Departments of 1 Surgery and 2 Pathology, University of Maryland School of Medicine and 3 Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201; 4 Department of Physiology, Virginia Commonwealth University, Richmond, Virginia 23298; and 5 Department of Medicine, School of Medicine, University of California, San Diego, California 92103
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ABSTRACT |
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Polyamines are required for the
early phase of mucosal restitution that occurs as a consequence of
epithelial cell migration. Our previous studies have shown that
polyamines increase RhoA activity by elevating cytosolic free
Ca2+ concentration ([Ca2+]cyt)
through controlling voltage-gated K+ channel expression and
membrane potential (Em) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated
[Ca2+]cyt activates Rho-kinase (ROK/ROCK)
resulting in myosin light chain (MLC) phosphorylation. Studies were
conducted in stable Cdx2-transfected intestinal epithelial
cells (IEC-Cdx2L1), which were associated with a highly differentiated
phenotype. Reduced [Ca2+]cyt, by either
polyamine depletion or exposure to the Ca2+-free medium,
decreased RhoA protein expression, which was paralleled by significant
decreases in GTP-bound RhoA, ROCK-1, and ROK proteins, Rho-kinase
activity, and MLC phosphorylation. The reduction of [Ca2+]cyt also inhibited cell migration after
wounding. Elevation of [Ca2+]cyt induced by
the Ca2+ ionophore ionomycin increased GTP-bound RhoA,
ROCK-1, and ROK
proteins, Rho-kinase activity, and MLC
phosphorylation. Inhibition of RhoA function by a dominant negative
mutant RhoA decreased the Rho-kinase activity and resulted in
cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the
specific inhibitor Y-27632 not only decreased MLC phosphorylation but
also suppressed cell migration. These results indicate that increase in
GTP-bound RhoA by polyamines via [Ca2+]cyt
can interact with and activate Rho-kinase during intestinal epithelial
restitution. Activation of Rho-kinase results in increased MLC
phosphorylation, leading to the stimulation of myosin stress fiber
formation and cell migration.
mucosal injury; intracellular calcium; Cdx2 gene; dominant negative mutant RhoA; cytoskeleton
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INTRODUCTION |
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THE SMALL GTPase Rho
functions as a molecular switch of various cellular processes by
shuttling between the inactive GDP-bound form and the active GTP-bound
form (11, 14). Rho exerts its distinct actions through
interactions with specific targets. Recently, numerous effector
molecules of Rho have been identified, including protein kinase N (PKN)
(3, 55), ROK/ROCK (20, 40), the myosin-binding subunit of myosin phosphatase (15),
p140mDia (56), citron and citron-kinase (19),
rhophilin (55), rhotekin (35), and rectifier
potassium channel (4). Among these effectors, ROK/ROCK is characterized as a downstream target of Rho and has been
implicated in the regulation of cell shape and dynamic reorganization of cytoskeletal proteins (1, 13, 25, 37, 44, 47). ROK/ROCK
is commonly divided into two isoforms, ROCK-I and ROCK-II, corresponding to ROK and ROK
, respectively. The active form of
Rho interacts with the COOH-terminal portion of the putative coiled-coil domain of ROK/ROCK and activates its phosphotransferase activity (20). Increasing evidence indicates that
activated ROK/ROCK regulates the phosphorylation of myosin light chain
(MLC) by the direct phosphorylation of MLC and by the inactivation of myosin phosphatase through the phosphorylation of myosin binding subunit (2, 15). MLC phosphorylation is crucial for the
actin-myosin interaction for the formation of stress fibers and
contractile rings in nonmuscle cells, thus resulting in cell migration
(2, 25).
Early mucosal restitution is a primary repair modality in the gastrointestinal tract and occurs as a consequence of epithelial cell migration to resealing of superficial wounds after injury (5, 6, 27, 41, 50). This rapid mucosal reepithelialization following superficial wounding is a complex process that includes the flattening, spreading, migrating, and repolarizing of differentiated columnar epithelial cells. Our previous studies (10, 30, 54) and others (5, 6, 38) have examined this process of intestinal epithelial cell migration in an in vitro system that resembles the early stage of mucosal healing in vivo, such as independence from cell division, complete dependence on cytoskeletal reorganization, and absolute requirement of polyamines. Although most of these studies employ undifferentiated intestinal epithelial cells (IEC-6 line) as a model, we (31) have recently demonstrated that differentiated intestinal epithelial cells (IEC-Cdx2L1 line) induced by forced expression of the Cdx2 gene, which encodes a transcription factor controlling intestinal epithelial cell differentiation (42, 46), migrate over the wounded edge much faster than undifferentiated parental IEC-6 cells. Increased migration of differentiated IEC-Cdx2L1 cells after wounding results, at least partially, from the activation of voltage-gated K+ (Kv) channels and the increase in the driving force for Ca2+ influx during restitution (32). Because the early rapid mucosal repair is function of differentiated intestinal epithelial cells from the surface of the mucosa in vivo, these differentiated IEC-Cdx2L1 cells provide an excellent in vitro model for restitution.
