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HORMONES AND SIGNALING

Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration

Departments of ¹Cell and Developmental Biology, ²Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, and ³Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee

Submitted 25 July 2007 ; accepted in final form 8 November 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.

Rac GTPase; wound healing; restitution

A number of soluble growth factors present in the lumen, including the epidermal growth factor receptor (EGFR) ligands, transforming growth factor- (TGF-), and EGF (36, 50), trefoil factor peptides (15), and TGF-β (16), accelerate restitution in in vitro and in vivo models of intestinal injury, suggesting these factors may be beneficial for patients with pathologies involving intestinal ulceration such as IBD. In particular, absence of EGFR ligands is associated with spontaneous or increased susceptibility to experimental IBD in several mouse models (17, 27, 47). Furthermore, administration of EGF-containing enemas has been reported to induce remission in patients with ulcerative colitis (44), demonstrating that EGFR-mediated wound healing is a promising therapeutic approach for IBD in humans. These findings emphasize the importance of studies to increase our understanding of the molecular mechanisms by which EGFR ligands enhance intestinal epithelial restitution and repair.

Following ligand binding, EGFR dimerizes, acquires increased tyrosine kinase activity, and becomes highly phosphorylated on C-terminal cytoplasmic tyrosine residues that provide a platform for recruitment and activation of downstream signaling modules including mitogen-activated protein kinase (MAPK) cascades (25, 39), phosphatidylinositol 3-kinase (PI3K) (37), phospholipase C (PLC)- (2), and Src family kinases (32). Using an in vitro model of wound healing, we have previously shown that EGF stimulates migration of young adult mouse colonic epithelial (YAMC) cells surrounding a wound in the monolayer (14, 21). Although EGFR-stimulated cell proliferation has been exhaustively characterized (reviewed in Refs. 13 and 41) the signaling pathways that promote restitution downstream of EGFR are not as well defined. The process of cell migration requires tight regulation of the actin cytoskeletal organization, which is controlled by members of the Rho family of small GTPases Cdc42, Rac, and Rho (reviewed in Ref. 34). Rho GTPases cycle between an active GTP-bound state and an inactive GDP-bound state, regulated by guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GTP for GDP and GTPase activating proteins (GAPs), which stimulate the intrinsic GTPase activities of Rac, Rho, or Cdc42. When Rho GTPases are activated, a C-terminal prenylation site promotes translocation to the plasma membrane (reviewed in Refs. 38 and 43) initiating signaling pathways involved in modification of the actin cytoskeleton through downstream effector proteins that specifically recognize and bind to the GTP-bound form of the GTPase.

Data from several model systems implicate Rho GTPases as critical regulators of wound healing (reviewed in Ref. 48). In fibroblast migration assays, formation of lamellipodia at the leading edge of cells requires Rac activation (29). In this regard, extension of lamellipodia is the first visible sign of restitution following intestinal epithelial erosion both in vivo and in vitro (19, 20). Additionally, expression of dominant negative Rac1 in the mouse small intestine slowed enterocyte cell migration along the crypt-villus axis, implicating this small GTPase in the normal turnover and maintenance of the intestinal epithelium (45). As rapid cell migration is critical for proper restitution in the gastrointestinal tract, we hypothesized that the small GTPase Rac is also required for EGF-stimulated intestinal epithelial wound closure. Here we demonstrate that Rac, but not the related Rho GTPase Cdc42, is required for EGF-stimulated wound closure via a signal transduction pathway dependent upon both PI3K and Src family kinases. These data link EGFR activation in intestinal epithelial cells to increased motility and wound closure requiring Rac activation.

	MATERIALS AND METHODS

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Cell culture. The conditionally immortalized YAMC cell line isolated from the colonic epithelium of H-2K^b-tsA58 (Immorto) mice has been previously described (49). EGFR-null mouse colonic epithelial (MCE) (EGFR^–/– MCE) cells were isolated in the Vanderbilt Digestive Disease Research Center Novel Cell Line Core from the colonic epithelium of EGFR-null heterozygous mice crossed with the Immorto mouse (46, 49). Cells were maintained on rat tail collagen- (Mediatech, Herndon, VA) coated plates (5 µg/cm²) in RPMI 1640 supplemented with 5% FBS, 5 U/ml mouse IFN- (Intergen, Norcross, GA), 100 U/ml penicillin and streptomycin, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenous acid (BD Biosciences, San Jose, CA) at 33°C (permissive conditions). Before all experiments, cells were serum starved for 16–18 h in RPMI 1640 containing 0.5% FBS and 100 U/ml penicillin and streptomycin with no interferon at 37°C (nonpermissive conditions).

