MEK1 restores migration of polyamine-depleted cells by retention and activation of Rac1 in the cytoplasm

Rajiv J. Vaidya, Ramesh M. Ray, and Leonard R. Johnson

Department of Physiology, The University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 25 June 2004 ; accepted in final form 13 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously showed that polyamines are required for proliferation and migration both in vivo and in a cultured intestinal epithelial cell (IEC-6) model. Wounding of the IEC-6 monolayer induced transient ERK activation, which was further enhanced by EGF. EGF stimulated migration in control and polyamine-depleted cells, but the degree of stimulation was significantly less in polyamine-depleted cells. Inhibition of MEK1 inhibited basal as well as EGF-induced ERK activation and migration. Expression of constitutively active (CA)-MEK and dominant-negative (DN)-MEK had significant effects on F-actin structure. CA-MEK increased stress fiber and lamellipodia formation, while DN-MEK showed loss of stress fibers and abnormal actin cytoskeletal structure. Unlike EGF, CA-MEK significantly increased migration of both control and polyamine-depleted cells. The most important and significant finding in this study was that polyamine depletion caused localization of Rac1 and RhoA to the nuclear as well as perinuclear regions. Interestingly, CA-MEK completely reversed the subcellular distribution of Rac1 and RhoA proteins in polyamine-depleted cells. Polyamine depletion increased Rac1 in the nuclear fraction and decreased it in the cytoplasmic and membrane fractions of vector-transfected cells. CA-MEK prevented accumulation of Rac1 in the nucleus. Polyamine depletion significantly decreased Rac1 activity during 6-h migration in vector-transfected cells. Cells transfected with CA-MEK had almost identical levels of activated Rac1 in all three groups. These results suggest that polyamine depletion prevents activation of Rac1 and RhoA by sequestering them to the nucleus and that expression of constitutively active MEK reverses this effect, creating the cellular localization required for activation.

epidermal growth factor; extracellular signal-regulated kinase; IEC-6 cells


CELL MIGRATION IS AN IMPORTANT and fundamental process during intestinal development, normal epithelial cell turnover, and disease conditions involving ulceration of the single cell layer lining of the gastrointestinal tract (10, 11). Cell migration requires an intact and functioning cytoskeleton, which consists of filamentous or F-actin, tubulin, and intermediate fibers. F-actin is formed by the polymerization of 42-kDa monomers, termed G-actin. F-actin along with associated binding proteins form the actin cortex, a dense network localized just inside the inner surface of the plasma membrane (1). Prominent, long filaments of actin traverse the cell as stress fibers during migration, and short filaments extend into the lamellipodia (31). Focal adhesions provide required attachment to the substratum, and the cytoskeleton exerts force on the extracellular matrix via stress fibers (3). In response to receptor signaling via integrins and the extracellular matrix (13) or soluble factors (34), cell migration is initiated. Soluble factor and integrin signaling are relayed to the cytoplasm by signal transduction pathways involving a subgroup of the Ras superfamily of small GTP-binding proteins (28). Rho and Rac regulate the polymerization of actin to produce stress fibers and lamellipodia, respectively (41, 42). Cdc42 has been shown to be responsible for the formation of filopodia (28).

Damage to the gastrointestinal epithelium can result from infection (ulcer), chemical agents (alcohol or other drugs), or mechanical forces (stretching), and immediate repair is required to restore the epithelial barrier against luminal antigens. Gastrointestinal repair is a continuous process that can be divided into two stages, namely, cell spreading or migration and proliferation. During the early restitution stage (conservatively, up to 12 h), viable cells bordering the lesions extend lamellipodia and migrate over the damaged area. During the later stage, migration continues and cell proliferation becomes the major repair factor. Cells that are polyamine deficient fail to migrate normally during the early phase of restitution (23, 36, 56) and throughout the process of repair. Polyamines are involved in the organization of the cytoskeleton, an essential component of cell migration. Polyamine-deficient Chinese hamster ovary cells lack actin filaments and microtubules (33). In vitro polyamines stimulate the rapid polymerization of G-actin and the formation of bundles from F-actin, indicating a possible direct effect of polyamines on cytoskeletal organization (14). Our laboratory (53, 54) has shown that the early phase of mucosal healing due to cell migration requires polyamines and that polyamine depletion inhibits migration in intestinal epithelial cells (IEC)-6 and led to numerous alterations in the cytoskeleton. When cells were depleted of polyamines by means of treatment with {alpha}-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), 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 (24). There was also a redistribution of tropomyosin from stress fibers to the actin cortex. Additional changes in response to polyamine depletion include a marked reduction in the formation of lamellipodia and a dissociation of actin from nonmuscle myosin II (55). While no changes occur in the absolute amounts of G- and F-actin, the association of actin with the sequestering protein thymosin {beta}4 is inhibited (22). Polyamine depletion also has been shown to decrease the rate of cell proliferation by G1 cell cycle arrest (40, 47).

