Cdk5 regulates activation and localization of Src during corneal epithelial wound closure

Chun Y. Gao1, Mary Ann Stepp2, Robert Fariss1 and Peggy Zelenka1,*

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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Recent studies have shown that Cdk5, a member of the cyclin-dependent-kinase family, regulates adhesion and migration in a mouse corneal epithelial cell line. Here, we extend these findings to corneal wound healing in vivo and examine the mechanism linking Cdk5 to cytoskeletal reorganization and migration. Cdk5 was overexpressed in the corneal epithelium of transgenic mice under control of the ALDH3 promoter. Elevated Cdk5 expression retarded corneal debridement wound closure in these animals and suppressed remodeling of the actin cytoskeleton. Conversely, the Cdk5 inhibitor, olomoucine, accelerated debridement wound healing in organ cultured eyes of normal mice, caused migrating cells to separate from the epithelial cell sheet, and increased the level of activated Src(pY416) along the wound edge. To explore the relationship between Cdk5 and Src in greater detail, we examined scratch-wounded cultures of corneal epithelial cells. Src was activated in cells along the wound edge and blocking this activation with the Src kinase inhibitor, PP1, inhibited wound closure by 85%. Inhibiting Cdk5 activity with olomoucine or a dominant negative construct, Cdk5T33, increased the concentration of Src(pY416), shifted its subcellular localization to the cell periphery and enhanced wound closure. Cdk5(pY15), an activated form of Cdk5, also appeared along the wound edge. Inhibiting Src activity with PP1 blocked the appearance of Cdk5(pY15), suggesting that Cdk5 phosphorylation is Src dependent. Cdk5 and Src co-immunoprecipitated from scratch-wounded cultures, demonstrating that both kinases are part of an intracellular protein complex. These findings indicate that Cdk5 exerts its effects on cell migration during corneal epithelial wound healing by regulating the activation and localization of Src.

Key words: Cyclin-dependent kinase, Src family kinase, Translocation, Wound healing, Transgenic mice


    Introduction
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 Introduction
 Materials and Methods
 Results
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 References
 
Cdk5 is a proline-directed protein kinase that is closely related to the cyclin-dependent kinases. Cdk5 and its activator, p35, are expressed primarily in terminally differentiated neurons, where they regulate neuronal functions such as migration, neurite outgrowth, exocytosis (Dhavan and Tsai, 2001Go), microtubule organization (Rashid et al., 2001Go; Xie et al., 2003Go) and synaptic-vesicle endocytosis (Tan et al., 2003Go). In addition, Cdk5 and p35 are expressed in a range of non-neuronal cell types, and several non-neuronal functions have also been established (Chen and Studzinski, 2001Go; Gao et al., 2002Go; Lazaro et al., 1997Go; Negash et al., 2002Go; Philpott et al., 1997Go).

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., 2002Go). 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, 1997Go). 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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Animal experimentation
All animal studies were performed in accordance with the NIH Guidelines for Care and Use of Laboratory Animals and the recommendations of the Association for Research in Vision and Ophthalmology. Mouse strain FVB/N was used throughout.

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, 1997Go) 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., 2002Go). 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., 1997Go). 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., 1997Go) 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, 1997Go). 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., 1993Go). 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., 2002Go). 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., 2002Go).

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, 1998Go). 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., 2002Go).


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 Materials and Methods
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 References
 