The natural polyamines spermidine and spermine and their precursor,
putrescine, are organic cations found in all eukaryotic cells
(43). It has been recognized for some time that the
control of cellular polyamines is a central convergence point for the multiple signaling pathways driving different epithelial cell functions
including cell motility, proliferation, and apoptosis (17, 18, 50). Studies from our laboratory (30, 31,
33, 54) and others (18, 21, 22) have shown that
polyamines are necessary for the stimulation of cell migration after
wounding and play a critical role in the maintenance of intestinal
mucosal integrity. The process of early mucosal restitution is
associated with a dramatic increase in polyamine synthesis both in vivo
(17, 50, 51) and in vitro (30, 31, 54) after
wounding, and depletion of cellular polyamines by inhibition of
ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine
biosynthesis, with D,L--difluoromethylornithine (DFMO) inhibits
cell migration and delays mucosal healing. Although little is known
about specific functions of polyamines in the regulation of cell
migration, these compounds have been shown to stimulate the expression
of Kv channel genes, induce membrane hyperpolarization, and increase
Ca2+ influx during restitution in intestinal epithelial
cells that do not express voltage-dependent Ca2+ channels
(54).
We (30) and others (34) have recently found that polyamines are necessary for the activity and synthesis of RhoA by raising cytosolic free Ca2+ concentration ([Ca2+]cyt) and that activation of RhoA plays an important role in polyamine-dependent intestinal epithelial cell migration after wounding. The aim of the current study was to further determine whether activated RhoA increases ROK/ROCK activity resulting in MLC phosphorylation. First, we examined whether manipulating RhoA activity by altering cellular polyamines, [Ca2+]cyt, or overexpression of the dominant negative mutant RhoA affected the expression of ROK/ROCK proteins and the Rho-kinase enzyme activity in differentiated IEC-Cdx2L1 cells. Second, we determined whether observed activation of ROK/ROCK regulated MLC phosphorylation in the presence or absence of polyamines. Third, we determined whether inhibition of ROK/ROCK activity by treatment with the specific inhibitor Y-27632 [(+)-R-trans-4-(1-aminomethyl)-N-(4-pyridyl) cyclohexanecarboxamide] altered cellular distribution of actomyosin stress filaments and decreased cell migration. Some of these data have been published in abstract form (29).
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MATERIALS AND METHODS |
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Materials.
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 primary antibody, an
affinity-purified mouse monoclonal antibody against ROK or ROCK-1,
was purchased from BD Biosciences (San Jose, CA). Specific antibodies
against RhoA and MLC kinase (MLCK) were from Santa Cruz Biotechnology
(Santa Cruz, CA). The specific rabbit polyclonal antibody against
nonmuscle myosin II was obtained from Biomedical Technologies
(Stoughton, MA). Rhotekin Rho binding domain assay kit and dominant
negative RhoA cDNA in pUSEamp were purchased from Upstate Biotechnology
(Lake Placid, NY). Y-27632 was purchased from Calbiochem (San Diego,
CA). Adeno-X Expression system was obtained from Clontech Laboratories
(Palo Alto, CA). [32P]orthophosphate and
[
-32P]ATP were obtained from Amersham (Arlington
Heights, IL). DFMO was purchased from Ilex Oncology (San Antonio, TX).
Cell culture and general experimental protocol.
The stable Cdx2-transfected IEC-6 cells were developed and
characterized by Suh and Traber (42) and were a kind gift
from Dr. Peter G. Traber (University of Pennsylvania, Philadelphia, PA). The expression vector, the LacSwitch System (Stratagene, La Jolla,
CA), was used for directing the conditional expression of
Cdx2, and isopropyl--D-thiogalactopyranoside
(IPTG) served as the inducer for the gene expression (46).
