3922 Research Article

IntroductionuPAR, the receptor for urokinase (uPA), allows cell-surfaceconversion of plasminogen to plasmin, thereby increasingpericellular proteolysis and extracellular matrix (ECM) degradation,which are important for cell migration and tissue remodeling (Blasiand Carmeliet, 2002).

In addition to regulating proteolysis, uPAR directly modulates celladhesion, differentiation, apoptosis, proliferation and migrationthrough non-proteolytic mechanisms (Alfano et al., 2006; Blasi andCarmeliet, 2002). Because uPAR lacks a cytosolic domain, it musttransmit intracellular signals through transmembrane proteins,including integrins, epidermal growth factor (EGF) receptor (EGFR),p130Cas (also known as BCAR1) and G-protein coupled receptors(reviewed by Blasi and Carmeliet, 2002). Moreover, uPAR directlyand specifically binds to the ECM protein vitronectin (VN), and thisinteraction is enhanced by uPA. Mutants of uPAR that are impairedin their binding of vitronectin (but not uPA) lose the ability to modifycell shape and to induce cell migration (Madsen et al., 2007).

Deletion of the uPAR gene in mice causes impairment ofneutrophil recruitment, abnormal migration and adhesion ofperitoneal macrophages (Gyetko et al., 1995; Gyetko et al., 1994;Simon et al., 2000), alteration of kidney membrane permeability,protection from proteinuria, accelerated renal fibrosis in obstructivenephropathy (Wei et al., 2007; Zhang et al., 2003), and bone-homeostasis impairment (Furlan et al., 2007). uPAR-knockout (KO)mice are also deficient in their hematopoietic stem-cell homeostasis(Marc Tjwa and Peter Carmeliet, University of Leuven, Belgium,unpublished).

High levels of uPAR in human cancers is an independentnegative prognostic marker (Stephens et al., 1999). The role of uPARin tumors is still not understood, nor is it understood whether andhow uPAR regulates cell growth in vivo. In cancer cell lines, uPARregulates cell proliferation, activating growth-promoting pathwaysthrough integrins and the EGFR (Aguirre Ghiso et al., 1999; Liuet al., 2002). Fast-growing and highly metastatic Hep3 epidermoidcarcinoma cells produce large amounts of uPAR. Silencing of uPARin Hep3 cells induces a dormancy state in vivo, which is reversedby restoring the uPAR levels (Yu et al., 1997). In these cells, uPARoverexpression induces constitutive EGFR signaling, which requiresuPAR–integrin-α5β1 interaction (Aguirre Ghiso et al., 1999; Liuet al., 2002). The EGFR might also represent the key moleculelinking uPAR to extracellular-signal-regulated kinase (ERK)activation (Jo et al., 2003; Repertinger et al., 2004).

During cutaneous wound healing, growth factors (includingEGF), cytokines and chemokines coordinate several processes, suchas inflammation, proliferation, migration and angiogenesis, whichare all required for appropriate tissue remodeling at the injury site(see Gillitzer and Goebeler, 2001; Werner and Grose, 2003).Resting keratinocytes adjacent to the wound margins are activatedand show increased proliferation, dissolution of cell-cell adhesions,detachment from the basement membrane, lateral migration andinvasion of the wounded area (Martin, 1997; Singer and Clark,1999).

We show that, in vivo, uPAR-KO mice have a delayed wound-healing response, and decreased keratinocytes proliferation andmigration. In vitro, primary keratinocytes showed deficiencies in

The urokinase receptor (uPAR) is involved in a series ofpathological processes, from inflammation to cancer. We haveanalyzed in detail the role of uPAR and the mechanismsinvolved in keratinocyte behavior during wound healing byexploiting uPAR-knockout (KO) mice. In vivo, uPAR-KO miceshowed delayed wound healing, with abnormal keratinocytemigration and proliferation. In vitro, unlike wild-type cells,primary uPAR-KO keratinocytes did not proliferate in responseto epidermal growth factor (EGF), their growth and migrationwere not inhibited by EGF-receptor (EGFR) inhibitors, and theydid not adhere to uncoated surfaces. Whereas EGFR levels inuPAR-KO keratinocytes were normal, there was no tyrosinephosphorylation upon addition of EGF, and its downstreamtargets, extracellular-signal-regulatedkinases 1 and 2 (ERK1/2),

were not activated. Re-introduction of mouse uPAR rescued allphenotypes. In vitro adhesion and migration defects wereassociated with the failure of uPAR-KO keratinocytes tonormally produce and secrete laminin-5 (LN5), an event thatrequires EGFR signaling. These results were confirmed in vivo,with LN5 being upregulated during wound healing in wild-typebut not in uPAR-KO epidermis.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/121/23/3922/DC1

Key words: EGF-receptor, Laminin-5, Keratinocytes, uPAR, Woundhealing

Summary

uPAR-deficient mouse keratinocytes fail to produceEGFR-dependent laminin-5, affecting migration in vivoand in vitroSilvia D’Alessio1, Laura Gerasi1 and Francesco Blasi2,*1Università Vita Salute San Raffaele and Istituto Scientifico H San Raffaele, via Olgettina 60, 20132 Milano, Italy2IFOM (Fondazione Istituto FIRC di Oncologia Molecolare), via Adamello 16, 20139 Milano, Italy*Author for correspondence (e-mail: blasi.francesco@hsr.it)

Accepted 11 September 2008Journal of Cell Science 121, 3922-3932 Published by The Company of Biologists 2008doi:10.1242/jcs.037549

Jour

nal o

f Cel

l Sci

ence

3923uPAR knockout and keratinocyte migration

adhesion, migration and proliferation. Moreover, in theabsence of uPAR, the EGFR and its signaling pathway werenot activated, partially explaining the proliferation defect.The deficient EGFR activation impairs laminin-5 (LN5)secretion and deposition both in vitro and in vivo, thusaffecting keratinocyte adhesion and migration.

ResultsSkin wound healing is delayed in uPAR-KO miceWe examined skin wound healing in wild-type (wt) anduPAR-KO littermates as described in the Materials andMethods. The average wound length in uPAR-KO micewas significantly increased at all time points whencompared with wt mice (supplementary material Fig. S1and Table S1). Moreover, whereas the healing time for wtmice was 11.2±1.4 days, in uPAR-KO littermates the valueincreased up to 15.9±2.1 days, representing a small butsignificant delay (P=0.00028).

Decreased keratinocytes migration and proliferation invivo justifies the uPAR-KO phenotypeFor histological analysis, uPAR-KO and wt skins werewounded and tissue samples collected 1, 3, 5 and 11 daysafter wounding. Frozen sections were immunostained forkeratin 5 and wound diameters measured at each timepoint. Fig. 1A shows immunostaining 5 days afterwounding. The arrows indicate the position of theadvancing epithelial edges and define the wound diameter.Measurements at various times after wounding showedthat closure of wound margins in uPAR-KO mice was significantlydelayed at all time points (Fig. 1B).

During wound healing, keratinocytes migrate and proliferatewithin the injury (Goldfinger et al., 1999; Martin, 1997; Parks, 1999;Pilcher et al., 1999). At 3 days after wounding in uPAR-wt mice,the epidermis at the margins of the wounds was thickened and awedge of basal keratinocytes invaded the provisional matrix of thewound bed (Fig. 2A, left panel). This hyperproliferation, however,was almost absent in uPAR-KO wounds, as judged by the numberof cell layers (Fig. 2A, right panel).

The measure of this distance and the thickness of the keratinocytelayers give an estimate of the actual distance covered bykeratinocytes and of their proliferation rate. As shown in Fig. 2A,the length of the epidermal tongue was strongly reduced in uPAR-KO wounds (202±42 μm; mean value ± s.e.m.) when comparedwith uPAR-wt wounds (592±89 μm; P<0.001). Indeed, both at 3(Fig. 2A) and 5 (Fig. 1A) days after injury, in uPAR-KO mice thetip of the epidermal tongue was still located very close to the initialwound edge, in the area just beneath the crust.

As shown in Fig. 2B, the decreased proliferation of uPAR-KOkeratinocytes was confirmed by counting the number of PCNA-positive cells 3 days after wounding, from the tip of the epidermaltongue (Fig. 2A, black arrow) to the start of the proliferatingkeratinocyte zone (Fig. 2A, blue arrow), followingimmunohistochemistry.

