. That
same pattern is shown in vector-transfected cells (Fig.
8). In cells, expressing constitutively
active Rac1, however, the cytoskeleton appeared immune to polyamine
depletion. There were no visible differences in stress fibers,
lamellipodia, or the actin cortex in polyamine-depleted V12-Rac1 cells
compared with controls or those treated with both DFMO and putrescine
(Fig. 8). Compared with vector-transfected control cells the V12-Rac1 cells showed more stress fibers and a decrease in cortical actin. Both
of these changes are indicative of more actively migrating cells.
Constitutively active Cdc42 expressing cells migrated at a rapid rate
compared with DFMO-treated vector cells, and as one would predict, the
actin cytoskeleton of polyamine-depleted cells was similar to V12-Rac1
cells (data not shown).

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Fig. 8.
Constitutively active Rac1 prevents the
effects of polyamine depletion on the F-actin cytoskeleton of IEC-6
cells. Vector (pMX-IRES-GFP) and constitutively active (V12)
Rac1-transfected cells were grown for 4 days as described earlier and
replated in 35-mm dishes (each containing a matrigel-coated glass
coverslip). Cells were allowed to attach and spread for 24 h in
control and DFMO- and DFMO plus putrescine (Put)-containing
medium. Cells were washed, fixed, and stained with Texas Red phalloidin
for the localization of F-actin. Representatives of three experiments
are shown.
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Activation of RhoA, Rac1, and Cdc42 in Normal and
Polyamine-depleted V12-Rac1, V14-RhoA, and F28L-Cdc42-expressing
Cells--
To understand the hierarchy of activation of the Rho
GTPases in epithelial cells, we determined the activities of RhoA,
Rac1, and Cdc42 in cells expressing constitutively active RhoA, Rac1, and Cdc42 grown to confluence for 4 days in control, DFMO, and DFMO
plus putrescine-containing medium. Results in Fig.
9 show that activated Rac1 is significantly
reduced in vector-transfected polyamine-depleted cells. V12-Rac1
(constitutively active)-expressing cells grown in the presence of DFMO
showed the expected increase in activated Rac1. Because V12-Rac1
prevented the cytoskeletal changes and the inhibition of migration
associated with polyamine depletion and because RhoA and Cdc42 are
essential to cell migration, we examined RhoA and Cdc42 activity in
Rac1-transfected cells. Vector-transfected cells had basal levels of
RhoA and Cdc42 activity essential for normal migration, whereas
polyamine depletion significantly decreased RhoA and Cdc42 protein
levels and activities. As expected, V12-Rac1 cells had increased RhoA
and Cdc42 activity in both control and polyamine-depleted cells.
Although, polyamine-depleted cells had low levels of RhoA and Cdc42
protein, a high proportion of existing RhoA and Cdc42 protein was in
the active state in Rac1-transfected cells compared with
Vector-transfected cells (Fig. 9). Thus, Rac1 activated RhoA and Cdc42
in the presence or absence of polyamines in IEC-6 cells. In contrast,
Rac1 activity was not increased in V14-RhoA-transfected cells, whereas
Cdc42 activation was stimulated (Fig. 10).
Constitutively active Cdc42 (F28L)-expressing cells had significant
increases in both RhoA and Rac1 activity in normal as well as
polyamine-depleted cells (Fig. 10).

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Fig. 9.
Constitutively active V12-Rac1 activates RhoA
and Cdc42 in polyamine-depleted IEC-6 cells. Extracts of vector
and V12-Rac1 transfected-cells grown as control (C), DFMO
(D), or DFMO plus putrescine (DP) groups for 4 days were subjected to the GST-PAK and GST-PKN pull-down assay
described in the under "Experimental Procedures." Western blot
analysis was performed using RhoA-, Rac1-, and Cdc42-specific
antibodies to determine the levels of activated Rac1, RhoA, and Cdc42
proteins as well as total protein. Representative Western blots from
three observations are shown.
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Fig. 10.
Activities of Rho GTPases in normal and
polyamine-depleted cells expressing constitutively active RhoA and
Cdc42. Vector and V14-RhoA- and F28L-Cdc42-ransfected cells were
grown for 4 days in control medium (C) and control medium
with DFMO (D) and DFMO plus putrescine (DP). Cell
extracts were used for pull-down assays for RhoA, Rac1, and Cdc42 as
described in Fig. 9. Western blot analysis was performed using RhoA-,
Rac1-, and Cdc42-specific antibodies to determine the levels of
activated Rac1, RhoA, and Cdc42 proteins. Representative Western blots
from three observations are shown.
