Ras-induced spreading and wound closure in ... - The FASEB Journal

©2005 FASEB

The FASEB Journal express article 10.1096/fj.04-3327fje. Published online September 16, 2005.

Ras-induced spreading and wound closure in human epidermal keratinocytes Michael Tscharntke,1 Ruth Pofahl,1 Thomas Krieg, and Ingo Haase Department of Dermatology, University of Cologne and Center for Molecular Medicine, University of Cologne (CMMC) Joseph-Stelzmann-Strasse 9 50924 Cologne, Germany 1

These authors contributed equally to this work.

Corresponding author: Ingo Haase, Department of Dermatology, University of Cologne and Center for Molecular Medicine, University of Cologne (CMMC) Joseph-Stelzmann-Strasse 9 50924 Cologne, Germany. E-mail: [email protected] ABSTRACT Although it is known that growth factor signaling cascades are active during epithelial wound healing, signals that regulate reepithelialization after wounding are not very well characterized. The small GTP binding protein Ras is a molecular switch involved in the regulation of signals originating from different growth factor receptors. We have investigated consequences of its activation in primary human keratinocytes. We provide evidence that activation of Ras can lead to shape changes of keratinocytes caused by rearrangements of the actin cytoskeleton that result in membrane protrusion and ruffling. Similar shape changes were found in the migrating tip of newly formed epithelium in mouse wounds. These cytoskeletal changes occur independently of keratinocyte terminal differentiation, and they can determine the speed of wound epithelialization in vitro. Using various mutant constructs and specific pharmacological inhibitors, we found that the effects of activated Ras on the cytoskeleton of keratinocytes are mediated by a phosphatidylinositol 3 kinase-independent activation of Rac. Our results suggest that growth factor-induced, Ras-mediated changes of keratinocyte shape may be an important mechanism that determines the speed of wound epithelialization. Key words: wound epithelialization • cell spreading • Rac1 • epidermis • wound healing

E

pidermal keratinocytes form a stratified epithelium that lines the outer surface of the skin: the epidermis. Continuous self-renewal of this tissue requires ongoing proliferation of basal keratinocytes that is stimulated by various growth factors. Wounding of the skin can lead to the loss of epidermal tissue and normally results in the activation of growth-promoting signaling pathways. These pathways increase proliferative potential of epidermal keratinocytes and accelerate regeneration of the lost tissue. Apart from increased proliferation, migration of keratinocytes is required for wound healing. We have recently found that epidermal growth factor (EGF) and insulin like growth factor 1 (IGF-1) can influence keratinocyte shape and thereby regulate different parts of the migration process (1).

Page 1 of 18 (page number not for citation purposes)

Many growth factors transmit intracellular signals via the small GTPase p21Ras (Ras) the product of the H-ras oncogene, to different intracellular effector pathways. Among the known Ras effectors are the Raf/MEK/ERK kinase cascade (ERK cascade), phosphatidylinositol-3 kinase (PI3-K), and RalGDS (2). Activation of ERK signaling results in increased proliferation and migration of keratinocytes in vitro and in vivo, and ERK has been found to be activated in samples of hyperproliferative skin diseases (3–7). Activity of Akt, a downstream effector of PI3K, is required for normal epidermal morphogenesis and the formation of epidermal appendages (8), and constitutive activation of PI3-K in vitro stimulates keratinocyte spreading (1). In vitro, Ras can either stimulate or inhibit growth of keratinocytes. Growth inhibitory signals resulting from constitutive activation of Ras in vitro were found to be mediated by the p19ARFp53 tumor suppressor pathway and could be overcome by simultaneous activation of CDK4 (9, 10). Recent in vivo studies using transgenic mice suggest, however, that in an in vivo context Ras functions as a positive regulator of keratinocyte growth and an inhibitor of terminal differentiation at least in the basal epidermal cell layer (11). It is well known that growth factor signaling cascades involving Ras are active during epithelial wound healing (12, 13); our knowledge on how these growth factors contribute to epidermal regeneration is, however, limited. Although thus far growth regulation has been considered the principal function of Ras in mammalian cells, its coupling to several different downstream signaling pathways suggests that other cellular processes are regulated by Ras. For epidermal wound repair, keratinocytes have to both proliferate in order to replace lost tissue and migrate in order to close the skin defect. We have shown previously that activation of the classical mitogenactivated protein (MAP) kinase (ERK) cascade is responsible for EGF-induced random motility of keratinocytes in vitro (1). Transforming growth factor α (TGFα) signaling involving ERK activation appears to be enhanced in epidermal cells close to the wound in vivo (3, 13). Apart form MAP kinases, there are other pathways regulating migration of keratinocytes, for example, those involving keratinocyte growth factor, IGF-1, and the protein kinase Akt (1, 14, 15). We have therefore set out to investigate in which ways Ras could participate in epidermal wound closure. Here we describe that Ras can regulate the organization of the cytoskeleton in keratinocytes and thereby contributes to spreading and wound epithelialization in vitro. MATERIALS AND METHODS Reagents and tissue samples Reagents were purchased from Sigma (Schnelldorf, Germany) unless stated otherwise. Brefeldin A and LY294002 were purchased from Calbiochem (Bad Soden, Germany). Toxin B was a kind gift of Klaus Aktories (Freiburg, Germany). Samples of human foreskins were collected from circumcisions in the Dept. of Urology, University of Cologne. Keratinocyte culture and retroviral infection Primary human keratinocytes were isolated from foreskins and cultured on a 3T3 fibroblast feeder layer in FAD medium as described previously (16). 3T3 fibroblasts, strain J2, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS). FAD medium and DMEM with low calcium concentration (FAD low Ca and DMEM low Ca)


