1 National Eye Institute, NIH, Building 7, 7 Memorial Drive MSC 0704, Bethesda, MD 20892, USA
2 Department of Anatomy and Cell Biology and Department of Ophthalmology, George Washington University Medical Center, 2300I Street NW, Washington, DC 20037, USA
* Author for correspondence (e-mail: zelenkap{at}nei.nih.gov)
Accepted 14 April 2004
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Summary |
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Key words: Cyclin-dependent kinase, Src family kinase, Translocation, Wound healing, Transgenic mice
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Introduction |
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Previous work from this laboratory has shown that Cdk5 strengthens cell-matrix adhesion and retards closure of an in vitro scrape wound in a mouse corneal epithelial cell line (Gao et al., 2002). To determine whether Cdk5 also influences epithelial cell migration during in vivo corneal debridement wound healing, we generated transgenic mice using the ALDH3 promoter to direct Cdk5 expression to corneal epithelial cells (Kays and Piatigorsky, 1997
). We have used these animals in conjunction with an in vitro organ culture model of debridement wound healing and scratch-wounded cultures of mouse corneal epithelial cells to explore the effects of Cdk5 on cell migration, cytoskeletal reorganization, and Src activation. The results indicate that Cdk5 regulates corneal epithelial wound healing by limiting the accumulation of active Src at the periphery of cells along the wound edge.
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Materials and Methods |
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Generation of transgenic mice
A mouse aldehyde dehydrogenase class 3 promoter HindIII-SalI fragment from pALDH3-CAT vector (-1050 to the ATG of exon 2) (Kays and Piatigorsky, 1997) consisting of 1050 bp of the 5' flanking sequence, the 40 bp untranslated exon 1, the 3.4 kb of intron 1 and 5 bp of exon 2, and a human Cdk5 SalI-Nco1 fragment containing coding sequence spanning nucleotides 25-903 generated by polymerase chain reaction (PCR) were ligated into pCAT basic vector that had been digested with HindIII and NcoI (Promega, San Luis Obispo, CA). The 6.6 kb ALDH3-Cdk5 or ALDH3-Cdk5T33 minigenes were excised by restriction endonuclease digestion (HindIII and PvuI) (Invitrogen Life Technologies, Carlsbad, CA) and purified from agarose gels with Geneclean II (Qbiogene, Carlsbad, CA). Transgenic mice were generated using standard strategies and founders were identified by PCR of genomic DNA. ALDH3-Cdk5T33 transgenic lines had very low levels of expression in the cornea and were discontinued
Protein isolation and immunoblotting
Protein isolation and immunoblotting of A6(1) mouse corneal epithelial cells was performed as previously described (Gao et al., 2002). For tissue studies, corneas were dissected from enucleated eyes of 8-10-week-old mice, washed in PBS and placed in Dispase II (Roche Diagnostics, Mannheim, Germany) solution at 2.4 units ml-1 for 1.5-2 hours at 37°C. Sheets of corneal epithelium were then carefully removed from the stroma and proteins were extracted as previously described (Gao et al., 1997
). Briefly, cells were lysed in PBSTDS [PBS containing 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS)] with 1 Complete-MiniTM protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN) per 10 ml. 25-50 µg of total cell extract were immunoblotted as previously described (Gao et al., 1997
) using anti-CDK5 (rabbit polyclonal C-8, sc-173, or mouse monoclonal DC-17, sc-249; Santa Cruz Biotechnology, Santa Cruz, CA), anti-cSrc (rabbit polyclonal, SC-18; Santa Cruz Biotechnology), or anti-pY416-cSrc (Cell Signaling Technology, Beverly, MA). Immunoreactive bands were detected by enhanced chemiluminescence (ECL-Plus; Amersham Biosciences, Piscataway, NJ) using horseradish-peroxidase-linked anti-rabbit IgG (Amersham Biosciences).
Corneal debridement wounding
Manual debridement wounds were created on the corneas of 8-10-week-old wild-type and ALDH3-Cdk5 transgenic mice as described (Stepp and Zhu, 1997). Mice were anesthetized with general anesthesia and their eyes numbed with a topical anesthetic. Corneas were then scraped with a dull scalpel to remove the epithelium within a 1.5-mm central corneal area, which had been demarcated with a dull trephine. Animals were euthanized immediately after wounding or after 12 hours. To measure the wound area, corneas were stained with a vital dye and photographed through a dissecting microscope. Images of 34 normal and ten transgenic eyes were digitized and analysed by image processing software. Each wound area was measured three times and the results were averaged. Average original wound area (t=0) was determined from measurements of six normal corneas that were stained immediately after debridement wounding. Healed areas were calculated by subtracting each wound area at 12 hours from the average original wound area. To avoid experimenter bias, each wound-healing experiment included both transgenic and normal animals, and the genotypes were masked until wound-area measurements were completed. Statistical analysis was performed using SigmaStat 2.03 (SPSS, Chicago, IL).
