Somatic Excision Demonstrates that c-Jun Induces Cellular Migration and Invasion through Induction of Stem Cell Factor

Cancer cells arise through sequential acquisition of mutationsin tumor suppressors and oncogenes. c-Jun, a critical componentof the AP-1 complex, is frequently overexpressed in diversetumor types and has been implicated in promoting cellular proliferation,migration, and angiogenesis. Functional analysis of candidategenetic targets using germ line deletion in murine models canbe compromised through compensatory mechanisms. As germ linedeletion of c-jun induces embryonic lethality, somatic deletionof the c-jun gene was conducted using floxed c-jun (c-jun^f/f)conditional knockout mice. c-jun-deleted cells showed increasedcellular adhesion, stress fiber formation, and reduced cellularmigration. The reduced migratory velocity and migratory directionalitywas rescued by either c-Jun reintroduction or addition of secretedfactors from wild-type cells. An unbiased analysis of cytokinesand growth factors, differentially expressed and showing lossof secretion upon c-jun deletion, identified stem cell factor(SCF) as a c-Jun target gene. Immunoneutralizing antibody toSCF reduced migration of wild-type cells. SCF addition rescuedthe defect in cellular adhesion, cellular velocity, directionalmigration, transwell migration, and cellular invasion of c-jun^–^/^–cells. c-Jun induced SCF protein, mRNA, and promoter activity.Induction of the SCF promoter required the c-Jun DNA-bindingdomain. c-Jun bound to the SCF promoter in chromatin immunoprecipitationassays. Mutation of the c-Jun binding site abolished c-Jun-mediatedinduction of the SCF promoter. These studies demonstrate anessential role of c-Jun in cellular migration through inductionof SCF.

INTRODUCTION

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Introduction

The c-jun proto-oncogene encodes a prototypical member of theAP-1 transcription factor family. c-Jun heterodimerizes withmembers of the Jun/Fos and MaF/Nr1 families through a leucinezipper motif. AP-1 proteins, in turn, regulate transcriptionalactivity of downstream target genes or interact directly toinfluence transcription through association with other transcriptionfactors (13, 23, 28, 39). Phosphorylation of the c-Jun N-terminalkinase (JNK) subgroup of mitogen-activated protein (MAP) kinasesplays a key role in responding to diverse stress signals (28).JNK activation has been linked to both the inhibition and inductionof cellular apoptosis and to the regulation of cellular migration(26, 68, 69).

The migration of cells plays a critical role in a broad varietyof biological processes including cellular development, tissuerepair, and metastasis of tumors (29, 34, 42). Initiation ofcellular migration requires cell substratum adhesion interactionand the sequential generation of membrane protrusions (36).Actin polymerization provides both protrusive activity and directionalityof cellular movement. New adhesive sites are sequentially establishedin the extended membranes. Motile cells constantly remodel transientadhesions at the leading edges (43). In fibroblasts, focal complexesform which mature into focal adhesions. Remodeling of thesefocal adhesions is important, as cell motility is a dynamicbalance between contractual forces driving the cell body forwardand detachment of the posterior edge of the cell from its substratum.

Cellular migration is induced by a variety of growth factorsand cytokines. Stem cell factor (SCF), and its receptor Kit,play pivotal roles in cellular migration as well as differentiation,proliferation, and migration (74). SCF exists as a secretedsoluble form and as a membrane-bound glycoprotein (15, 73).Complete absence of SCF in the mouse is embryonic lethal (15),and SCF binding to Kit induces cellular migration of diversecell types including neural stem cells and endothelial cells(33). SCF directly activates microvascular endothelial cellsto promote tumor angiogenesis (55). Intracellular kinases areactivated consequent upon ligand-induced dimerization and transphosphorylationof Kit, a type III receptor protein-tyrosine kinase (70). Therelative importance of SCF-induced JNK, Akt, and extracellularsignal-regulated kinase (ERK) activity to cellular migrationremains to be fully understood.

c-jun^–/^– mice die in gestation from cardiovascularand hepatic defects (12). Therefore, to examine the role ofc-Jun in cellular adhesion, migration, and directional persistence,transgenic mice carrying floxed c-jun alleles (c-jun^f/f) inwhich the gene was flanked by loxP sites were used herein. Acuteexcision of c-jun using Cre recombinase identified a key rolefor c-Jun in cellular adhesion. Within 48 h of c-jun excision,c-jun^–/^– fibroblasts were round and flat and demonstratedgreater cellular spreading. Cellular adhesion was enhanced inc-jun^–/^– murine embryonic fibroblasts (MEFs), associatedwith increased stress fiber formation and reduced migrationinto a wound. c-Jun induced both the velocity of cellular migrationas well as directional persistence. The reduced persistenceof migratory directionality of c-jun^–/^– cells wasreversed by the addition of medium from parental c-jun^+/+ cells.Using an unbiased array-based proteomic approach, subtractiveanalysis of cytokine and growth factors differentially secretedupon deletion of c-jun identified SCF as a cytokine secretedin response to c-Jun. SCF rescued the defective migration ofc-jun^–/^– cells, and SCF expression was induced byc-Jun. Together these studies demonstrate a key role for c-Junin cellular migration through induction of SCF.

MATERIALS AND METHODS

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Materials and Methods

Transgenic mice, expression plasmids, and promoter cloning. Transgenic animals carrying floxed c-jun alleles, c-jun^f/f,were previously described (71). All experimental procedureswith these mice were approved by the ethics committee of Georgetownand Thomas Jefferson University. Animals were examined for acoital plug to define the day zero of gestation whenever copulationwas detected. The MEFs were produced as previously described(1) and cultured following the 3T3 protocol (60).