Santos et al. (45) demonstrated that Rho is required for the migration of IEC-6 cells after wounding or in response to growth factor stimulation. Inactivation of Rho by either microinjection of Rho GDI, Clostridium botulinum C3 ADP-ribosyltransferase toxin (C3 toxin), or expression of DN RhoA (Rho T19N) inhibited migration and also altered the cytoskeleton in a manner identical to that induced by polyamine depletion (38). Our laboratory recently showed that the effect of polyamine depletion on cell migration and the actin cytoskeleton could be prevented by expression of CA Rac1. Our laboratory also has shown that in IEC-6 cells, Rac1 activates both RhoA and Cdc42 (37).

EGF plays a critical role in the protection and repair of gastrointestinal mucosa (6, 12, 16, 26, 29, 44). EGF is produced by the salivary glands (29) and by gastric mucosa (52), and EGF receptors are present in the epithelial cells of the gastrointestinal tract (25, 27, 32, 49), which indicates the importance of EGF signaling in gastrointestinal mucosal integrity. In the present study, we have shown that EGF increased the migration of both control and polyamine-depleted IEC-6 cells but did not completely restore migration to control levels in polyamine-depleted cells. Using a stable cell line expressing constitutively active (CA)-MEK, we have demonstrated that the sustained activation of MEK significantly increased the migration of IEC-6 cells with a concomitant extensive reorganization of actin cytoskeletal structure. Polyamine depletion caused Rac1 and RhoA to localize to nuclei and perinuclear regions and prevented their activation. Transfection with CA-MEK prevented the localization of Rac1 to the nucleus, allowing it to be activated and to restore the cytoskeletal structure and migration of polyamine-depleted cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Disposable cultureware was purchased from Corning Glass Works (Corning, NY). Media and other cell culture reagents were obtained from Invitrogen (Carlsbad, CA). Dialyzed fetal bovine serum (FBS) and other chemicals were purchased from Sigma (St. Louis, MO). We also obtained mouse anti-ERK (Zymed Laboratories, San Francisco, CA), rabbit anti-phospho-ERK (Cell Signaling Technology, Beverly, MA), mouse anti-Rac1 (Upstate Biotechnology, Lake Placid, NY), mouse anti-actin (Sigma, St. Louis, MO), and rabbit anti-hemagglutinin (HA) probe antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies and rhodamine-phalloidin were purchased from Molecular Probes (Eugene, OR). DFMO was a kind gift from Ilex Oncology (San Antonio, TX). EGF was purchased from BD Biosciences (Bedford, MA). U-0126, a specific MEK inhibitor was purchased from Promega (Madison, WI). Enhanced chemiluminescence Western blot detection reagent was purchased from PerkinElmer Life Sciences (Boston, MA). The IEC-6 cell line (catalog no. CRL 1592) was obtained from the American Type Culture Collection (Manassas, VA) at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (35). IEC-6 cells originate from intestinal crypt cells as judged by morphological and immunological criteria. They are nontransformed and retain the undifferentiated character of epithelial stem cells. The test for mycoplasma was performed routinely and was negative. All chemicals used were of the highest purity commercially available.

Cell culture. Stock cell culture was maintained in T-150 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate. The flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were passaged once weekly at 1:20 dilution, and the medium was changed three times weekly. The cells were restarted from original frozen stock after every seven passages.

General experimental protocols. The general protocol for the experiments and the methods used were similar to those described previously (40). In brief, IEC-6 cells were plated at 6.25 x 104 cells/cm2 in control medium consisting of DMEM supplemented with 5% dialyzed FBS, 10 µg/ml of insulin, and 50 µg/ml gentamicin sulfate or in control medium containing 5 mM {alpha}-DFMO or DFMO plus 10 µM putrescine (DP). Cells were grown at 37°C in a humidified atmosphere of 90% air-10% CO2. They were fed every other day and serum starved for 24 h before harvesting or before the cell migration assay.

Transfections. IEC-6 cells were transfected with CA-MEK-HA-Tag, dominant-negative (DN)-MEK-HA-Tag, and empty vector (Vector). Stable clones were isolated and characterized as described previously (4).

Cell migration assay. Cells were grown under control, polyamine-deficient (DFMO), and DP conditions in 35-mm plates. Cells were serum starved for 24 h before the experiment (during day 3). 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 performed perpendicularly to the marked line using a gel-loading microtip. Plates were washed, and the area of migration was photographed with a charge-coupled device camera using NIH Image software (version 1.58) at the intersection of the marked line and the 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). Each experiment was conducted three times in duplicate, and each plate was wounded twice. Therefore, the number of experiments conducted was considered to be 6, even though the results are the means of 12 observations.

