β1 integrin promotes keratinocyte cell survival through ... · through a mek/erk signaling...

IntroductionIn normal epithelial cells, inadequate or inappropriate celladhesion to the extracellular matrix (ECM) leads to aspecialized form of apoptosis known as anoikis (Frisch andFrancis, 1994; Frisch and Screaton, 2001). Integrins are themajor receptors for cell adhesion to the ECM (Hynes, 2002),and they are the principle receptors for transmitting outside-insignals from the ECM that inhibit anoikis and promote cellsurvival (Giancotti and Ruoslahti, 1999; Stupack and Cheresh,2002). Although a number of different integrins can promotecell survival, specific integrin-ligand interactions may berequired to inhibit anoikis in a distinct cell type or under specialcircumstances (Wary et al., 1996; Matter and Ruoslahti, 2001).Survival signals that are transduced by a particular integrinmay be regulated during tissue remodeling events throughchanges in the bioavailability of appropriate ECM ligands.

Adhesion-dependent survival of epithelial cells can beregulated through the extracellular signal-regulated kinase(ERK) family of mitogen-activated protein kinases (MAPkinases) (Frisch and Screaton, 2001). Upon activation of ERKby integrin-mediated signals, ERK translocates to the nucleuswhere it activates the transcription of genes that promote cellsurvival (Schulze et al., 2001). Activation of focal adhesionkinase (FAK) is an early step in many integrin-mediatedsurvival pathways (Frisch et al., 1996; Giancotti and Ruoslahti,

1999; Hanks et al., 2003). Activated FAK can promote cellsurvival through interactions with several downstream signaltransduction molecules, including p130 Crk-associatedsubstrate (CAS) and phosphatidylinositol 3-kinase (PI3K)(Frisch and Screaton, 2001; Hanks et al., 2003). Some of theseFAK-mediated survival pathways may involve ERK activation.For example, CAS/Crk coupling and ERK activation cansuppress apoptosis in some cells (Cho and Klemke, 2000). Inaddition, FAK interactions with PI3K may stimulate theRaf/MEK/ERK signaling cascade through activation of p21-activated kinase (PAK) (King et al., 1998; Eblen et al., 2002).FAK has also been linked to Ras-mediated Raf/MEK/ERKsignaling through direct binding interactions with the Grb2adaptor protein (Schlaepfer et al., 1994).

Despite an increased understanding of intracellular signalingpathways that promote integrin-mediated survival in manycell types, mechanisms whereby specific integrins regulatekeratinocyte survival in the epidermis, and the role ofMEK/ERK signaling in this process, remain unclear.Keratinocytes in the basal layer of the stratified epidermis areadhered to the basement membrane (BM) that separates theepidermis from the dermis (Burgeson and Christiano, 1997).Laminin-5 (LN-5) is the major adhesive ligand in the cutaneousBM, and mutations in the genes that encode each of the threesubunits that comprise the LN-5 trimer (α3, β3, or γ2) lead to

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Inadequate or inappropriate adhesion of epithelial cells toextracellular matrix leads to a form of apoptosis known asanoikis. During various tissue remodelling events, such aswound healing or carcinoma invasion, changes in thephysical properties, and/or composition of the extracellularmatrix, can lead to anoikis of epithelial cells that lackappropriate receptor-matrix interactions. Laminin-5 is themajor ligand for keratinocyte adhesion in the epidermis,and it also promotes keratinocyte survival in vivo and invitro. Integrins α3β1 and α6β4 are the major receptors forlaminin-5; however, specific roles for these integrins inkeratinocyte survival have not been determined. In thecurrent study, we exploited keratinocyte cell lines derivedfrom wild-type or α3 integrin knockout mice to reveal acritical role for α3β1 in protecting keratinocytes fromapoptosis upon serum withdrawal. We show that α3β1-mediated adhesion to laminin-5 extracellular matrix

inhibits proteolytic activation of caspase-3 and TUNEL-staining, both hallmarks of apoptosis. We also show thatα3β1-mediated adhesion activates focal adhesion kinase(FAK) and extracellular signal-regulated kinase (ERK),and that inhibition of either FAK or ERK signaling leadsto apoptosis of keratinocytes attached to laminin-5. α6β4-mediated adhesion to laminin-5 only partially protects cellsfrom apoptosis in the absence of α3β1, and α6β4 is notnecessary for cell survival in the presence of α3β1. Theseresults suggest that α3β1 is necessary and sufficient formaximal keratinocyte survival on laminin-5. We proposea model to address the potential importance of α3β1-mediated survival for migrating keratinocytes at theleading edge of a cutaneous wound.