The expression plasmids encoding adenovirus directing Cre (Ad-Cre)expression or control virus (Ad-Null) were previously described(66). The EcoR1 fragment of the rat c-jun DNA (53) was subclonedinto a retroviral expression vector, murine stem cell virus-internalribosomal entry site-green fluorescent protein (MSCV-IRES-GFP),to form MSCV-c-Jun-IRES-GFP. The murine SCF promoter was clonedby amplifying a 2-kb fragment from the 5' flanking region ofthe kit-ligand (Kitl) gene (Table 1 lists the oligonucleotideprimers), followed by its insertion into the SmaI site of thepGL3-basic luciferase reporter vector. The c-Jun wild type andc-Jun DNA^– were previously described (2). For creatingthe AP-1 deletion mutant of the SCF promoter, each of the AP-1sites (TGACCCTCA,–1421 to –1413; TGAGTAA, –1375to –1369; TGAATCA, –1050 to –1056; and TGAGTCA,–604 to –598) contained within the SCF promoterwere sequentially deleted by in vitro mutagenesis. By makinguse of oligonucleotide primers lacking the AP-1 sites, the pGL3-SCFpromoter plasmid was amplified using Phusion High-Fidelity DNApolymerase (Finnzymes Oy, Espoo, Finland) followed by DpnI digestionto remove original template plasmid. First, single-site deletionmutants were created, followed by deletion of the second, third,and fourth sites in subsequent rounds of amplifications anddigestions.

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TABLE 1. List of oligonucleotide primers used in this study^a

Reagents and antibodies. The kinase inhibitor for MAP kinase (PD98059), Jun N-terminalkinase (SP600125), and phosphatidylinositol 3 (PI3)-kinase (LY294002)were obtained from Calbiochem EMD Bioscience (San Diego, CA).The p38 MAP kinase inhibitor (SB203580) was from Tocris CooksonInc. (Ellisville, MO), epidermal growth factor (EGF) was obtainedfrom Biosource International (Camarillo, CA), transforming growthfactor ß (TGF-ß) was from Promega (Madison,WI), and interleukin-1 (IL-1) and tumor necrosis factor alphawere from Sigma-Aldrich (St. Louis, MO). SCF from PeprotechInc. (Rocky Hill, NJ), anti-c-Jun antibody (H-79; Santa CruzBiotechnology, Santa Cruz, CA), anti-GDI (RTG Sol, Gaithersburg,MD), anti-SCF antibody (R&D Systems, Minneapolis, MN), andrhodamine-anti-phalloidin and DAPI (4',6'-diamino-2-phenylindole)(both from Sigma-Aldrich Corp., St. Louis, MO) were also used.Biodipy 650/665 anti-phalloidin was from Molecular Probes, Inc.(Eugene, OR).

Cell culture, viral cell transduction, and reporter gene assays. Cells were maintained in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal bovine serum and 100 µg/ml(each) of penicillin and streptomycin and were cultured in 5%CO₂ at 37°C. Adenovirus propagation was previously described(65). Infection was done at a multiplicity of infection (MOI)of 20, cells were cultured overnight, and medium was changedprior to experimental analysis. Retroviral infections were conductedas previously described (65). Transfections were conducted usingGenejuice transfection reagent (EMD Biosciences, San Diego,CA) and Lipofectamine (Invitrogen Corp, Carlsbad, CA) as describedpreviously (7). Statistical analysis was conducted using a Mann-WhitneyU test.

Microscopy and phalloidin staining for F-actin quantitation. Immunopositive MSCV-IRES-GFP- and MSCV-c-jun-IRES-GFP-transducedcells were examined in six-well plates. Phase-contrast microscopyand fluorescent imaging were carried out using the 20x and 60xobjectives of an Olympus LSM-5 Meta Laser confocal scanningmicroscope. Rhodamine-phalloidin F-actin staining was conductedas previously described (35). F-actin quantitation was alsocarried out by fluorescence-activated cell sorting analysis(40). Briefly, confluent c-jun^f/f cells treated with adenoviruseswere harvested and washed with phosphate-buffered saline (PBS).Cell pellets were fixed with paraformaldehyde and permeabilizedwith Triton X-100. Following a wash with PBS, cells were stainedwith 0.6 µM biodipy 650/665 phalloidin (Molecular Probes,Eugene, OR) in PBS for 10 min at room temperature (64).

Cells were plated on fibronectin-coated glass coverslips andgrown to approximately 80% confluence, and scanning electronmicroscopy was conducted as described previously (35).

Cell adhesion assay. Ninety-six-well cell surface matrix-coated strip-well tissueculture plates (no coating, collagen I, collagen IV, poly-L-lysine,laminin, fibronectin, and vitronectin) were used for cell adhesionassays. Equal numbers of cells were seeded at the bottom ofeach coated well and allowed to adhere by incubating the platesat 37°C in 5% CO₂ for planned intervals. Strip-wells containingadherent cells were removed at 30 min, 1 h, 1.5 h, 2 h, 3 h,and 4 h; cells were fixed in 1% glutaraldehyde for 10 min andstained with 0.1% crystal violet for 30 min. Following PBS washes,100 µl of 0.5% Triton X-100 was added to each well tolyse the cells and extract dye by incubating the plates overnightat room temperature with gentle shaking. Quantitation of extracteddye was conducted by measuring the absorbance at 595 nm. Foreach cell surface matrix, the background was noted from a coatedwell with no cells seeded and subtracted to obtain the actualvalues of absorbance at 595.

Assays of cell motility, migration, and invasion. Cells were plated on plastic dishes coated with 10 µg/mlfibronectin and cultured overnight in DMEM containing 5% fetalbovine serum. Cell movements were monitored using a Zeiss invertedmicroscope. Video images were collected with a charge-coupled-devicecamera (model 2400) at planned intervals, digitized, and storedas images using Metamorph, version 3.5, software (18). Imageswere converted to QuickTime movies, and the positions of nucleiwere tracked to quantify cell motility. Cellular velocity wascalculated in micrometers using Metamorph software. The effectof kinase inhibitors on cell migration were determined afterculturing the cells with 25 µM PD98059, 25 µM LY294002,25 µM SB203580, 25 µM SP600125, 10 µM Y27632,10 µM H-1152, and 10 µM HA-1100 (6, 10, 27, 47,49, 67). Prior to an examination for effects on cell motility,analyses were conducted for 3 h. At this time point, the persistencefor migratory directionality was determined as a relative D/Tratio representing the ratio of the direct distance (D) fromstart point to end point divided by the total track distance(T) (18). Net displacements were measured every 15 min fromstart point to end point. Data from at least 100 cells werecollected for each set or treatment.