Cdk5 expression in transgenic mice
Cdk5 cDNA was cloned downstream of the ALDH3 promoter containing 1050 bp upstream of the transcription initiation site and extending through the normal translational start site (ATG) in exon 2, making this ATG the start site for translation of Cdk5 (Fig. 1A). Thus, the Cdk5 protein contained no additional amino acids at either the N-terminus or the C-terminus. A transgenic line was generated that expressed approximately three times the normal endogenous level of Cdk5 in the corneal epithelium (Fig. 1B). Cdk5 overexpression did not affect corneal epithelial morphology and did not appear to disrupt the normal pattern of Cdk5 localization as judged by immunofluorescence with a specific anti-Cdk5 antibody (Fig. 1C,D). Transgenic corneas were more intensely stained, consistent with the elevated levels of Cdk5 protein, and showed some accumulation of Cdk5 in the apical cytoplasm of basal and wing cells, which was not seen in wild-type animals (Fig. 1D). Cdk5 was highly concentrated along the basal aspect of the basal epithelial cells and along membranes of superficial cells in both normal and transgenic corneas (Fig. 1C,D), in good agreement with previous findings (Gao et al., 2002Go). In parallel experiments, immunostaining showed that the Cdk5 activating protein, p35, was also highly concentrated along plasma membranes in Cdk5 transgenic animals (not shown), as in the normal mouse cornea (Gao et al., 2002Go).



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Fig. 1. Transgene expression in corneal epithelia. (A) The ALDH3 promoter/Cdk5 construct, including the untranslated first exon and the translation start site in exon 2 (arrow). (B) Protein extracts were prepared from corneal epithelia isolated from wild-type (wt) and heterozygous ALDH3-Cdk5 transgenic (tg) littermates. Cdk5 expression was examined by immunoblotting using an anti-Cdk5-C-terminus-specific antibody. The lower panel shows Ponceau staining of protein in the same samples as a loading control. The experiment was performed in triplicate and images were quantified by densitometric scanning. The results indicated that Cdk5 expression in transgenic animals was elevated 3.0 times. (C) Immunofluorescence of Cdk5 in corneas of an adult normal mouse. Immunostaining was seen in all layers of the corneal epithelium (ep) and was most intense along the basal aspect of the basal cells (small arrows) in both normal and transgenic animals. In superficial cells, staining appeared to be associated with cell membranes (arrowhead). Keratocytes of the corneal stroma (s) were not significantly stained. (D) Immunofluorescence of Cdk5 in corneas of ALDH3-Cdk5 transgenic animals. Cdk5 accumulation was seen in apical region of basal and wing cells of the transgenic corneas (double arrow) as well as along the basal aspect of the basal cells (small arrows) and along membranes of the superficial cells (arrowhead). Bar, 100 µm (C,D). (E) Effect of transgene expression on corneal wound healing. A 1.5 mm debridement wound was made in the central cornea of age-matched wild-type and ALDH3-Cdk5 transgenic mice, at age 8-10 weeks. Initial wound areas were determined by image analysis of six eyes (four normal and two transgenic) immediately after wounding. Wound areas at 12 hours were determined by image analysis of 34 wild-type and ten transgenic eyes. The healed area was calculated by subtracting the average remaining wound area at 12 hours from the average initial wound area and was then expressed as a proportion of the average initial wound area. ALDH3-Cdk5 mice showed a significantly lower rate of wound healing than the wild type (P<0.01).

 

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., 1994Go; Veeranna et al., 1996Go). 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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Fig. 2. Effect of olomoucine on debridement wound healing in organ culture. A 1.5 mm debridement wound was made in corneas of wild-type mice and enucleated eyes were organ cultured for 12 hours in defined medium in the presence or absence of olomoucine (15 µM). Initial wound areas were measured immediately after wounding (20 eyes). To determine the effect of olomoucine, one eye of each animal was cultured with olomoucine and the other eye was cultured without, as a paired control (ten pairs). After 12 hours, eyes were stained and photographed. Each wound area was measured using ImagePro Plus image analysis software (Media Cybernetics, San Diego, CA). (A) Bar graph showing the average area healed as proportion of the original wound area. Wound healing was significantly enhanced by olomoucine (P<0.007). Error bars represent s.e.m. (B) Corneal debridement wound immediately after wounding. (C) Debrided cornea after 12 hours in organ culture. (D) Debrided cornea after 12 hours organ culture in the presence of 15 µM olomoucine.