Key words: α3β1 integrin, Keratinocyte, Apoptosis, Anoikis, ERK

Summary

α3β1 integrin promotes keratinocyte cell survivalthrough activation of a MEK/ERK signaling pathwayAsha Manohar*, Swati Ghosh Shome*, John Lamar, Lee Stirling, Vandana Iyer, Kevin Pumiglia andC. Michael DiPersio ‡

Center for Cell Biology and Cancer Research, Albany Medical College, MC-165, 47 New Scotland Avenue, Albany, New York, NY 12208, USA*These authors contributed equally to this work‡Author for correspondence (e-mail: [email protected])

Accepted 20 April 2004Journal of Cell Science 117, 4043-4054 Published by The Company of Biologists 2004doi:10.1242/jcs.01277

Research Article

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epidermal blistering in mutant mice and in patients withjunctional epidermolysis bullosa (Burgeson and Christiano,1997; Ryan et al., 1999; Kuster et al., 1997). Keratinocytes canbind to LN-5 through two integrin receptors, α3β1 and α6β4(Nguyen et al., 2000). Targeted null mutations in the genes thatencode the subunits for these integrins also lead to epidermalblistering, although the mechanisms of blistering are distinct(van der Neut et al., 1996; Dowling et al., 1996; Georges-Labouesse et al., 1996; DiPersio et al., 1997). Epidermaladhesion to BM laminins is critical for keratinocyte survival invivo, because mice lacking integrins α3β1 and α6β4 showincreased apoptosis in regions of detached epidermis (DiPersioet al., 2000b). A specific role for LN-5 in maintainingkeratinocyte survival is supported by studies in culturedkeratinocytes and mice with a targeted null mutation in theLAMA3gene (Ryan et al., 1999; Nguyen et al., 2000; Fujisakiand Hattori, 2002).

During wound healing, the cutaneous BM is broken downand keratinocytes are stimulated to migrate over a provisionalECM that is rich in fibronectin and dermal collagen (Grinnell,1992). Keratinocytes also secrete abundant LN-5 as theymigrate into the wound, which is thought to promote α3β1-mediated cell migration and provide the foundation for newBM during re-epithelialization (Goldfinger et al., 1999;Nguyen et al., 2000). Considering the pro-survival effects ofLN-5 discussed above, it is possible that adhesion to newlydeposited LN-5 also contributes to keratinocyte survival duringmigration into the provisional wound ECM. LN-5, α3β1 andα6β4 are also expressed at high levels in many invasivecarcinomas, suggesting possible roles for these adhesionproteins in promoting tumor cell invasion and survival, as well(Dajee et al., 2003; Felding-Habermann, 2003; Bartolazzi etal., 1994; Natali et al., 1993; Patriarca et al., 1998; Lohi et al.,2000; Pyke et al., 1995). Indeed, previous studies havesuggested that α6β4 promotes survival of normal ortransformed epithelial cells (Dowling et al., 1996; Weaver etal., 2002; Bachelder et al., 1999), possibly through activationof PI3K (Shaw et al., 1997). In contrast, the role of integrinα3β1 in regulating epithelial cell survival remains unclear.Aside from overlapping ligand-binding specificities, α3β1 andα6β4 appear to have distinct and separable functions inepidermal keratinocytes (Carter et al., 1990; DiPersio et al.,2000b; Nguyen et al., 2000), and the relative contributions ofthese two integrins to epithelial cell survival are likely to differboth in resting epithelia and during tissue remodeling.