Migration of cells across a membrane was determined using aBoyden chamber, as previously described (31, 32). A gradientof SCF was created through the addition of SCF (0.5 ng/ml) tothe lower chamber. Analysis of three-dimensional invasive activitywas conducted as previously described (44). A total of 10⁵ cellswere embedded in 100 µl of collagen in a 96-well plateand cultured for 24 h. The collagen-cell plugs were transferredto 24-well plates and embedded in 1 ml of collagen and culturedfor 5 days. Migration from the central plug into the surroundingcollagen was monitored by phase-contrast microscopy.

Cytokine array analysis. Mouse cytokine arrays spotted on nitrocellulose membranes wereobtained from Raybiotech (Norcross, GA). Conditioned mediumfrom Ad-Null- and Ad-Cre-treated c-jun^f/f cells was preparedby culturing cells in serum-free DMEM for 24 to 48 h. Membraneswere then processed according to the manufacturers' instructionsfor assessment of secreted cytokines and growth factors presentin conditioned medium.

Real-time PCR, ChIP assays, and enzyme-linked immunosorbent assay (ELISA). All gel-based PCRs and reverse transcription-PCRs (RT-PCRs)were done with an ExTaq DNA Polymerase kit (Takara Shuzo, Shiga,Japan) using the oligonucleotide primers listed in Table 1.RNA was extracted using a standard guanidinium isothiocyanatemethod, RQ1 DNase I (Promega, Madison, WI) treated, and phenol-chloroformextracted. RNA quantitation was done in an Agilent 2100 Bioanalyzer(Palo Alto, CA), and equal quantities were used for the reversetranscription reactions. Primers for all the genes includinghousekeeping control gene transcripts were either designed usingPrimer Express 5.1 (Applied Biosystems Inc., Foster City, CA)(Table 1) or referenced from Sugimoto et al. (54). Chromatinimmunoprecipitation (ChIP) analysis was performed accordingto a standard protocol provided by Upstate Biotechnology, Inc.,with minor modifications (14, 22). PCR amplifications were doneusing a Takara ExTaq Kit and oligonucleotide primers listedin Table 1. PCR amplification was carried out for a region notcontaining any AP-1 site within the SCF promoter to serve asa control (negative) for sonication.

For ELISA, cells were seeded at 80% of confluence, and the growthmedium was changed 24 h later to basal medium containing 0.1%bovine serum albumin after samples were washed with PBS. Forty-eighthours later, the conditioned medium was collected, and supernatantwas obtained by centrifugation at 2,000 rpm for 5 min, followedby filtration through a 0.45-µm-pore-size membrane filter.SCF in the conditioned medium was measured using a mouse SCFELISA kit (Raybiotech, Narcross, GA) in triplicate, as per themanufacturer's recommendations, and normalized by the totalprotein levels in the medium of each individual sample. TheTGF-ß and EGF ELISA assays were conducted using amouse TGF-ß Quantikine ELISA Kit (R&D Systems,Inc., Minneapolis, MN) (11) and a Mouse EGF Quantikine ELISAkit (R&D Systems, Inc., Minneapolis, MN) (8), respectively.Experiments were conducted at least three separate times.

Measurement of ROCK activation. Rho-associated protein kinase (ROCK) activation was assessedby a CycLex Rho kinase assay kit (Cyclex Co., Ltd., Nagano,Japan) which uses the myosin-binding subunit of myosin phosphataseas a substrate (59). To exclude the activity of other kinasesfrom the results, the absorbance values obtained from ROCK inhibitor-treatedlysates were subtracted from total absorbance.

RESULTS

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Results

Endogenous c-Jun inhibits cellular adhesion. In order to examine the role of c-Jun in guided migration, conditionalknockout mice in which the c-jun gene is flanked by loxP siteswere used. MEFs derived from these mice (c-jun^f/f) were cultured(60) and transduced with either adenovirus-expressing Cre oran equal titer of adenovirus-null virus. Analyses were conductedwithin 48 h of transduction with Ad-Cre. PCR and RT-PCR confirmedthe deletion of c-jun in DNA and the presence of expressed CremRNA transcripts in cellular RNA. Equal amounts of amplificationof RPL-19 in DNA and RPL-19 transcripts in RNA served as housekeepinggene controls for the PCR and RT-PCR amplification reactions(Fig. 1A and B). Expression of Cre recombinase resulted in deletionof c-Jun and loss of c-Jun protein by Western blot analysiswhen normalized to the protein loading control GDI (Fig. 1C).No inhibitory effect of either the Ad-Cre or Ad-null virus onproliferation of cells was observed, consistent with recentstudies (45). To determine whether c-Jun regulates cellulardiameter in suspension, the diameters of cells were assessedusing a Multisizer Z3 (Beckman Coulter Inc, Miami, FL). Celldiameters in suspension were identical for the c-jun^+/+ andc-jun^–/^– cells (Fig. 1D), but c-jun^–/^–cells adhered to tissue culture plates showed greater cellularspreading by phase-contrast microscopy (Fig. 1E); this observationwarrants further analysis.