had essentially the same composition as FAD medium and DMEM, respectively, but contained only 50 µM of calcium ions. Calcium ions were removed by incubating 50 ml FCS with 2 g Chelex resin (Bio-Rad, Munich, Germany) overnight on a rotating shaker followed by sterile filtration. The retroviral vector pLXSN containing the untagged cDNA for V12Ras as well as the plasmid vector pcDNA3 containing untagged variants of V12Ras that harbour point mutations in the effector domain were kindly provided by Dr. Julian Downward (Cancer Research UK). Constructs were subcloned into the retroviral vector pLXSN. Correct insertion was verified by direct DNA sequencing. Ecotropic producer lines were generated by transfecting GP+E packaging cells (17) using FUGENE (Roche, Mannheim, Germany) reagent and subsequent selection in 1 mg/ml G418 (Life Technologies, Karlsruhe, Germany) for 14 days. Supernatants were used to infect AM-12 amphotropic packaging cells (18). Clones of amphotropic packaging cells producing each retroviral vector were generated and isolated as described previously (19). For infection, fresh supernatant of AM12 packaging cells was passed through a syringe filter (0.45 µm pore size) and applied to keratinocyte cultures growing on a G418-resistant feeder layer 2 days after passaging in the presence of 8 µg/ml polybrene. After 4 h the supernatant was removed and keratinocytes were allowed to grow for 2 days before G418 at a concentration of 500 µg/ml was added for selection. Cells were held in selection medium for 10 days. Coating of cell ware, adhesion, and spreading assay For adhesion assays, keratinocytes were transiently transfected with V12Ras and plated onto collagen I (20 µg/ml), fibronectin (10 µg/ml), and poly-L-lysine (20 µg/ml) coated Permanox chamber slides (Invitrogen, Karlsruhe, Germany) in DMEM without supplements. Cells were allowed to adhere for 1 h, washed twice, and fixed with 4% paraformaldehyde. Slides were stained with an antibody against the HA-tag and with TRITC-phalloidin. Five hundred cells were counted per sample, and the proportion of HA-tag-positive cells adherent to collagen and fibronectin was calculated and compared with the proportion of HA-tag-positive cells adherent to poly- L-lysine. Two independent experiments were carried out. Spreading assays were carried out on collagen-coated Permanox chamber slides (Invitrogen) as previously described (1). Transient transfection of primary human keratinocytes Transient transfections were carried out with cDNAs encoding HA-tagged mutants of Ras in the expression vector pDCR (a generous gift of Dr. Craig Webb, Frederick, MD) and myc tagged mutants of Rac1, N17Rac1, and L61Rac1, which were kindly provided by Dr. Alan Hall (London, UK). Primary human keratinocytes were harvested when 60% confluent and 6 × 105 cells per sample were transferred to an electroporation cuvette. Transfection was carried out in an Amaxa nucleofector using solutions provided by the manufacturer. Best results were achieved when using 4 µg DNA per sample. Wound epithelialization in vitro Keratinocytes were plated on 30 mm cell culture dishes coated with 10 µg/ml collagen I and cultured in FAD low Ca. Cultures were treated with 4 µg/ml mitomycin C in DMEM containing Page 3 of 18 (page number not for citation purposes)