IEC-6 cells, derived from normal rat intestinal crypts, were
transfected with pOPRSVCdx2 by electroporation technique, and clones
resistant to selection medium containing 0.6 mg/ml G418 and 0.3 mg/ml
hygromycin B were isolated and screened for Cdx2 expression
by Northern blot, RNase protection assays, and electrophoretic mobility
shift assay. Stock-stable Cdx2-transfected IEC-6
(IEC-Cdx2L1) cells were grown in DMEM supplemented with 5%
heat-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate. Before experiments, cells were grown in DMEM containing 4 mM
IPTG for 16 days to induce cell differentiation as described in our
previous publications (31, 32) and by others (42, 46).
Recombinant adenovirus construction and infection. Adenoviral vectors were constructed using the Adeno-X Expression system (Clontech) according to the protocol recommended by the manufacturer (23). Briefly, the cDNA of human dominant negative mutant RhoA (DNMRhoA) was cloned into the pShuttle by digesting the pUSEamp(+)/DNMRhoA (T19N) with EcoR1/Xho1 and ligating the resulting fragments into the Xba1 site of the pShuttle vector. pAdeno-X/DNMRhoA (AdDNMRhoA) was constructed by digesting pShuttle constructs with PI-SceI/I-CeuI and ligating the resulting fragments into the PI-SceI/I-CeuI sites of the pAdeno-X adenoviral vector. Recombinant adenoviral plasmids were packaged into infectious adenoviral particles by transfecting human embryonic kidney (HEK)-293 cells by using LipofectAMINE PLUS reagent. The adenoviral particles were propagated in HEK-293 cells and purified on cesium chloride ultracentrifugation. Titers of the adenoviral stock were determined by standard plaque assay. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescence microscopy and Western blot analysis using anti-RhoA antibody. pAdeno-X, which was the recombinant replication-incompetent adenovirus carrying no cDNA insert (AdNull), was grown and purified as described above and served as a control adenovirus. Cells were infected by various concentrations of AdDNMRhoA or AdNull, and cell samples were collected for various measurements 72 h after the infection.
Measurement of [Ca2+]cyt. Details of the digital imaging methods employed for measuring [Ca2+]cyt are described in our previous publications (30, 32, 54). Briefly, IEC-Cdx2L1 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) under an atmosphere of 10% CO2 in air. 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). The concentration of [Ca2+]cyt was calculated from fura 2 fluorescence emission excited at 380 and 360 nm using the ratio method (28).
Determination of cellular GTP-bound RhoA. The GST-tagged fusion protein, corresponding to residues 7-89 of mouse rhotekin Rho binding domain, was used to determine the cellular GTP-bound RhoA by the affinity precipitation (pull down) protocol according to the manufacturer's instructions with slight modifications (36). Briefly, cells were lysed with cold Rho binding lysis buffer containing 50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). After brief sonication, cell lysates were clarified by centrifugation at 14,000 g at 4°C for 10 min. A small portion of supernatants (40 µg) was taken for determinations of protein concentrations by BCA reagent and total RhoA by Western blot analysis. Equal amounts of (500 µg) supernatants were incubated with 30 µg of glutathione-agarose slurry of rhotekin Rho binding domain for 45 min at 4°C on a rocker platform. The beads were then washed three times with a washing buffer comprising 50 mM Tris, pH 7.2, 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 0.1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. RhoA bound to agarose beads was solubilized in Laemmli's SDS sample buffer and boiled for 5 min. Each sample was analyzed by 15% SDS-PAGE, followed by Western immunoblotting with the specific antibody against RhoA. Specific bands were visualized with a chemiluminescence detection system (NEL-100; Du Pont NEN). The amount of GTP-bound RhoA was normalized for the total amount of RhoA (1/12.5) in each sample.
Measurement of ROK/ROCK enzyme activity.
ROK/ROCK enzyme activity was determined on immunoprecipitate from cell
extracts as described previously (24). Cells were rinsed
with ice-cold PBS containing 1 mM vanadate and 1 µM microcystin-LR, and cells lysates were sonicated at 4°C for 20 s and centrifuged for 10 min at 10,000 rpm. The supernatant was incubated for 1 h
with 100 µl of protein A/G plus agarose beads, and, after
centrifugation, the amount of protein was determined in the
supernatant. Equal amounts of proteins were immunoprecipitated with 2 µg of the antibody against ROK for 2 h and then with 100 µl
of protein A/G-agarose beads for another 1 h at 4°C. After the
incubation, supernatant was carefully removed, and the beads were
washed for four times with lysis buffer and two times with kinase
buffer. The ROK/ROCK kinase activity was measured in a reaction mixture
(final volume 50 µl) containing 20 mM
Tris · HCl, pH 7.5, 100 mM KCl, 0.1 mM DTT, 5 mM
MgCl2, 1 mM EDTA, 1 µM microcystin-LR, 1 mM ATP, and 10 µM myosin light chain-20 (MLC20). Reactions were
initiated by the addition of [
-32P]ATP to a final
concentration of 100 µM. After incubation at 30°C for 10 min,
25-µl aliquots were removed and added to phosphocellulose disks. The
disks were washed immediately for 10 min with 75 mM phosphoric acid and
then dried, and the 32P radioactivity was determined by
adding 5 ml of scintillation fluid to vials containing paper. ROK/ROCK
enzyme activity was expressed as counts per minute per milligram of
protein per minute.
