Department of Physiology, The University of Tennessee Health Science Center, Memphis, Tennessee
Submitted 25 June 2004 ; accepted in final form 13 October 2004
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ABSTRACT |
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epidermal growth factor; extracellular signal-regulated kinase; IEC-6 cells
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 -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
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.
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MATERIALS AND METHODS |
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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 -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 (WW0WWT). 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.
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RESULTS |
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DISCUSSION |
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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 1015 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 (3739). 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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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