50 µg/ml calcium ions (DMEM low Ca) without FCS for 2 h, then washed in phosphate-buffered saline (PBS), and the monolayer was wounded with a tip of a glass pipette. Cells were incubated in DMEM low Ca in the absence for 4–6 h. Cultures were fixed, permeabilized, and stained with TRITC-labeled phalloidin. Digital images were acquired as previously described (1). Protein extraction and Western blotting To analyze phosphorylation of ERK, we transfected keratinocytes with mutants of Ras and cultured them in FAD medium overnight. The next morning, FAD medium was removed and cells were starved for 4 h in DMEM without serum. Keratinocytes were lysed in situ in modified RIPA buffer containing 5 mM EDTA, 1% Triton X-100, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, 20 µM leupeptin, 1 mM PMSF, 0.5 mg/ml soybean trypsin inhibitor, 0.5 mM NaVO3, and 10 mg/ml p-nitrophenylphosphate; scraped from the dishes; and sonicated for 30 s at full power. Lysates were centrifuged at 14,000 × g for 10 min, and the supernatant was used for Western blot analysis. Equal amounts of protein were separated by SDS-PAGE and blotted onto Hybond-P PVDF membranes (Amersham, Freiburg, Germany). ERK phosphorylation was detected with antibodies specific for phosphorylated ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:1000). Blots were reprobed with antibodies to ERK2 (Santa Cruz Biotechnology; dilution 1:500) to check for equal loading of the lanes. Expression of Ras was detected using a pan Ras antibody (EMD Biosciences, San Diego, CA; dilution 1:50) and an antibody specific for the V12 mutation of Ras (EMD Biosciences; dilution 1:50). Protein bands were visualized with HRP coupled secondary antibodies on Hyperfilm using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, UK). Histopathology of skin wounds and immunostaining Wounding experiments were carried out as described previously (20) and after obtaining approval by the local authorities. Wounds were created on the back skin of mice using a 4 mm biopsy punch. Two days after they were wounded, mice were killed and wounds were excised immediately and fixed in 4% paraformaldehyde overnight. Further processing was done according to standard histopathological procedures. Giemsa-stained 6-µm sections of paraffinembedded wound tissue were photographed using a Leica microscope equipped with a digital camera (Hitachi). Staining of keratin 14 was performed as previously described (21). For staining of phosphorylated ERK1/2, 6-µm sections of frozen tissue were fixed in ice-cold acetone. After blocking with 10% normal goat serum in 0.2% fish skin gelatine (FSG) for 1 h, the sections were washed in PBS. Phospho-specific monoclonal rabbit anti-ERK1/2 antibody was obtained from Cell Signaling New England Biolabs (Beverly, MA) and used according to the protocol given by the manufacturer. Sections were counterstained with propidium iodide to visualize nuclei and mounted in Gelvatol. Fluorescent stainings were analyzed using a Leica TCS upright confocal laser-scanning microscope at excitation wavelengths of 488 and 543 nm. Transfected cells were fixed in 4% paraformaldehyde for 10 min at room temperature, followed by permeabilization with 0.5% Triton X-100 for 5 min at room temperature. Immunostaining was carried out using antibodies against the HA tag (Roche, Basel, Switzerland; dilution 1:1000) and myc tag (9B11, New England Biolabs; dilution 1:500). Secondary antibodies coupled to


Alexa 488 (Molecular Probes, Eugene, OR) at a dilution of 1:500 or 1:1000 were used for visualization. Polymerized actin was stained using TRITC-labeled phalloidin. FACS analysis Keratinocytes were trypsinized and resuspended in DMEM containing 10% FCS. The primary antibody against H2kk (PE-labeled, BD Biosciences, San Jose, CA) was diluted 1:100 and incubated on ice for 10 min in the dark. Cells were washed three times in PBS and analyzed using a FACS Calibur (Becton-Dickinson, Franklin Lakes, NJ). Time-lapse video microscopy Primary human keratinocytes were transfected as described and plated onto collagen I (20 µg/ml)-coated six-well plates (Falcon, BD Biosciences). Sixteen hours after plating, the medium was changed and cells were kept in DMEM without serum throughout the experiment. The surface of the medium was overlaid with mineral oil to avoid evaporation. Cells were observed for 18 h in an incubator chamber attached to an IX81 Olympus microscope at 37°C, 5% CO2, and 60% humidity. Frames were taken every 5 min using an OBS CCD FV2T camera (Olympus). For visualization and quantitation of cell movements, the computer programs OBS Cell R (Olympus) and DIAS (Solltech, Oakdale, IA) were used. RESULTS We have activated Ras signaling in keratinocytes by means of retroviral infection and transient transfection of human primary epidermal keratinocytes with mutant H-Ras constructs (22). These mutants all encode the activating G12→V mutation of Ras (V12Ras) in combination with either the wild-type or a mutated effector domain of Ras that selectively impairs binding to specific downstream effectors: V12Ras35S (carrying the T35→S mutation that enables signaling to the ERK cascade but not to PI3-K and RalGDS), V12Ras40C (carrying the Y40→C mutation that enables signaling to PI3-K but not to ERK and RalGDS), and V12Ras37G (carrying the E37→G mutation that enables signaling to RalGDS but not to PI3-K and ERK). In transient transfection studies, constructs were attached to a C-terminal HA tag; for retroviral gene transfer, untagged constructs were used. For transient transfection, we applied a new, commercially available technique that allows efficient transduction of primary cells. To optimize transfection conditions, we transfected a construct encoding the H2kk cell surface protein into primary human keratinocytes. Under optimized conditions, using program 28, we achieved a transfection efficiency of ~50% as determined by FACS analysis with a FITC-labeled antibody against H2kk (Fig. 1A). Primary human keratinocytes expressing H2kk were viable and displayed no morphological alterations as compared with untransfected cells (data not shown), indicating that the transfection procedure itself does not modify spreading behavior of keratinocytes. When mutants of Ras were transfected, we performed immunostaining of the HA tag in order to distinguish transfected from untransfected cells (Fig. 1C, Fig. 3). For experiments in which high transduction efficiencies were required, we infected primary human keratinocytes with retroviruses harboring the mutant constructs of Ras. This resulted in