Western blot analysis.
Cell samples, placed in SDS sample buffer, were sonicated and then
centrifuged (12,000 rpm) at 4°C for 15 min. The supernatant from cell
samples was boiled for 5 min and then subjected to electrophoresis on
7.5% or 12.5% SDS-PAGE gels according to Laemmli (16).
After the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 1×
phosphate-buffered saline/Tween 20 [PBS-T: 15 mM
NaH2PO4, 80 mM Na2HPO4,
1.5 M NaCl, pH 7.5, and 0.5% (vol/vol) Tween 20]. Immunological
evaluation was then performed for 1 h in 1% BSA/PBS-T buffer
containing the specific antibody (1 µg/ml) against RhoA, ROK,
ROCK-1, or MLC protein. The filters were subsequently washed with
1 × PBS-T and incubated for 1 h with the second antibody
conjugated to peroxidase by protein cross-linking with 0.2%
glutaraldehyde. After extensive washing with 1× PBS-T, the
immunocomplexes on the filters were reacted with chemiluminescence
reagent and then exposed to autoradiography film.
Assay for MLC phosphorylation. The MLC phosphorylation was assessed by using a method described previously (39). Before the addition of [32P]orthophosphate, cells were rinsed with balanced salt solution without phosphate (BSS: 10 mM HEPES buffered with Tris, pH 7.4, 140 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, and 1.5 mM CaCl2) and then incubated with 4 ml of BSS containing 0.2 mCi/ml [32P]orthophosphate at 37°C for 3 h. After the incubation, cells were lysed with RIPA buffer containing protease inhibitors. Equal amounts of proteins (500 µg) were taken from each sample and immunoprecipitated with anti-MLC antibody for 2 h, followed by the addition of protein L-agarose beads, the incubation was continued for another hour. Beads were washed with lysis buffer and the proteins suspended in Laemmli buffer, followed by the separation of proteins on 15% SDS-PAGE gel. Phosphorylated MLC (P-MLC) bands were analyzed by autoradiography. Densities of bands corresponding to P-MLC were measured by a densitometric analysis.
Nonmuscle myosin II staining. The immunofluorescence procedure was carried out according to the method of Vielkind and Swierenga (48) with minor changes (30, 32). The primary antibody recognizes the 200-kDa nonmuscle myosin II in immunoblots of IEC-Cdx2L1 cell extracts and does not cross-react with other cytoskeletal proteins (31). Nonspecific slides were incubated without antibody to nonmuscle myosin II. Slides were viewed through a Zeiss confocal microscope (model LSM410).
Measurement of cell migration. The migration assays were carried out as described in our earlier publications (30-32). Cells were plated at 6.25 × 104/cm2 in DMEM-dFBS on 60-mm dishes thinly coated with Matrigel according to the manufacturer's instructions. To initiate migration, we scratched the cell layer with a single-edge 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 are reported as the number of migrating cells per millimeter of scratch.
Statistics. All data are expressed as means ± SE from six dishes. Autoradiographic and immunofluorescence labeling experiments were repeated three times. The significance of the difference between means was determined by analysis of variance. The level of significance was determined using Duncan's multiple-range test (12).
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RESULTS |
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Effects of cellular polyamines and [Ca2+]cyt on GTP-bound RhoA in differentiated IEC-Cdx2L1 cells. Our previous studies have shown that reduced [Ca2+]cyt concentration by either polyamine depletion or the removal of extracellular Ca2+ significantly decreased expression of total RhoA protein in undifferentiated intestinal epithelial cells (parent IEC-6 line) and that elevation of [Ca2+]cyt by the Ca2+ ionophore ionomycin increased RhoA expression regardless of the presence or absence of cellular polyamines (30, 32). Because cellular RhoA has the GDP-bound inactive form and the GTP-bound active form (14, 26), the current study was further to determine whether alteration of cellular polyamines and [Ca2+]cyt affected levels of GTP-bound RhoA in differentiated intestinal epithelial cells (IEC-Cdx2L1 line).