Organ culture experiments were performed as previously described (Stepp et al., 1993). Briefly, 8-10-week-old wild-type mice were euthanized and subjected to corneal-debridement wounding as described above. The eyes were enucleated and cultured for 12 hours with or without 15 µM olomoucine (Calbiochem, San Diego, CA) in Medium 500 (Cascade Biologies, Portland, OK), supplemented with L-glutamine (60 µg ml-1), penicillin (20 units ml-1) and streptomycin (20 mg ml-1) at 37°C in a humidified atmosphere of 95% air and 5% CO2. Wound areas were determined as described for in vivo experiments.
Scrape wounding of cultured A6(1) corneal epithelial cells was performed as previously described (Gao et al., 2002). Briefly, confluent cultures in 60 mm2 culture dishes were scratch wounded using a plastic pipette tip and then cultured for an additional 4 hours in the presence of platelet-derived growth factor (PDGF) before fixation and staining. Where indicated, olomoucine (15 µM) or PP1 (10 µM) were added 24 hours before wounding and replenished at the time of wounding. Cultures that were to be harvested for biochemical studies were treated similarly except that they were grown in 75 cm2 culture flasks and multiple scratch wounds were made with the tip of a cell scraper. Transfections were performed as described (Gao et al., 2002
).
Immunofluorescence and confocal microscopy
Whole eyes from wild type or ALDH3-Cdk5 transgenic mice were fixed and stained with rhodamine-phalloidin, anti-Cdk5 (C-8, Santa Cruz), and/or activated anti-Src (pY416, Cell Signaling Technology) using a previously described protocol (Danjo and Gipson, 1998). After staining, the eyes were enucleated and transected posterior to the corneal limbus under a dissecting microscope. Corneal tissues were washed in PBS and whole-mounted on Superfrost microscope slides (Erie Scientific Company, Portsmouth, NH) for laser scanning confocal microscopy (Leica TCS SP2, Leica Microsystem, Germany). Immunofluorescence of A6(1) corneal epithelial cells was performed as previously described (Gao et al., 2002
).
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Results |
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Retarded wound healing in ALDH3-Cdk5 transgenic mice
To study the effect of Cdk5 on corneal cell migration in vivo, 1.5 mm debridement wounds were made in the central corneas of normal and Cdk5 transgenic mice, and wound areas were measured after 12 hours. As shown in Fig. 1E, corneal wound healing was retarded by about 40% compared with normal littermate controls. This finding was highly significant statistically (P=0.001). Examination of transgenic corneas 1 weeks and 2 weeks after wounding showed no abnormalities associated with restratification (not shown). Thus, overexpression of Cdk5 in the corneal epithelium seems to have a specific effect on the migration phase of wound closure.
Effect of olomoucine on wound healing in organ culture
To test whether Cdk5 activity regulates cell migration in the normal cornea, we used the Cdk5 kinase inhibitor olomoucine to block endogenous Cdk5 activity (Glab et al., 1994; Veeranna et al., 1996
). Corneas of normal mice (10 weeks old) were subjected to debridement wounds (1.5 mm) and eyes were cultured in the presence or absence of olomoucine (15 µM) for 12 hours in defined media. Wound areas were analysed by image analysis and the extent of healing was determined by subtracting the unhealed area at 12 hours from the initial wound area. In olomoucine-treated eyes, the healed area at the end of 12 hours was more than twice as great (P=0.007) as in the normal controls (Fig. 2A). In addition, the wound edge appeared ragged and disorganized in the presence of olomoucine compared with untreated controls (Fig. 2B-D), suggesting that inhibition of Cdk5 might also affect the morphology and cytoskeletal organization of migrating cells.