detectable expression of mutant proteins, as determined by Western blotting with a pan-Ras antibody and an antibody specifically recognizing Ras protein with the G12→V mutation (Fig. 1B). Expression of V12Ras following either transient transfection or retroviral gene transfer resulted in a dramatic change of keratinocyte shape: cells appeared much larger and showed an increased number of ruffles when compared with untransfected cells or with cells infected with the empty vector control (Fig. 1C, and data not shown). Simultaneously, transduced keratinocytes showed a gross reduction in the amount of detectable polymerized actin: a ring of actin cables adjacent to the cytoplasmic membrane that was present in untransfected cells disappeared in V12Rasexpressing cells (Fig. 1C). Overall, these cells appeared darker than untransfected cells when stained with TRITC-labeled phalloidin, a stain for polymerized actin, indicating that they contained a reduced amount of polymerized actin. To analyze whether V12Ras-induced spreading leads to enhanced adhesion of keratinocytes, we carried out adhesion assays in V12Ras-transfected keratinocytes on collagen I and fibronectin. Counting of V12Ras-expressing and -nonexpressing keratinocytes adherent to collagen I or fibronectin revealed that the proportion of adherent V12Ras-expressing keratinocytes was increased on both substrates (1.6-fold on collagen I and 1.5-fold on fibronectin) as compared with the unspecific adhesion substrate poly-L-lysine (1-fold). This indicates that expression of V12Ras facilitates specific adhesion of keratinocytes to extracellular matrix components that are abundantly present in skin wounds. Since V12Ras-transfected cells seemed to dissolve ring-like actin cables at the cell periphery and actively protrude their plasma membrane, we determined the speed of cell spreading by measuring the area covered by a single cell within 40 min after plating onto collagen I-coated surfaces (1). Analysis of 200 attached cells per sample revealed increased numbers of large cells in the V12Ras-transfected population (Fig. 1D). This indicates that expression of V12Ras accelerated spreading of keratinocytes on collagen I. Although V12Ras-expressing cells seemed to contain less polymerized actin than respective controls, Ras-induced spreading could be abrogated by incubation of cells with cytochalasin D, an inhibitor of actin polymerization (Fig. 1E). Shape changes of keratinocytes upon activation of Ras have been observed previously and were interpreted by others as indicative of senescence or growth arrest that is followed by entry of the terminal differentiation program (5, 9). To test whether the shape changes observed were associated with terminal differentiation, we induced growth arrest in primary keratinocytes by treating them with 4 µg/ml mitomycin C for 2 h. These cells did not show gross morphological alterations 24 h after treatment but enlarged during subsequent days and started to express the terminal differentiation marker involucrin (Fig. 2A). By day 5 after mitomycin C treatment, >90% of keratinocytes were involucrin-positive (data not shown). When we treated these cells with cytochalasin D, they remained large and spread out (Fig. 2A, upper panel) although actin filaments in these cells were fragmented (Fig. 2A, lower panel), suggesting that keratinocyte morphology after mitomycin-induced growth arrest is not solely maintained by reorganization of the actin cytoskeleton. In contrast, keratinocyte shape changes after V12Ras transfection could be completely inhibited by incubation with cytochalasin D (Fig. 1E, 2B). To further investigate a possible correlation between keratinocyte shape changes upon Ras activation and terminal


keratinocyte differentiation, numbers of transfected and untransfected keratinocytes that expressed the terminal differentiation marker involucrin were scored 30 h after transient transfection with V12Ras. At this time, V12Ras-induced shape changes were already present (see Fig. 1). Results in Table 1 show that the number of involucrin-positive keratinocytes was lower in the V12Ras-transfected than in the -untransfected population, indicating that until 30 h after transfection V12Ras did not stimulate initiation of the terminal differentiation program. To analyze the influence of V12Ras on keratinocyte growth in our system, we infected primary human keratinocytes with retroviruses encoding for mutants of Ras or empty vector and analyzed their growth over 14 days. Growth curves in Fig. 2C show that V12Ras-expressing keratinocytes did not proliferate less than empty vector controls, although spreading of keratinocytes on collagen-coated dishes was greatly enhanced (Fig. 2B). These results demonstrate that V12Rasmediated keratinocyte shape changes are not the result of an induction of terminal differentiation. Activated Ras can stimulate several different intracellular pathways by binding to distinct downstream effector molecules. Among these pathways is the classical MAP kinase cascade. We analyzed phosphorylation of extracellular regulated kinase (ERK) 1 and 2 using an antibody that specifically detects the phosphorylated forms of these kinases. Expression of V12Ras and V12Ras35S, but not V12Ras37G, V12Ras40C, or V12Rac, an activated version of the small GTP binding protein Rac1, resulted in a pronounced activation of MAP kinase signaling (Fig. 3A, 3B). To test which Ras-related pathways were involved in the observed shape changes, we carried out spreading assays with keratinocytes transfected or infected with the different mutants of Ras. Keratinocytes expressing V12Ras35S and V12Ras37G were not enlarged and did not show enhanced spreading when plated onto collagen-coated dishes (Fig. 3C–E). In contrast, V12Ras40C induced keratinocyte spreading, although not as extensively as V12Ras (Fig. 3C, 3F). As for V12Ras, V12Ras40C-induced spreading could be inhibited by incubation with cytochalasin D (data now shown). In single keratinocytes, activation of Ras dramatically accelerated cell spreading. To investigate whether this effect had an impact on the ability of keratinocytes to close a defect within a cell monolayer, we carried out in vitro assays of wound epithelialization using keratinocytes retrovirally infected with different mutants of Ras that were growth arrested by mitomycin C treatment before the experiment. Transduction of keratinocytes with V12Ras stimulated wound epithelialization and resulted in a faster wound closure as compared with the empty vector control neo (Fig. 4A−F). A similar result was obtained with keratinocytes expressing V12Ras40C. Although V12Ras35S and V12Ras37G also facilitated wound closure, this effect was less pronounced as compared with V12Ras and V12Ras40C. We conclude that stimulation of Ras in keratinocytes leads to a proliferation-independent acceleration of wound closure in vitro. Whereas different downstream signaling pathways contribute to this effect, reepithelialization is most efficiently stimulated by effectors that are also activated by V12Ras40C. To investigate whether accelerated wound closure upon Ras activation was due to increased cell migration, we analyzed random migration of V12Ras-transfected keratinocytes over 18 h. Quantitation of speed and distance of migration revealed no significant differences between V12Ras-expressing and wild-type cells. The average path lengths of 10 V12Ras expressing keratinocytes and 11 controls during 1055 min observation time were 155.8 ± 51.8 µm and 188.9 ± 33.5 µm, respectively. This shows that accelerated wound closure in vitro, although it