Exposure of differentiated IEC-Cdx2L1 cells to 5 mM DFMO for 4 days, which totally inhibited ODC activity (31, 32), almost completely depleted cellular polyamines (data not shown), which was associated with a decrease in K+ channel activity, depolarized Em, and reduction of [Ca2+]cyt (data not shown). The reduction of [Ca2+]cyt in polyamine-depleted cells was accompanied by a significant decrease in RhoA expression (Fig. 1, A and B). Levels of GTP-bound RhoA and total RhoA in DFMO-treated cells were decreased by ~55% and ~40%, respectively. Addition of 5 µM spermidine to the cultures containing DFMO not only prevented the reduction of [Ca2+]cyt (data not shown) but also restored GTP-bound RhoA and total RhoA to normal. Levels of [Ca2+]cyt, GTP-bound RhoA, and total RhoA in cells treated with DFMO plus spermidine were similar to those of control cells. On the other hand, exposure to 1 µM ionomycin increased [Ca2+]cyt in DFMO-treated cells (data not shown). Consistent with the effect on [Ca2+]cyt, treatment with ionomycin also increased levels of GTP-bound RhoA in polyamine-deficient cells (Fig. 1, A and B). Furthermore, removal of extracellular Ca2+ from the culture medium completely prevented the restoration of GTP-bound RhoA by exogenous spermidine. Neither the Ca2+-free medium nor ionomycin altered cell attachment and cell viability in control and DFMO-treated cells (data not shown). These results indicate that polyamines increase levels of GTP-bound RhoA, at least partially, through Ca2+ in differentiated intestinal epithelial cells.
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Effect of induced RhoA by polyamines on ROK/ROCK activity.
It has been shown that ROK/ROCK proteins in epithelial cells are
isoforms ROK and ROCK-1 (2, 13, 20). To determine the
potential downstream effector of RhoA during intestinal epithelial restitution, we tested the possibility that induced RhoA by polyamines via Ca2+ increased ROK
and ROCK-1 expression. As shown
in Fig. 2, treatment of cells with DFMO
decreased ROK
and ROCK-1 protein expression by ~60% and decreased
the Rho-kinase activity by ~45%. This inhibitory effect of polyamine
depletion on ROK/ROCK was completely prevented by the activation of
RhoA through exogenous spermidine and elevation of
[Ca2+]cyt concentration by ionomycin.
Expression of ROK
and ROCK-1 proteins and the Rho-kinase enzyme
activity in cells treated with DFMO plus either spermidine or ionomycin
were similar to those observed in control cells. In contrast, decreased
RhoA activity caused by exposure to the Ca2+-free medium
prevented the restoration of ROK
and ROCK-1 levels and the
Rho-kinase activity in cells treated with DFMO plus spermidine.
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Effect of ROK/ROCK activity on MLC phosphorylation.
To define the mechanism through which ROK/ROCK activation regulates
cellular distribution of cytoskeleton, we examined the role of ROK/ROCK
in MLC phosphorylation. Levels of MLC and P-MLC proteins were measured
when ROK/ROCK activity was manipulated in differentiated IEC-Cdx2L1
cells. Consistent with the inhibitory effect on ROK/ROCK activity,
polyamine depletion by DFMO decreased levels of MLC protein and
phosphorylation of MLC (Fig. 4).
Expression of MLC protein was decreased by ~30% in DFMO-treated
cells (Fig. 4A), and levels of P-MLC were decreased by
~45% (Fig. 4, B and C). Elevation of
[Ca2+]cyt induced by ionomycin or exogenous
spermidine given with DFMO not only reversed the inhibitory effect on
polyamine depletion on MLC expression but also restored MLC
phosphorylation to normal levels. There were no significant differences
in P-MLC levels between controls and cells treated with DFMO plus
spermidine or ionomycin. On the other hand, decreased
[Ca2+]cyt by removal of extracellular
Ca2+ from the culture medium prevented the restoration of
P-MLC by spermidine in polyamine-deficient cells. Levels of P-MLC in
cells treated with DFMO plus spermidine and then exposed to the
Ca2+-free medium were similar to those observed in cells
treated with DFMO alone. These data suggest that activation of
RhoA/Rho-kinase signaling pathway following elevation of
[Ca2+]cyt enhances MLC phosphorylation, thus
resulting in actomyosin stress fiber formation.