Rac1 activation assay. Biological activity of Rac1 protein was analyzed using pull-down assays performed according to the method of Kranenburg et al. (20). Glutathione S-transferase-p21-activated kinase (GST-PAK) fusion protein was prepared by lysing the bacteria (Escherichia coli BL21-DE-3pLysE strain transformed with GST-PAK plasmid 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. Cell lysate was sonicated and clarified by performing centrifugation at 10,000 g for 15 min. The fusion protein was recovered by the addition of glutathione-agarose beads to the supernatant. The beads were washed three times in cell lysis buffer and resuspended before the addition of the cell lysates (100 µg). After 1 h of tumbling at 4°C, beads were washed with lysis buffer and the amount of Rac1 protein bound to GST-PAK protein was analyzed by performing SDS-PAGE and Western blot analysis using a Rac1-specific antibody. Proteins (20 µg) from each sample were resolved using SDS-PAGE to determine the level of total Rac1 protein.

Western blot analysis. Protein samples were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane using electroblotting. The membranes were then probed with appropriate primary and secondary antibodies. Immunocomplexes were visualized using enhanced chemiluminescence detection reagent. Immunoblotting for ERK and phospho-ERK was performed using the Odyssey Infrared Imaging System according to the manufacturer's protocols (LI-COR Biosciences, Lincoln, NE). Goat anti-mouse Alexa Fluor 680 (Molecular Probes) and goat anti-rabbit IRDye 800 (Rockland Immunochemicals, Gilbertsville, PA) were used to detect total ERK and phospho-ERK.

Immunocytochemistry. Cells were seeded onto coverslips coated with poly-L-lysine (BD Labware, Bedford, MA) and grown as described earlier. Cells were fixed with 3.7% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and washed with PBS. Coverslips were blocked with 3% BSA in PBS for 20 min and then incubated with primary antibody for 2 h. Coverslips were then washed with 0.1% BSA in PBS for 20 min, followed by a 2-h incubation with an appropriate fluorescent dye-conjugated secondary antibody. Coverslips were mounted on glass slides and observed using a Nikon Diaphot inverted microscope with appropriate filters.

Cell fractionation. Cells were grown under control, DFMO, and DFMO plus putrescine conditions for 3 days. Cells were serum starved for 24 h before the experiment. Cell monolayers were washed twice with DPBS. Cells were harvested in ice-cold cell lysis buffer (in mM: 50 HEPES, pH 7.5; 50 NaCl, 1 MgCl2, 2 ethylenediamine tetraacetic acid, and protease inhibitors), and cell lysates were centrifuged at 1,500 g for 10 min to collect the nuclear fraction. The resulting supernatant was centrifuged at 15,000 g for 10 min, and the heavy membrane fraction pellet was resuspended in cell lysis buffer. The supernatant was used as the cytoplasmic fraction. Equal amounts of protein were used for the detection of Rac1 using Western blot analysis.

Statistics. Data are means ± SE. All experiments were repeated three times (n = 3). Western blots are representative of three experiments. ANOVA with appropriate post hoc testing was used to determine the significance of the differences between means. P < 0.05 was regarded as statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
MAP kinase activation increases migration of control and polyamine-depleted IEC-6 cells. To determine the effect of EGF on the migration of control and polyamine-depleted IEC-6 cells, we grew cells in control medium and medium containing DFMO, and the cell migration assay was performed as described in MATERIALS AND METHODS. EGF significantly increased migration of IEC-6 cells in the control condition (65%) compared with cells not treated with EGF. EGF increased migration of polyamine-depleted cells by 100% but failed to completely restore the migration of polyamine-depleted cells to that of control cells treated with EGF (Fig. 1).



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Fig. 1. EGF increases migration of control and polyamine-depleted intestinal epithelial (IEC-6) cells. IEC-6 cells were grown in medium ± 5 mM {alpha}-difluoromethylornithine (DFMO) for 4 days. Confluent monolayers were wounded with a gel-loading tip in the center of plates, marked to localize the wound site, washed, and incubated with medium ± DFMO and ± 10 ng/ml EGF. A: plates were photographed immediately to record the wound width (0 h) and then again at the marked wound location after 6 h of incubation. Representative images of 3 experiments are shown. B: quantitative analysis of migration showing wound width covered compared with initial wound size (0 h) using NIH Image software analysis. Values are means ± SE. *P < 0.05, significantly different from EGF.

 
Increased migration in response to EGF prompted us to think that wounding modulates MAPK in IEC-6 cells. Therefore, we determined the time course of phosphorylation of ERK1/2 in control, polyamine-depleted (DFMO), and DP conditions. Wounding caused a transient increase in ERK1/2, which returned to basal levels within 30 min in all three conditions, but EGF caused sustained activation of ERK1/2 for up to 30 min in all three conditions (Fig. 2).