Similar to wt, at day 15 all uPAR-KO wounds were completelyre-epithelialized; however, the newly formed epidermis could stillbe distinguished from normal epidermis by its increased thickness,particularly that of the basal keratinocyte layer (not shown).Indeed, day-15 frozen sections that were stained with anti-keratin-5 antibody showed that, in uPAR-wt skin, the keratinocyte multi-layer was thinner than that of uPAR-KO mice, indicating that,

whereas wt cells were reacquiring their quiescent state, uPAR-KO keratinocytes were still in their active proliferative state (Fig.2B). These data indicate that, in vivo, upon skin injury, uPAR-KO keratinocyte migration and proliferation are delayed. Thesedata also indicate that uPAR is necessary for a proper wound-healing time.

The effects observed in uPAR-KO mice did not depend onabnormal acute inflammation or abnormal neoangiogenesis.Quantification of neutrophils, mast cells and macrophages byimmunohistochemistry and of microvessel density byimmunofluorescence at various time points after wounding failedto show any significant difference between wt and uPAR-KO mice(supplementary material Fig. S2a,b; Fig. S3a,b; Table S2 and S3;and data not shown).

Overall, these results indicate that the wound-healing deficiencyobserved in uPAR-KO mice is due to abnormal keratinocytebehavior and not to abnormal wound-induced inflammation orangiogenesis.

The absence of uPAR reduces keratinocytes proliferation invitroOur observations in vivo are consistent with previously publisheddata showing that uPAR mRNA is expressed by migratingkeratinocytes at the edge of wounds (Romer et al., 1994). To testthe role of uPAR in keratinocyte proliferation, primary keratinocyteswere isolated from 2-day-old wt and uPAR-KO littermates. Theabsence of uPAR in cells derived from the KO mice was confirmedby western blot analysis (not shown).

Both uPAR-KO and wt primary keratinocytes were grown in thepresence of either 1% or 8% Chelex-treated fetal calf serum (FCS)and their growth monitored every day over a 7-day period. As shownin Fig. 3A, uPAR-expressing keratinocytes grew significantly faster

Fig. 1. Skin wound healing is delayed in uPAR-KO mice. uPAR-wt and -KO skins werewounded, and cross-sections of skin samples were collected 3, 5 and 11 days later.(A) Representative wt and uPAR-KO cross-sections of 5-day-old wounds stained forkeratin 5 (K5) are shown. Arrows indicate the position of the advancing epithelial edgesand the wound diameter. Below the arrows are reported the mean wound diameters 5days after injury. (B) Average wound diameters (± s.d.) from 10 wt and 13 KO mice foreach time point (Student’s t-test).

Jour

nal o

f Cel

l Sci

ence

3924

than uPAR-KO cells in the presence of 1% FCS. The differencebetween the growth rate of wt and uPAR-KO keratinocytes waseven higher in the presence of 8% FCS (P<0.001) (Fig. 3B).

uPAR is required for EGFR and ERK1/2 activation in culturedkeratinocytesTo activate primary keratinocytes in vitro, several growth factors,such as EGF, bFGF, PDGF, TGFβ and HGF, were tested. Amongthese, only EGF differentially affected the proliferation of uPAR-wt vs uPAR-KO keratinocytes (not shown). In the presence of 1%FCS, EGF increased the growth rate of primary uPAR-wtkeratinocytes. By contrast, EGF did not promote growth of uPAR-KO cells (Fig. 3C,D). These results suggest that the mitogenicactivity of EGF on keratinocytes requires uPAR.

To support these observations, we measured the effect of aspecific inhibitor of EGFR tyrosine-kinase activity, AG1478, onthe basal and EGF-induced growth of both wt and uPAR-KOkeratinocytes. AG1478 did not alter EGFR expression (not shown),but inhibited both basal and EGF-induced proliferation of uPAR-wt keratinocytes, while having no effect on uPAR-KO cells (Fig.3C,D).

To further investigate the role of uPAR in modulating EGFactivity, we performed western blot analysis on total orimmunoprecipitated cell extracts of primary keratinocytes grownin the presence or absence of EGF. As shown in Fig. 3E, uPAR-wtand -KO cells had comparable levels of EGFR. However, uPAR-KO keratinocytes showed lower levels of both basal and EGF-induced tyrosine phosphorylation of the EGFR. The two receptorscould be co-immunoprecipitated both in the presence and absenceof EGF (Fig. 3F). This confirms previous results that showed anEGF-independent interaction between uPAR and the EGFR (Liu etal., 2002; Mazzieri et al., 2006). We therefore conclude that theabsence of uPAR does not alter EGFR expression; however, thelack of uPAR-EGFR interaction affects EGFR activationindependently of the presence of its natural ligand, EGF.

The absence of uPAR also prevented phosphorylation of ERK1and ERK2 (also known as MAPK3 and MAPK1, respectively, and

Journal of Cell Science 121 (23)

collectively referred to as ERK1/2 owing to their high homology)– downstream effectors of EGFR signaling – even in the presenceof EGF (Fig. 3G). These data suggest that uPAR is required forEGF-induced ERK1/2 activation in murine keratinocytes.

As shown in Fig. 3H, AG1478 was able to block the basal and EGF-induced activation of both EGFR and ERK1/2 in wtkeratinocytes, whereas uPAR co-immunoprecipitation remainedunaltered, meaning that uPAR-EGFR interaction is independentfrom the EGFR activity.

Similar results were obtained in the presence of a MEK1inhibitor, UO126 (data not shown). Overall, the data indicate thatuPAR interacts with the EGFR, and that this interaction modulatesEGFR activation and ERK1/2 phosphorylation, which are bothrequired for keratinocyte growth.

Murine, but not human, uPAR rescues the EGF response inuPAR-KO keratinocytesTo further demonstrate the requirement of uPAR in keratinocyteproliferation and EGFR activation, uPAR-KO keratinocytes wereinfected with either a murine or human uPAR retroviral expressionvector (Fig. 4A). An empty vector was used as a negative control.

As shown in Fig. 4B and C, overexpression of murine uPARrescued both basal and EGF-induced cell proliferation, as well asEGF-induced ERK1/2 phosphorylation (Fig. 4D). However, humanuPAR did not rescue any of the uPAR-mediated phenotypes (Fig.4D), unless cells were treated with human uPA (supplementarymaterial Fig. S4b). Because the binding of uPA to human and murineuPAR is species specific (Estreicher et al., 1989), this resultsuggests that endogenous uPA is normally required for the EGFresponse in murine keratinocytes.

uPAR is essential for keratinocyte adhesion, spreading andmigration in vitroIn the skin, the basement membrane underlying keratinocytes iscomposed of collagen IV (ColIV), LN5 and laminin-10 (LN10).The basement membrane not only supports the skin architecturebut is also required for growth, migration and differentiation of

Fig. 2. The absence of uPAR delays keratinocyte migration and proliferation in vivo. (A) uPAR-wt and -KO paraffin-embedded cross-sections of 3-day-old woundsstained with hematoxylin and eosin. The length of the epidermal tongue was measured blindly at �10 magnification in eight sections per wound in eight mice pergenotype by computer-assisted morphometry from the tip of the epidermal tongue (black arrowhead) to the start of the proliferating keratinocyte zone (bluearrowhead). (B) uPAR-wt and -KO paraffin-embedded cross-sections of 5-day-old wounds were immunostained using an anti-PCNA antibody and counterstainedwith hematoxylin. Measurements of PCNA-positive nuclei are reported. The numbers (± s.d.) refer to the average of eight wounds per genotype. (C) wt and uPAR-KO frozen cross-sections of 15-day-old wounds stained for keratin 5 (K5) are shown. Arrows indicate the thickness of the epidermis.

Jour

nal o

f Cel

l Sci

ence

3925uPAR knockout and keratinocyte migration

keratinocytes (Carter et al., 1990; Frank and Carter, 2004; Hamelerset al., 2005; Nguyen et al., 2000a; Nguyen et al., 2000b; Nguyenet al., 2000c). Moreover, migration of keratinocytes during re-epithelialization of cutaneous wounds is regulated by several ECMcomponents – including collagens, fibronectin (FN) and laminin(Kim et al., 1992; Larjava et al., 1993; MacNeil, 1994); theseproteins modulate keratinocyte adhesion, and therefore theirmigrating and proliferating capabilities.

To compare the adhesion properties of wt and uPAR-KOkeratinocytes, cells were seeded on glass coverslips that were coatedwith either ColIV, FN, VN or LN5. Cell adhesion was quantitated,as described in the Materials and Methods, 8 hours after plating.The adhesion of wt and uPAR-KO keratinocytes on differentsubstrates was not significantly different. However, an importantdifference was observed in the absence of exogenous substrates (Fig.5A). Whereas 75% of uPAR-wt cells adhered and spread, only 5-

10% of the uPAR-KO keratinocytes adhered to (but did not spreadon) a glass coverslip (Fig. 5B).