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Association of observed changes in RhoA/Rho-kinase activity and rates of cell migration. Polyamine depletion by DFMO significantly inhibited cell migration (from 398 ± 8 to 93 ± 4 cells/mm, n = 6, P < 0.05) in differentiated IEC-Cdx2L1 cells, which was completely prevented by spermidine given with DFMO (from 93 ± 4 to 393 ± 10 cells/mm, n = 6, P < 0.05). Removal of extracellular Ca2+ from the culture medium blocked the restoration of cell migration by spermidine in DFMO-treated cells (from 393 ± 10 to 90 ± 3 cells/mm, n = 6, P < 0.05), whereas an increase in [Ca2+]cyt induced by ionomycin promoted cell migration in the absence of cellular polyamines (from 86 ± 3 to 138 ± 5 cells/mm, n = 6, P < 0.05). These results indicate that alterations in RhoA/Rho-kinase signaling are associated with changes in intestinal epithelial cell migration.
Effect of inhibition of ROK/ROCK activity by its specific inhibitor
Y-27632 on cell migration.
To elucidate the role of ROK/ROCK activation in the process of
polyamine-dependent cell migration during restitution, we carried out
three complementary relevant experiments using the specific ROK/ROCK
inhibitor Y-27632 (49). The first study was performed to
confirm the specific inhibitory effect of Y-27632 on ROK/ROCK in
differentiated IEC-Cdx2L1 cells. Figure
5, A and B, shows
that administration of Y-27632 at the concentration of 50 µM for
6 h not only decreased levels of ROK protein but also inhibited the Rho-kinase activity in control cells and cells treated with DFMO
plus spermidine. In contrast, treatment with Y-27632 at the same dose
did not inhibit expression of MLCK. Consistently, inhibition of
ROK/ROCK by Y-27632 was associated with a significant decrease in MLC
phosphorylation (Fig. 5C). These results clearly indicate that Y-27632 is a specific inhibitor for ROK/ROCK in intestinal epithelial cells.
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Effect of inhibition of ROK/ROCK on cellular distribution of
nonmuscle myosin II.
To investigate the mechanism by which ROK/ROCK activation mediates cell
migration, we examined the effects of changes in ROK/ROCK activity on
cellular distribution of nonmuscle myosin II in control and
polyamine-deficient IEC-Cdx2L1 cells. In control migrating cells (Fig.
7A), there were long stress
fibers that traversed the cytoplasm, and a thick network of cortical
myosin II fibers was observed just inside the plasma membrane.
Inhibition of ROK/ROCK activity by Y27632 at the concentration of 75 µM during the period of cell migration significantly inhibited the
formation of myosin II stress fibers in control cells (without DFMO)
(Fig. 7, A vs. B). Myosin II stress fibers were
sparse and devoid of long stress fiber formation. Consistent with the
inhibitory effect on ROK/ROCK, polyamine depletion by DFMO also
inhibited formation of myosin stress fibers in IEC-Cdx2L1 cells (Fig.
7, A vs. C), and features of myosin
distribution were similar to those observed in control cells
exposed to Y27632 (Fig. 7, B vs. C). On the other
hand, restoration of ROK/ROCK activity by exogenous spermidine given together with DFMO reversed the inhibitory effect of polyamine depletion on myosin stress fiber formation. The distribution of nonmuscle myosin II in cells grown in the presence of DFMO plus spermidine was indistinguishable from that in control cells (Fig. 7,
A vs. D). In contrast, inhibition of ROK/ROCK by
either Y27632 or removal of extracellular Ca2+ after
wounding completely prevented the restoration of the distribution of
nonmuscle myosin II by exogenous spermidine in polyamine-deficient cells (Fig. 7, D vs. E and F). These
results indicate that ROK/ROCK activation following elevation of
[Ca2+]cyt induced by polyamines regulates
cellular distribution of nonmuscle myosin II and enhances actomyosin
stress fiber formation during restitution after wounding.