correlated with cell shape changes, was not due to stimulation of random migration in V12Rasexpressing keratinocytes. To analyze whether similar changes in keratinocyte shape could also be observed in vivo, we created a wound at the back of a mouse. The mouse was killed 2 days after wounding and the wound tissue subjected to histopathological analysis. Whereas epidermal keratinocytes in unwounded interfollicular epidermis and the epithelium of the wound edge exhibited a typical cuboidal shape, keratinocytes at the tip of the newly formed epithelial tongue were flat and spread out (Fig. 4G). Furthermore, staining of wounded epithelium with an antibody against activated ERK revealed strong nuclear staining of phosphorylated ERK1/2 in keratinocytes at the wound edge and partly in the epithelial tongue. Since many extracellular stimuli lead to the activation of ERK in a Ras-dependent fashion, these data suggest that 1) factors present in the wound environment lead to the activation of Ras in wound edge keratinocytes and 2) Ras induced shape changes do not only occur in vitro but also in vivo in migrating epidermal cells. PI3-K is an effector of Ras in keratinocytes and is thought to be stimulated by expression of V12Ras40C (11). It has been shown to induce spreading in different cell types, including human keratinocytes, and to mediate IGF-1-induced spreading and wound epithelialization (1). We therefore analyzed V12Ras-stimulated spreading of keratinocytes in the presence of the specific PI3-K inhibitor LY294002. Surprisingly, incubation with LY294002 did not inhibit V12Rasstimulated spreading (Fig. 5C), although phosphorylation of its target Akt upon treatment of keratinocytes with IGF-1 was abrogated (ref 1 and data not shown). This indicates that activation of PI3-K is not required for the observed cell shape changes after stimulation of Ras. We have also analyzed functions of Arf6, another small GTP binding protein that regulates cytoskeletal rearrangements and spreading of epithelial cells (23), in the context of our experiments. Inhibition of Arf6 function using Brefeldin A did not change spreading or wound epithelialization of V12Ras-expressing keratinocytes (data not shown). Another downstream effector of Ras known to be involved in the regulation of cytoskeletal actin dynamics is the small GTP binding protein Rac1. To test possible functions of Rac1 in V12Rasinduced keratinocyte spreading, we inhibited signaling via the small GTP binding proteins Rac and Cdc42 by incubating V12Ras-transfected primary keratinocytes with Clostridium difficile toxin B, an inhibitor of Rac and Cdc42 (24). Incubation with toxin B dramatically inhibited spreading of V12Ras-expressing keratinocytes (Fig. 5A). In addition, to analyze the role of Rac more specifically, we inhibited Rac signaling by expression of a dominant-negative mutant. Primary human keratinocytes expressing V12Ras from a retroviral promoter were transiently transfected with a plasmid encoding the dominant-negative mutant of Rac1, N17Rac1, and spreading was determined as described. Cell spreading was reduced in V12Ras-infected keratinocytes expressing N17Rac1 (Fig. 5B). We also expressed a constitutively active mutant of Rac1, L61Rac1, in primary human keratinocytes using transient transfection. Cells transfected with this mutant showed enhanced spreading when plated onto collagen-coated dishes, similarly to V12Ras-transfected cells (Fig. 5D). This indicates that activity of Rac1 is sufficient to induce keratinocyte spreading and is required for V12Ras-stimulated spreading.