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FIG. 1. Somatic excision of c-jun in mouse embryo fibroblasts induces a spread cellular morphology. (A) Schematic outline of the genomic wild-type (c-jun), floxed (c-jun^f/f), and deleted c-jun (c-jun) locus with loxP sites ( ${blacktriangleleft}$ ) and PCR primer binding sites ( $->$ ). (B) PCR showing excision of c-jun^f/f alleles (I). Lanes 1 to 4, wild-type c-jun^+/+; lanes 5 to 8, c-jun^f/f DNA. All sample lanes were treated with Ad-Cre at various MOIs of virus as indicated. RT-PCR from the RNA shows expression of Cre in Ad-Cre treated fibroblast cells (II). (C) Western analysis of (c-jun^f/f) cells with no virus, Ad-Null-, and Ad-Cre-treated (MOI of 20) cells analyzed for the presence of c-Jun protein in the lysates; GDI was used as loading control. (D) Cell diameter measurements (in suspension) of Ad-Null- and Ad-Cre-treated c-jun^f/f cells determined on cells in suspension. (E) Spread morphology of Ad-Null- and Ad-Cre-treated c-jun^f/f cells growing in culture adhering to the tissue culture plate.

To examine the morphology of cells deleted of c-Jun, scanningelectron microscopy was conducted (Fig. 2A). Fibroblasts demonstratedan elongated bipolar fibroblastoid shape, either in the absenceof adenovirus or in the presence of a control adenovirus. Theaddition of adenovirus-expressing Cre resulted in both c-junexcision and a change in cellular morphology. Cells deletedof c-jun were rounder and flatter (Fig. 2A). Stress fiber formationassessed by phalloidin (Fig. 2B) confirmed the spread morphologyand demonstrated a peripheral distribution of stress fibersin c-jun^–/– cells (Fig. 2B). In order to examinewhether the round morphology of c-jun^–/^– cells correlatedwith increased cellular adhesion, assays were conducted on distinctsubstrata (Fig. 2C). Adhesion analyses were conducted at severaltime points. In order to determine the effect of substratumadhesion, a comparison was made between c-jun^–/^–and c-jun^+/+ cells at 30 and 240 min. c-jun^–/^– cellswere more adherent than c-jun^+/+ cells at 240 min. For easeof comparison of the effect of the substratum upon the increasein adhesion, at initial contact (30 min) was normalized to 100%.A relative increase in adhesion was observed in c-jun^+/+ versusc-jun^–/^– for cells plated on laminin and vitronectin(Fig. 2C and D).

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FIG. 2. c-Jun regulates cellular spreading and adhesion. (A) Scanning electron micrographs of c-jun^f/f cells treated with either no virus, Ad-Null, or Ad-Cre. Note the Ad-Cre-treated c-jun^f/f cells show a flattened appearance after c-jun excision. (B) Rhodamine-phalloidin staining for F-actin of untreated, Ad-Null, and Ad-Cre-treated c-jun^f/f cells. Peripheral staining of F-actin is observed in Ad-Cre-treated cells (deleted of c-jun). Note that c-jun^–/^– cells show greater spreading in comparison to c-jun^+/+ cells (untreated or Ad-Null-treated cells). Magnification, x600 (60x objective and 10x eyepiece). (C) Cellular adhesion assay comparing c-jun^f/f MEFs treated with either Ad-Null or Ad-Cre. Cells were plated on distinct substrata as indicated and analysis was conducted at six time points (30 min to 240 min). The data are shown as mean ± standard deviations of three separate experiments. (D) The substratum-induced adhesion is shown as difference in adhesion at 4 h and 30 min in Ad-Cre- versus Ad-Null-treated c-jun^f/f cells.

c-Jun enhances cellular migratory velocity. The speed of wound closure represents the combination of severalfactors including migratory velocity and persistence of migratorydirectionality (18). Video microscopy was undertaken of woundclosure (Fig. 3A and B) to determine a detailed analysis ofthe cellular velocity of c-Jun-deficient cells (Fig. 3C). Woundclosure was delayed in c-jun^–/^– cells (Fig. 3B).The c-jun^–/^– cells showed reduced ( $~$ 45%) cellularvelocity (Fig. 3C) (P < 0.05).

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FIG. 3. c-jun excision reduces cellular migration rates. Wound healing assay of untreated, Ad-Null-, or Ad-Cre-treated c-jun^f/f cells. (A) Cells were imaged every 15 min by video microscope for 24 h. Representative images at 0, 12, and 24 h are shown. c-jun^–/^– cells showed reduced wound closure at 24 h. Graphs show wound size (B), migration velocity (C), and PMD (D) at 0, 12, and 24 h. D/T calculations were based on data collected after analysis of video images for cell movement every 15 min for 3 h. Data are shown as means ± standard error of the means for more than three separate experiments. *, P < 0.05.

The relatively greater defect in wound closure rate comparedwith the reduction in cellular velocity of c-jun^–/^–cells suggested that a defect in directional persistence maycontribute to defective wound closure. Analysis was thereforeconducted of the persistence of migratory directionality (PMD).Representative examples of cell movement plated on fibronectin-coatedslides tracked at 15-min intervals over 3 h were used to determinethe role for c-Jun in persistence of migratory directionality(Fig. 3D). The quantitation of PMD was undertaken using a relativeD/T ratio, representing the ratio of direct distance from thestart point to the endpoint divided by the total track distance(18). c-Jun-deficient cells showed a 66% reduction in persistenceof directionality (Fig. 3D).

To determine whether c-Jun was sufficient to reverse the defectin cellular migration, c-jun^–/^– MEFs were transducedwith a retroviral expression vector encoding c-Jun. c-jun^f/fcells were transduced with Ad-Cre or Ad-control and then sequentiallytransduced with a retrovirus expression vector encoding IRES-GFPor c-Jun-IRES-GFP. Reanalysis was conducted 48 h posttransductionwith the retrovirus encoding c-Jun. c-Jun expression reversedthe defect in cellular velocity and migratory directionality(Fig. 4).

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FIG. 4. c-Jun rescues the migration defect of c- jun^–/^– cells. c-jun^f/f cells transduced with Ad-Cre to excise c-jun^f/f 48 h postexcision were retransduced with retrovirus expressing either GFP or c-Jun. Video microscopy demonstrated induction of migration by c-Jun expression and rescue of cellular migration in c-jun^–/^– cells. (A) Path tracings of cellular movements. (B) Quantitation of cellular velocity. (C) Persistence of directionality of migration. EB, error bar; SEM, standard error of the mean.