Antibodies, growth factors, and inhibitors. FITC-conjugated goat anti-mouse secondary antibody was from Jackson ImmunoResearch Labs (West Grove, PA). Mouse monoclonal Rac and rabbit polyclonal EGFR antibodies were from Upstate (Charlottesville, VA). Mouse monoclonal EGFR antibody conjugated to phycoerythrin, EGFR (528) PE, (Santa Cruz Biotechnology, Santa Cruz, CA) was used for fluorescence-activated cell-sorting (FACS) analysis. Mouse monoclonal hemagglutinin (HA)-Tag (262K), rabbit monoclonal pY1173 EGFR, rabbit polyclonal phospho-Akt (Ser 473) and pY925 FAK antibodies, and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody were from Cell Signaling Technology (Beverly, MA). Anti-active ERK1 and 2 polyclonal antibody was from Promega (Madison, WI). Mouse monoclonal actin antibody was from Sigma-Aldrich (St. Louis, MO). Mouse monoclonal Cdc42 antibody and HRP-conjugated goat anti-mouse IgG was from BD Transduction Laboratories (San Diego, CA). Murine EGF was a gift from Stanley Cohen (Vanderbilt University, Nashville, TN). Rac1 inhibitor NSC23766, U0126, wortmannin, and LY294002 were from Calbiochem (San Diego, CA). PP1 and PP2 were from Biomol (Plymouth Meeting, PA). Src inhibitor CGP77675 was a gift from Anna Suter (Novartis, Basel, Switzerland).

Generation of stable cells. EGFR^–/– MCE cells were stably transfected with pcDNA3.1/Zeo vector control (Invitrogen, Carlsbad, CA), pcDNA3.1/Zeo/wild-type EGFR, or pcDNA3.1/Zeo/kinase dead EGFR-K721R by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Zeocin-selected pools of cells were stained with anti-EGFR 528-PE antibody (50 µl/5 x 10⁶ cells). PE (EGFR) positive cells were sorted at the Veterans Affairs Medical Center Flow Cytometry Special Resource Center (Nashville, TN) by using a Becton-Dickinson FACSAria. Stable pools of cells expressing EGFR were maintained in 200 µg/ml Zeocin.

siRNA and transient transfections. Nontargeting, mouse Rac1, and mouse Cdc42 SMARTpool small interfering (si)RNA was from Dharmacon RNA Technologies (Lafayette, CO). YAMC cells were transfected at 50% confluence with 50 nM nontargeting, 50 nM Rac1, or 50 nM Cdc42 SMARTpool siRNA by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations for siRNA transfections. Twenty-four hours after transfection, the cells were serum starved for 16–18 h, and migration assays were performed as described below. Dominant negative Akt constructs were a gift from Dr. Joseph Testa. YAMC cells were transfected with vector control or dominant negative Akt1 or Akt2 using Lipofectamine 2000 according to the manufacturer's recommendations.

Migration assays. Cells were seeded onto 35-mm plates coated with 2.5 µg of human fibronectin (BD Bioscience, Bedford, MA). Six circular wounds were made in each confluent plate by using a rotating silicon tip as previously described (14). The cells were washed twice with PBS and incubated at nonpermissive conditions with or without 10 ng/ml EGF. For migration assays with the use of inhibitors, cells were pretreated with inhibitor for 45 min before wounding, and then fresh inhibitor was added after wounding. Wounds were photographed at 0 and 8 h after wounding, and the size of each wound was determined using ImageJ software (National Institutes of Health, Bethesda, MD). The percent wound closure after 8 h was calculated. Values shown are the average wound closure rates for all six wounds ± SD.