To determine directly whether α3β1-mediated adhesionregulates keratinocyte survival, we cultured keratinocyte celllines derived from wild-type or α3-null mice on LN-5 ECM,and then compared them for susceptibility to apoptosis inducedby serum withdrawal. We demonstrate that the presenceof α3β1 inhibits proteolytic activation of caspase-3 andsuppresses apoptosis in serum-starved keratinocytes. We alsoshow that the pro-survival effects of α3β1 occur throughactivation of FAK and at least partly through MEK-dependentactivation of ERK. Our findings therefore reveal a novel rolefor α3β1 in suppressing caspase-3 activation and apoptosisthrough a MEK/ERK signaling pathway. Although α6β4 wasnecessary and sufficient for cell attachment to LN-5 in theabsence of α3β1, α6β4-mediated adhesion was not sufficientto suppress caspase-3 activation fully. Furthermore, blockingα6β4-mediated adhesion in cells that express α3β1 did not

induce caspase-3 activation, indicating that α3β1 is moreeffective than α6β4 in promoting keratinocyte survival. Ourfindings distinguish the survival promoting functions of thesetwo LN-5-binding integrins and illustrate the importance ofspecific integrin-ligand interactions for adhesion-dependentsurvival of epithelial cells.

Materials and MethodsMouse keratinocyte (MK) cell cultureThe MK+/+ cell line (MK-1.16) and MK–/– cell line (MK-5.4.6) werederived from keratinocytes isolated from wild-type or α3 integrinknockout mice, respectively (DiPersio et al., 2000a). MK–/– cells werestably transfected with a full-length human α3 cDNA (a gift fromMartin Hemler, Dana-Farber Cancer Institute, Boston, MA); α3-transfectants express high levels of α3β1 on the cell surface, asdescribed previously (DiPersio et al., 2000a). MK growth mediumconsisted of Eagle’s Minimum Essential Medium (EMEM;BioWhittaker, Walkersville, MD) supplemented with 4% FBS(BioWhittaker) from which Ca2+ had been chelated, 0.05 mM CaCl2,0.4 µg/ml hydrocortisone (Sigma, St Louis, MO), 5 µg/ml insulin(Sigma), 10 ng/ml EGF (Invitrogen Corporation, Carlsbad, CA),2×10–9 M T3 (Sigma), 10 units/ml interferon γ (INFγ; Sigma), 100units/ml penicillin and 100 µg/ml streptomycin (Invitrogen), and L-Glutamine (Invitrogen). Stocks of MK cell lines were maintained at33°C, 8% CO2, on tissue culture plates coated with 30 µg/mldenatured rat tail collagen (Cohesion, Palo Alto, CA). Forexperiments, MK cells were sub-cultured on LN-5 ECM preparedfrom the human squamous cell carcinoma line SCC-25 (Rheinwaldand Beckett, 1981), as described previously (DiPersio et al., 2000a).

TUNEL analysisMK cells were cultured in serum-free EMEM, 0.05 mM CaCl2, onLN-5 ECM for two days, then fixed in 4% paraformaldehyde.Apoptotic cells were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) using the ApoptosisDetection System, Fluorescein (Promega, Madison, WI) followed bydirect visualization on an Olympus BX60 fluorescence microscope.To quantify the number of apoptotic cells, flow cytometry of TUNEL-positive cells was performed according to the manufacturer’s protocol.For each condition, 10,000 cells were analyzed by flow and thepercentage of TUNEL-positive cells was determined. As a positivecontrol for detection of DNA fragmentation, cells were treated withDNase I before TUNEL. As a negative control, terminal transferasewas omitted from the TUNEL reaction.