In the absence of exogenous substrates, adhesion and spreadingof keratinocytes depends on the ability of the cells to secrete anddeposit their own matrix, in particular LN5 (Frank and Carter, 2004;Hintermann and Quaranta, 2004; Nguyen et al., 2000a; Nguyen etal., 2000b; Nguyen et al., 2000c). Our data therefore suggest thatuPAR-KO keratinocytes might have a defect in the productionand/or secretion of an ECM substrate.

We also carried out a long-term in vitro wound assay withconfluent monolayers of wt and uPAR-KO keratinocytes culturedon ColIV- or LN5-coated surfaces. After scrape wounding, themigration of keratinocytes was measured at different time points.Fig. 5C shows representative examples of long-term migration onexogenous LN5. Wt cells migrated into the scraped area and closedthe wound within 24 hours. By contrast, uPAR-KO keratinocytes

Fig. 3. uPAR promotes keratinocyteproliferation in vitro and is required forEGFR and ERK1/2 activation in culturedcells. (A,B) Primary keratinocytes from 2-day-old newborn wt and uPAR-KOlittermates were grown in S-MEMcontaining 1% (A) or 8% (B) chelexedFCS, and their growth rate measured everyday over a 7-day period. Values areexpressed as absorbance at 650 nm afterstaining with 0.1% crystal violet in 200 mM MES, pH 6.0. The mean (± s.d.) oftriplicate samples is reported. (C,D) Semi-confluent primary wt (C) and uPAR-KO(D) keratinocytes were grown in S-MEM1% chelexed FCS in the presence orabsence of EGF (10 ng/ml) and/or AG1478(10 μM). The growth rate was measuredevery day over a 7-day period as describedabove. (E) The EGFR levels were evaluatedby western blotting of wt and uPAR-KOcell lysates using an anti-EGFR antibody.(F) Semi-confluent primary wt and uPAR-KO keratinocytes were serum-starved for18 hours and then stimulated with EGF (10 ng/ml) for 15 minutes. Cell lysateswere immunoprecipitated with an antibodyagainst EGFR and immunoblotted withanti-phospho-Tyr (pTyr), anti-EGFR andanti-uPAR antibodies. (G) Wt and uPAR-KO cell lysates were immunoblotted usingantibodies against phospho-ERK1/2(pERK1/2) and ERK2 (totERK). (H) Semi-confluent wt and uPAR-KO primarykeratinocytes were serum-starved for 18hours and stimulated with EGF (10 ng/ml)for 15 minutes. Cells were pre-treated for20 minutes with AG1478 (10 μM). Proteinextracts were then immunoblotted forphospho-ERK1/2 and ERK2 orimmunoprecipitated with an anti-EGFRantibody and immunoblotted for phospho-Tyr, uPAR and EGFR.

Jour

nal o

f Cel

l Sci

ence

3926

did not close the wound. Similar results were obtained on ColIV(data not shown). One possible explanation of these results, amongothers, might be the inefficient production and/or secretion of LN5.Scrape wounding might remove exogenous LN5 and therefore cellmigration could depend on newly produced and secreted LN5, whichmight be deficient in uPAR-KO cells.

Migration of wt and uPAR-KO keratinocytes towards differentexogenous substrates was also performed using the modifiedBoyden chamber assay, as described in the Materials and Methods.

Journal of Cell Science 121 (23)

uPAR-KO keratinocyte migration was significantly decreased onColIV, FN and VN, but not on exogenous LN5 (Fig. 5D). Thedifferences were statistically significant (Fig. 5D; *P≤0.051,**P≤0.0013, Student’s t-test). These results might be explainedby the fact that, even in the presence of exogenous collagen orFN, migration of keratinocytes would still require endogenousLN5, as previously described (DiPersio et al., 1997; Nguyen etal., 2000a; Nguyen et al., 2000b; Zhang and Kramer, 1996). Thesedata further support the hypothesis that uPAR-KO keratinocytes

Fig. 4. Murine uPAR rescues the EGF response inuPAR-KO keratinocytes. (A) uPAR-KO keratinocyteswere infected with either a murine or human uPAR(muPAR or huPAR, respectively) retroviral expressionvector and the expression analyzed by western blot usingan anti-uPAR antibody (kindly provided by StevenRosenberg) that recognizes both the human and mousereceptor. An empty vector was used as a negativecontrol. (B,C) The growth rate of infected uPAR-KOkeratinocytes was monitored in the presence (C) orabsence (B) of exogenous EGF (10 ng/ml). (D) Semi-confluent puromycin-selected cells were serum-starvedfor 18 hours and then stimulated with EGF (10 ng/ml)for 15 minutes. After protein extraction, the levels ofhuPAR, muPAR, active ERK (pERK1/2) and total ERK(totERK2) were determined by western blot analysis ontotal cell lysates.

Fig. 5. uPAR is required for keratinocyte adhesionand migration in vitro. (A) Wt and uPAR-KOprimary keratinocytes were seeded on ColIV-, FN-,VN- or LN5-coated 24-well plates, or in the absenceof any exogenous substrate. After 8 hours, thenumber of adherent cells was quantitated by stainingwith 0.1% crystal violet in water. Values areexpressed as absorbance at 650 nm and the mean ±s.d. of triplicate samples is reported. (B) Wt anduPAR-KO primary keratinocytes were seeded for 12hours in the absence of an exogenous substrate.Representative phase-contrast images (�20) ofdeposited cells are shown. (C) Wt and uPAR-KOkeratinocytes were seeded on LN5- or ColIV-coateddishes and grown to confluency. Cell surfaces werescraped with a pipette tip in either a single stripe or agrid pattern. The wounds were incubated in S-MEMcontaining 1% chelexed FCS and photographed atvarious times, as indicated, with the indentationmarks aligned. The lines indicate the wound edge atthe start of the experiment (t=0). (D) Haptotaxis of wtand uPAR-KO keratinocytes was analyzed by 48-well Boyden chamber assay in S-MEM 1% chelexedserum through FN-, ColIV-, VN- or LN5-coated 8-μm-pore-size polycarbonate filters at 37°C for 6hours. Results, expressed as the mean number ofmigrated cells ± s.d. from triplicate samples, arerepresentative of at least three experiments.

Jour

nal o

f Cel

l Sci

ence

3927uPAR knockout and keratinocyte migration

are unable to produce and secrete sufficientamounts of LN5, and therefore would beimpaired in adhesion, spreading and migrationon uncoated surfaces.

To exclude possible differences in the levelsand/or expression of integrins, we measured thelevels of integrins for the different substrates usedand found no difference between the twogenotypes (data not shown). For example, weexamined integrinα3β1 – one of the two receptorsfor LN5 – which is specific for cell-ECM contacts.Western blot analysis on cell lysates revealedsimilar levels of expression of both α3 and β1integrin subunits in wt and uPAR-KO cells (datanot shown), indicating that the uPAR-KO defectin keratinocyte adhesion and migration was notcaused by changes in the expression of the LN5-binding integrin.

uPAR regulates the deposition of LN5Overall, the above data prompted us to investigatethe production and secretion of LN5 in uPAR-KOkeratinocytes. To verify whether the absence ofuPAR affected the intrinsic capacity ofkeratinocytes to produce LN5, we performedimmunoblotting and reverse transcriptase (RT)-PCR analysis on suspended or adherent wt anduPAR-KO keratinocytes. In suspension, i.e. in theabsence of adhesive stimuli, an equal amount ofLN5 protein (Fig. 6A) and mRNA (Fig. 6B) wasfound in wt and uPAR-KO keratinocytes, indicatingthat the loss of uPAR expression did not affect theproduction of LN5 in the absence of an adhesivestimuli. Infection of uPAR-KO keratinocytes witha murine uPAR cDNA retroviral vector had noeffect on LN5 production (Fig. 6A,B).

However, when keratinocytes were analyzed for the depositionof LN5 upon adhesion on ColIV, very different results wereobtained. Cells were allowed to adhere by seeding on a ColIV-coatedsurface for 45 minutes; the secreted matrix was then scraped offthe plates, size-separated by SDS-PAGE and immunoblotted withan anti-LN5 (γ2 chain)-specific antibody. As shown in Fig. 6A,densitometric quantification of the immunoblots showed a consistentincrease in the amount of deposited LN5-γ2 protein in wtkeratinocytes. By contrast, LN5 deposition was very much reducedin uPAR-KO cells. This may well be due to a difference in LN5-γ2 gene activation, as judged by the measurement of the specificmRNA by semi-quantitative RT-PCR (Fig. 6B). Similar data wereobtained when cells were allowed to seed on glass (data not shown).Importantly, re-introduction of murine uPAR rescued the uPAR-KO phenotype both at the protein and mRNA level (Fig. 6A,B).