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Fig. 2. Wounding and EGF increase ERK1/2 activation in IEC-6 cells. Cells were grown in medium ± 5 mM DFMO or DFMO + 10 µM putrescine (PUT) for 4 days, wounded, washed, and incubated with medium ± EGF for 0, 5, 15, and 30 min. At the specified times, cells were lysed in the presence of protease inhibitors. Proteins (10 µg) from each sample were separated on SDS-PAGE gel, followed by Western blot analysis using antibodies specific for ERK and phospho-ERK1/2. Signal detection was performed using the LI-COR Odyssey infrared imaging system. Representative Western blots from 3 observations are shown.

 
The finding that both wounding and EGF increased ERK activation and migration suggested a role for the MAP kinase pathway in migration. To investigate this hypothesis, we used U-0126, a specific inhibitor of MEK that prevents MEK1 from promoting both threonine and tyrosine phosphorylation (9, 30), and studied its effects on migration. As shown in Fig. 3, inhibition of MEK significantly inhibited wound- and EGF-induced migration in control, DFMO-grown, and DP-grown cells. Activation of MAPK by EGF increased migration by 30% compared with untreated cells grown in control medium. U-0126 significantly inhibited basal as well as EGF-induced migration (40–50%) (Fig. 3A). Although EGF increased migration of polyamine-depleted cells, migration reached only the level of control cells without EGF. Addition of exogenous putrescine along with DFMO restored migration to the level of control, indicating that the observed effects were due to polyamine depletion and not to DFMO. Interestingly, U-0126 significantly inhibited migration of polyamine-depleted cells in both the presence and absence of EGF compared with the control and DP groups. These experiments provided direct evidence that MEK is essential for the migration of control as well as polyamine-depleted cells. To validate the inhibitory effect of U-0126, we determined the levels of phosphorylated ERK1/2 in control, DFMO-grown, and DP-grown cells. U-0126 completely inhibited wound- and EGF-induced ERK 1/2 phosphorylation in all three groups without any effect on total protein levels (Fig. 3B).



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Fig. 3. Effect of MEK1 inhibition on migration. Cells were grown in medium ± 5 mM DFMO or DFMO + 10 µM putrescine for 4 days. Confluent monolayers were wounded with a gel-loading tip in the center of plates marked to localize the wound site, washed and were left untreated (UT) or treated with DMSO (V), EGF (E), U-0126 (U), and EGF + U-0126 (UE). A: quantitative analysis of migration showing wound width covered compared with initial wound size (0 h) using NIH Image software analysis. Cells were treated for 6 h. Values are means ± SE of 6 observations. *P < 0.05, significantly higher than the respective UT. #P < 0.05, significantly lower than the respective EGF treated. B: after wounding and treatment for 30 min, 10 µg of protein from each sample were separated on SDS-PAGE gel, followed by Western blot analysis using antibodies specific for ERK and phospho-ERK1/2. Signal detection was performed using the LI-COR Odyssey infrared imaging system. Representative Western blots from 3 observations are shown.

 
Polyamine depletion has no effect on migration of IEC-6 cells expressing CA-MEK1. Our finding that activation of the ERK/MAPK pathway significantly increased migration of control and polyamine-depleted cells, and that EGF did not restore migration of polyamine-depleted cells to the level of control cells treated with EGF, led us to predict that sustained activation of MAPK might restore migration of polyamine-depleted cells. To test this hypothesis, we used stable IEC-6 cell lines expressing HA-tagged CA-MEK and DN-MEK, which were characterized and reported in a previous study by our laboratory (4). Figure 4 shows the actin cytoskeleton and HA tag localization in these cells. Empty vector-transfected cells did not stain for the HA tag (Fig. 4). Cells transfected with CA-MEK and DN-MEK showed robust expression of recombinant proteins. Cells transfected with CA-MEK showed characteristic nuclear localization of HA tag, indicating expression of constitutively active MEK1 protein. In contrast, HA tag was localized throughout the cytoplasm in the cells transfected with DN-MEK. Nuclear and nonnuclear localization of HA-Tag-CA-MEK and HA-Tag-DN-MEK, respectively, indicated that these cells expressed recombinant proteins and confirmed their biological activity. Furthermore, cells expressing CA-MEK exhibited significant spreading and F-actin stress fiber formation, characteristics of actively migrating cells, which were less prominent in vector cells. In contrast, cells expressing DN-MEK showed significant loss of actin stress fibers essential for maintenance of cell shape, size, and migration. Dense F-actin foci in the cells transfected with DN-MEK colocalized with intense HA tag staining in the cytoplasm.



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Fig. 4. Actin cytoskeletal structure of cells transfected with constitutively active and dominant-negative MEK1. Empty vector (Vector), constitutively active (CA-MEK), and dominant-negative (DN-MEK)-MEK1-transfected cells were grown on coverslips, washed, fixed, and processed for localization of hemagglutinin (HA) tag using anti-HA tag antibody and rhodamine-conjugated phalloidin for visualization of F-actin. Arrows indicate stress fibers, asterisks indicate the nucleus, and arrowheads indicate HA tag.