Preparation of MK cell lysates for analysis of caspase-3MK cells were trypsinized from stock plates and resuspended inserum-free EMEM, 0.05 mM CaCl2. For most experiments, cells werepre-incubated in suspension culture for 90 minutes at 33°C, thenplated on LN-5 ECM at a sub-confluent density of approximately6.25×105 cells/35 mm well, or equivalent. One hour after plating,unattached cells were removed by gentle rinsing. To induce apoptosis,cells were cultured in serum-free EMEM, 0.05 mM CaCl2 for anadditional 24 or 48 hours, as indicated in the figure legends. Forexperiments in which MEK was inhibited, cells were treated with thepharmacological inhibitor U0126 (Calbiochem, San Diego, CA) at afinal concentration of 10 µM, or with an equivalent volume of DMSOas a control. U0126 was added to cells during the pre-incubationperiod before attachment to LN-5 ECM; for 48 hour time points,U0126 was replenished in the medium after the first 24 hours ofculture. For experiments in which integrin α6β4 was blocked, cellswere pre-treated 15 minutes before plating with the rat anti-α6monoclonal antibody GoH3 (BD Pharmingen, San Diego, CA) or

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with rat IgG2a isotype control antibody (BD Pharmingen) at aconcentration of 5 µg/ml, then cultured in the presence of 5 µg/mlGoH3 or control antibody for 24 hours. For experiments in which cellswere kept in suspension, cells were cultured in serum-free mediumfor 24 hours in wells coated with 1% agarose to prevent adhesion.Apoptotic cells that had detached during the 24 hour culture periodwere collected from the medium by centrifugation, and cell lysateswere combined for detached and attached cells. For α6β4-blockingexperiments and corresponding controls, only attached cells werelysed to focus on cells that were adhered through α3β1 or α6β4. Celllysates were prepared in Cell Lysis Buffer (1% Triton X-100, 20 mMTris pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 2.5 mMsodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4,1 µg/ml leupeptin, and 2 mM PMSF), then sonicated for 15 secondsand clarified by centrifugation at 20,800 g for 10 minutes. Proteinconcentrations were determined using the Pierce BCA Protein AssayKit (Pierce, Rockford, IL). Caspase-3 activation was monitored bywestern blot, as described below.

Adenoviral infection of MK cellsHA-tagged Ras V12 and β-galactosidase were cloned into pAdTrack,as described (Meadows et al., 2001). Replication-defective adenovirusencoding a GFP-FRNK fusion protein was a generous gift from DrAllen Samarel (Heidkamp et al., 2002). The day before infection,1.73106 MK–/– cells or 1.03106 MK+/+ cells were seeded ontocollagen-coated 10 cm dishes in serum-containing growth medium.MK cells were infected for 24 hours with adenovirus expressing eitherHA-tagged Ras V12 or β-galactosidase as a control (multiplicity ofinfection=70), or with adenovirus expressing GFP-FRNK or GFPas a control (multiplicity of infection=350). Infected cells weretrypsinized and sub-cultured on LN-5 ECM at a density ofapproximately 1×106 cells per 35 mm well, and cultured in serum-free EMEM to induce apoptosis, as described above. GFPfluorescence was visualized on an Olympus IX70 invertedmicroscope.

Analysis of adhesion-dependent signal transductionAdhesion-dependent signaling was assayed essentially as describedpreviously (Aplin and Juliano, 1999). For most experiments, MK cellcultures were serum-starved for 4-6 hours in serum-free medium(EMEM, 0.05 mM Ca2+, 0.5% heat-inactivated BSA). Cells were thenremoved from plates with trypsin, treated with 1 mg/ml trypsininhibitor and pelleted. Cells were washed once, resuspended in serum-free medium, and incubated in suspension at 33°C, 8% CO2, for 30minutes. Cells were then either kept in suspension as a control, orplated at sub-confluent densities onto LN-5 ECM and allowed toattach for times indicated in the figures. After incubation, MK celllysates were prepared in Cell Lysis Buffer and quantitated asdescribed above. Phosphorylation of ERK1/2 and FAK were assayedby western blot, as described below. ERK1/2 kinase activity wasassayed using an in vitro kinase assay to detect ERK-mediatedphosphorylation of a GST-Elk-1 recombinant fusion protein (p44/42MAP Kinase Assay Kit; Cell Signaling Technology, Beverly, MA).To assay phosphorylation of p130CAS, cell monolayers were lysed inmodified RIPA buffer (1% NP-40, 0.25% deoxycholate, 50 mM TrispH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 20 µg/mlaprotinin, 12.5 µg/ml leupeptin and 2 mM PMSF) and quantified.Aliquots of cell lysate (125 µg) were pre-cleared with anti-mouseIgG-conjugated agarose beads (Sigma), then immunoprecipitated with3 µl of mouse monoclonal anti-p130CAS antibody (TransductionLaboratories, Lexington, KY) followed by incubation with 30 µl ofanti-mouse IgG-agarose beads overnight at 4°C. Samples werewashed with RIPA buffer, resolved by reducing 10% SDS/PAGE, andtransferred to PVDF membranes (Bio-Rad, Hercules, CA) for westernblotting, as described below.