Immunofluorescence. YAMC cells cultured on fibronectin-coated glass chamber slides were wounded as described above, then washed twice with PBS, and incubated with or without 10 ng/ml EGF for 10 min. Cells were fixed in 4% paraformaldehyde-PBS and permeabilized with 0.4% Triton X-100 in PBS. The cells were then stained with a monoclonal Rac antibody (1:50) followed by anti-mouse FITC secondary. Coverslips were mounted by using the Prolong Antifade Kit (Molecular Probes) and imaged on a Zeiss Axiovert 200 microscope.

Cell lysates, SDS-PAGE, and Western blot analysis. Cells were washed twice with cold PBS and lysed in Rac lysis buffer [50 mM Tris·HCl (pH 7.5), 10 mM MgCl₂, 200 mM NaCl, 1% (vol/vol) Nonidet P-40, 5% (vol/vol) glycerol] with protease and phosphatase inhibitor cocktails (Sigma). Lysates were cleared by centrifugation, protein concentrations were determined with the D_C protein assay (Bio-Rad Laboratories, Hercules, CA), and 25 µg of each sample were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked in 5% (wt/vol) nonfat dry milk in Tris-buffered saline (50 mM Tris, 138 mM NaCl, 2.7 mM KCl, pH 8.0) with 0.05% Tween 20 (TBST) for 1 h. Membranes were probed with primary antibody at 4°C overnight, washed in TBST (6 x 5 min), incubated with HRP-conjugated secondary antibody for 1 h, and then washed in TBST (6 x 5 min). Bound HRP was detected by using Western Lightning enhanced chemiluminescence kit (Perkin-Elmer, Boston, MA).

Rac activation assays. Rac activation was assayed as previously described (7). Briefly, the p21-binding domain (PBD) of PAK fused to glutathione S-transferase (GST) was expressed from pGEX-PBD, a gift from Josephine Adams (University College of London, London). Recombinant GST-PBD was purified with glutathione-Sepharose 4B beads (Pharmacia, Piscataway, NJ). Immediately after treatment, cells were lysed in Rac lysis buffer, and 1 mg of total lysate was nutated with GST-PBD beads at 4°C for 1 h. Beads were collected by centrifugation and were washed three times with washing buffer [25 mM Tris·HCl (pH 7.6), 1 mM DTT, 30 mM MgCl₂, 40 mM NaCl, 1% (vol/vol) Nonidet P-40] and twice with washing buffer without Nonidet P-40. Proteins were eluted by boiling beads in 2x Laemmli sample buffer for 5 min, separated on a 15% SDS-polyacrylamide gel, and blotted for Rac.

Statistical analysis. Statistical significance between control and test conditions was determined using a paired two-sample Student's t-test with a confidence level of 0.05. All data presented are representative of at least three independent experiments.

	RESULTS

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EGF stimulates Rac membrane translocation and increased lamellipodia formation. We have previously reported that YAMC cells treated with EGF show increased rates of wound closure in an assay that mimics ulceration in vivo (14, 21). As lamellipodia formation is typically the first visible sign of restitution of experimental wounds (19), we hypothesized that EGF stimulates cell migration through increased lamellipodia formation. To test this idea, circular wounds were made in confluent monolayers of YAMC cells, and cultures were incubated with or without EGF for 10 min. The percentage of cells exhibiting visible lamellipodia at the wound margin was visualized by phase contrast microscopy and quantified. As shown in Fig. 1A, EGF stimulates a twofold increase in the percentage of cells with lamellipodia at the wound margin (35.97 ± 7.53 vs. 17.98 ± 4.48%).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Epidermal growth factor (EGF) stimulates increased lamellipodia formation and Rac membrane translocation in young adult mouse colonic epithelial (YAMC) cells. A: YAMC cells were wounded by using a rotating silicon tip, treated with or without EGF (10 ng/ml in this and all subsequent experiments) for 10 min, and the percent of cells at the wound margin with lamellipodial extensions was quantified. B: YAMC cells were wounded as in A, fixed, and stained for Rac (green) and nuclei labeled with DAPI (blue). Images were taken at x60. C: enlarged portion of image in B showing cells with lamellipodia at the wound margin in EGF-treated culture. *P < 0.001 compared with control. Arrowheads, Rac translocation to the leading edge.