 
Expression of CA-MEK restores actin cytoskeleton structure of polyamine-depleted cells. Polyamine depletion inhibited migration of the vector-transfected cells by 47%. However, in cells transfected with CA-MEK, the migration increase was twofold that in control cells transfected with vector. Expression of CA-MEK restored migration of polyamine-depleted cells to a level comparable to that of cells transfected with CA-MEK grown in control medium, and it was significantly higher than that found in vector-transfected cells (Fig. 5A). The addition of exogenous putrescine with DFMO prevented the effects of polyamine depletion on migration. We further confirmed that transfection with CA-MEK increased the rate of migration of polyamine-depleted cells by plating cells transfected with CA-MEK and vector together at a 1:1 ratio in DFMO-containing medium for 4 days. Six hours after wounding, cells were fixed and stained for HA tag and F-actin. Figure 5B depicts the localization of the HA tag and F-actin at the migrating edge. Most cells at the leading edge of the monolayer were HA tag-positive and had almost no cortical actin. In contrast, slowly migrating cells were devoid of HA tag staining. The absence of HA tag staining and the presence of thick cortical actin indicated that these more slowly migrating cells had been transfected with vector and that cells transfected with CA-MEK were migrating more rapidly. Thus our results indicate that constitutive activation of MEK is sufficient to restore migration of polyamine-depleted cells. These observations clearly indicate that the MEK-ERK pathway plays an important role in the regulation of migration.



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Fig. 5. Polyamine depletion has no effect on the migration of cells expressing constitutively active (CA-MEK)-MEK1. A: empty vector (Vector), CA-, and DN-MEK1-transfected cells were grown in medium ± 5 mM DFMO or DFMO + 10 µM putrescine for 4 days, wounded, washed, and incubated with fresh medium for 6 h. Quantitative analysis of migration showed wound width covered compared with initial wound size (0 h) using NIH Image software analysis. Values are means ± SE of 6 observations. #P < 0.05, significantly different from vector-transfected cells. B: cells transfected with vector and CA-MEK were grown together (1:1) in medium containing 5 mM DFMO for 4 days, wounded, and allowed to migrate for 6 h. The cells were then washed, fixed, and processed for localization of HA tag using anti-HA tag antibody and rhodamine-conjugated phalloidin for visualization of F-actin. Arrows indicate actin cortex, and arrowheads indicate HA tag.

 
Previous reports from this laboratory have shown that polyamine depletion alters the actin cytoskeletal structure. Inhibition of migration is attributed to the presence of dense cortical actin bundles that fail to reorganize into actin stress fibers essential for the migration (37, 38). In the present study, because expression of CA-MEK restored the migration of polyamine-depleted cells, we examined the organization of the actin cytoskeleton at the migrating edge of the wounded cell monolayer using rhodamine-conjugated phalloidin, which specifically binds to F-actin. Figure 6 portrays F-actin staining of control and polyamine-depleted cells transfected with vector and CA-MEK at the migrating edge (6 h after wounding). It is apparent from actin staining that vector-transfected cells migrated without significant loss of cell-cell adherence, indicated by their migration as a sheet or monolayer, while cells transfected with CA-MEK lost cell-cell contact and migrated as individual or solitary cells, evident by large gaps between the cells (Fig. 6). Cells transfected with CA-MEK contained remarkable lamellipodia and stress fibers, a characteristic feature of cell spreading and migration. In contrast, cells transfected with vector had short stress fibers and significantly less spreading than did cells transfected with CA-MEK. More important, polyamine-depleted cells transfected with CA-MEK, which migrated faster than polyamine-depleted vector cells, also showed actin remodeling similar to that seen in control cells transfected with CA-MEK. Unlike DFMO-treated cells transfected with vector, polyamine-depleted cells transfected with CA-MEK were able to reorganize cortical actin to form extensive stress fibers typical of actively migrating cells.



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Fig. 6. Expression of CA-MEK restores actin cytoskeletal structure of polyamine-depleted cells. Cells transfected with vector (A) and CA-MEK (B) were grown in medium ± 5 mM DFMO or DFMO + 10 µM putrescine for 4 days, wounded, washed, and incubated with fresh medium for 6 h. Cells were then fixed and stained with rhodamine-conjugated phalloidin for visualization of F-actin. Enlarged areas (boxed areas in A and B) show F-actin organization at the wound edge. Images are representative of 3 observations. Arrows indicate stress fibers, and arrowheads indicate lamellipodia.