Western blottingEqual amounts of MK cell lysates (10 µg to 20 µg) were resolved byreducing 10% SDS/PAGE and transferred to nitrocellulosemembranes. For western blot, primary antibodies were used at thefollowing concentrations: rabbit polyclonal anti-caspase-3 (CellSignaling Technology), 1:1000; rabbit polyclonal anti-keratin 14(Covance Inc., Richmond, CA), 1:10,000; rabbit polyclonal anti-FAK(phospho-Tyr397) (BioSource International, Camarillo, CA), 1:1000;rabbit polyclonal anti-FAK (Upstate Biotechnology, Lake Placid,NY), 1:1000; rabbit polyclonal anti-phospho-ERK1/2 (Cell SignalingTechnology), 1:1000; rabbit polyclonal anti-ERK1/2 (Promega),1:5000; mouse monoclonal anti-p130CAS (TransductionLaboratories), 1:1000; rabbit polyclonal anti-GFP (Santa CruzBiotechnology, Santa Cruz, CA), 1:1000; mouse monoclonal anti-phospho-tyrosine 4G10 (Upstate Biotechnology), 1:1000; mousemonoclonal anti-HA-tag (Covance), 1:1000. Peroxidase (HRP)-conjugated secondary antibodies were used at the followingconcentrations: goat anti-rabbit IgG (Cell Signaling Technology),1:2000; goat anti-rabbit IgG (Pierce), 1:15,000; goat anti-mouse IgG(Pierce), 1:15,000. Chemiluminescence was performed using theSuperSignal Kit (Pierce).

ResultsMK cells that lack α3β1 integrin show increasedsusceptibility to apoptosis upon serum deprivationTo determine whether α3β1 integrin plays a role inkeratinocyte survival, we exploited keratinocyte cell linesderived from mice that were either wild-type (MK+/+ cells) orhomozygous for a null mutation in the gene for the α3 integrinsubunit (MK–/– cells) (DiPersio et al., 2000a). MK cells werecultured on LN-5 ECM for two days, either in the presence ofserum and growth factor/hormonal supplements to promotesurvival, or in the absence of serum and supplements to induceapoptosis. α3-null keratinocytes adhere to LN-5 ECM throughα6β4 integrin (DiPersio et al., 1997) (also shown in Fig. 3).Fluorescent staining for TUNEL-positive cells revealed verylittle apoptosis in cultures of MK+/+ cells attached to LN-5ECM under serum-free conditions (Fig. 1A,B). In contrast,TUNEL-positive cells were readily detectable in cultures ofα3β1-deficient MK–/– cells attached to LN-5 ECM underserum-free conditions (Fig. 1C,D). Restoration of α3β1integrin expression in MK–/– cells through stable transfectionwith a cDNA encoding the α3 integrin subunit reduced thenumber of apoptotic cells to levels seen in MK+/+ cells (Fig.1E,F). Calculation of the proportion of fluorescent cells fromat least 400 cells per culture revealed that 7.5% of MK–/– cellswere TUNEL-positive, compared with 0.5% of MK+/+ cellsand 1.0% of α3-transfected MK–/– cells.

