Role of β-Pix in Corneal Epithelial Cell Migration on Fibronectin

Abstract

Purpose.: Corneal epithelial migration during wound healing is important for maintenance of corneal transparency, and fibronectin plays a key role in regulation of the adhesion and migration of corneal epithelial cells. The role of β-Pix in intracellular signaling that underlies the stimulatory effects of fibronectin on the adhesion and migration of corneal epithelial cells was examined.

Methods.: Simian virus 40–transformed human corneal epithelial (HCE) cells were cultured on fibronectin or on bovine serum albumin as a control. The localization and tyrosine phosphorylation of β-Pix were examined by immunofluorescence and immunoprecipitation analyses, respectively. The actin cytoskeleton and focal adhesions were detected by staining of cells with rhodamine-phalloidin and antibodies to phosphotyrosine, respectively. The effects of depletion of β-Pix on HCE cell adhesion and migration on fibronectin were investigated by cell transfection with a small interfering RNA specific for β-Pix mRNA.

Results.: Fibronectin induced the tyrosine phosphorylation of β-Pix as well as its apparent accumulation at focal adhesions in HCE cells. Depletion of β-Pix inhibited the effects of fibronectin on remodeling of the actin cytoskeleton and the formation of focal adhesions. It also inhibited the migration of HCE cells on fibronectin in an in vitro model of wound healing, but it did not affect cell adhesion to fibronectin.

Conclusions.: β-Pix contributes to the regulation of the formation of focal adhesions as well as that of cell migration by fibronectin in HCE cells. This protein therefore likely plays an important role in signal transduction underlying corneal epithelial wound healing.

The corneal epithelium plays an essential role in maintenance of corneal homeostasis, with the healing of corneal epithelial wounds being important for restoration of corneal transparency. In response to injury of the corneal epithelium, the remaining epithelial cells adhere to and migrate over a fibronectin matrix deposited at the wound site to cover the area of the defect. ^{1 –3} Fibronectin also promotes the adhesion and migration of corneal epithelial cells in organ culture or in monolayer culture in vitro. ^4,5 A peptide (PHSRN) derived from the second cell-binding domain of fibronectin also promotes the migration of corneal epithelial cells in culture. ⁶ Corneal epithelial cells upregulate the expression of integrin chains that serve as receptors for fibronectin during wound healing. The outside-in signaling induced by the adhesion of the cells to fibronectin promotes the clustering of integrins as well as the formation of focal adhesions that link to the actin cytoskeleton and thereby regulate cell adhesion and migration. ^5,7  

Small GTPases of the Rho family, including Rho, Rac, and Cdc42, act as molecular switches to control the remodeling of the actin cytoskeleton that underlies various cellular processes including adhesion, migration, and proliferation. ⁸ We have previously shown that lysophosphatidic acid activates Rho and thereby promotes the migration of corneal epithelial cells. ⁹ We also found that Rac1 is activated in corneal epithelial cells plated on fibronectin, and that the activated Rac1 induces remodeling of the actin cytoskeleton and the formation of focal adhesions. ⁵ Rac1 was also shown to contribute to the regulation of corneal epithelial cell adhesion and motility by fibronectin. 

Small GTPases of the Rho family cycle between inactive (GDP-bound) and active (GTP-bound) forms, with these transitions being regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. ¹⁰ β-Pak–interacting exchange factor (β-Pix) is a GEF for Rac1 and Cdc42 ^11,12 and has been shown to regulate cell motility induced by lysophosphatidic acid. ¹³ It has remained unknown, however, whether β-Pix contributes to signaling activated by fibronectin in corneal epithelial cells during wound healing. We have therefore now examined the possible role of β-Pix in the regulation of corneal epithelial cell adhesion and migration by fibronectin. 