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Effect of Cdk5 and olomoucine on cytoskeletal organization during wound healing
To test whether Cdk5 did indeed affect cytoskeletal organization along the wound edge, eyes of normal and ALDH3-Cdk5 transgenic animals were placed in organ culture after debridement wounding. In the normal cornea after 12 hours in culture, the usual cobblestone organization of the epithelium was interspersed with rows of elongated cells perpendicular to the wound edge (Fig. 3A). The cytoplasm of elongated cells was diffusely stained with rhodamine-phalloidin, suggesting active remodeling of the actin cytoskeleton (Fig. 3A). By contrast, epithelia of Cdk5 transgenic animals retained their cobblestone appearance, had few if any elongated cells near the wound margin and showed almost no diffuse actin staining (Fig. 3B).
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To test the effect of Cdk5 inhibition on cell morphology and actin organization, eyes of wild-type mice were organ cultured with or without olomoucine following corneal debridement wounding. After 12 hours, corneal whole mounts were prepared and stained with rhodamine-phalloidin. In the absence of olomoucine, the debridement wound edge appeared to be continuous and the epithelial sheet appeared to migrate as a whole (Fig. 4A), consistent with the higher-magnification view shown in Fig. 3A. A similar pattern of actin staining was seen in control cultures incubated with DMSO, the vehicle used for olomoucine (results not shown). By contrast, in the presence of olomoucine, the wound edge was irregular, cells at the wound edge seemed loosely associated with one another and individual cells appeared to have separated completely from the epithelial sheet (Fig. 4B).
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The detachment of migrating cells in the presence of olomoucine suggested that Cdk5 might promote cell-cell adhesion during wound healing. Because previous studies have indicated that Cdk5/p35 actually reduces cell-cell adhesion (Kwon et al., 2000; Negash et al., 2002
), we considered the possibility that Cdk5 might have an additional, novel function during wound healing. To explore this possibility, we examined the effect of olomoucine on activated Src, a tyrosine kinase known to regulate multiple aspects of migration and cell-cell adhesion (Frame et al., 2002
). For this, corneas that had been incubated with and without olomoucine were immunostained with an antibody specific for the activated form of Src (pY416). This revealed a band of active Src several cells wide along the wound edge (Fig. 4C). Interestingly, culturing the corneas in the presence of olomoucine appeared to intensify active Src staining along the edge of this band, in cells immediately adjacent to the wound edge (Fig. 4D).
Redistribution of c-Src to the wound edge in A6(1) cells
To examine this effect in greater detail, we turned to a mouse corneal epithelial cell line that we have previously used to study the effect of Cdk5 on cell migration during closure of in vitro scrape wounds (Gao et al., 2002). A6(1) cells were grown to confluence, scratch wounded and then cultured with or without olomoucine. Wounding led to activation of Src in a band several cells wide along the wound edge, as in organ-cultured corneas (compare Fig. 5A and Fig. 4C). Moreover, olomoucine altered the subcellular localization of active Src in cells immediately adjacent to the wound edge (Fig. 5B). This, too, resembled the situation in organ-cultured corneas (compare Fig. 5B and Fig. 4D), suggesting that Cdk5 plays a similar role in regulating Src localization in both systems.
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Higher magnification showed that active Src was localized to the cell periphery and to the perinuclear region in the absence of olomoucine (Fig. 5C), as previously reported in fibroblasts (Fincham et al., 1996) and keratinocytes (Fincham et al., 1996
). By contrast, in the presence of olomoucine, active Src was concentrated at the cell periphery and staining in the perinuclear region was almost absent from most cells (Fig. 5D). Olomoucine-treated cells also showed multiple lamellipodia, consistent with their enhanced rate of wound closure. Staining with rhodamine-phalloidin showed corresponding changes in the organization of the cytoskeleton. In the absence of olomoucine, actin was primarily cortical and was organized into large bundles, seen as points or elongated ovals in the confocal section (Fig. 5E). In the presence of olomoucine, these large bundles of actin were replaced by a network of fine fibrils throughout the cytoplasm and by fibers of smaller diameter (Fig. 5F). These findings suggest that inhibition of Cdk5 promotes the peripheral localization of active Src, and enhances cytoskeletal changes that are usually associated with Src activity (Frame et al., 2002
), including dissolution of stress fibers and formation of lamellipodia.
As an additional test of the ability of Cdk5 to regulate the subcellular localization of Src(pY416), A6(1) cells were transiently transfected with GFP-Cdk5 or GFP-Cdk5T33. Immunostaining for active Src demonstrated that Src(pY416) was primarily localized to the perinuclear region of cells that expressed GFP-Cdk5 compared with untransfected cells in the same field (Fig. 6A,B). Conversely, in cells that expressed GFP-Cdk5T33, Src(pY416) was present at high concentrations even in the tips of very long cell processes (Fig. 6C,D).