To confirm our quantification of apoptotic cells, weperformed flow cytometric analysis of TUNEL-positive cellsin cultures of α3-null MK–/– cells and α3-transfected MK–/–

cells. α3-transfected MK–/– cells cultured in the presence ofserum did not contain a detectable population of TUNEL-positive cells (Fig. 1G, MKα3, +serum). About 1% ofuntransfected MK–/– cells were TUNEL-positive under theseconditions (Fig. 1G, MK–/–, +serum), suggesting that absenceof α3β1 results in only a slight increase in apoptosis in thepresence of serum. Serum deprivation caused only a smallinduction of apoptosis in α3-transfected MK–/– cells (Fig. 1G,MKα3, –serum). In contrast, serum deprivation inducedapoptosis in α3-null MK–/– cells to levels that were much

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higher than those seen in α3-transfected MK–/– cells (Fig. 1G,compare black bars). Almost 7% of α3-null MK–/– cells wereapoptotic after two days of adhesion to LN-5 ECM underserum-free conditions, compared with only 0.3% of α3β1-expressing MK cells (Fig. 1G), demonstrating that absence ofα3β1 caused a greater than 20-fold increase in the number ofapoptotic cells. Importantly, this approach provides a minimumestimate of the number of apoptotic cells, because DNAfragmentation is a relatively late event in apoptosis, and asignificant proportion of cells that have committed to apoptosismay not be detected by TUNEL-staining. The increase in

apoptosis seen in MK–/– cultures is significant, because evensmall changes in the proportion of apoptotic cells can havedevastating effects over time on tissue development orhomeostasis (Jacobson et al., 1997). These data demonstratethat absence of α3β1 significantly increases the sensitivity ofMK cells to apoptosis under serum-free conditions.

MK cells that lack α3β1 integrin show increasedactivation of caspase-3 upon serum deprivationCaspase-3 is an effector caspase involved in the execution ofapoptotic pathways in a variety of cell types, and its activationis a convenient and widely used readout of apoptosis(Nicholson, 1999). Caspase-3 is activated through proteolyticconversion of the full-length, inactive zymogen (35 kDa) toactivated 17 kDa and 12 kDa subunits (Nicholson et al., 1995).To determine whether keratinocyte apoptosis caused by absenceof α3β1 is accompanied by increased activation of caspase-3,we monitored cleavage of caspase-3 by immunoblot of MK celllysates with an antiserum that recognizes the 35 kDa pro-form,as well as the 17 kDa activated fragment and cleavageintermediates. The 35 kDa pro-form of caspase-3 was detectedeasily in both MK+/+ and MK–/– cells cultured on LN-5 ECMin the presence of serum, but cleaved caspase-3 wasundetectable under these conditions (Fig. 2A, +serum). MK cellculture in serum-free medium induced cleavage of caspase-3 tothe 17 kDa fragment and a cleavage intermediate of ~25 kDa,indicative of increased apoptosis. A considerably higher levelof caspase-3 cleavage occurred in MK–/– cells than in MK+/+

cells, as indicated by the higher ratio of cleaved to uncleavedcaspase-3 in the latter cells (Fig. 2A, serum-free). Stabletransfection of MK–/–cells with α3 integrin suppressed caspase-3 activation under serum-free conditions (Fig. 2A, serum-free,α3). Quantification of bands in Fig. 2A corresponding tozymogen and cleaved forms revealed that 79.0% of the totalcaspase-3 was cleaved in MK–/– cells, compared with 17.2% inMK+/+ cells and 18.4% in α3-transfected MK–/– cells. MK cellsthat were cultured in suspension for 24 hours showed highlevels of cleaved caspase-3 whether or not they expressed α3β1(Fig. 2B), indicating that the protective effects of α3β1expression require cell adhesion to LN-5 ECM. Taken together,these data demonstrate that α3β1-mediated adhesion to LN-5suppresses caspase-3 activation and subsequent apoptosis thatis induced by serum withdrawal.

Cell adhesion through α6β4 integrin is not required forMK cell survival on LN-5 ECM when α3β1 integrin isexpressedA considerable proportion of MK–/– cells that were adhered toLN-5 ECM under serum-free conditions remained negative forTUNEL staining over the time course of our survival assays(Fig. 1). Furthermore, MK–/– cells showed considerably higherlevels of activated caspase-3 when cultured in suspension thanthey did when attached to LN-5 ECM (Fig. 2). As adhesion ofα3-null mouse keratinocytes to LN-5 ECM is completelydependent on α6β4 integrin (DiPersio et al., 1997), theseobservations suggest that α6β4 can also contribute to MK cellsurvival. Indeed, previous studies have demonstrated a role forα6β4 in promoting survival of both normal and transformedepithelial cells (Bachelder et al., 1999; Weaver et al., 2002),