Methods

Materials

A mixture of Dulbecco's modified Eagle's medium (DMEM) and nutrient mixture F-12 as well as reduced serum medium (OPTI-MEM), trypsin-EDTA, gentamicin, fetal bovine serum, cationic liposome-based reagent(Lipofectamine 2000), and both a small interfering RNA (siRNA) specific for β-Pix mRNA and a control scrambled siRNA were obtained from Invitrogen-Gibco (Carlsbad, CA). Fibronectin was obtained from Roche (Basel, Switzerland). Bovine serum albumin (BSA), cholera toxin, bovine insulin, recombinant human epidermal growth factor, and a protease inhibitor cocktail were obtained from Sigma-Aldrich (St. Louis, MO). Basic fibroblast growth factor (bFGF) was obtained from R&D Systems (Minneapolis, MN). Plastic culture dishes (100-mm diameter) and 24-well plates were obtained from Corning (Corning, NY), and 35-mm glass-bottom culture dishes were from Iwaki (Tokyo, Japan). Mouse monoclonal antibodies to phosphotyrosine were obtained from UBI (Temecula, CA), those to β-Pix were from Millipore (Billerica, MA), and those to β-actin were from Sigma-Aldrich. Alexa Fluor 488–labeled goat antibodies to mouse immunoglobulin G, rhodamine-phalloidin, and carbocyanine dimer stain (TOTO-3) were obtained from Invitrogen. Protein G–conjugated beads (Sepharose 4B), horseradish peroxidase–conjugated goat secondary antibodies, and detection reagents were obtained from GE Health Care (ECL Plus; Little Chalfont, UK). 

Cells and Cell Culture

Simian virus 40–immortalized human corneal epithelial (HCE) cells ¹⁴ were provided by RIKEN Biosource Center (Tokyo, Japan). They were cultured in supplemented hormonal epithelial medium (SHEM), which consists of DMEM/F-12 supplemented with 15% heat-inactivated fetal bovine serum, bovine insulin (5 μg/mL), cholera toxin (0.1 μg/mL), recombinant human epidermal growth factor (10 ng/mL), and gentamicin (40 μg/mL). The epithelium of the rabbit cornea was removed mechanically as previously described. ¹⁵ For experiments, HCE cells were plated at a density of 2 × 10⁴ cells per 35-mm dish, 5 × 10⁵ cells per 100-mm dish, or 5 × 10⁴ cells per well in 24-well plates, all of which had been coated with fibronectin (10 μg/mL) plus 1% BSA or with 1% BSA alone (control). 

Immunoblot Analysis

HCE cells incubated in 100-mm dishes as well as rabbit corneal epithelium were lysed on ice in 0.5 mL of a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% nonionic, nondenaturing detergent (Nonidet P-40; Roche), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na₃VO₄, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000g for 15 minutes at 4°C, and the resulting supernatants were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. The separated proteins were transferred electrophoretically to a nitrocellulose membrane. After blocking of nonspecific sites with 5% skim milk, the membrane was incubated for 1 hour at room temperature with primary antibodies at a dilution of 1:1000 in washing buffer [20 mM Tris-HCl (pH 7.4), 5% skim milk, 0.1% Tween 20]. The membrane was washed in washing buffer, incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibodies (1:1000 dilution in washing buffer), washed again, incubated with detection reagents (ECL Plus) for 5 minutes, and then exposed to film. 

Immunoprecipitation

HCE cells incubated in 100-mm dishes were lysed on ice in 0.5 mL of a solution containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MgCl₂, 10% glycerol, 1 mM EGTA, 1 mM NaF, 1% nonionic, nondenaturing detergent (Nonidet P-40), 100 μM Na₃VO₄, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000g for 10 minutes at 4°C, and the resulting supernatants (100 μg of protein) were incubated for 1 hour at 4°C in a final volume of 200 μL with antibodies to β-Pix (1:100 dilution) and 20 μL of protein G (Sepharose) beads. The beads were then separated by centrifugation and washed twice with cell lysis buffer, and the bound proteins were subjected to immunoblot analysis as described above. 