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DISCUSSION |
Although polyamines are required for such basic cell processes as
proliferation and migration, the actual mechanism of their action at
the cellular and molecular levels is poorly understood. There is
agreement, however, that the effects of these compounds depend on their
strong positive charges. Putrescine, spermidine, and spermine contain
2, 3, or 4 amine groups, respectively, with pK values above 9, so they
are polycations at physiological pH and strongly bind to negatively
charged macromolecules, particularly nucleic acids and proteins. The
fact that these are not point charges but are fixed along a flexible
carbon chain allows polyamines to interact with macromolecules in
structurally specific ways. Spermine molecules, for example, bind to
phosphate groups of the DNA helix, occupying the small groove and
stabilizing the helix by binding the two strands together (31, 32). The
most frequently studied mechanisms of action of polyamines concern
membranes (17) and growth-regulated genes (33). Polyamines stabilize
cell membranes (34) including those of organelles such as the Golgi
(35). The roles of polyamines in regulating membrane-bound
enzymes, the transport of ions and metabolites, Ca2+
homeostasis, protein kinase C, and membrane fusion have been reviewed
in detail (17). Spermine and spermidine block the inward rectifying
K+ channel and, thus, perhaps act as physiological
regulators of its activity (36, 37). Wang and co-workers (38, 39) have recently shown that polyamines may increase K+
channel-mediated Ca 2+ influx, which is required for
cell migration, and concluded that this is related to the levels of
RhoA protein. There is also accumulating evidence that polyamines can
influence the phosphorylation and, hence, the activation of kinases
(40).
The most important single finding in our study is that the activation
of Rac1 and, to a lesser extent, Cdc42 allowed the migration of
polyamine-depleted intestinal epithelial cells. In fact, the depletion
of polyamines had no discernible effect on either the migration or the
cytoskeletal structure of cells constitutively expressing Rac1
activity. As shown in Fig. 2 polyamine depletion inhibited the
activities of all three Rho- GTPases. We have previously shown that
RhoA activity is essential for cell migration of this same cell line
(26). However, unlike Rac activity, RhoA activity is not sufficient for
migration as polyamine depletion inhibited cell migration in cells
constitutively expressing activated RhoA to the same extent that it
inhibited migration of wild type and vector-transfected cells (27).
These results imply that polyamines are required for the direct
activation of Rac1 or for a step upstream from, and leading to, the
activation of Rac1. Once Rac is activated cell migration is independent
of polyamines.
Rao et al. (39) show that increased Ca2+ influx
results in the synthesis of RhoA and propose that
polyamine-dependent cell migration is mediated by the
expression of RhoA. They found decreased RhoA protein levels in
polyamine-depleted cells similar to our current finding (Fig. 1) and to
our previous report (27), but they did not measure the activity of
RhoA, so it is not known whether Ca2+ influx was correlated
with the actual activity of the protein. Signal transduction pathways
are characteristically regulated by changes in the activation of
preexisting proteins rather than by stimulating their synthesis or
decreasing their degradation in response to rapid changes in extra- and
intracellular signals. Fig. 9 shows that polyamine depletion decreased
the level of RhoA protein in constitutively active Rac1 cells to the
same extent that it did in the control vector-transfected cells.
Because these cells migrated normally, it means that the levels of RhoA
protein have little to do with the inhibition of migration after
polyamine depletion.
Interestingly, in Fig. 2, Rac1 protein levels remained normal in
polyamine-depleted cells, whereas the levels of RhoA and Cdc42
decreased to 60 and 45% that of normal, respectively. Thus, protein
levels of Rac1 remain normal throughout the time course of polyamine
depletion (Fig. 3A), and Rac1 is affected only by an
approximate 60% inhibition of activity occurring between day 3 and day
4 of exposure to DFMO. It is between days 3 and 4 that spermine levels
decrease significantly (6). At this same time there are similar
decreases in the activities of RhoA and Cdc42. This time course
parallels that for the significant decrease in cell migration, which
occurs after incubation with DFMO (27). Earlier Yuan et al.