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To determine whether the activation of Src along the wound edge plays a role in wound healing, confluent cultures of A6(1) cells were scratch wounded and allowed to heal in the presence or absence of the Src kinase inhibitor PP1. Wound areas were determined by image analysis at time zero and after 12 hours (Fig. 7A-D). The results indicated that inhibition of Src activity reduced wound healing by approximately 85% (Fig. 7E), demonstrating that Src activity is indeed an important determinant of the rate of wound closure.
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To examine the effect of inhibiting Cdk5 on the level of Src activation, multiple scratch wounds were made in each confluent cell culture to increase the number of cells in which Src is activated. Control and experimental cultures were wounded identically. Using olomoucine to inhibit endogenous Cdk5 activity in scratch-wounded cultures and immunoblotting with an antibody specific for Src(pY416) showed that the amount of active Src is significantly increased when Cdk5 is blocked (Fig. 8A,B). Inhibiting Cdk5 activity by transiently transfecting cells with the dominant negative Cdk5T33 construct caused a similar increase in the level of active Src, although the magnitude of the effect was somewhat less owing to the limited efficiency of transient transfection (Fig. 8C). Co-immunoprecipitation experiments showed that Cdk5 is part of an intracellular protein complex containing Src, some of which is in the active, phosphorylated form (Fig. 8D). Thus, Cdk5 and active Src are in close physical proximity in corneal epithelial cells. Moreover, Cdk5 activity does not appear to be required for this association, because Cdk5 and Src(pY416) also co-immunoprecipitated in the presence of olomoucine (Fig. 8D).
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Cdk5(Y15) is a known substrate for the Src-family kinase Fyn (Sasaki et al., 2002), and so the close association between Src(pY416) and Cdk5 suggested that Src might affect the phosphorylation state of Cdk5 during wound healing. To examine this possibility, A6(1) cultures were scratch wounded and cultured in the presence or absence of the Src inhibitor PP1 or the inactive analog PP3. Immunostaining with a phosphorylation-specific antibody to Cdk5(pY15) showed that Cdk5 was phosphorylated on Y15 in cells along the wound edge in control cultures that were incubated in the presence of the inactive analog PP3 (Fig. 9A). Similar effects were seen in control cultures without PP3 (not shown). By contrast, treating the cultures with PP1 almost entirely suppressed phosphorylation of Cdk5(Y15) (Fig. 9B). Controls in which the Cdk5(Y15) antibody was omitted showed no fluorescence (Fig. 9C). Phalloidin staining of F-actin in these cultures indicated that PP1 also suppressed the characteristic reorganization of the cytoskeleton along the wound edge (Fig. 9D,E). In the presence of PP1, actin staining was primarily cortical, there was little diffuse staining and the cells formed few lamellipodia (Fig. 9E). Control cultures that received neither PP3 nor PP1 were indistinguishable from those that received PP3 (Fig. 9F). These findings demonstrate that inhibiting Src activity and overexpressing Cdk5 (Fig. 3B) have similar effects on the actin cytoskeleton.
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Discussion |
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Although in vitro scrape-wound closure mimics many aspects of in vivo wound healing, there are important differences, which might be related to differences in extracellular matrix components and integrins (Stepp et al., 1993). In particular, the corneal epithelium migrates as a sheet in the intact eye, whether in vivo or in organ culture, whereas cultured A6(1) corneal epithelial cells migrate as single cells (Gao et al., 2002
). A continuous `purse-string' actin cable also forms around the wound edge in vivo (Danjo and Gipson, 1998
) but not in organ culture or cell cultures (Dalton and Steele, 2001
). Instead, the pattern of F-actin staining along the wound edge suggests that healing in these culture systems occurs though synchronous crawling of multiple rows of cells as seen in MDCK epithelial cell sheets (Fenteany et al., 2000
). The finding that Cdk5 has a similar effect on wound closure in all these systems suggests that it might act on common regulatory elements that regulate cytoskeletal organization during cell migration.