Cell Transfection with siRNA

Cells (5 × 10⁵) were seeded in 100-mm dishes and cultured for 24 hours to 50–60% confluence in SHEM. Each siRNA (final concentration of 100 nM) was mixed with cationic liposome-based reagent (Lipofectamine 2000; 5 μL) and diluted to a final volume of 500 μL with cationic liposome-based reagent (OPTI-MEM) before addition to the cells in 4.5 mL of the medium. After incubation with siRNA for 3 hours, the cells were incubated for an additional 6 hours in SHEM, replated on culture dishes, and cultured for 72 hours in SHEM before experiments. 

Cell Adhesion Assay

Cells incubated in 24-well plates for 60 minutes in unsupplemented DMEM/F-12 were washed twice with Ca²⁺- and Mg²⁺-free phosphate-buffered saline [PBS(–)] and then exposed to 0.05% trypsin-EDTA for 30 minutes at 37°C. The number of cells released by trypsin-EDTA was then determined with the use of a cell counter (Beckman Coulter, Brea, CA). 

Immunofluorescence Microscopy

Cells incubated in 35-mm glass-bottomed culture dishes were fixed for 15 minutes at 37°C with 3.7% formalin, washed twice with PBS(–), and incubated for 1 hour at room temperature with 1% BSA in PBS(–). The cells were then incubated for 1 hour at room temperature with antibodies to phosphotyrosine or to β-Pix [1:100 dilution in PBS(–) containing 1% BSA], washed with PBS(–), and incubated for 1 hour with Alexa Fluor 488–conjugated secondary antibodies (1:1000 dilution) and rhodamine-phalloidin (1:100 dilution) in PBS(–) containing 1% BSA. They were then examined with a laser confocal microscope (LSM5; Carl Zeiss, Hallbergmoos, Germany). 

Wound Closure Assay

Cells were seeded in coated 24-well plates and cultured for 72 hours in SHEM before incubation for 6 hours in unsupplemented DMEM/F-12. The cell monolayer was then scraped with the narrow end of a micropipette tip to generate a wound ∼0.1 cm in width. Cells were fixed in PBS(–) containing 3.7% formalin at various times thereafter, and phase-contrast images were acquired with a Zeiss Axiovert inverted microscope equipped with a charge-coupled device camera (Carl Zeiss). The wound area in each image was determined by computerized planimetry with NIH Image ver. 1.62f software. 

Statistical Analysis

Quantitative data are presented as mean ± SD. Differences were analyzed with Dunnett's test. A P value of <0.05 was considered statistically significant. 

Results

We first examined the effects of fibronectin on the localization of β-Pix and on the actin cytoskeleton in HCE cells (Fig. 1). The cells were deprived of serum and then cultured on fibronectin plus BSA or on BSA alone for 60 minutes before analysis by immunofluorescence microscopy. Immunostaining for β-Pix revealed immunoreactivity to be present in large dotlike structures associated with large bundles of F-actin (revealed by staining with rhodamine-phalloidin) at the cell periphery in HCE cells plated on fibronectin plus BSA. In contrast, HCE cells plated on BSA alone exhibited few such β-Pix–positive dotlike structures and a thinner rim of F-actin staining. Staining with antibodies to phosphotyrosine also revealed numerous large dotlike structures, corresponding to focal adhesions, that were associated with bundles of F-actin at the cell periphery in HCE cells plated on fibronectin plus BSA. The similarity in the patterns of β-Pix and phosphotyrosine staining thus suggested that fibronectin might induce the redistribution of β-Pix to focal adhesions linked to the actin cytoskeleton in HCE cells. 

Figure 1.