Rac activation and expression are required for EGF-stimulated intestinal cell migration. To test the hypothesis that Rac activation is required for EGF-stimulated cell migration, we treated YAMC cells with EGF and measured wound closure after 8 h in the presence or absence of a Rac1 pharmacological inhibitor, NSC23766, which has been shown to specifically inhibit Rac activation without interfering with Cdc42 or Rho (22). The Rac1 inhibitor completely blocked EGF-stimulated wound closure compared with cells treated with vehicle alone (Fig. 2A). To determine the specificity of Rac regulation of EGF-stimulated migration, YAMC cells were transfected with nontargeting, Rac1, or Cdc42 siRNA pools and subjected to migration assays. Western blot analysis of the cells used in the migration assay confirmed that Rac1 and Cdc42 protein levels were decreased by siRNA transfection (data not shown). EGF-stimulated wound closure was completely blocked in cells transfected with Rac1 siRNA but not in cells transfected with either nontargeting or Cdc42 siRNA (Fig. 2B). Interestingly, both Rac1 inhibitor and Rac1 siRNA decreased basal cell migration, demonstrating that Rac regulates both basal as well as EGF-stimulated migration. In cells transfected with Cdc42 siRNA, basal cell migration was inhibited, but EGF-stimulated migration was not, suggesting that EGF signaling can bypass a requirement for Cdc42.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Rac activation and expression are required for EGF-stimulated cellular migration. A: YAMC cells were pretreated for 1 h with 50 µM Rac inhibitor NSC23766, then wounded, and exposed to EGF in the presence or absence of the Rac inhibitor. Wounds were photographed after 0 and 8 h, and percent wound closure was calculated. B: YAMC cells were transfected with either 50 nM nontargeting, 50 nM Rac1, or 50 nM Cdc42 small interfering (si)RNA and then wounded and measured as above. C: YAMC cells were transfected with indicated siRNA constructs, and Rac activation was assessed by using glutathione S-transferase (GST)-p21-binding domain (PBD) beads to isolate GTP-bound Rac with or without 3 min EGF exposure. Total lysates were prepared for Western blot analysis for Rac, Cdc42, or actin as a loading control. *P < 0.001 compared with matched untreated cells. @P < 0.01 compared with vehicle control (A) or nontargeting control (B) cells.

EGFR kinase activity is required for EGF-stimulated Rac activation and migration. To determine the kinetics of Rac activation, YAMC cells were treated with EGF for 0 to 10 min, and GTP-bound Rac levels were assessed. As shown in Fig. 3A, EGF stimulated a robust and rapid activation of Rac within 30 s that was sustained up to 6 min. EGF also promoted Akt and ERK1 and 2 phosphorylation in these cells. Interestingly, detectable activation of these proteins was first observed only after Rac activation, suggesting that the signaling pathways that activate Rac are upstream or independent of cell proliferation (ERK1 and 2) and cell survival (Akt) pathways.

View larger version (28K): [in this window] [in a new window]   Fig. 3. EGF rapidly stimulates Rac activation in YAMC but not EGFR–/– mouse colonic epithelial (MCE) cells. A: YAMC cells were treated with EGF for the indicated times, and Rac activation was assessed as in Fig. 2. The total lysate was blotted for Rac to confirm equal protein levels before the GST-PBD pulldown assay. Activation of Akt and ERK1 and 2 in the total lysate were detected using phospho-specific antibodies. B: YAMC and EGF receptor (EGFR)–/– MCE cells were exposed to EGF for the indicated times, and Rac activation was assessed as in A.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Expression of wild-type, but not kinase dead EGFR, in EGFR–/– MCE cells restores EGF-stimulated Rac activation and migration. YAMC cells and EGFR–/– MCE cells stably expressing vector, wild-type, or kinase dead EGFR were treated with EGF for 3 min. A: EGFR and ErbB2 were immunoprecipitated and immunocomplexes were subjected to SDS-PAGE and Western Blot analysis using anti-phosphotyrosine antibody. B: Rac activation was assessed as in Fig. 2. The total lysate was blotted for Rac to confirm equal protein levels before the GST-PBD pulldown assay and for EGFR to compare EGFR expression levels in the stable cell lines. Akt and ERK1 and 2 activation in the total lysate were determined as in Fig. 3. C: YAMC cells and EGFR–/– MCE cells stably expressing vector alone, wild-type EGFR, or kinase dead EGFR were wounded with a rotating silicon tip and incubated with or without EGF for 8 h. Percent wound closure was determined by time lapse microscopy as above. *P < 0.001 compared with matched control. pY, phosphotyrosine.