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DISCUSSION |
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Precise regulation of epithelial restitution to reseal superficial wounding is critical for the maintenance of gastrointestinal mucosal integrity under physiological and pathological conditions. Cellular polyamines are known to be an important regulator of intestinal mucosal restitution, but specific functions of polyamines in epithelial cell migration are largely undefined. We (30, 54) have recently reported that polyamines increase RhoA activity by altering [Ca2+]cyt concentration through regulation of K+ channels and that depletion of cellular polyamines by DFMO inhibits K+ channel expression, reduces [Ca2+]cyt by downregulating the driving force for Ca2+ influx, decreases RhoA activity, and suppresses cell migration in undifferentiated parent IEC-6 cells. However, the exact mechanisms by which activated RhoA regulates epithelial cell migration during restitution remain to be demonstrated. In particular, whether downstream effectors of Ca2+-induced RhoA after activation of K+ channels by increased polyamines are important in this process is unclear. Because ROK/ROCK is identified as one of the downstream targets of RhoA and is involved in Rho-induced formation of actomyosin stress fibers and focal adhesions (13, 25, 37), we have elucidated the role of this molecule in the regulation of polyamine-dependent intestinal epithelial cell migration after wounding.
To extend our previous findings, we employed differentiated IEC-Cdx2L1 cells as a model in the current study and have demonstrated that RhoA is also implicated in the signaling pathway of differentiated intestinal epithelial cell migration after wounding. Differentiated IEC-Cdx2L1 cells highly express Kv1.1 and Kv1.5 channels, which is associated with an increase in whole cell K+ currents, membrane hyperpolarization, and a rise in [Ca2+]cyt (32). The migration rates in differentiated IEC-Cdx2L1 cells are about four times those of parental IEC-6 cells. Basal levels of total RhoA and GTP-bound RhoA in differentiated IEC-Cdx2L1 cells are higher than those observed in parental IEC cells (data not shown). Consistent with the observations in parental IEC-6 cells (30, 34), the activation of RhoA also absolutely requires cellular polyamines in these differentiated epithelial cells. As shown in Fig. 1, reduction of [Ca2+]cyt by polyamine depletion with DFMO decreased levels of GTP-bound RhoA. Elevation of [Ca2+]cyt levels in polyamine-deficient cells by the Ca2+ ionophore ionomycin prevented the inhibitory effect of DFMO on RhoA activity. Although polyamines regulate intestinal epithelial cell migration through multiple signaling pathways (52, 53), the current findings indicate that the polyamine's action is mediated, at least partially, by [Ca2+]cyt.
The most important findings reported in this article are that
Ca2+-induced RhoA following activation of K+
channels by increased polyamines activates ROK/ROCK activity in
differentiated IEC-Cdx2L1 cells. An increase in GTP-bound RhoA by
elevation of [Ca2+]cyt induced ROK and
ROCK-1 protein expression and stimulated the Rho-kinase activity,
whereas inhibition of RhoA by either reduced
[Ca2+]cyt (Fig. 2) or a dominant negative
mutant RhoA (Fig. 3) decreased ROK/ROCK activity. Although the precise
mechanisms that account for activation of ROK/ROCK by Rho are unclear
in general, GTP-bound RhoA has been shown to recognize at least two
different types of target interfaces, the coiled coil domain of
ROK/ROCK and the NH2-terminal regulatory domain of PKN
(3, 20, 40, 55). Because GTP-bound RhoA slightly induces
autophosphorylation of ROK/ROCK but dramatically increases the
Rho-kinase activity (1), it is unlikely that GTP-bound
RhoA stimulates ROK/ROCK activity by altering its autophosphorylation.
The Ca2+-induced RhoA following activation of
K+ channels by increased polyamines might directly interact
with the COOH-terminal portion of the coiled domain of ROK/ROCK and result in a conformational change of ROK/ROCK, leading to activation of
the Rho-kinase toward selective substrates such as MLC or the myosin
binding subunit of myosin phosphatase. Further studies are clearly
necessary to understand the exact mechanisms involved in the
RhoA-induced ROK/ROCK activation in intestinal epithelial cells.
The observations from the current study further indicate that changes in ROK/ROCK activity alter MLC phosphorylation in differentiated IEC-Cdx2L1 cells. Inhibition of ROK/ROCK by either polyamine depletion with DFMO or treatment with its specific inhibitor Y27632 was associated with a significant decrease in levels of P-MLC, whereas the restoration of ROK/ROCK activity in DFMO-treated cells by exogenous spermidine or ionomycin returned P-MLC levels to near normal (Figs. 4 and 5). MLC has been identified as a substrate of ROK/ROCK in muscle and nonmuscle cells, and activation of ROK/ROCK phosphorylates MLC at Ser-19 and Thr-18, the same sites that are phosphorylated by MLC kinase (2). It has been shown that phosphorylation of MLC at Ser-19 stimulates actin-activated myosin ATPase and plays an important role in the regulation of cellular motility, cellular contraction, and cytokinesis (2, 9, 14). When constitutively active forms of Rho or ROK/ROCK are introduced into nonmuscle cells, an enhancement of MLC phosphorylation is detected, which is associated with increases in the stress fiber formation and focal adhesions (2, 15). Because recent studies have demonstrated that ROK/ROCK also regulates MLC phosphorylation through the inactivation of myosin phosphatase, the exact contribution of these two pathways to the elevation of P-MLC following RhoA-induced ROK/ROCK activation by increased polyamines in intestinal epithelial cells is not clear at present.