These findings indicate that uPAR is required for the adhesion-induced expression and deposition of LN5.

EGFR regulates wt keratinocyte migration by controlling LN5depositionAdhesion to LN5 is important for proper keratinocyte migration invitro, and in vivo during re-epithelialization of cutaneous wounds(Frank and Carter, 2004; Hamelers et al., 2005; Nguyen et al., 2000a;Nguyen et al., 2000b; Zhang and Kramer, 1996). To find a linkbetween the EGFR and LN5 phenotypes in uPAR-KO keratinocytes,

we analyzed the migration of wt and uPAR-KO keratinocytes ondifferent exogenous substrates in the presence or absence of thespecific inhibitor of EGFR tyrosine-kinase activity, AG1478, usinga modified Boyden chamber assay. As shown in Fig. 7A, AG1478inhibited the migration of wt cells seeded on ColIV, FN or VN, buthad no effect on uPAR-KO keratinocytes. Moreover, AG1478 didnot alter the migration of either wt or uPAR-KO keratinocytesseeded on LN5-coated filters. Similar results were obtained in thepresence of a MEK1 inhibitor (data not shown). The lack ofinhibition by AG1478 on uPAR-wt keratinocyte migration in thepresence of exogenous LN5 contrasts with the inhibitory effectobserved on cells seeded on other substrates. Therefore, these datasuggest that the exogenous supply of LN5 bypasses the requirementfor the EGFR-MAPK pathway in LN5 deposition and, therefore,in cell spreading and migration.

To test this hypothesis, wt and uPAR-KO keratinocytes were firstgrown in suspension and then seeded on a ColIV-coated surface inthe presence or absence of AG1478. After 45 minutes of incubation,cells and secreted matrices were collected and analyzed as describedabove. RT-PCR analysis (Fig. 7B) showed that AG1478 preventedthe upregulation of LN5 mRNA in wt cells, but had no effect in uPAR-KO cells. Likewise, immunoblotting (Fig. 7C) showed that AG1478drastically decreased the amount of deposited LN5 in wt keratinocytesand had no effect in uPAR-KO keratinocytes. Similar results wereobtained in the presence of UO126 (supplementary material Fig. S4c).

Fig. 6. Production and secretion of LN5 by uPAR-wt and -KO keratinocytes. (A) Suspended (susp.)wt or uPAR-KO keratinocytes, or uPAR-KO keratinocytes infected with a murine uPAR (muPAR)retrovirus, were seeded on a ColIV-coated surface. After 45 minutes, attached cells were detachedwith 10 mM EDTA and secreted LN5 was detached from the surface with SDS-sample buffer. As acontrol, suspended cells were lysed. The samples were immunoblotted with anti-LN5 (γ2 chain)antibody. (Right) The amounts of secreted LN5 (γ2 chain) levels in the western blot werequantitated relative to levels in the suspended wt sample (=100%) ± s.d. The values are expressedas the average of at least three experiments. (B) Suspended wt or uPAR-KO keratinocytes, or uPAR-KO keratinocytes infected with a muPAR retrovirus, were seeded on a ColIV-coated surface. After45 minutes, attached cells were detached for mRNA isolation and the amount of LN5 (γ2 chain)mRNA and actin (control) measured by semi-quantitative RT-PCR. The histogram represents theincrease in LN5 mRNA levels relative to the suspended wt sample (=100%). The values areexpressed as the average of at least three independent experiments. Error bars represent the s.d.

Jour

nal o

f Cel

l Sci

ence

3928

These findings indicate that, similar to uPAR, the EGFR-MAPKpathway affects expression and deposition of LN5.

Unlike wt, uPAR-KO mice do not upregulate LN5 (γ2 chain)expression in vivo during wound healingWe next investigated whether the LN5-deposition defect, observedin uPAR-KO keratinocytes in vitro, was also detectable in vivo.Immunofluorescent staining of intact skin sections with an anti-LN5 (γ2 chain) antibody revealed no differences in LN5 depositionbetween wt and KO mice (data not shown). However, 3 days afterfull-thickness skin excision wounds, the same type ofimmunostaining revealed that LN5 was overexpressed in wt butnot in uPAR-KO skin (Fig. 8A).

We also collected wounded skin of wt and uPAR-KO mice atdifferent times after incision, and performed immunoblottinganalysis on skin extracts with an anti-LN5 (γ2 chain) antibody (Fig.8B). Unwounded skin was used as time-zero control (0 days). At3 and 5 days after wounding, uPAR-wt mice showed induction ofLN5 expression, whereas the level of LN5 remained unchanged inuPAR-KO skins. The tissue-extract data confirm the results obtainedby immunofluorescence. We can therefore conclude that both invitro and in vivo induction of LN5 expression and depositionrequires uPAR.

DiscussionCutaneous wound repair comprises invasion of inflammatory cellsand fibroblasts, neo-angiogenesis, proliferation and migration ofkeratinocytes, contraction, and remodeling of the scar tissue (Martin,1997; Parks, 1999; Pilcher et al., 1999). In normal skin keratinocytes,expression of mRNA for uPA and its receptor, uPAR, is undetectableby in situ hybridization (Lund et al., 1996). After wounding, uPA anduPAR mRNA are expressed, with uPAR being confined tokeratinocytes at the leading edge of the wound (Romer et al., 1994).

Wounding dramatically activates resting epidermal keratinocytesadjacent to the wound margin to a hyperproliferative, migratory

Journal of Cell Science 121 (23)

and invasive state that allows invasion and successful re-epithelialization of the wound bed. This includes increasedproliferation of basal keratinocytes, dissolution of cell-cell adhesion,detachment of keratinocytes from the basement membrane, lateralmigration into the wounded area and invasion of the provisionalmatrix of the wound bed (Martin, 1997; Singer and Clark, 1999).

The EGFR pathway is important in skin wound healing and inskin cancer. Human and mouse squamous skin carcinomasoverexpress EGFR ligands and mouse squamous skin tumors alsodisplay constitutive activation of the EGFR kinase (Rho et al., 1994;Xian et al., 1995). Following injury, a transient elevation of EGFRand its ligand in the skin and other epithelia is believed to contributeto the migration and proliferation of keratinocytes that are adjacentto wound margins (Nanney et al., 2000; Stoll et al., 1997; Wernerand Grose, 2003).

We investigated the role of uPAR in skin wound repair. uPAR-KO mice show delayed wound healing when compared with wtmice (Fig. 1A). The histology of the wounds correlates with theirgross appearance, and closure of the wound margins is in factsignificantly delayed in the absence of uPAR.

In uPAR-KO epidermis, several events that are crucial to woundhealing are altered – in particular, keratinocyte proliferation andmigration (Fig. 2A,B). Other processes, such as neutrophil andmacrophage recruitment or neo-vessel formation, are not affected(supplementary material Fig. S2 and S3). However, our data showthat uPAR contributes to, but is not essential for, wound repair, aswound re-epithelialization is completed in uPAR-KO skins as well(Fig. 1A,B). This suggests a functional overlap between the uPA-uPAR system and other proteases, such as matrix metalloproteases(MMPs), which are expressed by the leading-edge keratinocytes(Martin, 1997; Singer and Clark, 1999). Proteases play a cooperativeand crucial role in wound healing. In fact, wound healing is stronglydelayed in the absence of plasminogen, but the additional inhibitionof all MMPs together prevents closure of the wound (Lund et al.,1999; Romer et al., 1996a; Romer et al., 1996b). The requirement

Fig. 7. EGFR regulates the migration of uPAR-wt keratinocytesby controlling LN5 deposition. (A) Migration of keratinocyteswas analyzed by 48-well Boyden chamber assay in S-MEM 1%chelexed serum with or without AG1478 (10 μM) through FN-,ColIV-, VN- or LN5-coated 8-μm-pore-size polycarbonatefilters at 37°C for 6 hours. The mean number of migrated cells ±s.d. from triplicate samples is representative of at least threeexperiments. (B,C) Suspended wt and uPAR-KO keratinocyteswere seeded on a ColIV-coated surface with or without AG1478(10 μM). After 45 minutes, attached cells were detached with 10mM EDTA, used for mRNA isolation, and subjected to RT-PCRusing LN5 (γ2 chain) primers (B), whereas secreted LN5 wasdetached from the surface with SDS-sample buffer andimmunoblotted (IB) with anti-LN5 (γ2 chain) antibodies (C).Actin was used as a control. The amounts of secreted LN5 (γ2chain) (protein and mRNA) were quantitated and expressedrelative to levels in wt samples (=100%) ± s.d., and arerepresentative of at least three experiments.