 

Effects of fibronectin on the distribution of β-Pix and on the actin cytoskeleton in HCE cells. Cells were incubated in unsupplemented DMEM/F-12 for 24 hours before plating in the same medium on glass-bottomed culture dishes that had been coated with fibronectin (FN) plus BSA (middle and bottom panels) or with BSA alone (top panels). The cells were incubated for 60 minutes and then fixed and stained with rhodamine-phalloidin (red) to detect actin filaments as well as with antibodies to β-Pix (green) or with those to phosphotyrosine (p-Tyr, green) to detect focal adhesions. Nuclei were detected by staining with carbocyanine dimer stain (TOTO-3; blue). Scale bar, 10 μm. Data are representative of three independent experiments.

We next examined whether the tyrosine phosphorylation of β-Pix is induced in response to the adhesion of HCE cells to fibronectin. Immunoprecipitation analysis revealed that the amount of tyrosine-phosphorylated β-Pix was indeed markedly increased 60 minutes after seeding of HCE cells on fibronectin plus BSA compared with that apparent for cells plated on BSA alone (Fig. 2A). With the use of immunoblot analysis, we also confirmed that β-Pix is expressed in the normal rabbit corneal epithelium (Fig. 2B), rendering unlikely the possibility that its expression in HCE cells was related to cell immortalization. 

Figure 2.

 

Effect of fibronectin on the tyrosine phosphorylation of β-Pix in HCE cells and expression of β-Pix in the rabbit corneal epithelium. (A) Cells were incubated in unsupplemented DMEM/F-12 for 24 hours before plating in the same medium in dishes that had been coated with fibronectin plus BSA or with BSA alone. The cells were incubated for 60 minutes, after which cell lysates were prepared and subjected to immunoprecipitation (IP) with antibodies to β-Pix. The resulting precipitates were subjected to immunoblot (IB) analysis with antibodies to phosphotyrosine and to β-Pix. (B) A lysate of rabbit corneal epithelium was subjected to immunoblot analysis with antibodies to β-Pix (lane 2). Lysis buffer alone was analyzed as a negative control (lane 1). All data are representative of three independent experiments.

To investigate the possible role of β-Pix in the adhesion and migration of HCE cells, we depleted the endogenous protein by RNA interference (RNAi). We thus transfected HCE cells with an siRNA specific for β-Pix mRNA or with a control siRNA and then cultured the cells for 72 hours. Immunoblot analysis revealed that transfection of HCE cells with β-Pix siRNA resulted in a concentration-dependent decrease in the amount of endogenous β-Pix compared with that apparent in cells transfected with the control siRNA (Fig. 3). 

Figure 3.

 

Depletion of β-Pix by RNAi in HCE cells. Cells were transfected with β-Pix siRNA (final concentration of 50 or 100 nM) or a control siRNA (100 nM) and were cultured for 72 hours after transfection. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to β-Pix and to β-actin (loading control). Data are representative of three independent experiments.

We examined the effect of depletion of β-Pix by RNAi on the morphology of HCE cells cultured on fibronectin plus BSA. Immunofluorescence analysis revealed that phosphotyrosine staining, corresponding to focal adhesions, was decreased markedly at the periphery of cells transfected with the β-Pix siRNA compared with that apparent in those transfected with the control siRNA (Fig. 4). Indeed, depletion of β-Pix reduced both the apparent size of focal adhesions as well as the thickness of the rim of F-actin staining at the cell periphery. 

Figure 4.

 

Effects of β-Pix depletion by RNAi on the morphology of HCE cells plated on fibronectin. Cells transfected with β-Pix or control siRNAs (100 nM) and cultured for 72 hours as in Figure 3 were incubated in unsupplemented DMEM/F-12 for 6 hours before plating in the same medium on dishes coated with fibronectin plus BSA. After incubation for 60 minutes, the cells were fixed and subjected to immunofluorescence staining with antibodies to phosphotyrosine (green) as well as to staining with rhodamine-phalloidin (red) and with carbocyanine dimer stain (TOTO-3; blue). Scale bar, 10 μm. Data are representative of three independent experiments.