 

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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Fig. 3. Localization of F-actin in wild-type and transgenic corneas during wound healing in organ culture. Debridement wounds were made in corneas of wild-type and ALDH3-Cdk5 transgenic mice. Eyes were placed in organ culture for 12 hours and then fixed and stained with rhodamine-phalloidin. Corneas were dissected and whole mounted for laser scanning confocal fluorescence microscopy. (A) Rhodamine-phalloidin staining of the wound edge in a wild-type cornea. Open arrowheads indicate rows of cells with diffuse actin staining oriented perpendicular to the wound edge. Elongated cells are indicated by solid arrowheads. Points of intense actin staining are often located at the junctions of three or more cells (arrow). (B) Rhodamine-phalloidin staining of the wound edge in an ALDH3-Cdk5 cornea shows cobblestone organization along the wound edge, with few, if any, elongated cells. Cortical actin staining is distributed uniformly around the cell periphery and very few cells show diffuse staining. Bar, 100 µm.

 

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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Fig. 4. Effect of olomoucine on F-actin and Src activation at the wound edge. Debridement wounds were made in corneas of wild-type mice, eyes were organ cultured for 12 hours, fixed and double-stained with rhodamine-phalloidin (F-actin) and antibody to activated Src (Src pY416). Stained tissues were dissected, whole mounted and examined by confocal microscopy. (A) F-Actin staining in the absence of olomoucine shows a sharp, regular wound edge, with actin staining at cell-cell boundaries and along the wound edge. (B) In the presence of olomoucine, the wound edge is disorganized and cells have separated from the epithelial cell sheet (arrows). F-Actin staining is seen in a broader band of cells along the wound edge than in untreated corneas. (C) Immunostaining of active Src (Src pY416) shows that Src is activated in a narrow band of cells adjacent to the wound edge. (D) In the presence of olomoucine, immunostaining of active Src suggests that inhibiting Cdk5 augments the activation of Src in many cells along the wound edge (arrows). Bar, 125 µm.

 

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., 2000Go; Negash et al., 2002Go), 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., 2002Go). 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., 2002Go). 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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Fig. 5. Effect of olomoucine on Src activation and F-actin in scratch-wounded cultures. Confluent cultures of A6(1) cells were scratch wounded then cultured for 12 hours with or without 15 µM olomoucine. After 12 hours, cultures were double stained with antibody for active Src (pY416) and rhodamine-phalloidin. (A) Active Src is seen in a band of cells along the wound edge. (B) In the presence of olomoucine, the distribution of active Src is altered. Intense staining is seen immediately adjacent to the wound edge. (C) A higher magnification view of cells shown in (A). Active Src is localized to the cell periphery (arrow) and the perinuclear region (arrowhead). (D) Higher magnification of cells shown in (B). In the presence of olomoucine, immunostaining of active Src is enhanced in lamellipodia along the cell periphery (arrow) and diminished in the perinuclear region (arrowhead). (E) Rhodamine-phalloidin staining of same cells as (C) shows that F-actin is organized into thick bundles that appear as large points or elongated ovals in the optical section (arrows). (F) Rhodamine-phalloidin staining of cells shown in (D). In the presence of olomoucine, actin staining is more diffuse and sectioned actin fibers are narrower. Lamellipodia are not strongly stained for F-actin (arrows). Bar, 250 µm (A,B), 25 µm (C,D).

 

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., 1996Go) and keratinocytes (Fincham et al., 1996Go). 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., 2002Go), 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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Fig. 6. Effect of Cdk5 and Cdk5T33 on localization of active Src in A6(1) cells. A6(1) corneal epithelial cells were transiently transfected with EGFP-Cdk5 (A,B) or EGFP-Cdk5T33 (C,D) and then cultured overnight. Cells were immunostained with antibody specific for active Src(pY416) and rhodamine-tagged secondary antibody. (A) EGFP fluorescence showing a cell transfected with EGFP-Cdk5. (B) Rhodamine fluorescence of the same field showing the localization of active Src in transfected and untransfected cells. In the EGFP-Cdk5-transfected cell, active Src is located primarily in the perinuclear region (arrow), with little active Src in cell processes. By contrast, cell processes are well stained in untransfected cells in the same field (arrowhead). (C) EGFP fluorescence of a cell transfected with EGFP-Cdk5T33. (D) Rhodamine fluorescence of the same field, showing the localization of active Src in transfected and untransfected cells. In the EGFP-Cdk5T33-transfected cell, cell processes are well stained, with high concentrations of active Src in cell processes (arrowhead). Active Src is also present in lamellipodia (double arrow). Bar, 25 µm.