The effect of c-Jun on the cellular area of adherent cells wasnext assessed. Deletion of c-jun increased the cellular area(Fig. 5A and C). Reintroduction of c-jun by viral transduction,reduced the cellular area compared to that of wild-type controlcells (Fig. 5A and C). Studies were undertaken to determinewhether c-Jun was capable of rescuing the defect in cellularmigration, or whether the defect in migration was being maintainedby secondary effects, independent of c-Jun. Reintroduction ofc-jun into the c-jun^–/^– cells also rescued the abundanceof c-Jun protein, which was very similar to c-jun^+/+ cells,as assessed by Western blot analysis (Fig. 5B). Compared withc-jun^+/+ cells, c-jun^–/^– cells displayed a threefoldincrease in cellular area. Reintroduction of c-Jun into c-jun^–/^–cells restored the cellular area to that of c-jun^+/⁺ cells (Fig.5C).

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FIG. 5. c-jun rescues cellular morphology of c-jun^–/^– cells. IRES-GFP- or c-Jun-IRES-GFP-expressing MSCV vectors were transfected into untreated, Ad-Null-, or Ad-Cre-treated c-jun^f/f cells. (A) Fluorescence microscopy of c-jun^–/^– (c) versus cells rescued with c-jun (f). (B) Western blot analysis of the MEFs either deleted of c-jun or rescued with c-jun. GFP is expressed by the MSCV vectors used. (C) Cellular area determined using NIH Image software. EB, error bar; SEM, standard error of the mean.

SCF is secreted in response to endogenous c-Jun and promotes cellular migration. Earlier coculture experiments of human keratinocytes and c-jun^–/^–fibroblasts identified IL-1 as a c-Jun target gene regulatingkeratinocyte differentiation through paracrine secretion (56).To consider the possibility that c-Jun may regulate the secretionof paracrine factors, which in turn contribute to fibroblastmigration, conditioned medium from c-jun^+/+ cells was addedto the c-jun^–/^– cells. The conditioned medium ofc-jun^+/+ cells rescued the migratory characteristics of c-jun^–/^–cells including cellular migration velocity and PMD (Fig. 6A).The medium from c-jun^+/+ cells restored the defective PMD ofc-jun^–/^– cells, indicating a key role for secretedfactors in defective migration of c-jun^–/^– cells(Fig. 6). Neither IL-1 nor tumor necrosis factor alpha rescuedthe PMD defect of c-jun^–/^– cells (data not shown).

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FIG. 6. Conditioned medium from c-jun^+/+ cells rescues the defective migratory response of c-jun^–/^– cells. (A) Path tracings from PMD assays conducted on untreated (a), Ad-Null (b), Ad-Cre (c), and Ad-Cre c-jun^f/f cells treated with conditioned medium (d) from c-jun^+/+ cells. Treatment of Ad-Cre-infected cells with conditioned tissue culture medium (CM) from wild-type cells rescued their defective cellular migration (cellular velocity) (B) and persistence of direction (C) (D/T ratio). EB, error bar; SEM, standard error of the mean.

In view of the ability of conditioned medium from c-jun^+/+ cellsto rescue the defective migration of c-jun^–/^– cells,an unbiased proteomic analysis was conducted to screen for additionalcandidate cytokines and growth factors contributing to cellularmigration. A cytokine and growth factor array approach was used,conducting subtractive comparison of medium from c-jun^+/⁺ andc-jun^–/^– cells. Multiplicative quantitative studiesdemonstrated that the abundance of most proteins was unalteredby c-Jun deletion (Fig. 7A; see Fig. S1 in the supplementalmaterial). The abundance of SCF was, however, reduced by 35%(Fig. 7A). In order to determine the mechanisms by which c-Junregulated SCF abundance, an ELISA was conducted for secretedSCF in conditioned medium (Fig. 7B). c-jun deletion reducedSCF protein abundance from 500 pg/ml to 200 pg/ml (Fig. 7B).The mRNA levels of the secreted (KL-1) and membrane-bound (KL-2)forms of SCF were determined by real-time quantitative RT-PCRfrom c-jun^f/f cells treated with Ad-Null and Ad-Cre (Fig. 7C).The relative quantitation data obtained from real-time quantitativeRT-PCR shown in the Fig. 7C was normalized to expression ofthe housekeeping gene 18S rRNA using ABI SDS 2.1 software. SCFmRNA levels were reduced more than 80% in c-jun^–/^–cells compared with the wild-type control (Fig. 7C). Severalother known c-Jun target genes assessed as a form of positivecontrol also showed reduced expression in c-jun^–/^–cells, including twist homolog 1, twist homolog 2, and twistneighbor (Fig. 7D).

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FIG. 7. c-jun induces SCF secretion and expression. (A) The supernatant of c-jun^+/+ and c-jun^–/^– cells was analyzed by cytokine and growth factor arrays. Supernatant from c-jun^+/+ cells that rescued a c-jun^–/^– cellular migration defect was assessed. Representative data of three separate experiments are shown. Mean data of the SCF abundance are shown (see Fig. S1 in the supplemental material for names of the proteins assayed in the array). (B) ELISA for SCF abundance in conditioned medium from c-jun^f/f cells treated with no virus, Ad-Null, and Ad-Cre. (C) Real-time RT-PCR quantification of c-jun, SCF (KL-1, soluble form; KL-2, membrane-bound form) transcripts of KL gene for SCF. (D) Real-time RT-PCR quantitation of known c-jun target genes (twist 1, twist 2, and twist neighbor). In all real-time quantitative RT-PCR assays, the data for the gene of interest were normalized to the expression of transcripts for the 18S rRNA housekeeping control gene.