Fig. 1.Absence of α3β1 from keratinocytes leads to increasedapoptosis upon serum deprivation. Wild-type MK+/+ cells (A,B), α3-null MK–/– cells (C,D), or α3-transfected MK–/– cells (E,F) werecultured on LN-5 ECM in serum-free medium for two days.Apoptotic cells were identified by TUNEL staining (A,C,E);corresponding phase images are shown (B,D,F, respectively).Examples of apoptotic cells are indicated with arrows in C and D.(G) Flow cytometry was performed and the percent of TUNEL-positive cells was determined for MK–/– cells (MK–/–), or α3-transfected MK–/– cells (MKα3) grown in the presence (white bars)or absence (black bars) of serum.


and increased apoptosis was reported in the epidermis of β4-deficient mice (Dowling et al., 1996). To determine whetherblocking α6β4 increases apoptosis in α3β1-expressing MKcells, cells were cultured on LN-5 ECM in serum-free mediumfor 24 hours, then treated with either anti-α6 function blockingantibody GoH3 or with an isotype control antibody. Adhesionof α3-null MK–/– cells to LN-5 ECM was almost completelyinhibited by GoH3 (Fig. 3B), but not by control antibody (Fig.3A), confirming that adhesion to LN-5 ECM is mediated byα6β4 in these cells. We determined that cell surface levels ofα6β4 are the same in MK+/+ and MK–/– cells using surfaceiodination (DiPersio et al., 2000a) and flow cytometry (D.Choma and C. M. DiPersio, unpublished). Therefore, we usedthe same GoH3 treatment to block α6β4 in α3β1-expressingMK cells. Blocking α6β4 had no effect on cell adhesion orspreading of either MK+/+ cells (Fig. 3C,D) or α3-transfectedMK–/– cells (Fig. 3E,F), indicating that α3β1 was sufficient tomediate adhesion and spreading on LN-5 ECM. To comparelevels of apoptosis, cells were lysed and assayed by westernblot for cleavage of caspase-3. Blocking α6β4 with GoH3 didnot induce caspase-3 activation above background levels seenin IgG control-treated cells for either MK+/+ cells or α3-transfected MK–/– cells (Fig. 3G). Taken together, results fromFigs 2 and 3 indicate that α6β4-mediated adhesion to LN-5ECM may contribute to keratinocyte survival in the absenceof α3β1-mediated adhesion, but that it is not necessary formaximal survival of cultured keratinocytes that are adhered toLN-5 through α3β1.

MK cells that lack α3β1 integrin show reduced signalingthrough focal adhesion kinaseFocal adhesion kinase (FAK) has been shown to play animportant role in integrin-dependent cell survival (Frisch et al.,

1996; Frisch and Screaton, 2001). Auto-phosphorylation ofFAK at Tyr-397 is a critical initiating event in FAK-mediatedsignal transduction pathways (Cary and Guan, 1999). Todetermine whether increased apoptosis in MK–/– cells wascorrelated with reduced FAK signaling, we plated MK cells onLN-5 ECM in serum-free medium and assayed for FAKactivation by immunoblot with an antibody specific forphosphorylation at Tyr-397 (Fig. 4A, upper panel). Control

Fig. 2.Absence of α3β1 from keratinocytes leads to increasedcaspase-3 activation upon serum deprivation. (A) Wild-type MK+/+

cells (+/+ lanes), α3-null MK–/– cells (–/– lanes), or α3-transfectedMK–/– cells (α3 lanes) were cultured on LN-5 ECM for 24 hours inthe presence of serum (+serum) or in the absence of serum (serum-free). Cell lysates were assayed for caspase-3 activation by westernblot. (B) Cells were kept in suspension for 24 hours in the absence ofserum (serum-free, susp.), then assayed for caspase-3 activation as inA. Migratory positions of pro-caspase-3 and cleaved forms ofcaspase-3 are indicated. Results are representative of more than fourindependent experiments.