DISCUSSION Since it is known as an oncogene, the small GTP binding protein Ras has been the subject of extensive investigations. The majority of this work has focused on the growth regulatory properties of Ras. In this study, we describe an effect of Ras activity on keratinocyte shape: activation of Ras leads to accelerated protrusion of the plasma membrane and formation of large lamellipodial structures. Similar shape changes in keratinocytes have been observed previously upon Ras activation. In these studies, enlargement of keratinocytes after retroviral infection with an activated mutant of Ras was speculated to be associated with the induction of terminal differentiation (9). Here we provide evidence that Ras-induced keratinocyte shape changes occur independently of terminal keratinocyte differentiation. This conclusion is based on the following findings: 1) Ras-induced shape changes can be inhibited by treatment with cytochalasin D, an inhibitor of actin polymerization, whereas shape changes accompanying terminal differentiation of keratinocytes following mitomycin C-induced growth arrest cannot be reversed by cytochalasin D treatment; 2) expression of an activating mutant of Ras does not result in an increased expression of the terminal differentiation marker involucrin; and 3) primary human keratinocytes show increased proliferation after infection with a retroviral vector encoding an activating mutant of Ras. This suggests that keratinocyte spreading following Ras activation and growth arrest/induction of terminal differentiation are distinct processes. Although V12Ras-expressing keratinocytes contained less polymerized actin than did controls, shape changes were also dependent on actin polymerization. This indicates that Ras-induced spreading was accompanied by dynamic changes of the actin cytoskeleton. Using mutants of the Ras effector domain, we have found that protrusion of the cell membrane in keratinocytes expressing activated Ras is not a function of ERK activation or stimulation of Ral GDS. Our data also suggest that keratinocyte spreading following Ras activation may be the consequence of PI3-K-independent stimulation of the small GTPase Rac. The molecular mechanism mediating PI3-K-independent Rac activation by Ras in cooperation with the GEF Tiam 1 has been recently proposed (25). An alternative pathway that could be relevant in the context of our results is mediated by PREL1, a new protein that can bind to activated Ras and signals to the actin cytoskeleton via the Ena/VASP complex (26). In the future, it will be interesting to investigate these pathways in more detail. There are several conceivable biological functions of Ras-induced keratinocyte spreading. These include wound closure, morphogenesis of epidermal appendages, and invasive properties of epidermal tumors. Using an in vitro wound healing assay, we have shown that activation of Ras can lead to accelerated reepithelialization of wounded keratinocyte monolayers in vitro. This effect is apparently not due to stimulated random migration of keratinocytes. Interestingly, a mutant of Ras that stimulates Rac (V12Ras40C) is more efficient in inducing wound closure than a mutant that stimulates the Raf/ERK cascade (V12Ras35S). This is consistent with our previous results showing that IGF-1, which induces keratinocyte spreading and random motility, stimulates reepithelialization of wounded keratinocyte monolayers more efficiently than EGF, which also stimulates motility but does not induce spreading. These findings may suggest that the capacity of epidermal keratinocytes to close a wound is more determined by their ability to spread than by random cell motility.


Epithelialization studies in vitro often focus on the movement of single epithelial cells. Wound reepithelialization in skin in vivo, however, occurs in a way that formations of wound-edge keratinocytes approach each other as sheets in which individual cells partly maintain cell-cell contacts. Therefore, random keratinocyte migration cannot be the parameter that best predicts the ability of a keratinocyte population to close a skin wound. On the basis of our in vitro findings, we suggest that keratinocyte spreading is an early event in wound healing that determines the efficiency of wound reepithelialization. This is supported by our morphological analysis of a reepithelializing wound showing that flattening and spreading indeed occur in fresh murine skin wounds 2 days after wounding. Molecular dissection of signaling pathways that facilitate keratinocyte spreading could therefore be useful for the understanding of pathogenic mechanisms involved in wound-healing disturbances as well as for the identification of new therapeutic targets. ACKNOWLEDGMENTS This work was supported by the research network program SFB589 of Deutsche Forschungsgemeinschaft; by the Center for Molecular Medicine, University of Cologne; and by the Koeln Fortune Program, Faculty of Medicine, University of Cologne. We are grateful to Carien Niessen for critical discussions and to Klaus Aktories and Alan Hall for providing materials. REFERENCES 1.

Haase, I., Evans, R., Pofahl, R., and Watt, F. M. (2003) Regulation of keratinocyte shape, migration and wound epithelialization by IGF-1- and EGF-dependent signalling pathways. J. Cell Sci. 116, 3227–3238

2.

Downward, J. (2003) Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11–22

3.

Haase, I., Hobbs, R. M., Romero, M. R., Broad, S., and Watt, F. M. (2001) A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis. J. Clin. Invest. 108, 527–536

4.

Haase, I., and Hunzelmann, N. (2002) Activation of epidermal growth factor receptor/ERK signaling correlates with suppressed differentiation in malignant acanthosis nigricans. J. Invest. Dermatol. 118, 891–893

5.

Chaturvedi, V., Cesnjaj, M., Bacon, P., Panella, J., Choubey, D., Diaz, M. O., and Nickoloff, B. J. (2003) Role of INK4a/Arf locus-encoded senescent checkpoints activated in normal and psoriatic keratinocytes. Am. J. Pathol. 162, 161–170

6.

Hobbs, R. M., Silva-Vargas, V., Groves, R., and Watt, F. M. (2004) Expression of activated MEK1 in differentiating epidermal cells is sufficient to generate hyperproliferative and inflammatory skin lesions. J. Invest. Dermatol. 123, 503–515

7.

Scholl, F. A., Dumesic, P. A., and Khavari, P. A. (2004) Mek1 alters epidermal growth and differentiation. Cancer Res. 64, 6035–6040 Page 10 of 18 (page number not for citation purposes)

8.