To determine whether the reduction in SCF abundance in c-jun^–/^–cells contributed to the defect in cellular migration, c-jun^–^/^–cells were treated with SCF. The addition of 0.5 ng/ml SCF completelyreversed the defect in cellular migration (Fig. 8A) velocityand directionality (Fig. 8 B and C, bars 7 versus 8). Immunoneutralizingantibody to SCF was used to examine its ability to inhibit migrationof c-jun^+/+ cells. SCF immunoneutralization reduced cellularvelocity and persistence of migratory directionality by 40%(Fig. 8B and C, bars 4 versus 6). Collectively, these studiessuggest that the reduced SCF production in c-jun^–/^–contributes to the defect in velocity and directionality ofcellular migration. To determine whether the increased cellularadhesion observed in c-jun^–/^– cells (Fig. 2) wasa function of defective SCF secretion, adhesion assays wereconducted. Addition of SCF reduced cellular adhesion of c-jun^–/^–cells particularly when cells were plated on laminin or vitronectin(Fig. 8D).

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FIG. 8. SCF rescues migration defect of c-jun^–/^– cells. (A) Path tracings for c-jun^f/f cells treated with Ad-Cre and either an immunoneutralizing antibody to SCF (IN- ${alpha}$ SCF) (1 µg/ml) or the addition of SCF (0.5 ng/ml). Data for more than three experiments are shown. Data for quantitation of cell migration parameters, including velocity of migration (B) and the D/T ratio to assess persistence of migratory directionality (C) are shown. (D) Cellular adhesion assays were conducted as described in the legend of Fig. 2, with the addition of SCF (0.5 ng/ml) on various matrices (collagen I, laminin, and vitronectin). Substratum adhesion is shown as the difference between 4 h and 30 min. EB, error bar; SEM, standard error of the mean.

Transmigration or migration across a cellular membrane was nextassessed using a Boyden chamber. c-jun^–/^– cellswere defective in transmigration compared with c-jun^+/+ cells(Fig. 9A). The addition of SCF (0.5 ng/ml) promoted transmigrationof c-jun^–/^– cells and fully rescued the defect intransmigration, while immunoneutralizing antibody to SCF reducedmigration of c-jun^+/+ cells by 40% (Fig. 9B). The restorationof c-jun^–/^– cellular transmigration by SCF is consistentwith a role of reduced SCF production leading to a migrationdefect. In order to determine the role of c-jun in cellularinvasion, the ability of cells to invade a collagen matrix inthree dimensions was assessed (44). The invasive phenotype displayedby cells negotiating three-dimensional extracellular matrixbarriers is a more complex form of cellular behavior and maymore closely resemble the in vivo function of c-Jun. Invasionof collagen was assessed by the detection of refractile cellsdistal to the cellular margin after 5 days. Deletion of c-junvirtually abolished the ability of cells to invade the collagenin a three-dimensional matrix (Fig. 10B). Addition of SCF (0.5ng/ml) rescued the invasive phenotype of c-jun-deficient cells.Immunoneutralizing antibody to SCF reduced the number of cellsinvading collagen by 23% (Fig. 10E versus F).

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FIG. 9. SCF rescues the transwell cellular migration defect of c-jun^–/^– cells. c-jun^f/f cells were treated with Ad-Cre and analyzed for transwell migration. Cells traversing the membrane were stained blue with crystal violet and appear lighter in the images. The effect of immunoneutralizing antibody to SCF or addition of SCF (0.5 ng/ml) on transwell migration was quantitated for more than five separate experiments. (B) Data are shown as the means ± standard deviations (SD). *, P < 0.05. IgG, immunoglobulin G; IN- ${alpha}$ SCF, immunoneutralizing antibody to SCF; EB, error bar.

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FIG. 10. SCF rescues three-dimensional collagen invasion defect of c-jun^–/^– cells. c-jun^f/f cells were treated with Ad-Null or Ad-Cre and analyzed for invasion within three-dimensional collagen gels with or without SCF (0.5 ng/ml), immunoglobulin G (IgG), or immunoneutralizing antibody against SCF (IN- ${alpha}$ SCF). The edge of the embedded island of fibroblasts is marked by a dashed line on each panel. Invasion was quantitated in three-dimensional collagen gels after 5 days in the presence of SCF or following the addition of immunoneutralizing antibodies against SCF. EB, error bar; SD, standard deviation.

SCF is a direct target gene induced by c-Jun. To determine whether the SCF gene was a direct transcriptionaltarget of c-Jun, a 2.0-kb fragment of the murine SCF promoterwas cloned and linked to a luciferase reporter gene. Sequencingof the SCF promoter demonstrated the presence of a canonicalAP-1 binding site(s) (Fig. 11). The activity of the murine SCFpromoter was reduced by 80% in c-jun^–/^– cells whennormalized for transfection efficiency using the Renilla luciferasereporter gene, pRL-LUC (Fig. 11A). Transfection of c-jun^–/^–cellswith an expression vector for c-Jun induced SCF-Luc reporteractivity sixfold compared with the effect of expression of aDNA-binding-defective mutant of c-Jun (c-Jun DNA^–) (Fig.11B). In order to determine whether the SCF promoter functionedas direct transcriptional target, deletion mutation of the SCFpromoter AP-1 sites was conducted. Comparison was made betweenthe SCF-wt and SCF-AP-1 mutant promoters. c-Jun induced theSCF promoter but failed to induce the SCF-AP-1 mutant promoter(Fig. 11C). The SCF promoter was 5- to 10-fold more active thanthe luciferase vector control in the presence of c-jun whilethe SCF-AP-1 mutant reporter conveyed basal activity to theluciferase vector under identical conditions. The finding suggeststhat the AP-1 sites function as both basal level and c-Jun-responsiveenhancer elements.