We next investigated the effect of β-Pix depletion on the migration of HCE cells cultured on fibronectin plus BSA. Cell migration in a wound closure assay was found to be inhibited by transfection with the β-Pix siRNA compared with that apparent for cells transfected with the control siRNA (Fig. 5A). Whereas cells transfected with the control siRNA had covered ∼74 and ∼98% of the original wound area after 12 and 24 hours, respectively, those transfected with the β-Pix siRNA had covered only ∼7 and ∼21% of the wound area at these times (Fig. 5B). 

Figure 5.

 

Effect of β-Pix depletion by RNAi on the migration of HCE cells on fibronectin in a wound healing assay. (A) Cells transfected with β-Pix or control siRNAs were replated in 24-well plates coated with fibronectin plus BSA and cultured for 72 hours. They were then incubated in unsupplemented DMEM/F-12 for 6 hours before scratch wounding. The cells were fixed at 0, 12, and 24 hours after wounding, and the wounded area was examined by phase-contrast microcopy. Scale bar, 200 μm. (B) Quantitation of the remaining area of the wound at the indicated times after wounding. Data are mean ± SD of triplicates from an experiment that was repeated three times with similar results. *P < 0.05 versus the corresponding value for cells transfected with the control siRNA (Dunnett's test).

We examined the effect of β-Pix depletion on the adhesion of HCE cells to fibronectin. The extent of cell adhesion did not differ between cells transfected with β-Pix siRNA and those transfected with the control siRNA (Fig. 6). 

Figure 6.

 

Effect of β-Pix depletion by RNAi on the adhesion of HCE cells to fibronectin. Cells transfected with β-Pix or control siRNAs and cultured for 72 hours as in Figure 3 were incubated in unsupplemented DMEM/F-12 for 6 hours before plating in the same medium in 24-well plates coated with fibronectin plus BSA. After incubation for 60 minutes, the number of adherent cells was determined. Data are mean ± SD from three independent experiments.

Finally, we investigated the effect of bFGF on β-Pix localization in and the morphology of HCE cells. Immunofluorescence analysis revealed that bFGF induced the formation of lamellipodia and filopodia as well as markedly increased the extent of β-Pix staining at the cell periphery (Fig. 7). 

Figure 7.

 

Effects of bFGF on the distribution of β-Pix and on the morphology of HCE cells. Cells were incubated in unsupplemented DMEM/F-12 for 24 hours before plating in the same medium on glass-bottomed culture dishes that had been coated with BSA alone. The cells were then incubated for 60 minutes in the absence or presence of bFGF (10 ng/mL), fixed, and stained with antibodies to β-Pix (green) as well as with rhodamine-phalloidin (red) and carbocyanine dimer stain (TOTO-3; blue). Scale bar, 10 μm. Data are representative of three independent experiments.

Discussion

We have shown that fibronectin induced the redistribution of β-Pix to focal adhesions in association with the formation of such structures and the accumulation of F-actin at the cell periphery in HCE cells. Fibronectin also increased the tyrosine phosphorylation of β-Pix in HCE cells, as revealed by immunoprecipitation and immunoblot analysis. Depletion of endogenous β-Pix by RNAi inhibited the formation of focal adhesions in HCE cells plated on fibronectin as well as the migration of cells on fibronectin in a wound closure assay. Depletion of β-Pix did not affect the adhesion of HCE cells to fibronectin, however. Our results thus suggest that signaling by β-Pix mediates the effect of fibronectin on HCE cell migration. 

Fibronectin induces cell adhesion and migration in various cell types. ^5,16 We have previously shown that fibronectin activates Rac1 and induces the formation of focal adhesions in association with membrane ruffling at the cell periphery in HCE cells. ⁵ Moreover, activated Rac1 contributes to the stimulatory effects of fibronectin on the adhesion and motility of HCE cells. β-Pix functions as a GEF for the small GTPases Rac1 and Cdc42. ^17,18 We have now shown that ablation of endogenous β-Pix by RNAi inhibited the migration of HCE cells on fibronectin without affecting cell adhesion to fibronectin. Depletion of β-Pix also disrupted the formation of large focal adhesions and the accumulation of actin at the periphery of HCE cells plated on fibronectin, although the formation of numerous smaller focal adhesions remained apparent. These results suggest that a signaling pathway mediated by β-Pix and Rac1 contributes to the migration of corneal epithelial cells during wound healing. 