 
Effect of MEK activation on RhoA and Rac1 localization and Rac1 activation. Soluble factors and integrins relay signals to the cytoskeleton by signal transduction pathways involving a subgroup of the Ras superfamily of small GTP-binding proteins Rho, Rac, and Cdc42 (28). Activation of these proteins by GTP binding and their subcellular localization is crucial to migration. Previously, researchers at our laboratory showed that polyamine depletion decreased Rac1 activity and that Rac1 activated RhoA and Cdc42 in IEC-6 cells (37). Rac1 activity proved to be necessary and sufficient for migration of polyamine-depleted IEC-6 cells. In this study, we determined the effects of expression of CA-MEK on the localization of RhoA and Rac1. RhoA and Rac1 antibodies used for immunolocalization were tested to ascertain that they did not cross react with other cellular proteins. Western blot analysis of the whole cell extract showed single bands at 21 kDa representing RhoA and Rac1 without nonspecific cross reaction with other cellular proteins in control, DFMO-grown, and DP-grown cells (Fig. 7A). The results shown in Fig. 7A confirmed the specificity and suitability of these antibodies for immunolocalization studies as well as previous observations at our laboratory that polyamine depletion decreases RhoA protein levels without any effect on Rac1 protein levels (38). Figure 7B shows that Rac1 (4 and 6) and RhoA proteins (7 and 9) were localized throughout the cytoplasm in cells transfected with vector grown in control and DP media and that aggregates of RhoA at the cell periphery were distinguishable. Interestingly, in polyamine-depleted cells (DFMO group), the subcellular localizations of Rac1 (5) and RhoA (8) were significantly altered compared with the control and DP groups. Figure 7B (8) also shows a perinuclear and nuclear localization of the RhoA protein in polyamine-depleted cells transfected with vector and significantly less RhoA localized at the cell periphery. In contrast, cells transfected with CA-MEK showed that a significant fraction of RhoA was localized in large aggregates throughout the cytoplasm with relatively more toward the cell periphery (Fig. 7C, 46). To our surprise, a significant fraction of Rac1 in polyamine-depleted cells transfected with vector was localized in the nucleus (Fig. 7B, 5), but it was uniformly distributed in the cytoplasm in the control and DP groups (Fig. 7B, 4 and 6). Expression of CA-MEK decreased nuclear and increased cytoplasmic localization of Rac1 in polyamine-depleted cells (Fig. 7C, 5). Unlike polyamine-depleted cells transfected with vector (Fig. 7B, 5), Rac1 was localized in the protrusions of DFMO-treated cells transfected with CA-MEK (Fig. 7C, 5). In fact, polyamine depletion had no discernible effect on the localization of either Rac1 or RhoA in cells transfected with CA-MEK. Although immunolocalization using specific antibodies showed changes in the subcellular localization of RhoA and Rac1, we determined the levels of Rac1 in membrane, nucleus, and cytoplasmic fractions of cells transfected with vector and CA-MEK grown under control, DFMO, and DP conditions. The results in Fig. 8 show that polyamine depletion decreased the levels of Rac1 protein in the cytoplasmic and membrane fractions, while it increased those levels in the nuclear fraction of the vector-transfected cells. In contrast, in cells transfected with CA-MEK, the membrane fraction showed significantly higher Rac1 protein levels in the control, DFMO, and DP groups. Unlike cells transfected with vector, polyamine depletion had no effect on the levels of Rac1 in the membrane, nuclear, and cytoplasmic fractions of cells transfected with CA-MEK. Polyamine depletion had no effect on levels of total Rac1 protein in either group.



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Fig. 7. Effect of polyamine depletion on RhoA and Rac1 localization. IEC-6 cells were grown in medium ± 5 mM DFMO or DFMO + 10 µM putrescine for 4 days, total cell extracts were prepared, and equal amounts of protein were separated on SDS-PAGE gels, followed by Western blot analysis using antibodies specific for RhoA and Rac1. Representative Western blots from 3 observations are shown (A). Cells transfected with Vector (B) and CA-MEK (C) were grown on coverslips in medium ± 5 mM DFMO or DFMO + 10 µM putrescine for 4 days. The cells were then washed, fixed, and processed for the visualization of F-actin with rhodamine-conjugated phalloidin (1–3) and the localization of Rac1 (4–6) and RhoA (7–9) by specific antibodies. Alexa-Fluor 488-conjugated secondary antibodies were used for the detection of Rac1 and RhoA. A representative image from each group is shown. Arrows indicate actin cortex, and arrowheads indicate Rac1 and RhoA. C, control; D, DFMO; DP, DFMO + putrescine.

 


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Fig. 8. Rac1 distribution in subcellular fractions. Cells transfected with vector and CA-MEK were grown in medium ± 5 mM DFMO or DFMO + 10 µM putrescine for 4 days, and cell lysates were prepared, which were subjected to membrane, nuclear, and cytosolic fractionation as described in MATERIALS AND METHODS. Equal amounts of protein were separated on SDS-PAGE gels, followed by Western blot analysis using antibody specific for Rac1. Representative Western blots from 3 observations are shown.