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FIG.11. c-jun induces SCF expression and binds to SCF promoter in ChIP assays. Schematic representation of murine SCF gene promoter linked to luciferase reporter gene and luciferase assay for cloned SCF promoter in c-jun^+/+ and c-jun^–/^– cells, effect of expression vectors coding wild-type c-Jun or DNA-binding-defective c-Jun mutant. As shown in panel A, the activity of the SCF promoter was assessed in c-jun^+/+ or c-jun^–/^– cells, and the luciferase activity was normalized for transfection efficiency through cotransfection with the Renilla luciferase reporter (psv-Renilla-LUC). (B) Activity of the SCF promoter was determined in the presence of cotransfected expression vector for either wild-type c-Jun or the DNA-binding-defective c-Jun mutant. Luciferase reporter activity is shown of transfections conducted in c-jun^–/^– cells. Data are means ± standard error of the mean of more than five separate transfections. (C) The SCF promoter AP-1 elements were mutated in the context of the full-length promoter. Activities of the SCF wild-type (WT) and SCF-AP-1 mutant promoters were compared in the presence of cotransfected the c-Jun expression vector. (D) ChIP assay showing recruitment of c-jun to the AP-1 sites in the SCF promoter. (E) Schematic representation of mechanism by which c-jun induction of SCF promotes guided migration.

To determine whether c-Jun bound the SCF promoter in the contextof local chromatin, ChIP assays were conducted. Oligonucleotideprimers designed to the proximal SCF promoter (designated "negative"in Fig. 11D) failed to amplify genomic SCF sequence in the presenceof the c-Jun immunoprecipitate, indicating the efficiency ofsonication. Amplification of SCF promoter sequences was observedwith primers directed to the AP-1 site and three other AP-1like elements in the SCF promoter. ChIP assays conducted onc-jun^f/f cells treated with Ad-Cre abrogated c-Jun occupancyat the SCF promoter AP-1 site (Fig. 11D). Thus, c-Jun inducesSCF protein and mRNA abundance and c-Jun induces the SCF promoterthrough a DNA-binding-dependent mechanism.

DISCUSSION

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Discussion

The current studies demonstrate an essential role for c-Junin regulating the velocity of cellular migration and directionalpersistence. Wound healing is known to induce the expressionof AP-1 transcription factors (16). c-Jun together with Jun-B,c-Fos, and FosB is induced within 1 h of wounding of cornealepithelium (50). It has been proposed that AP-1-dependent activationof a subset of genes including keratins and integrins, in turn,contributes to the epithelial cell migration response to wounding(4, 5). The AP-1 transcription factors induce production ofmatrix metalloproteases and plasminogen activator, providinga mechanism by which cells move into the extracellular matrix.Herein, video microscopy demonstrated a reduction in the migratoryvelocity of cells acutely deleted of c-jun. The defect in migratorypersistence of c-jun^–/^– cells was rescued throughsupernatant derived from fibroblasts expressing c-Jun or withthe addition of SCF, consistent with a role for c-Jun in regulatingautocrine secretion of factors that promote cellular migration.c-jun deletion reduced SCF protein abundance, mRNA, and promoteractivity. c-Jun expression induced the SCF promoter and boundthe SCF promoter's AP-1 sites in ChIP assays. These findingsdefine a new role for c-jun in regulating SCF expression and,thereby, cellular invasion.

SCF induced migration of c-jun^–/^– cells, suggestingthat the receptor, Kit, maintains normal function in the absenceof c-Jun and confirming the biological importance of SCF asa c-Jun target gene. In addition to serving as a growth factorfor various cell types, including hematopoietic stem cells,mast cells, melanocytes, and germ cells, SCF has chemotacticproperties for endothelial cells, functioning as a proangiogenicfactor (55). Complete absence of SCF, which is encoded by themouse steel locus (S1), or Kit kinase, leads to embryonic orperinatal lethality from anemia. In humans, gain-of-functionmutations of c-kit occur in human cancers (gastrointestinalstromal tumors, mystocytomas, T-cell lymphomas, and dysgerminomas[19]), and paracrine or autocrine activation of kit has beenimplicated in other human malignancies including ovarian andlung cancers (25). The correlation between Kit expression andfunctional measurements of pluripotentiality suggests that kitmay be a useful marker for stem cells (38). SCF expression isupregulated in tumors in a grade-dependent manner, and highSCF expression correlates with short patient survival (55).Tumor cells with the strongest SCF expression are found withinthe tumor-infiltrating border, and c-jun is expressed predominantlyat the infiltrating tumor edge (63). Collectively, these studiesare consistent with a model in which c-Jun-mediated SCF productionmay contribute to tumor progression.

Both TGF-ß and EGF have been implicated in cellularmigration during Drosophila development (72). Neither TGF-ßor EGF were represented on the arrays used. TGF-ßand EGF production was reduced in c-jun^–/^– MEFs,as determined by ELISA, approximately 25% and 60%, respectively(see Fig. S1B in the supplemental material). We examined thepossibility that TGF-ß or EGF may also promote migrationof c-jun^–/^– cells. Although addition of supraphysiologicalconcentrations of EGF (10 ng/ml) and TGF-ß (10 ng/ml)rescued in part the PMD defect of c-jun^–/^– cells,physiological concentrations (EGF at 20 pg/ml and TGF-ßat 100 pg/ml) did not rescue the migration defect (see Fig.S1C to E in the supplemental material). These findings indicatethat in cells acutely deleted of c-jun, the receptor signalingpathway for TGF-ß and EGF remains intact. Furthermore,these studies suggest that supraphysiological or pathologicalbut not physiological concentrations of EGF and TGF-ßmay play a role in c-jun-mediated migration. TGF-ßis a known target of c-Jun in mammalian cells (62). The currentfindings are consistent with previous studies in Drosophilamelanogaster, in which a key role for c-jun kinase in cellularmigration is evidenced by dorsal closure. Mutations of hemipterousand basket, the Drosophila homologs of human JNKK and JNK, respectively,are associated with failed closure of the dorsal cuticle. DrosophilaJNK is activated transiently immediately prior to dorsal closure,facilitating amnio serosa contraction in adhesion complexesin adjacent cells (37). It has been proposed this activity maycontribute to the anterior movement of dorsal epithelium. DrosophilaJNK activity persists in leading-edge epithelial cells to promoteongoing expression of the TGF-ß homolog decapentaplegia(52). decapentaplegia is known to promote cellular migrationin an autocrine fashion at the epithelial cell edge (17). Thefindings herein that the c-jun^–/^– cellular migratorydefect is rescued by supraphysiological concentrations of TGF-ßsuggests that the loss of TGF-ß production in c-jun^–/^–cells may contribute to defective migration in murine fibroblastsunder a subset of circumstances.