 

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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Fig. 7. Effect of Src on scratch-wound closure in A6(1) cells. (A) Scratch wounds were made in confluent cultures of A6(1) cells. (B) After 24 hours in culture, the wound was largely occupied by migrating cells. (C) Scratch wounds were made in confluent cultures of A6(1) cells in the presence of the Src-family-kinase inhibitor PP1. (D) After 24 hours in the presence of PP1, very few cells had migrated into the wound area. The dark spots in the lower left quadrant of each panel are ink spots used to identify the region of interest. (E) Quantitative image analysis of the proportion of the original wound area occupied by migrating cells at the end of 24 hours in the absence or presence of PP1. In three experiments, the occupied area decreased from 54±6% to 7.5±1.6%. The difference between the groups is significant (P<0.001).

 

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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Fig. 8. Immunoblotting of Src and active Src in A6(1) cells with and without olomoucine. (A) Multiple scratch wounds were made in confluent cultures of A6(1) cells. Scratch-wounded cultures were incubated in the absence or presence of 15 µM olomoucine and cell lysates were prepared. Next, 50 µg whole cell lysate (WCL) was loaded into each lane of a 12% SDS-polyacrylamide gel and immunoblotted with antibodies against Src or active Src (pY416). (B) The results were quantified by densitometry and averaged (n=5). Olomoucine treatment increased the level of Src(pY416) more than 2.5 times (P=0.04). Error bars=s.e.m. (C) A6(1) cells were transiently transfected with the dominant negative construct EGFP-Cdk5T33. (Left) Whole cell lysate (50 µg) was immunoblotted with antibodies against Src or active Src (pY416). Quantification by densitometry indicated that the level of Src(pY416) was elevated 1.8±0.3 times (n=3) in transiently transfected cells. (Right) 50 µg whole cell extract was immunoblotted with anti-Cdk5 antibody to demonstrate expression of EGFP-Cdk5T33 (single arrow, Cdk5; double arrow, EGFP-Cdk5). (D) A(6)1 cells were incubated in the absence or presence of olomoucine. Cell extracts from subconfluent cultures were immunoprecipitated with anti-Cdk5 antibody and the precipitated proteins were immunoblotted for Src and active Src(pY416) (left) or Cdk5 (right). Both Src and active Src(pY416) were detected in Cdk5 immunoprecipitates (arrow). An unidentified, more rapidly migrating band was occasionally detected by this antibody but does not correspond to Src(pY416) according to manufacturer's technical data sheet. Immunoblotting with Cdk5 antibody confirmed that immunoprecipitation was effective.

 

Cdk5(Y15) is a known substrate for the Src-family kinase Fyn (Sasaki et al., 2002Go), 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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Fig. 9. Confluent cultures of A6(1) cells were scratch wounded then cultured with or without the Src inhibitor PP1 or the inactive analog PP3. After 24 hours, cultures were double stained with antibody for phosphorylated Cdk5(pY15) (A-C) and rhodamine phalloidin (D-F). (A) In the presence of PP3, Cdk5(pY15) immunofluorescence was observed in a band of cells along the wound edge. (B) In the presence of PP1, little or no Cdk5(pY15) immunofluorescence was detected along the wound edge. (C) Omission of the primary antibody to Cdk5(pY15) showed no detectable immunofluorescence. (D) Rhodamine-phalloidin staining of cells shown in (A) showed polymerization of F-actin along the wound edge, with diffuse cytoplasmic staining and formation of numerous lamellipodia (arrows). (E) Rhodamine-phalloidin staining of cells shown in (B) showed that the actin cytoskeleton was primarily cortical (open arrow) and few lamellipodia were formed. (F) Control cultures incubated without PP3 or PP1 were indistinguishable from those incubated with PP3. Numerous lamellipodia were observed (arrows).