The RhoA-induced ROK/ROCK activation plays a critical role in the process of intestinal epithelial cell migration during early epithelial restitution. Decreased ROK/ROCK activity by Y27632 not only decreased MLC phosphorylation (Fig. 5) but also inhibited normal cell migration after wounding (without DFMO) (Fig. 6A). These findings are consistent with data from others (8, 45), who have found that ROK/ROCK activation is necessary for hepatic stellate cell migration after wounding and that expression of the dominant negative mutant ROK/ROCK inhibits wound-induced cell migration in Madin-Darby canine kidney (MDCK) epithelial cells. An interesting and extended finding obtained from the current study is that decreased ROK/ROCK activity by Y27632 also prevents the restoration of intestinal epithelial cell migration by exogenous spermidine in polyamine-deficient cells (Fig. 6B). Our previous studies (30) have demonstrated that RhoA expression is regulated by cellular polyamines and that Ca2+-induced RhoA activation is necessary for polyamine-dependent intestinal epithelial cell migration. Together, the current observations and our previous findings (30-32, 54) strongly support the possibility that polyamines are required for the stimulation of cell migration after wounding in association with their ability to activate RhoA/Rho-kinase signaling pathway through control of [Ca2+]cyt concentration.
Results presented in Fig. 7 show that ROK/ROCK activation stimulates intestinal epithelial cell migration at least partially by altering cellular distribution of the cytoskeletal proteins. It is well known that cellular distribution and formation of cytoskeletal proteins are highly regulated by Rho and its downstream effectors in different types of cells (11, 25, 26). In the current study, decreases in ROK/ROCK activity by either treatment with Y27632 or exposure to the Ca2+-free medium in control cells and cells treated with DFMO plus spermidine resulted in reorganization of actomyosin filaments. The numbers of long stress fibers of myosin II decreased significantly, and in some cells they disappeared completely from cytoplasm, as observed in cells treated with DFMO alone (Fig. 7). Although the exact mechanisms by which inactivation of ROK/ROCK by polyamine depletion results in reorganization of myosin II are obscure, the MLC phosphorylation seems to play an important role in this process. Inhibition of ROK/ROCK activity was associated with a decrease in levels of P-MLC (Figs. 4 and 5), and the MLC phosphorylation has been shown to induce myosin-actin interaction in certain type of cells (2). It is possible that decreased MLC phosphorylation in polyamine-deficient cells reduces the interaction between myosin and actin, thereby leading to inhibition of stress fiber formation.
On the basis of current findings and our previous studies
(30-32, 54), we propose a model delineating the role
of ROK/ROCK activation following increased polyamines in the process of
intestinal epithelial cell migration after wounding (Fig.
8). In this model, increased polyamines
enhance K+ channel expression, cause membrane
hyperpolarization, raise [Ca2+]cyt
concentration by enhancing the driving force for Ca2+
influx, and increase RhoA activity, leading to ROK/ROCK activation. The
resultant activation of ROK/ROCK increases MLC phosphorylation, resulting in stimulation of stress fiber formation and cell migration during restitution. In contrast, depletion of cellular polyamines inactivates ROK/ROCK by reducing [Ca2+]cyt
through downregulation of K+ channel activity,
decreases MLC phosphorylation, and inhibits stress fiber formation,
thus leading to inhibition of cell migration.
|
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ACKNOWLEDGEMENTS |
---|
This work was supported by National Institutes of Health Grants DK-57819 and DK-61972 (to J.-Y. Wang), DK-28300 (to K. S. Murthy), and HL-54043 and HL-64945 (to J. X.-J. Yuan); by a Department of Veterans Affairs Merit Review Grant (to J.-Y. Wang); and by a Baltimore Research Education Foundation Pilot Grant (to J. N. Rao). J.-Y. Wang is a Research Career Scientist for the Department of Veterans Affairs Medical Research Service.
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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.
First published December 4, 2002;10.1152/ajpcell.00371.2002
Received 15 August 2002; accepted in final form 27 November 2002.
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