Mouse embryonic eyelid closure requires both the proliferationof epithelial cells and the migration of epithelial sheets (51,57). Defective eyelid closure has been demonstrated in micedefective in genes encoding growth factors and their receptors,including EGF receptor (EGFR), TGF- ${alpha}$ , activinßB, andMEKK1. The ablation of genes encoding activinßB, theTGF-ß family, and ROCK I/II results in defective eyelidclosure (58, 61). Thus, both the TGF-ß activin, MEKK1,and JNK pathway and the TGF- ${alpha}$ /EGF ${alpha}$ and ERK pathway contributeto active stress fiber formation and cellular migration. Thedestruction of either pathway in vivo appears to result in thefailure of eyelid closure in mice. As the rescue of PMD in c-jun^–/^–cells with EGF and TGF-ß occurred acutely, the receptorsand corresponding signaling pathway of these cells remainedfunctional.

Our results contrast with studies of more prolonged c-jun excisionin keratinocytes of these mice. K14-Cre; K5-Cre-2; c-jun^EP miceresulted in a failure of eyelid fusion during embryogenesis,and mice were born with the "eyes open" phenotype (30, 71).c-jun-deficient keratinocytes expressed less phosphorylatedEGFR (71), suggesting that a reduction in EGFR function mayhave contributed to defective migration. These differences mayeither relate to a cell-type-specific function of c-jun or reflectacute (this study) versus chronic (30, 71) effects of c-Jundeletion. Acute deletion of the retinoblastoma (Rb) gene inprimary quiescent cells is sufficient for cell cycle reentryand has a phenotypic consequence that is different from germline Rb functional inactivation (45). Such differences may bedue to functional compensation by Rb-related proteins. Somaticexcision of c-jun in the central nervous system blocks perineuronalsprouting, lymphocyte recruitment, and microglial activation.c-jun-deficient motor neurons show reduced target muscle reinervation(41). c-jun-deficient neurons expressed reduced galanin, ${alpha}$ 7ß1,and CD44, the expression of which contributes to normal neuronalregeneration (41). It will be of interest to assess furtherthe paracrine secretory deficiencies of c-jun^–/^–keratinocytes to better understand the cell-type-specific functionof c-jun in cellular migration.

Kit activation induces multiple downstream signaling pathwaysincluding JNK, ERK, Akt, and PI3-kinase. In the current studiesmultiple intracellular kinase signaling pathways contributedto the migration of wild-type MEFs including the PI3-kinase,ERK, JNK, and MAP kinase pathways (see Fig. S2 in the supplementalmaterial), consistent with previous studies implicating thesekinases in fibroblast migration (20, 21). The PI3-kinase pathwayhas been implicated in the cellular migratory response to growthfactor signals including EGF (46, 48). MAP kinase activity isknown to accelerate the immediate cell motility in some, butnot all, cells (9, 24). The role of candidate intracellularsignaling pathways was determined. Specific inhibitors for eachof the MAP kinase pathways and PI3-kinase activity were usedat the dose shown in many prior studies to inhibit the relevantpathway (6) without directly affecting viability (cellular viabilitywas confirmed in the current studies in the presence of inhibitor).The Jun kinase inhibitor SP600125 reduced basal PMD of the cells(see Fig. S2A and C in the supplemental material). This concentrationof SP600125 is consistent with the dose used in previous studies(6) and did not affect cell viability. The PI3-kinase inhibitorLY 294002 did not affect PMD but blocked Ras activation of amultimeric AP-1 reporter gene in our previous studies (3). Boththe p38 and ERK inhibitors (SB203580 and PD98059) reduced basallevel PMD. The ROCK inhibitor Y27632 did not affect wild-typeMEF migration but enhanced c-jun^–/^– MEF migration.A similar rescue of migration was observed with specific ROCKinhibitors, H-1152 and H-1100 (see Fig. S3A to D in the supplementalmaterial). The mechanism by which c-jun deletion and reductionin SCF leads to altered responsiveness to ROCK inhibitors remainsto be determined.

Imatinib (Gleevec) is a small-molecule inhibitor of signalingby SCF through c-Kit, in addition to blocking Abl and platelet-derivedgrowth factor signaling. As SCF promotes tumor cell proliferationand angiogenesis and c-Jun is frequently overexpressed in abroad array of human tumors, SCF inhibitors may thus provideadjunctive value in c-jun-expressing tumors. Given the importantrole of c-Jun as a bona fide oncogene (13, 23) and the commonfinding of c-Jun overexpression in tumors, it will be importantto assess further the relative importance of SCF secretion mediatedby c-Jun in regulating tumor metastases.

ACKNOWLEDGMENTS

We thank M. Karin for plasmids and helpful advice. Figure 11Ewas designed by Chenguang Wang.

This work was supported in part by R01CA70896, R01CA75503, R01CA86072,R01CA86071 (R.G.P.). The Kimmel Cancer Center was supportedby an NIH Cancer Center Core grant P30CA56036 (R.G.P). Thisproject is funded in part from the Ralph and Marian C. FalkMedical Research Trust and a grant from Pennsylvania Departmentof Health (R.G.P.).

The Pennsylvania Department of Health specifically disclaimsresponsibility for analyses, interpretations, or conclusions.

FOOTNOTES

* Corresponding author. Mailing address: The Kimmel Cancer Center, Departments of Cancer Biology and Medical Oncology, Thomas Jefferson University, 233 South 10th St., Philadelphia, PA 19107. Phone: (215) 503-5649. Fax: (215) 503-9334. E-mail: Richard.Pestell{at}jefferson.edu.

Published ahead of print on 4 December 2006.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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