The Rho family of small GTPases contributes to the regulation of cell adhesion and migration through remodeling of the actin cytoskelton. ^19,20 These proteins interact with various effectors to mediate intracellular signaling. ^21,22 Rho and its effector ROCK play an important role in cell adhesion and migration during corneal epithelial wound healing. ^9,23 bFGF stimulates the migration of corneal endothelial cells through activation of Cdc42 and inactivation of Rho. ²⁴ We also found that Rac1 and PAK, an effector of Rac and Cdc42, are activated in HCE cells plated on fibronectin, with this activation contributing to regulation of the adhesion and migration of HCE cells by fibronectin. ⁵ The activation of Rac1 and its localization to focal adhesions were found to be regulated by β-Pix in HEK 293 cells. ²⁵ Members of the Rho family of GTPases have been shown to be expressed at a relatively high level in the epithelium of the mouse cornea. ²⁶ We have now found that β-Pix, a GEF for Rac1 and Cdc42, contributes to the stimulatory effect of fibronectin on the migration of HCE cells, but it does not appear to mediate the promotion of HCE cell adhesion by fibronectin. These results suggest that cross-talk among signaling pathways mediated by Rho family proteins may be required for the promotion of the adhesion and migration of HCE cells by fibronectin. 

We have shown that β-Pix contributes to the migration of HCE cells on fibronectin. A role for β-Pix in cell migration has been demonstrated in various cell types. ^{13,27 –29} Although HCE cells have been immortalized by simian virus 40 and may exhibit the increased rates of cell cycle progression and cell migration compared with primary human corneal epithelial cells, they are widely studied as a model of the latter cells because of the limited availability of human corneal tissue and the short lifespan of the primary cells. Further studies with normal corneal epithelial cells will be necessary to confirm a physiological role for β-Pix in the migratory response of corneal epithelial cells to fibronectin. We have also shown that fibronectin induces the tyrosine phosphorylation of β-Pix as well as its accumulation at focal adhesions in HCE cells. Endothelin-1 induces the phosphorylation of β-Pix as well as its translocation to focal adhesions to activate Rho family GTPases in endothelial cells. ¹⁷ The tyrosine phosphorylation cycle of β-Pix at tyrosine-422 contributes to the assembly and disassembly of focal adhesion complexes in Src-transformed cells. ³⁰ A β-Pix–interacting protein also undergoes tyrosine phosphorylation in NIH 3T3 fibroblasts and contributes to cell spreading on fibronectin. ³¹ Moreover, bFGF induces the phosphorylation of β-Pix and its translocation to lamellipodia in neuronal cells. ³² We have also now shown that bFGF induced the redistribution of β-Pix to lamellipodia and filopodia in HCE cells. We further showed that depletion of endogenous β-Pix by RNAi markedly reduced the size of focal adhesions formed in HCE cells exposed to fibronectin. These observations suggest that the phosphorylation of β-Pix induced by fibronectin results in its translocation to focal contacts in HCE cells, where it may contribute to the local turnover of these structures during wound healing. 

In conclusion, we have shown that β-Pix mediates the regulation of cell motility by fibronectin in HCE cells. Further clarification of the mechanism of β-Pix signaling in these cells should provide greater insight into corneal epithelial wound healing as well as a possible basis for the development of new approaches to the treatment of corneal epithelial defects. 

Footnotes

 Supported in part by Grant No. 21791687 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Footnotes

 Disclosure: K. Kimura, None; S. Teranishi, None; T. Orita, None; H. Zhou, None; T. Nishida, None

The authors thank Shizuka Murata and Yukari Mizuno for technical assistance.