One key regulator of cytoskeletal organization during migration is the cytoplasmic tyrosine kinase Src, which regulates adhesion, cytoskeletal organization and migration through its effects on the Rho family of small GTPases (Frame et al., 2002). Src activity promotes the dissolution of Rho-dependent focal adhesions and stress fibers (Brouns et al., 2001
) while promoting Rac-dependent functions, such as formation of lamellipodia and remodeling of the actin cytoskeleton (Klemke et al., 1998
; Kiyokawa et al., 1998
; Etienne-Manneville and Hall, 2002
). Because Src activation occurs in the perinuclear region of the cell (Kaplan et al., 1995
) whereas the Src substrates involved in migration and adhesion are located at or near sites of cell-matrix or cell-cell contact (Etienne-Manneville and Hall, 2002
), translocation of active Src to the cell periphery is clearly an important element in controlling its effects.
The present findings suggest that Cdk5 exerts its effects on corneal epithelial cell migration and wound closure through negative regulation of Src. Src is activated along the leading edge of scratch-wounded cultures of corneal epithelial cells and in corneal debridement wounds in organ-cultured eyes. A similar pattern of Src activation was previously observed in scratch-wounded keratinocyte cultures (Yamada et al., 2000). In accordance with previously reported functions of Src (Frame et al., 2002
), Src activation in corneal epithelial cells is associated with dissolution of focal adhesions, remodeling of the actin cytoskeleton and formation of lamellipodia. Inhibition of Src activity not only blocks these effects but also strongly inhibits corneal wound closure. We have found that Cdk5 activity suppresses Src activation and opposes Src-dependent effects on cytoskeletal reorganization and corneal wound closure. Inhibiting Cdk5 activity increases both the level of Src(pY416) and the proportion found at the cell periphery. At the same time, inhibiting Cdk5 activity promotes focal-adhesion dissolution, increases actin remodeling and lamellipodium formation, and enhances corneal wound closure. Moreover, overexpressing Cdk5 produces the opposite effects, providing additional confirmation of the inverse relationship between Cdk5 and Src.
Interestingly, we also observed that inhibiting Cdk5 with olomoucine causes corneal epithelial cells near the wound edge to detach from their neighbors and to migrate as single cells, suggesting that Cdk5 might promote cell-cell adhesion in the migrating corneal epithelial sheet. This observation is particularly surprising in view of previous evidence from neurons (Kwon et al., 2000) and lens epithelial cells (Negash et al., 2002
) indicating that Cdk5 disrupts, rather than promotes, cell-cell adhesion. Disruption of cell-cell adhesion has been attributed to the ability of Cdk5/p35 to bind to ß-catenin and to reduce its association with N-cadherin (Kwon et al., 2000
; Negash et al., 2002
), a prominent cadherin in both neurons and lens (Lagunowich et al., 1992
; Leong et al., 2000
; Xu et al., 2002
). Interestingly, the predominant cadherins in the corneal epithelium are E-cadherin and P-cadherin (Xu et al., 2002
), rather than N-cadherin, suggesting that the effect of Cdk5 on cell-cell adhesion might depend on the particular cadherins involved. Moreover, Src has been shown to disrupt E- and P-cadherin-dependent adhesion in epithelial cells (Owens et al., 2000
), suggesting that the positive effect of Cdk5 on cell-cell adhesion in the cornea might also be exerted through negative regulation of Src.
Although it is not yet known how Cdk5 exerts its negative effect on Src, direct phosphorylation of Src at S75 is an interesting possibility (Kato and Maeda, 1999). Although phosphorylation at this site does not directly affect Src activity (Kato and Maeda, 2003
), resultant changes in Src structure might affect the stability of the active state by changing its accessibility to phosphatases (Kato and Maeda, 1999
). Alternatively, Cdk5-dependent phosphorylation of Src at S75 might regulate the transport and subcellular localization of active Src.
Just as Src is a potential substrate of Cdk5, Cdk5 is also a potential target of Src. Cdk5(Y15) can be phosphorylated by the Src family kinase Fyn (Sasaki et al., 2002) and the related tyrosine kinase Abl (Zukerberg et al., 2000
). Moreover, phosphorylation at this site increases Cdk5 activity up to sevenfold in neurons (Zukerberg et al., 2000
). Thus, the Cdk5-dependent regulation of Src activity might be a negative feedback mechanism initiated by Src itself. In support of this possibility, we have observed that PP1, a specific inhibitor of Src-family kinases, blocks Cdk5 phosphorylation on Y15, as indicated by immunofluorescence. However, further studies will be required to confirm this result and to determine whether Src-dependent phosphorylation of Cdk5(Y15) is associated with an increase in Cdk5 kinase activity in this cell type.
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Acknowledgments |
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