Jour

nal o

f Cel

l Sci

ence

3929uPAR knockout and keratinocyte migration

for plasmin and MMPs in wound healing has been ascribed to theneed for keratinocytes to dissect their way through the fibrin-richmatrix. The deletion of uPAR, however, does not affect fibrinolysis(Bugge et al., 1996). Therefore, our unexpected delayed woundhealing cannot be ascribed simply to deficient proteolysis. BecauseuPAR is a direct signaling regulator (Blasi and Carmeliet, 2002),these deficiencies probably depend on abnormal signaling.

uPAR is required for EGFR-induced keratinocyte proliferationin vitroThe in vitro experiments show that uPAR is required for keratinocyteproliferation, the response to exogenous EGF and activation of theEGFR. However, uPAR does not determine EGFR level. Notice thatuPAR and EGFR were co-immunoprecipitated in wt keratinocytesand that this interaction was not affected by EGF. uPAR is alsorequired for the activation of the ERK-MAPK pathway, a downstreameffector of EGFR signaling, independently of EGF. A similarconclusion has been reached for murine embryonic fibroblasts andMDA-MB 231 breast-cancer cells (Jo et al., 2007).

Thus, uPAR does not alter EGFR expression but affects itsactivation state. Indeed, EGFR- and MAPK-specific inhibitors blockboth basal and EGF-induced proliferation in wt but not in uPAR-KO cells, demonstrating that keratinocyte growth and EGFmitogenic activity require both uPAR and ERK1/2 phosphorylation.

Our data suggest that a uPAR-dependent autocrine or paracrinesignal activates EGFR and ERK1/2 phosphorylation. Indeed,because uPA-binding to uPAR is species specific (Estreicher et al.,1989), the ability of murine, but not human, uPAR to rescue themitogenic activity of EGF in uPAR-KO keratinocytes reveals aninvolvement also of uPA in this effect. The stimulation by humanuPA of the growth of uPAR-KO keratinocytes that were infectedwith retroviral vectors expressing human uPAR further supports theinvolvement of the uPAR ligand in regulating keratinocyte growth.

What is not clear is the physical connection between uPAR andEGFR. Co-immunoprecipitation of uPAR and EGFR, observed inthis and other papers, is not sufficient to demonstrate a directinteraction. Indeed, the co-immunoprecipitation in humanmonocytes of uPAR with integrins, transmembrane receptors andintracellular signaling molecules such as Src (Bohuslav et al., 1995)suggests that the co-immunoprecipitation represents a subcellularcolocalization of various proteins. This is a crucial issue forunderstanding the nature of the uPAR-dependent activity of severalmolecules.

uPAR is required for keratinocyte adhesion and migration invitroAdhesion of basal keratinocytes to the basement membrane (ColIV,LN5 and LN10) not only supports skin architecture but is alsorequired for growth, migration and differentiation (Estreicher et al.,1989; Hintermann and Quaranta, 2004; Nguyen et al., 2000a;Nguyen et al., 2000b; Zhang and Kramer, 1996). LN5 is the majoradhesive ligand present in the quiescent epidermal basementmembrane and, after injury, is rapidly transcribed (Ryan et al., 1994),translated and deposited into the basement membrane bykeratinocytes at the wound edge (Goldfinger et al., 1998; Lampeet al., 1998). This contrasts with the delayed expression of otherbasement-membrane components, such as type VII collagen (Lampeet al., 1998) and heparin sulphate proteoglycan (Oksala et al., 1995).Some studies also support the idea that LN5 contributes to migrationon other substrates, such as collagen or FN (DiPersio et al., 1997;Nguyen et al., 2000a; Nguyen et al., 2000b; Ryan et al., 1994).

We found that uPAR-KO keratinocytes adhered to and spread onvarious exogenous substrates, but not on uncoated surfaces such asglass, on which the cells had to deposit their own substrate. However,adhesion and motility was rescued in the presence of exogenous LN5.Thus, uPAR-KO keratinocytes might be deficient in LN5 depositionand secretion. Indeed, after initial adhesion on ColIV, whereas wtcells consistently increased the amount of LN5 (γ2 chain) proteinand mRNA, uPAR-KO cells did not. These findings indicate thatuPAR is required for increased expression and deposition of LN5upon adhesion of keratinocytes to an exogenous substrate.

Keratinocytes spread on LN5 using α3β1 integrin (Carter et al.,1990; Carter et al., 1991; DiPersio et al., 2000). However, thekeratinocyte defect is not explained by a reduced expression of α3β1integrin in uPAR-KO cell lysates, because the level of integrin α3and β1 subunits was not decreased. By contrast, in the absence ofuPAR, the deficient LN5 deposition can be ascribed to the reducedactivation of the EGFR signaling pathway.

LN5 (γ2 chain) is as a marker of invading tumor cells (Ono et al.,2002), and it has been suggested that EGFR influences the invasive

Fig. 8. uPAR upregulates LN5 (γ2 chain) in vivo during wound healing.(A) Representative wt and uPAR-KO cross-sections of 3-day-old woundsstained for LN5 (γ2 chain) and counterstained with hematoxylin and eosin(HE). (B) Wounded skins of wt and uPAR-KO mice were collected at differenttimes after incision, lysed and immunoblotted with an anti-LN5 (γ2 chain)antibody. The immunoblot shows one representative experiment, whereas thehistogram shows the average values obtained with eight wt and seven uPAR-KO mice. The amounts of LN5 (γ2 chain) are expressed as fold increaserelative to the uninjured uPAR-KO mice (left-most bar, arbitrary value of 1) ±s.d.

Jour

nal o

f Cel

l Sci

ence

3930 Journal of Cell Science 121 (23)

activity of tumor cells by stimulating the expression of LN5-γ2 (Katohet al., 2002; Richter et al., 2005; Veitch et al., 2003). Indeed, a specificEGFR inhibitor, AG1478, inhibits LN5 deposition at thetranscriptional and/or post-transcriptional level, and consequentlyinfluences LN5-dependent keratinocyte migration. Because the sameresults were obtained with the MEK1 inhibitor UO126, we proposethat, in wt cells, EGFR regulates LN5 deposition (and spreading,migration and proliferation) through ERK1/2.

Coincidence of in vitro and in vivo phenotypes in uPAR-KO skinThe results obtained with primary keratinocytes, i.e. decreasedproliferation, migration and inability to upregulate LN5 productionafter wounding, were also observed in vivo. Moreover, woundeduPAR-KO skin displayed aberrant keratinocyte proliferation,revealed by the thinness of the migrating keratinocyte layer, thereduced number of PCNA-positive cells and a delay of thekeratinocyte scar to return to normal thickness. Although othersignaling systems are certainly involved in wound healing, ourresults highlight the novel in vivo role of uPAR in this process; thisrole can be largely explained by the requirement of uPAR for theactivation of EGFR and its relative signaling pathways.

Materials and MethodsReagentsThe murine polyclonal anti-uPAR antibody was kindly provided by Steven Rosenberg(University of California, Berkeley, CA). MOPC-21 IgG was from Sigma-Aldrich(St Louis, MO); monoclonal anti-murine α3 and β1 from Chemicon International(Temecula, CA); Tyrphostin AG1478 from Calbiochem (San Diego, CA); polyclonalanti-EGFR and anti-laminin γ2 (C-20), and monoclonal anti-total ERK2 antibodiesfrom Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal anti-phosphotyrosineantibody from Upstate Biotechnology (Lake Placid, NY); polyclonal antibodydetecting phosphorylated ERK1 and ERK2 from Cell Signaling Technology (Beverly,MA); polyclonal anti-keratin-5 from Covance (Berkeley, CA); and horseradish-peroxidase-conjugated anti-mouse and rabbit IgG antibodies from GE Healthcare.