View larger version (29K): [in this window] [in a new window]   Fig. 5. EGF-stimulated Rac activation requires PI3K but not MEK1 and 2 or Akt activity. A: YAMC cells were incubated with 50 µM LY294002, 200 nM wortmannin, or 10 µM U0126 for 1 h before 3 min EGF exposure. Rac activation was analyzed as in Fig. 2. Phospho-Y1173 EGFR, phospho-Akt, and phospho-ERK1 and 2 were determined by Western blot analysis of total lysates. B: YAMC cells were transfected with vector, dominant negative Akt1 (DN Akt1), or dominant negative Akt2 (DN Akt2) and then exposed to EGF for 3 min, and Rac activation was assessed as in A. Hemogglutinin (HA)-Akt and phospho-Akt levels were detected by Western blot analysis. C: quantification of Rac activation levels in inhibitor and DN Akt experiments. Values represent the average of at least 3 independent experiments. *P < 0.001 vs. control.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Src family kinases are required for EGF-stimulated Rac activation and cell migration. Serum-starved YAMC cells were incubated with 2 µM CGP77675, 12.5 µM PP1, or 7.5 µM PP2 for 1 h. A: cells were treated with EGF for 3 min. Rac activation was assessed as above. Total lysates were blotted for Rac to confirm equal protein levels before the GST-PBD pulldown assay. Activation of focal adhesion kinase (FAK), EGFR, and ERK1 and 2 were detected by using the indicated phospho-specific antibodies. B: wounds were prepared as above, and percent wound closure was determined with or without EGF. *P < 0.001 compared with matched untreated cells. @P < 0.01 compared with vehicle untreated cells.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Src and PI3K both contribute to maximal Rac activation in response to EGF. YAMC cells were incubated with inhibitors as above or with both 50 µM LY294002 and 2 µM CGP77675 for 1 h. A: cells were exposed to EGF for 3 min. Rac activation was analyzed using GST-PBD beads to isolate GTP-bound Rac. Activation of EGFR, FAK, and Akt were detected in the total lysate by using phospho-specific antibodies as indicated. B: cell monolayers were wounded with or without EGF for 10 min, fixed, and subjected to immunofluorescence localization analysis for Rac as in Fig. 1. The percentage of cells at the wound margin with Rac at the leading edge was counted. C: percentage of cells at the wound margin with lamellipodial extensions was quantified. *P < 0.01 vs. control.

	DISCUSSION

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In response to injury, epithelial cells at the wound margin sense the loss of neighboring cells, extend lamellipodia, and begin to migrate into the denuded area to close the wound (26). Although EGF stimulates increased migration in YAMC cells in our wound closure assay, lamellipodial extension and cell movement into the denuded area do still occur in the absence of exogenous growth factor, albeit with reduced kinetics (Fig. 1, A and B; Fig. 2, A and B, untreated cells). Scrape wounding alone weakly activates Rac in YAMC cells, presumably only in the cells adjacent to the wound margin (R. Dise and D. Polk, unpublished observations), yet this activation appears to be important for restitution because both Rac inhibitor and Rac siRNA significantly decrease basal migration (Fig. 2), as do Src or PI3K inhibitors (Fig. 6) (18). Thus Src, PI3K, and Rac are effectors of both growth factor stimulated and basal migration, unlike some EGF-stimulated pathways such as p38 MAPK, which, at least in the YAMC cell model, is required for growth factor-induced but not basal wound healing (21). We hypothesize that one mechanism whereby EGF promotes colonic epithelial restitution is via more rapid and robust Rac activation in cells surrounding the wound margin because they both sense the denuded area and take up ligand, reinforcing signaling downstream of EGFR to enhance cellular migration. Thus EGFR stimulates both signals shared with basal restitution mechanisms and those unique to growth factor-accelerated wound healing (e.g., p38 MAPK). However, it is still unclear whether the signals intermediate of EGFR and Rac are similar to those by which mechanical wounding activates Rac and whether EGFR plays any role in the latter response, especially given that intestinal epithelial cells lacking EGFR do undergo restitution, despite an inability to respond to EGF (21). Interestingly, studies have demonstrated that filopodial extensions direct the formation of lamellipodia, suggesting that Cdc42 is upstream of Rac activation (30) in unstimulated migration. Consistent with this idea, we observed that transfection of cells with Cdc42 siRNA significantly inhibits basal cell migration in untreated cells (Fig. 2B). However, Cdc42 siRNA did not attenuate migration in cells exposed to EGF, implying that this small GTPase, unlike Rac, is dispensable for growth factor-induced restitution. Similarly, we were unable to detect Cdc42 activation in response to EGF (data not shown), and Cdc42 siRNA did not inhibit EGF-stimulated Rac activation (Fig. 2C), again uncoupling growth factor-induced Rac activation and migration from Cdc42.