Peng, X. D., Xu, P. Z., Chen, M. L., Hahn-Windgassen, A., Skeen, J., Jacobs, J., Sundararajan, D., Chen, W. S., Crawford, S. E., Coleman, K. G., et al. (2003) Dwarfism, impaired skin development, skeletal muscle atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2. Genes Dev. 17, 1352–1365

9.

Roper, E., Weinberg, W., Watt, F. M., and Land, H. (2001) p19ARF-independent induction of p53 and cell cycle arrest by Raf in murine keratinocytes. EMBO Rep. 2, 145–150

10. Lazarov, M., Kubo, Y., Cai, T., Dajee, M., Tarutani, M., Lin, Q., Fang, M., Tao, S., Green, C. L., and Khavari, P. A. (2002) CDK4 coexpression with Ras generates malignant human epidermal tumorigenesis. Nat. Med. 8, 1105–1114 11. Dajee, M., Tarutani, M., Deng, H., Cai, T., and Khavari, P. A. (2002) Epidermal Ras blockade demonstrates spatially localized Ras promotion of proliferation and inhibition of differentiation. Oncogene 21, 1527–1538 12. Brown, G. L., Nanney, L. B., Griffen, J., Cramer, A. B., Yancey, J. M., Curtsinger, L. J. d., Holtzin, L., Schultz, G. S., Jurkiewicz, M. J., and Lynch, J. B. (1989) Enhancement of wound healing by topical treatment with epidermal growth factor [see comments]. N. Engl. J. Med. 321, 76–79 13. Nanney, L. B., and King, L. E. J. (1996) Epidermal growth factor and transforming growth factor-alpha. In The Molecular and Cellular Biology of Wound Repair (Clark, R. A. F., ed) pp. 171–194, Plenum, New York 14. Tsuboi, R., Sato, C., Kurita, Y., Ron, D., Rubin, J. S., and Ogawa, H. (1993) Keratinocyte growth factor (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes. J. Invest. Dermatol. 101, 49–53 15. Ando, Y., and Jensen, P. J. (1993) Epidermal growth factor and insulin-like growth factor I enhance keratinocyte migration. J. Invest. Dermatol. 100, 633–639 16. Watt, F. M. (1998) Cultivation of human epidermal keratinocytes with a 3T3 feeder layer. In Cell Biology: A Laboratory Handbook (Celis, J. E., ed) Vol. 1, pp. 113–118, Academic Press, London 17. Markowitz, D., Goff, S., and Bank, A. (1988) A safe packaging line for gene transfer: separating viral genes on two different plasmids. J. Virol. 62, 1120–1124 18. Markowitz, D., Goff, S., and Bank, A. (1988) Construction and use of a safe and efficient amphotropic packaging cell line. Virology 167, 400–406 19. Zhu, A. J., and Watt, F. M. (1996) Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes. J. Cell Sci. 109, 3013–3023


20. Eckes, B., Colucci-Guyon, E., Smola, H., Nodder, S., Babinet, C., Krieg, T., and Martin, P. (2000) Impaired wound healing in embryonic and adult mice lacking vimentin. J. Cell Sci. 113, 2455–2462 21. Pasparakis, M., Courtois, G., Hafner, M., Schmidt-Supprian, M., Nenci, A., Toksoy, A., Krampert, M., Goebeler, M., Gillitzer, R., Israel, A., et al. (2002) TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2. Nature 417, 861–866 22. White, M. A., Nicolette, C., Minden, A., Polverino, A., Van Aelst, L., Karin, M., and Wigler, M. H. (1995) Multiple Ras functions can contribute to mammalian cell transformation. Cell 80, 533–541 23. Santy, L. C., and Casanova, J. E. (2001) Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J. Cell Biol. 154, 599–610 24. Barbieri, J. T., Riese, M. J., and Aktories, K. (2002) Bacterial toxins that modify the actin cytoskeleton. Annu. Rev. Cell Dev. Biol. 18, 315–344 25. Lambert, J. M., Lambert, Q. T., Reuther, G. W., Malliri, A., Siderovski, D. P., Sondek, J., Collard, J. G., and Der, C. J. (2002) Tiam1 mediates Ras activation of Rac by a PI(3)Kindependent mechanism. Nat. Cell Biol. 4, 621–625 26. Jenzora, A., Behrendt, B., Small, J. V., Wehland, J., and Stradal, T. E. (2005) PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins. FEBS Lett. 579, 455–463 Received December 15, 2004; accepted August 3, 2005.


Table 1 Involucrin expression in primary human keratinocytes transfected with V12Ras or left untransfected

Total V12Ras+ Involucrin+ V12Ras+/Involucrin+ V12Ras–/Involucrin+

Number of cells 923a 261 105 27 78

Percentage of total cells 100 28 11 3 8

a

All cells were double stained with a monoclonal antibody against the HA tag (V12Ras) and a polyclonal antibody against human involucrin.