Cell culturePrimary keratinocytes from 2-day-old newborn wt and uPAR-KO mice (Malliri etal., 2002) were cultured in media containing 0.02 mM CaCl2 (Hennings et al., 1980).To remove Ca2+ from serum, an analytical grade chelating resin, Chelex 100, wasused (Bio-Rad, Hercules, CA). Ca2+ concentration was then measured on each serumbatch with an atomic absorption spectrophotometer, kindly provided by Maria Tringali(University of Bicocca, Milan) (Perkin Elmer, Waltham, MA). Dermis and epidermiswere separated overnight by 0.05% trypsin-EDTA (0.02%) and minced in S-MEM(Invitrogen, Milan, Italy) supplemented with 1.2 mM CaCl2. Cell suspensions werefiltered through a 40 μm Cell Strainer (BD Biosciences, Bedford, MA) and distributedin ColIV (Roche, Mannheim, Germany)-coated dishes. Keratinocytes were culturedin S-MEM with 8% or 1% chelexed FCS (Sigma-Aldrich, St Louis, MO), 0.02 mMCaCl2 and 100 IU/ml penicillin/streptomycin (Invitrogen). For proliferation assays,cells were seeded at 30% confluency on ColIV-coated dishes and grown in 1%chelexed FCS-S-MEM with or without EGF (10 ng/ml) (Cambrex, East Rutherford,NJ), bFGF (Peprotech, Rocky Hill, NJ), HGF (Peprotech, Rocky Hill, NJ), PDGF(Peprotech, Rocky Hill, NJ), TGFβ (Peprotech, Rocky Hill, NJ), AG1478 (10 μM;Calbiochem San Diego, CA) or UO126 (10 μM; Promega, Madison, WI). Growthrate was measured every day over a 7-day period by washing with phosphate-bufferedsaline (PBS), fixing in 500 μl PBS containing 11% gluteraldehyde for 10 minutes atroom temperature, rinsing with water, air-drying and staining with 300 μl 0.1% crystalviolet (Sigma) in 200 mM MES, pH 6.0 (Sigma). After extensive rinsing, extractionwas performed with 500 μl 10% acetic acid (20 minutes at room temperature) and650 nm absorbance was measured. Values are expressed as the mean absorbance at650 nm of triplicate samples ± s.d. Rac-11P cells, kindly provided by ArnoudSonnenberg (Netherlands Cancer Institute, Amsterdam, The Netherlands), werecultured in DMEM supplemented with 10% bovine calf serum and were seeded 24hours before use, to obtain a final density of 100%.

Retroviral vectors and viral infectionpBabe-puro-h-uPAR (human uPAR), or pBabe-puro-m-uPAR (mouse uPAR) weretransfected into Phoenix packaging cells, and 24 hours later fresh viral supernatantswere collected, filtered (0.45 μm), supplemented with 5 μg/ml polybrene and usedto infect uPAR-KO keratinocytes. Cells (5�105) were plated 48 hours before infectionand selected for 4 days in puromycin (2 μg/ml; Sigma-Aldrich, St Louis, MO) afterfour rounds of infection (4 hours each) over a 2-day period. Cells expressing pBabe-puro empty vector were used as control.

Coating of dishes with ECM moleculesAll ECM proteins except LN5 were coated to culture dishes overnight at 4°C at 10μg/ml (FN, Roche; VN, Promega) or 20 μg/ml (ColIV). A LN5 matrix was obtainedby culturing Rac-11P cells to confluency (Delwel et al., 1993), after which cells weredetached with 10 mM EDTA in PBS containing a mix of protease inhibitors (Completeprotease inhibitor cocktail tablets; Roche) at 4°C. Before use, the dishes were washedtwice with PBS.

Immunoprecipitation and western blottingFor immunoprecipitation (IP), cells grown in 10-cm-diameter dishes were lysed in500 μl of buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2,10% glycerol, 1% Nonidet P-40 (Sigma) and protease inhibitors. Extracts werecentrifuged at 18,000 g for 10 minutes at 4°C, quantitated by Bio-Rad protein assay(Bio-Rad, Hercules, CA) and pre-cleared with lysis buffer-containing protein-Gagarose beads for 1 hour at 4°C. Pre-cleared lysates (0.5-1 mg) were incubated withantibodies against murine EGFR (1 μg/ml in lysis buffer) overnight at 4°C. Afterwashing with lysis buffer, the immunoprecipitates were resuspended in SDS-samplebuffer and immunoblotted with the indicated antibodies.

For immunoblotting, cell lysates (50-80 μg) from tissue-cultured cells or from miceback skin, or immunoprecipitated proteins were boiled for 5 minutes and resolvedby SDS-PAGE. Proteins were electro-transferred to polyvinylidene difluoridemembranes (Millipore, Billerica, MA), blocked with Tris-buffered saline (TBS)containing 5% non-fat dried milk and 0.02% Tween 20 for 1 hour at room temperatureand probed using the indicated antibodies. For LN5-production analysis, secretedmatrix was scraped off the plates, size-separated by SDS-PAGE and immunoblottedwith an anti-LN5 (γ2)-specific antibody. Specific binding was detected using an anti-rabbit, anti-goat or anti-mouse horseradish-peroxidase-linked F(ab�)2 fragment(1:5000, GE Healthcare) followed by an enhanced chemiluminescence methodaccording to the manufacturer’s instructions (Pierce Chemical, Rockford, IL). Whenindicated, before protein extraction, cells were serum-starved for 18 hours and pre-incubated for 20 minutes at 37°C in humidified air with 5% CO2.

Adhesion assays24-well microplates were coated overnight at 4°C with ColIV, FN, VN or LN-5 (seesection ‘Coating of dishes with ECM molecules’) or PBS. Plates were washed withPBS and saturated with 1% (wt/vol) BSA (Roche), for 2 hours at 37°C to block non-specific adhesion. Keratinocytes were detached with EDTA and suspended insupplement-free S-MEM. 6�104 cells/well were plated in triplicate and incubatedfor 6-12 hours at 37°C. Non-adherent cells were removed with PBS, and cell adhesionestimated in a colorimetric assay, using 0.1% crystal violet (Sigma) in 200 mM MES,pH 6.0 (Sigma). Absorbance was measured at 650 nm.

Migration and in vitro scrape-wound assaysMigration was assessed by using a 48-well microchemotaxis chamber (Neuro Probe,Gaithersburg, MD). 1% chelexed FCS–S-MEM (26 μl) was placed in the bottomcompartment and 200 μl of cell suspension (125,000 cells/ml) added to the topcompartment. The two compartments were separated by a FN-, ColIV-, VN- or LN5-coated 8-μm-pore-size polycarbonate filter (Neuro Probe) and incubated at 37°C inhumidified air with 5% CO2 for 6 hours. Thereafter, the filter was removed, scrapedand stained with Diff-Quick (Dade Diagnostic, Aguada, Puerto Rico), and migratedcells counted by light microscopy. Results, expressed as the mean ± s.d. from triplicatesamples represent at least three experiments. The significance of each migration assaywas evaluated using the t-test, assuming unequal variances. P-values lower than 0.05were considered statistically significant. Potential inhibitors were pre-incubated withcells for 30 minutes at 37°C and the assay was performed as described above.

For in vitro scrape-wound assays, keratinocytes were grown to confluency on LN5-or ColIV-coated dishes. Medium was aspirated and the cell-coated surface was scrapedwith a pipette tip in either a single stripe or a grid pattern. The scrape-wounded surfacewas washed with PBS to remove debris and then the wounds were allowed to healin 1% chelexed FCS–S-MEM and photographs taken at various times, as indicated,with the indentation marks aligned.

mRNA isolation and RT-PCRTotal cellular RNA was isolated in vivo from excised wounds and in vitro fromkeratinocyte suspension or after a 45-minute adhesion on ColIV-coated dishes, usingTrizol Reagent (Invitrogen), and cDNA was synthesized by RT-PCR on 1 μg RNAusing the Superscript RT-PCR system kit (Invitrogen). Specific transcripts wereamplified with the following primers (TIB MOLBIOL, Genova, Italy): LN5 (γ2 chain)(forward, 5�-AACCAGCAAGTGAGTTACGG-3�; reverse, 5�-CCATTGTGAC -AGGGACATGG-3�); actin (forward, 5�-GGCATCCTGACCCTGAAGT-3�; reverse,5�-CGGATGTCAACGTCACACTT-3�) (TIB MOLBIOL) using the standard PCRprotocol for the EuroTaq kit (Euroclone, Pero, Italy). PCR products were resolvedby 1.5% agarose-gel electrophoresis and stained with ethidium bromide.

Wound repair in vivoAdult uPAR-wt and uPAR-KO littermates were anesthetized with isoflurane, shavedand one full-thickness skin excision wound (1 cm in length) was cut with small scissorson either side of the dorsal midline of each mouse. The wounds were left untreated

Jour

nal o

f Cel

l Sci

ence

3931uPAR knockout and keratinocyte migration

and the mice were killed at different time points after injury. Wounds were eitherembedded for sectioning or excised with ~6 mm of the epidermal margins for RNAisolation or protein extraction. For each time point, wound diameters (distance betweenepithelial rims) were determined in at least five mice per genotype bothmacroscopically and microscopically. Cryo- and paraffin sections across the middleof the wounds were stained: the cryosections (5 μm) with keratin 5 (K5), LN5 (γ2chain) and DAPI, and the paraffin sections (7 μm) with hematoxylin and eosin oracidic toluidine blue (Sigma) (to identify mast cells) using standard methods. LN5(γ2 chain) and K5 primary antibodies were visualized with FITC-labeled secondaryantibodies (Molecular Probes, Eugene, Oregon) and images were collected using aLeica microscope. The length of the epidermal tongue was measured blindly in K5-stained sections, at 3 days after incision (eight sections per wound; n=8 per genotype)by computer-assisted morphometry from the tip of the epidermal tongue to the startof the zone of proliferating keratinocytes using Adobe Photoshop software.