Akt phosphorylation in response to growth factor-stimulated PI3K activation has been well defined (reviewed in Ref. 11), and PI3K and Akt activity are often thought to be equivalent. EGFR ligands activate PI3K, increasing cellular membrane PI-3,4,5-P₃, which initiates membrane localization of proteins containing plekstrin homology (PH) domains, such as Akt. In our studies, pharmacological inhibition of PI3K with LY294002 and wortmannin in YAMC cells decreases both Akt and Rac activation in response to EGF (Figs. 5 and 7). However, EGF-stimulated Rac activation in YAMC cells is maximal before maximal Akt activation (Fig. 3A), and a dominant-negative Akt fails to attenuate Rac activation (Fig. 5B). Thus PI3K appears to mediate Rac activation via a yet to be identified alternative substrate.

EGFR activates another small GTPase, Ras, resulting in increased proliferation through activation of the ERK MAPK cascade (12). In agreement with our previous findings showing that ERK MAPK signaling is not required for colonic epithelial migration (21), pharmacological inhibition of the MAPK cascade using a MEK1 and 2 inhibitor, U0126, did not alter Rac activation in response to EGF (Fig. 5A). These data indicate that the signaling pathways upstream of Rac activation in cultured MCE cells are part of a proliferation-independent phase of wound restitution, mimicking the in vivo response to injury.

Using three different pharmacological Src inhibitors, we demonstrate a requirement for Src family kinase activity in Rac activation. Previous work from our laboratory has shown that Src, Yes, and Fyn are expressed in YAMC cells and become activated in response to EGF (21). Because Src inhibitors regulate different family members to varying degrees, we attempted to provide further specificity to our analysis by using siRNA pools. However, we were not able to completely knock down individual family members, though some inhibition of Rac activation was observed with both Src and Fyn siRNA (data not shown). One possible function for Src family members in this context is stimulation of a Rac-specific GEF in intestinal epithelial cells. Data suggest that the most important factor influencing GTPase activation is the regulation of GEFs (42). Presently 85 mammalian GEFs have been identified, most of which contain the characteristic PH domain. Although a small number of GEFs have been well characterized, most are either novel or little is known about their GTPase specificity, making identification of the specific GEF involved in a particular cellular process problematic. Furthermore, although GEF activation is thought to be tightly regulated, a common mechanism of regulation has not been established. Vav2, a GEF for Rho, Rac, and Cdc42, has been shown to coimmunoprecipitate with activated EGFR and is tyrosine phosphorylated in response to EGF treatment of HeLa cells (33). Although Vav2 is expressed in YAMC cells, we were unable to detect either tyrosine phosphorylation of Vav2 or association of Vav2 with EGFR in response to EGF in YAMC cells (data not shown). An attractive hypothesis consistent with our present data is that the GEF responsible for Rac activation in MCE cells may be activated by Src family kinase tyrosine phosphorylation to relieve an intramolecular inhibition and expose the Dbl homology domain to facilitate exchange, similar to regulation of the hematopoietic-specific GEF Vav1 (1). Future experiments are required in order to focus on identifying the Src family member involved in Rac activation and identification of potential tyrosine phosphorylated GEFs in these cells that may be Src substrates. Alternatively, it is also possible that regulation of guanine nucleotide dissociation inhibitors (GDIs) or GAPs for Rac may be regulated by Src kinases, or that the role of Src in Rac activation is through Src-specific phosphorylation on EGFR since EGF stimulates phosphorylation of EGFR on tyrosines 845 and 1101 as Src kinase substrates (9). It is possible that these tyrosine residues activate signaling pathways involved in Rac activation, resulting in the requirement for Src family kinases to phosphorylate EGFR but not the EGF or other targets directly to activate Rac.