Fig. 1

Figure 1. Transduction of primary keratinocytes with mutants of Ras. A) FACS profile of primary human keratinocytes left untransfected (black line) or transfected with a plasmid-encoding H2kk surface protein (green line) using the Amaxa technique. Staining against H2kk was performed using a PE-labeled monoclonal antibody. Percentage of gated cells: M1 black line, 93%; M1 green line, 45%; M2 black line, 7,1%; M2 green line, 55%. B) Western blot analysis of expression of mutated Ras in retrovirally infected primary human keratinocytes using an antibody against pan Ras (top) or an antibody specifically recognizing the V12 mutation of Ras (bottom). C) Confocal images of primary human keratinocytes transfected with V12Ras. Green, HA tag; red, phalloidin staining. Scale bars: 20 µm (left and middle) and 15 µm (right). Arrows point to membrane ruffles. D) Histogram showing the size distribution of 400 keratinocytes spreading on collagen I -coated surfaces. Black bars, untransfected cells; red bars, cells transfected with V12Ras. E) Size distribution of keratinocytes expressing V12Ras and treated with 0.1% DMSO (vehicle control, red bars) or with 4 µM cytochalasin D (black bars). Page 14 of 18 (page number not for citation purposes)

Fig. 2

Figure 2. Ras-induced cytoskeletal changes are not related to terminal differentiation. A, B) Confocal pictures of primary human keratinocytes. A) keratinocytes were growth arrested by treatment with mitomycin C, cultured for 5 days, and treated with DMSO (left panel) or 4 µM cytochalasin D (right panel) for 1 h. Green staining shows expression of involucrin (upper panel); red staining is for polymerized actin using TRITC-phalloidin. Note that cell shape is maintained in cytochalasin D-treated cells, although actin fibers are fragmented. B) Single keratinocytes spreading on collagen I infected with empty retroviral vector LXSN (neo) or LXSN with V12Ras (V12Ras) and treated with 0.1% DMSO or 4 µM cytochalasin D for 30 min. C) Growth curves of primary human keratinocytes retrovirally infected with different variants of V12Ras. x-axis, day of culture; y-axis, cell number per well. Data points are mean ± SD of triplicates.


Fig. 3

Figure 3. Regulation of spreading by Ras-dependent signaling pathways. A, B) Western blot analysis with a phosphorylation-specific antibody against ERK (upper panels). Loading controls showing ERK expression (lower panels). A) Primary human keratinocytes were infected with empty retroviral vector or vectors encoding V12Ras or V12Rac. B) Primary human keratinocytes were transfected according to the Amaxa protocol with different mutants of Ras as indicated. C) Microscopic pictures of keratinocytes expressing V12Ras35S (left), V12Ras37G (middle), or V12Ras40C (right). Green staining shows HA tag; red staining is with TRITC-phalloidin for polymerized actin. Scale bars: 20 µm. D–F) Histograms showing the size distribution of 200 keratinocytes per sample spreading on collagen I after transfection with V12Ras35S (D), V12Ras37G (E), or V12Ras40C (F). x-axis shows cell size in arbitrary units; y-axis shows cell number. Page 16 of 18 (page number not for citation purposes)

Fig. 4

Figure 4. Epithelialization of wounded keratinocyte monolayers, keratinocyte shape in wounded skin, and activation of ERK1/2 in wound edge epithelium. A–F) Microscopic pictures showing primary human keratinocytes retrovirally infected with control vector neo (A), V12Ras (B), V12Ras-35S (C), V12Ras-37G (D), and V12Ras-40C (E). Cells were grown to confluence, growth arrested by mitomycin C treatment, and wounded with a glass pipette. Cultures were fixed after 6 h and stained with TRITC-phalloidin. F) Width of the nonepithelialized surface after 6 h in arbitrary units. Bars represent mean ± SD of 40 individual measurements at different points of the scratch wound. Labeling of the bars corresponds to the labeling of the microscopic pictures. G–I) Immunostaining for keratin 14 (G, green) and phosphorylated ERK1/2 (H and I, green) in wound edge epithelium (H) and normal epidermis (I). Note that keratinocytes in the epithelial tongue (G, blue arrow) are flat and spread out as compared with keratinocytes of the wound edge epithelium (G, white arrow). H, I) Red line marks the epidermal basement membrane. H) Note the nuclear staining pattern of activated ERK1/2. Page 17 of 18 (page number not for citation purposes)

Fig. 5

Figure 5. Regulation of V12Ras induced spreading by different downstream signaling pathways. Histograms show the size distribution of primary human keratinocytes transfected with V12Ras (A, C) or L61Rac (D) or retrovirally infected with V12Ras (B). A, C) Cells were treated with 0.1% DMSO as vehicle (black bars) or specific inhibitors (red bars) and then allowed to spread on collagen I for 40 min in the presence of vehicle or inhibitors. The following inhibitors were used: toxin B (20 ng/ml) for 14 h and LY294005 (25 µM) for 20 min. B) Keratinocytes expressing V12Ras from a retroviral promoter were left untransfected (black bars) or transfected with a dominant-negative mutant of Rac1, N17Rac (red bars), and then allowed to spread on collagen I for 40 min. D) Keratinocytes were transfected with a vector encoding L61Rac (red bars) or empty vector (black bars) and allowed to spread on collagen I for 40 min.