 
Because activation of Rac1 occurs in the cytoplasm in response to the availability of upstream activators leading to its translocation to the membrane, and because polyamine depletion decreases Rac1 protein levels in the membrane fraction (Fig. 8), we determined the activity of Rac1 in cells transfected with vector and CA-MEK. Figure 9A show significantly high levels of activated Rac1 (GTP-Rac1) protein during 6 h of migration in cells transfected with vector grown under control conditions, while polyamine depletion significantly decreased the levels of active Rac1 in these cells. Expression of CA-MEK increased the levels of activated Rac1 in polyamine-depleted cells to the level of control cells (Fig. 9B). Cells grown in DP conditions were identical to controls, indicating that observed effects were due to the lack of polyamines and not to the presence of DFMO.



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Fig. 9. Expression of CA-MEK restores Rac1 activation in polyamine-depleted cells. Empty vector- (A) and CA-MEK-transfected (B) cells were grown in medium ± 5 mM DFMO or DFMO + 10 µM putrescine for 4 days. Confluent monolayers were wounded with a gel-loading tip, washed, and incubated with medium ± DFMO or DFMO + 10 µM putrescine. After 3 and 6 h, cell extracts were prepared and subjected to glutathione S-transferase-p21-activated kinase fusion protein pull-down assay as described in MATERIALS AND METHODS (Rac1 activation assay). Active (GTP-Rac1) and total Rac1 were detected by using Rac1-specific antibody. Representative Western blots from 3 observations are shown, along with densitometric analysis. *Significantly different from respective control and 0 h.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
During the renewal of skin and intestine, epithelial cells migrate up from a basal layer and crypts, respectively. Regulation of the migration of intestinal epithelial cells in response to wounding is important for the maintenance of gastrointestinal mucosal integrity under physiological and pathological conditions. Regeneration and repair of intestinal tissue are associated with proliferation and migration of epithelial cells from the adjacent mucosa (50). The regulation of mucosal growth is unique because it not only depends on normal blood factors but also is influenced by trophic hormones released from the mucosa and by other factors present within the digestive tract. Polyamines are known to play a critical role in the migration of intestinal epithelial cells, but the specific role of polyamines in migration is undefined. Earlier publications from our laboratory (37, 38, 45) established the role of Rho family GTPases in the regulation of migration and showed that Rac1, like RhoA, is essential for migration, but that Rac1 activity, unlike RhoA, is sufficient for migration.

The injury-induced increases in the production of EGF (18, 57) and overexpression of epidermal growth factor receptor (EGFR) have been observed during mucosal repair (48). EGF also stimulates the proliferation and migration of epithelial cells (2, 5). Previously, our laboratory showed that polyamine depletion attenuated mucosal repair in an in vivo rat model. The present study investigates the role of the EGF signaling pathway in cell migration to elucidate upstream events and the role of polyamines in the regulation of those events.

In the present study, we found that wounding transiently increased ERK1/2 activation and that EGF increased the migration in control and polyamine-depleted cells. However, in polyamine-depleted cells, EGF stimulated migration only to the level observed in untreated control cells (Fig. 1). U-0126, a strong inhibitor of MEK, inhibited basal and EGF-mediated ERK phosphorylation (Fig. 3), indicating that activation of MAPK is required for migration. Xie et al. (58) reported that the EGF-mediated disassembly of focal adhesions depended on ERK activation. Klemke et al. (17) reported that activated ERK can associate with and phosphorylate myosin light chain kinase, thereby increasing its kinase activity and enhancing cell migration. Thus ERK activation plays an important role by regulating the cellular machinery required for migration. In IEC-6 cells, we have observed that upon EGF treatment, EGFR downregulation occurs within 10–15 min (unpublished observations). Although EGF was present in the medium throughout the 6-h migration period, ERK1/2 activity decreased over time (Fig. 2), suggesting that EGFR downregulation prevents sustained activation of ERK. We predict that downregulation of EGFR and the transient nature of ERK activation might be rate-limiting factors in the migration of polyamine-depleted cells.