(41) showed that restoration of migration was most effectively
achieved by spermine and to a lesser extent by spermidine and that
putrescine was ineffective. These observations clearly establish the
role of polyamines on the synthesis of RhoA and Cdc42 proteins and
their activities. It is also important to note that normal migrating
IEC-6 cells had high levels of active Rac1 compared with RhoA and Cdc42
(Fig. 1) and that polyamine depletion decreased active protein levels
significantly below basal levels (Fig. 2). Fig. 7 depicts results that
are important controls regarding our findings on Rac1 and Cdc42
activity. First, polyamine depletion had little effect on the
activities of either protein in cells transfected with the
corresponding constitutively active constructs. Second, DFMO caused a
decrease in enzyme activity in cells transfected with vector alone that
was similar to the inhibition produced in wild-type cells. And, third,
polyamine depletion had no effect on vector-transfected cells that had
been activated with GTP
S. This indicates that the absence of
polyamines does not interfere with the ability of the Rho GTPases to be activated.
Another important finding in the current studies is the demonstration
for the first time that Rac1 and Cdc42 activities are essential for
optimal epithelial cell motility. Expression of dominant negative Rac1
resulted in an approximate 40% inhibition of cell migration, whereas
dominant negative Cdc42 expression inhibited motility by ~70% (Fig.
6). On the other hand, cells transfected with constitutively active
Rac1 migrated more than twice as fast as control cells transfected with
vector alone, and cells expressing active Cdc42 migrated ~30% faster
than controls. Taken together with our previously reported results with
Rho (26), these data mean that each of the three Rho GTPases is
required for optimal migration of intestinal epithelial cells.
Using fibroblasts, Hall and co-workers (12) show that all of the Rho
GTPases are involved in the formation of focal adhesion complexes. In
addition, Rho was linked to stress fiber formation (15), whereas Rac
and Cdc42 were shown to be required for the formation of lamellipodia
and filopodia, respectively (12). There is also evidence that Rac1 is
involved in stress fiber formation as well (16). As indicated in the
introduction, there is significant cross-talk between the Rho family
GTPases. Cdc42 can activate Rac and, thus, induce filopodia in
association with lamellipodia (41). Rac can activate Rho, although this
does not appear to be a strong response in fibroblasts (15, 16).
Nothing is known about the hierarchy of activation of the Rho GTPases
in epithelial cells, and our previous data (26, 27) along with that
reported here are the first to correlate the activity of the Rho
GTPases directly to the migration of any cell type. Work previously
done on fibroblasts has correlated the activity of this group of
proteins with cytostructural changes and with the formation of focal
adhesions, lamellipodia, or filopodia, not with actual cell migration
(42).
Intestinal epithelial cells expressing constitutively active Rac1 had
increased basal levels of active RhoA and Cdc42, indicating that Rac1
activates RhoA and Cdc42 (Fig. 9). Constitutively active RhoA-expressing cells had increased Cdc42 activity, whereas Rac1 activities were unchanged (Fig. 10). Activation of Cdc42 increased basal levels of active Rac1 and RhoA proteins in both normal and polyamine-depleted cells. Active Cdc42 may directly activate RhoA. These observations clearly indicate that Rac1 is upstream of RhoA and
that RhoA may be downstream of Cdc42. The position of Cdc42 in this
hierarchy is difficult to assign due to cross-talk. However, active
Rac1 cells depleted of polyamines migrate at the normal rate, whereas
active Cdc42 cells have a depressed rate of migration in the absence of
polyamines (Fig. 6B).
Our finding that Rac1 activation is not only essential but also
sufficient for cell migration allows a number of important conclusions
to be made about the involvement of the polyamines in migration. First,
polyamines either activate Rac1 directly or, more likely, are required
for a process necessary for the activation of Rac. In other words, they
must act upstream of Rac. Second, polyamines are not required for
migration because of their ability to interact directly with
cytoskeletal elements. Thus, the known ability of spermidine and
spermine to induce F-actin formation and actin fiber bundling in
vitro (20) is not required for cell motility, since these effects
would be downstream from Rac. Third, polyamines are required for
activation of Rac1 and not for the maintenance of levels of Rac
protein. Rac protein levels were unaffected by polyamine depletion.
Fourth, Rac1 activates RhoA, seen in the constitutively active Rac1
cells depleted of polyamines (Fig. 9). This was predictable since RhoA
activity is essential for migration (26, 27). Thus, the activation of
RhoA is downstream from Rac1 and does not require polyamines directly.
Fifth, although polyamine depletion decreases the levels of Rho
protein, this has no effect on migration. Cells expressing constitutively active Rac1 migrated normally despite their decreased amount of RhoA protein. And, finally, Rac is necessary for a step in
the migration process in addition to the activation of RhoA.