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A previous study from this laboratory indicated that Cdk5 regulates the migration of cultured corneal epithelial cells during in vitro scrape-wound closure (Gao et al., 2002Go). The present work demonstrates that Cdk5 has a similar effect on corneal debridement wound healing in transgenic mice that overexpress Cdk5 in the corneal epithelium. Although the limited amount of tissue precluded direct kinase assays in transgenic corneal epithelia, several lines of evidence argue that the effect on wound healing is due to an increase in Cdk5/p35 kinase activity. First, direct kinase assays have confirmed that a comparable level of Cdk5 overexpression increases Cdk5 kinase activity and decreases cell migration in transfected corneal epithelial cells (Gao et al., 2002Go). Second, inhibiting Cdk5 kinase activity with the dominant negative mutation Cdk5T33 increases the rate of wound closure in corneal epithelial cells in vitro (Gao et al., 2002Go). Finally, inhibiting Cdk5 activity with olomoucine (Glab et al., 1994Go) enhances cell migration in the intact eye in organ culture. Olomoucine inhibits Cdk5 kinase activity with a 50% inhibitory concentration (IC50) of 3 µM (Vesely et al., 1994Go). Although Cdk1 (Cdc2) and Cdk2, both of which have an IC50 of 7 µM (Vesely et al., 1994Go), might also have been inhibited at the concentration of olomoucine used in these experiments (15 µM), Cdk5 appears to be the relevant kinase, because the dominant negative mutation Cdk5T33 produced the same effects in cultured cells. Moreover, Cdk1 and Cdk2 are activated only during cell cycle progression (Nigg, 1995Go), which is suppressed during closure of a small debridement wound (Suzuki et al., 2003Go).

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., 1993Go). 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., 2002Go). A continuous `purse-string' actin cable also forms around the wound edge in vivo (Danjo and Gipson, 1998Go) but not in organ culture or cell cultures (Dalton and Steele, 2001Go). 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., 2000Go). 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., 2002Go). Src activity promotes the dissolution of Rho-dependent focal adhesions and stress fibers (Brouns et al., 2001Go) while promoting Rac-dependent functions, such as formation of lamellipodia and remodeling of the actin cytoskeleton (Klemke et al., 1998Go; Kiyokawa et al., 1998Go; Etienne-Manneville and Hall, 2002Go). Because Src activation occurs in the perinuclear region of the cell (Kaplan et al., 1995Go) 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, 2002Go), 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., 2000Go). In accordance with previously reported functions of Src (Frame et al., 2002Go), 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., 2000Go) and lens epithelial cells (Negash et al., 2002Go) 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., 2000Go; Negash et al., 2002Go), a prominent cadherin in both neurons and lens (Lagunowich et al., 1992Go; Leong et al., 2000Go; Xu et al., 2002Go). Interestingly, the predominant cadherins in the corneal epithelium are E-cadherin and P-cadherin (Xu et al., 2002Go), 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., 2000Go), 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, 1999Go). Although phosphorylation at this site does not directly affect Src activity (Kato and Maeda, 2003Go), resultant changes in Src structure might affect the stability of the active state by changing its accessibility to phosphatases (Kato and Maeda, 1999Go). 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., 2002Go) and the related tyrosine kinase Abl (Zukerberg et al., 2000Go). Moreover, phosphorylation at this site increases Cdk5 activity up to sevenfold in neurons (Zukerberg et al., 2000Go). 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.


    Acknowledgments
 
We thank C. Lutz and C. Wright for technical assistance, J. Piatigorsky for providing the ALDH3 promoter and A6(1) corneal epithelial cells, as well as for reading the manuscript, and members of the NEI Central Transgenic Facility for generating the transgenic mouse lines.


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