Rac activation has long been described as a key mediator of lamellipodial extension and cell migration. In recent years, significant progress has been made with regard to the exact mechanisms by which Rac participates in these processes. For example, direct binding to multifunctional scaffold molecules such as IQ motif and GAP-related domain containing protein 1 (IQGAP1) has been demonstrated to link Rac with rearrangements of the actin cytoskeleton, which promotes lamellipodial protrusion (6). IQGAP1 appears to serve as both regulator and effector of Rac1 and Cdc42; for example, it can suppress the intrinsic GTPase activity of Cdc42 (23), effectively promoting or sustaining Cdc42-driven signaling. In turn, IQGAP1 can function downstream of Rho family GTPases, driving cytoskeletal rearrangement through interaction with the Arp2 and 3 complex and neural Wiskott-Aldrich syndrome protein (N-WASP) (8, 28). Although it is not currently known whether EGF signaling through Rac is regulated by PI3K and/or Src at the level of an IQGAP-associated complex, the possibility is intriguing, given recent results implicating direct binding of IQGAP1 to Rac in response to VEGF in neural progenitor (4) and endothelial cells (51). Interestingly, in the study from Yamaoka-Tojo and colleagues (51), IQGAP1 served as a scaffold for a complex including Rac1 and the VEGF type 2 receptor; this complex appeared to be required for Akt phosphorylation, cell migration, and cell growth in response to reactive oxygen species. Whether this is a common mechanism shared by other growth factor receptors such as EGFR remains to be determined. IQGAP1 has been identified as an EGFR-binding protein in a proteomic screen (10), but the physiological significance of this interaction is as yet untested.

Our data indicate a requirement for both Src family kinases and PI3K signaling for maximal Rac activation and extension of lamellipodia in response to EGF. Since GEF activation requires translocation to the plasma membrane through PH domains, an attractive hypothesis in this regard is that PI3K recruits the GEF to the plasma membrane, bringing it into proximity with activated EGFR and Src, where it can be further activated through phosphorylation. The synergistic effect of inhibiting both Src and PI3K suggests that these pathways contribute independently to activation of Rac (Fig. 7). Because inhibition of PI3K does not block Src activity (Fig. 7), it is unlikely that PI3K is upstream of Src, and similarly inhibition of Src family kinases does not block Akt phosphorylation, demonstrating that PI3K activation is independent of Src tyrosine kinase activity. Thus our data suggest a model in which PI3K and Src work in parallel to fully activate Rac. Additionally, we find that pharmacological inhibition of PLCs, protein kinase Cs (PKCs), and p38 MAPK does not affect EGF-stimulated Rac activation (data not shown). Together, these data demonstrate a novel pathway whereby EGFR signaling increases wound closure through the coordinated activation of two different kinase cascades that induce colonic epithelial cell motility via Rac. These data contribute to our understanding of the complex molecular mechanisms regulating this small GTPase family member, which is critical to the maintenance of an intact intestinal monolayer in vitro and in vivo with implications for colonic epithelial diseases such as ulcerative colitis.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institutes of Health Awards R1-DK-054993 and K01-DK-077956, the Vanderbilt Digestive Disease Research Center, P30-DK-058404, and a Research Fellowship Award from the Crohn's and Colitis Foundation of America.

	ACKNOWLEDGMENTS

 
We thank members of the Polk Laboratory, especially Wei Tong, for technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. Brent Polk, 1025 MRBIV, 2215 Garland Ave., Nashville, TN 37232-0696 (e-mail: d-brent.polk{at}vanderbilt.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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