ImmunohistochemistryImmunohistochemistry was performed using anti-macrophage F4/80 or anti-PCNAantibodies (Serotec, Raleigh, North Carolina), with a biotinylated secondary antibody,an ABC reagent (Vector Laboratories, Burlingame, CA), diaminobenzidine (Sigma-Aldrich) and hematoxylin counterstain. Blood vessels were identified using an anti-CD31 primary antibody (PharmMingen, San Diego, CA), an Alexa-Fluor-488secondary antibody (Molecular Probes) and DAPI counterstain (Vector Laboratories).Blood vessels were counted in CD31-labeled sections in a �20 microscopic fieldcentered on the wound, in at least eight wounds per genotype, for each time point.Macrophages, hyperproliferative keratinocytes and mast cells were counted insections following immunohistochemistry or histochemistry. At least three high-powerfields immediately adjacent to the wound site were quantified and averaged in eightwounds per genotype for each time point.

This work was supported by grants from the AICR (Association forInternational Cancer Research), AIRC (Italian Association for CancerResearch) and the Italian Ministry of Health. The authors thankRoberta Mazzieri (TIGET Institute, San Raffaele, Milan) for her helpwith the editing of the manuscript and stimulating discussions; ArnoudSonnenberg from the Netherlands Cancer Institute (Amsterdam, TheNetherlands) for providing Rac-11P cells; and Maria Tringali(University of Bicocca, Milan) for her help with Ca2+ measurementsin cell culture serum.

ReferencesAguirre Ghiso, J. A., Kovalski, K. and Ossowski, L. (1999). Tumor dormancy induced

by down regulation of urokinase receptor in human carcinoma involves integrin andmapk signaling. J. Cell Biol. 147, 89-104.

Alfano, D., Iaccarino, I. and Stoppelli, M. P. (2006). Urokinase signaling through itsreceptor protects against anoikis by increasing BCL-xL expression levels. J. Biol. Chem.281, 17758-17767.

Blasi, F. and Carmeliet, P. (2002). uPAR: a versatile signalling orchestrator. Nat. Rev.Mol. Cell. Biol. 3, 932-943.

Bohuslav, J., Horejsi, V., Hansmann, C., Stockl, J., Weidle, U. H., Majdic, O., Bartke,I., Knapp, W. and Stockinger, H. (1995). Urokinase plasminogen activator receptor,beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes.J. Exp. Med. 181, 1381-1390.

Bugge, T., Flick, M., Danton, M., Daugherty, C., Romer, J., Dano, K., Carmeliet, P.,Collen, D. and Degen, J. (1996). Urokinase-type plasminogen activator is effective infibrin clearance in the absence of its receptor or tissue-type plasminogen activator. Proc.Natl. Acad. Sci. USA 93, 5899-5904.

Carter, W., Kaur, P., Gil, S., Gahr, P. and Wayner, E. (1990). Distinct functions forintegrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigenin a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes.J. Cell Biol. 111, 3141-3154.

Carter, W., Ryan, M. and Gahr, P. (1991). Epiligrin: a new cell adhesion ligand for integrinalpha 3 beta 1 in epithelial basement membranes. Cell 65, 599-610.

Delwel, G., Hogervorst, F., Kuikman, I., Paulsson, M., Timpl, R. and Sonnenberg, A.(1993). Expression and function of the cytoplasmic variants of the integrin alpha 6 subunitin transfected K562 cells: activation-dependent adhesion and interaction with isoformsof laminin. J. Biol. Chem. 268, 25865-25875.

DiPersio, C., Hodivala-Dilke, K., Jaenisch, R., Kreidberg, J. and Hynes, R. (1997).alpha3beta1 Integrin is required for normal development of the epidermal basementmembrane. J. Cell Biol. 137, 729-742.

DiPersio, C. M., van der Neut, R., Georges-Labouesse, E., Kreidberg, J. A.,Sonnenberg, A. and Hynes, R. O. (2000). alpha3beta1 and alpha6beta4 integrinreceptors for laminin-5 are not essential for epidermal morphogenesis and homeostasisduring skin development. J. Cell Sci. 113, 3051-3062.

Estreicher, A., Wohlwend, A., Belin, D., Schleuning, W. D. and Vassalli, J. D. (1989).Characterization of the cellular binding site for the urokinase-type plasminogen activator.J. Biol. Chem. 264, 1180-1189.

Frank, D. and Carter, W. (2004). Laminin 5 deposition regulates keratinocyte polarizationand persistent migration. J. Cell Sci. 117, 1351-1363.

Furlan, F., Galbiati, C., Jorgensen, N., Jensen, J., Mrak, E., Rubinacci, A., Talotta,F., Verde, P. and Blasi, F. (2007). Urokinase plasminogen activator receptor affectsbone homeostasis by regulating osteoblast and osteoclast function. J. Bone Miner. Res.22, 1387-1396.

Gillitzer, R. and Goebeler, M. (2001). Chemokines in cutaneous wound healing. J. Leukoc.Biol. 69, 513-521.

Goldfinger, L., Stack, M. and Jones, J. (1998). Processing of laminin-5 and its functionalconsequences: role of plasmin and tissue-type plasminogen activator. J. Cell Biol. 141,255-265.

Goldfinger, L., Hopkinson, S., deHart, G., Collawn, S., Couchman, J. and Jones, J.(1999). The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinatelyregulate wound healing in cultured epithelial cells and in the skin. J. Cell Sci. 112, 2615-2629.

Gyetko, M. R., Todd, R. F., 3rd, Wilkinson, C. C. and Sitrin, R. G. (1994). The urokinasereceptor is required for human monocyte chemotaxis in vitro. J. Clin. Invest. 93, 1380-1387.

Gyetko, M. R., Sitrin, R. G., Fuller, J. A., Todd, R. F., 3rd, Petty, H. and Standiford,T. J. (1995). Function of the urokinase receptor (CD87) in neutrophil chemotaxis. J.Leukoc. Biol. 58, 533-538.

Hamelers, I. H., Olivo, C., Mertens, A. E., Pegtel, D. M., van der Kammen, R. A.,Sonnenberg, A. and Collard, J. G. (2005). The Rac activator Tiam1 is required for(alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration. J. CellBiol. 171, 871-881.

Hennings, H., Michael, D., Cheng, C., Steinert, P., Holbrook, K. and Yuspa, S. (1980).Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell 19, 245-254.

Hintermann, E. and Quaranta, V. (2004). Epithelial cell motility on laminin-5: regulationby matrix assembly, proteolysis, integrins and erbB receptors. Matrix Biol. 23, 75-85.

Jo, M., Thomas, K. S., O’Donnell, D. M. and Gonias, S. L. (2003). Epidermal growthfactor receptor-dependent and -independent cell-signaling pathways originating fromthe urokinase receptor. J. Biol. Chem. 278, 1642-1646.

Jo, M., Thomas, K. S., Takimoto, S., Gaultier, A., Hsieh, E. H., Lester, R. D. and Gonias,S. L. (2007). Urokinase receptor primes cells to proliferate in response to epidermalgrowth factor. Oncogene 26, 2585-2594.

Katoh, K., Nakanishi, Y., Akimoto, S., Yoshimura, K., Takagi, M., Sakamoto, M. andHirohashi, S. (2002). Correlation between laminin-5 gamma2 chain expression andepidermal growth factor receptor expression and its clinicopathological significance insquamous cell carcinoma of the tongue. Oncology 62, 318-326.

Kim, J., Zhang, K., Chen, J., Wynn, K., Kramer, R. and Woodley, D. (1992). Mechanismof human keratinocyte migration on fibronectin: unique roles of RGD site and integrins.J. Cell Physiol. 151, 443-450.

Lampe, P., Nguyen, B., Gil, S., Usui, M., Olerud, J., Takada, Y. and Carter, W. (1998).Cellular interaction of integrin alpha3beta1 with laminin 5 promotes gap junctionalcommunication. J. Cell Biol. 143, 1735-1747.

Larjava, H., Salo, T., Haapasalmi, K., Kramer, R. and Heino, J. (1993). Expression ofintegrins and basement membrane components by wound keratinocytes. J. Clin. Invest.92, 1425-1435.