Sustained ERK2 activation mediates scattering in SK-N-MC cells in response to the Ret (rearranged during transfection) proto-oncogene and fibroblast growth factor (51), and colony dispersion in SCC-12F cells (21). In fibroblasts, U-0126 blocks EGF-mediated migration (46). These findings suggest that ERK1/2 inhibition alone is sufficient to prevent migration. CA-MEK cells showed HA tag staining localized exclusively to the nucleus, unlike the cytoplasmic localization observed in DN-MEK cells (Fig. 4). The results shown in Fig. 4 confirmed that CA- and DN-MEK cells indeed express recombinant MEK proteins in IEC-6 cells. Expression of CA-MEK significantly altered the actin cytoskeleton structure with extensive stress fibers and lamellipodia, characteristic features of the actively migrating cell. In contrast, DN-MEK expression disorganized the actin structure with significant loss of stress fibers and altered cell shape (Fig. 4). An intact and dynamic cytoskeleton is essential for maintaining the shape and adherence of cells to the substratum. A previous report from our laboratory showed that expression of DN-MEK increased detachment and induced subsequent spontaneous apoptosis or anoikis in IEC-6 cells (4). Our laboratory also has shown that polyamine depletion prevents reorganization of the cortical actin cytoskeleton into stress fibers and decreases integrin focal adhesion kinase (FAK) signaling and subsequent activation of Rho family GTPases (37–39). Because CA-MEK-transfected cells demonstrated extensive actin cytoskeletal remodeling (Fig. 6B), we predicted that sustained MEK activation might restore actin remodeling and migration in polyamine-depleted cells. As expected, CA-MEK expression completely prevented cortical actin formation observed in the polyamine-depleted vector-transfected cells, with a concomitant increase in stress fibers and lamellipodia (Fig. 7). Cell migration depends on reorganization of the actin cytoskeleton and development of cell polarity, enabling cells to make new adhesive contacts at their leading edge and break existing contacts at their trailing edge. This process is associated with actin reorganization and in some cases active membrane ruffling at the leading edge, an event mediated by the small GTPase Rac (15, 42). A contractile force at the rear of the cell develops and can be regulated by actin/myosin motor function (7, 8).

Evidence suggests that subcellular localization of Rho family GTPases is crucial for migration. A major function of Rho family GTPases is to regulate the organization of the actin cytoskeleton. Filopodia, lamellipodia, and stress fibers are regarded as components of the typical phenotypes of activated Cdc42, Rac, and Rho, respectively (15, 19, 42, 43). EGF increased migration of polyamine-depleted IEC-6 cells and CA-MEK increased Rac1 activity and migration, suggesting that steps leading to Rac activation, i.e., guanine nucleotide exchange factor activity, might not be affected by polyamine depletion. Immunocytochemical localization of Rac1 and RhoA in vector- and CA-MEK-transfected cells showed significantly different subcellular distributions of these proteins. RhoA in control and polyamine-depleted cells was present in the cytoplasm, and small aggregates were distributed toward the periphery of the cells (Fig. 7B, 4 and 6). Cells transfected with CA-MEK had larger and more evenly distributed RhoA aggregates throughout the cytoplasm in control as well as in polyamine-depleted cells. In contrast, and surprisingly, Rac1 was localized predominantly in the nucleus in polyamine-depleted vector-transfected cells (Fig. 7B, 5), unlike control cells and those grown in DP, in which Rac1 was evenly distributed throughout the cytoplasm. Increased Rac1 protein in the nuclear fraction and decreased levels in the cytoplasmic and membrane fractions of cells depleted of polyamine and transfected with vector support our immunofluorescence observations. Expression of CA-MEK increased Rac1 activity in control and polyamine-depleted cells as evidenced by its increased association with the membrane fractions and increased levels of GTP-Rac1 (Figs. 8 and 9B). However, cells transfected with vector had significantly low levels of Rac1 associated with the membrane and low levels of Rac1 activity during the 6-h migration period (Figs. 8 and 9A). These results support our assumption that decreased activation of Rac1 in polyamine-depleted cells might be due to the unavailability of Rac1 in the cytoplasm for activation by upstream exchange factors. More significantly, in polyamine-depleted cells transfected with CA-MEK, the cytoplasm and cell protrusions showed prominent Rac1 staining in all three groups. Furthermore, decreased levels of Rac1 in the cytoplasmic and membrane fractions of polyamine-depleted cells suggest that the recruitment of Rac1 to the membrane might be reduced as a result of retention in the nucleus in response to polyamine depletion. These results clearly demonstrate that sustained MAPK activation is important for the actin cytoskeletal organization essential for the migration of intestinal epithelial cells. Earlier we showed that transfection with constitutively active Rac1 and RhoA increased migration of control cells, but only Rac1 activation restored migration of polyamine-depleted cells. Furthermore, Rac1 activated RhoA, indicating that Rac1 is an important and rate-limiting factor for migration in IEC-6 cells.

In summary, we conclude that polyamine depletion sequesters Rac1 by translocating it to the nucleus, away from activating factors and other cytoplasmic upstream activating signals such as integrins and FAK. Constitutive activation of MEK in polyamine-depleted cells restored the cytoskeletal structure and migration, probably by preventing the translocation of Rac1 to the nucleus and promoting its distribution and activation into protrusive structures, leading to downstream activation of RhoA and Cdc42. The stimulation of migration by EGF and elucidation of the mechanism by which MEK regulates intracellular distribution and aggregation in actively migrating cells needs further exploration.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52784 and the Thomas A. Gerwin Endowment.


    ACKNOWLEDGMENTS
 
We sincerely acknowledge Mary Jane Viar for critically reading the manuscript and Greg Short and Danny Morse for help in preparation of the figures.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. M. Ray, Dept. of Physiology, The Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (E-mail: rray{at}physio1.utmem.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.


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