Liu, D., Ghiso, J. A. A., Estrada, Y. and Ossowski, L. (2002). EGFR is a transducer ofthe urokinase receptor initiated signal that is required for in vivo growth of a humancarcinoma. Cancer Cell 1, 445-457.

Lund, L., Eriksen, J., Ralfkiaer, E. and Romer, J. (1996). Differential expression ofurokinase-type plasminogen activator, its receptor, and inhibitors in mouse skin afterexposure to a tumor-promoting phorbol ester. J. Invest. Dermatol. 106, 622-630.

Lund, L., Romer, J., Bugge, T., Nielsen, B., Frandsen, T., Degen, J., Stephens, R. andDano, K. (1999). Functional overlap between two classes of matrix-degrading proteasesin wound healing. EMBO J. 18, 4645-4656.

MacNeil, S. (1994). What role does the extracellular matrix serve in skin grafting andwound healing? Burns 20 Suppl. 1, S67-S70.

Madsen, C. D., Ferraris, G. M., Andolfo, A., Cunningham, O. and Sidenius, N. (2007).uPAR-induced cell adhesion and migration: vitronectin provides the key. J. Cell Biol.177, 927-939.

Malliri, A., van der Kammen R. A., Clark, K., van der Valk, M., Michiels, F. andCollard, J. G. (2002). Mice deficient in the Rac activator Tiam1 are resistant to Ras-induced skin tumours. Nature 417, 867-871.

Martin, P. (1997). Wound healing-aiming for perfect skin regeneration. Science 276, 75-81.

Mazzieri, R., D’Alessio, S., Kenmoe, R. K., Ossowski, L. and Blasi, F. (2006). Anuncleavable uPAR mutant allows dissection of signaling pathways in uPA-dependentcell migration. Mol. Biol. Cell 17, 367-378.

Nanney, L., Paulsen, S., Davidson, M., Cardwell, N., Whitsitt, J. and Davidson, J.(2000). Boosting epidermal growth factor receptor expression by gene gun transfectionstimulates epidermal growth in vivo. Wound Repair Regen. 8, 117-127.

Nguyen, B., Gil, S. and Carter, W. (2000a). Deposition of laminin 5 by keratinocytesregulates integrin adhesion and signaling. J. Biol. Chem. 275, 31896-31907.

Nguyen, B., Ryan, M., Gil, S. and Carter, W. (2000b). Deposition of laminin 5 in epidermalwounds regulates integrin signaling and adhesion. Curr. Opin. Cell Biol. 12, 554-562.

Nguyen, D. H., Webb, D. J., Catling, A. D., Song, Q., Dhakephalkar, A., Weber, M.J., Ravichandran, K. S. and Gonias, S. L. (2000c). Urokinase-type plasminogenactivator stimulates the Ras/Extracellular signal-regulated kinase (ERK) signalingpathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase,Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with thetransient phosphorylation of ERK in urokinase-treated cells. J. Biol. Chem. 275, 19382-19388.

Jour

nal o

f Cel

l Sci

ence

3932 Journal of Cell Science 121 (23)

Oksala, O., Salo, T., Tammi, R., Hakkinen, L., Jalkanen, M., Inki, P. and Larjava, H.(1995). Expression of proteoglycans and hyaluronan during wound healing. J. Histochem.Cytochem. 43, 125-135.

Ono, Y., Nakanishi, Y., Gotoh, M., Sakamoto, M. and Hirohashi, S. (2002). Epidermalgrowth factor receptor gene amplification is correlated with laminin-5 gamma2 chainexpression in oral squamous cell carcinoma cell lines. Cancer Lett. 175, 197-204.

Parks, W. (1999). Matrix metalloproteinases in repair. Wound Repair Regen. 7, 423-432.Pilcher, B., Wang, M., Qin, X., Parks, W., Senior, R. and Welgus, H. (1999). Role of

matrix metalloproteinases and their inhibition in cutaneous wound healing and allergiccontact hypersensitivity. Ann. N. Y. Acad. Sci. 878, 12-24.

Repertinger, S., Campagnaro, E., Fuhrman, J., El-Abaseri, T., Yuspa, S. and Hansen,L. (2004). EGFR enhances early healing after cutaneous incisional wounding. J. Invest.Dermatol. 123, 982-989.

Rho, O., Beltran, L. M., Gimenez-Conti, I. B. and DiGiovanni, J. (1994). Alteredexpression of the epidermal growth factor receptor and transforming growth factor-αduring multistage skin carcinogenesis in SENCAR mice. Mol. Carcinog. 11, 19-28.

Richter, P., Bohmer, F., Hindermann, W., Borsi, L., Hyckel, P., Schleier, P., Katenkamp,D., Kosmehl, H. and Berndt, A. (2005). Analysis of activated EGFR signalling pathwaysand their relation to laminin-5 gamma2 chain expression in oral squamous cell carcinoma(OSCC). Histochem. Cell Biol. 124, 151-160.

Romer, J., Lund, L., Eriksen, J., Pyke, C., Kristensen, P. and Dano, K. (1994). Thereceptor for urokinase-type plasminogen activator is expressed by keratinocytes at theleading edge during re-epithelialization of mouse skin wounds. J. Invest. Dermatol. 102,519-522.

Romer, J., Bugge, T., Pyke, C., Lund, L., Flick, M., Degen, J. and Dano, K. (1996a).Impaired wound healing in mice with a disrupted plasminogen gene. Nat. Med. 2, 287-292.

Romer, J., Bugge, T., Pyke, C., Lund, L., Flick, M., Degen, J. and Dano, K. (1996b).Plasminogen and wound healing. Nat. Med. 2, 725.

Ryan, M., Tizard, R., VanDevanter, D. and Carter, W. (1994). Cloning of the LamA3gene encoding the alpha 3 chain of the adhesive ligand epiligrin. Expression in woundrepair. J. Biol. Chem. 269, 22779-22787.

Simon, D. I., Wei, Y., Zhang, L., Rao, N. K., Xu, H., Chen, Z., Liu, Q., Rosenberg, S.and Chapman, H. A. (2000). Identification of a urokinase receptor-integrin interactionsite. Promiscuous regulator of integrin function. J. Biol. Chem. 275, 10228-10234.

Singer, A. and Clark, R. (1999). Cutaneous wound healing. N. Engl. J. Med. 341, 738-746.

Stephens, R., Nielsen, H., Christensen, I., Thorlacius-Ussing, O., Sorensen, S., Dano,K. and Brunner, N. (1999). Plasma urokinase receptor levels in patients with colorectalcancer: relationship to prognosis. J. Natl. Cancer Inst. 91, 869-874.

Stoll, S., Garner, W. and Elder, J. (1997). Heparin-binding ligands mediate autocrineepidermal growth factor receptor activation In skin organ culture. J. Clin. Invest. 100,1271-1281.

Veitch, D., Nokelainen, P., McGowan, K., Nguyen, T., Nguyen, N., Stephenson, R.,Pappano, W., Keene, D., Spong, S., Greenspan, D. et al. (2003). Mammalian tolloidmetalloproteinase, and not matrix metalloprotease 2 or membrane type 1 metalloprotease,processes laminin-5 in keratinocytes and skin. J. Biol. Chem. 278, 15661-15668.

Wei, Y., Tang, C.-H., Kim, Y., Robillard, L., Zhang, F., Kugler, M. C. and Chapman,H. A. (2007). Urokinase receptors are required for {alpha}5beta1 integrin-mediatedsignaling in tumor cells. J. Biol. Chem. 282, 3929-3939.

Werner, S. and Grose, R. (2003). Regulation of wound healing by growth factors andcytokines. Physiol. Rev. 83, 835-870.

Xian, W., Kiguchi, K., Imamoto, A., Rupp, T., Zilberstein, A. and DiGiovanni, J. (1995).Activation of the epidermal growth factor receptor by skin tumor promoters and in skintumors from SENCAR mice. Cell Growth Differ. 6, 1447-1455.

Yu, W., Kim, J. and Ossowski, L. (1997). Reduction in surface urokinase receptor forcesmalignant cells into a protracted state of dormancy. J. Cell Biol. 137, 767-777.

Zhang, G., Kim, H., Cai, X., Lopez-Guisa, J., Alpers, C., Liu, Y., Carmeliet, P. andEddy, A. (2003). Urokinase receptor deficiency accelerates renal fibrosis in obstructivenephropathy. J. Am. Soc. Nephrol. 14, 1254-1271.

Zhang, K. and Kramer, R. (1996). Laminin 5 deposition promotes keratinocyte motility.Exp. Cell Res. 227, 309-322.

Jour

nal o

f Cel

l Sci

ence