Fig. 3.α6β4-mediated adhesion is not required for keratinocytesurvival on LN-5 ECM in the presence of α3β1. α3-null MK–/– cells(A,B), wild-type MK+/+ cells (C,D), or α3-transfected MK–/– cells(E,F) were seeded onto LN-5 ECM in serum-free medium in thepresence of 5 µg/ml of anti-α6 blocking antibody GoH3 (B,D,F) orrat IgG2a isotype control antibody (A,C,E). Adherent cells werephotographed approximately two hours after plating. Note thatMK–/– cells fail to spread due to the lack of α3β1 (A), and GoH3completely eliminated MK–/– cell adhesion (B). (G) 24 hours afteradhesion, the cells shown in panels A and C-F were lysed andassayed for caspase-3 activation by western blot as described in Fig.2 (upper panels); cell lines and antibody treatments are indicatedabove the lanes. Filters were stripped and re-probed for keratin 14(lower panels, K14). Results are representative of two independentexperiments, one of which was performed in duplicate.

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blots for total FAK showed that FAK protein levels weresimilar under all conditions (Fig. 4A, lower panel). Asexpected, FAK activation in MK+/+ cells was adhesion-dependent because phospho-FAK was detected in adherentMK+/+ cells (Fig. 4A, LN-5 ECM, +/+ lanes), but it was barelydetectable in non-adherent MK+/+ cells (Fig. 4A, suspended,+/+ lanes). In contrast, FAK activation was reduced markedlyin α3β1-deficient MK–/– cells adhered to LN-5 ECM (Fig. 4A,LN-5 ECM, –/– lanes). Adhesion-dependent FAK activationwas completely restored in MK–/– cells transfected with humanα3 (Fig. 4A, α3 lanes). Phospho-FAK levels remainedsuppressed in α3-null cells, relative to α3β1-expressing cells,under serum-free conditions for 24 hours (Fig. 4B) or 48 hours(not shown), consistent with the time course of MK–/– cellapoptosis in our cell survival assays.

Tyrosine phosphorylation of the adaptor protein CAS servesas an additional readout for FAK activation, because CASbinds directly to FAK and is subsequently phosphorylated bySrc family kinases that bind to active FAK at phospho-Tyr-397(Cary and Guan, 1999). As expected, phosphorylation of CASwas also adhesion-dependent in MK+/+ cells (Fig. 4C, +/+lanes). In contrast, adherent MK–/– cells expressed barelydetectable levels of phospho-CAS (Fig. 4C, –/– lanes), whichwere completely restored upon transfection with α3 (Fig. 4C,α3 lanes). Interestingly, we observed lower levels of total CASprotein in α3-null MK–/– cells compared with MK+/+ cells orα3-transfected MK–/– cells (Fig. 4C, lower panel). Importantly,however, comparison of band intensities for total and phospho-CAS between α3-expressing MK cells and MK–/– cellsindicates that detectable CAS protein in MK–/– cells is

completely non-phosphorylated, reflecting reduced FAKfunction in these cells. Reduced levels of total CAS protein inMK–/– cells may reflect an additional level at which α3β1regulates FAK/CAS signaling, and the mechanism of thisregulation is the subject of a separate study. Taken together,results in Fig. 4 show that absence of α3β1 causes decreasedFAK signaling in MK cells adhered to LN-5 under serum-freeconditions, consistent with loss of FAK-mediated signalingpathways that promote epithelial cell survival.

Exogenous expression of FRNK, an inhibitor of FAKsignaling, induces MK cell apoptosis on LN-5 ECMFAK-related non-kinase (FRNK) is an autonomouslyexpressed product of the FAK gene that consists of only the C-terminal region of FAK and acts as a competitive inhibitor ofFAK-mediated signal transduction from focal adhesions (Caryand Guan, 1999). To determine the effects of inhibiting FAKfunction on keratinocyte survival, we infected wild-type MK+/+

cells with an adenovirus encoding a green fluorescent (GFP)-FRNK fusion protein, which was shown previously to disruptFAK signaling and induce anoikis in rat ventricular myocytes(Heidkamp et al., 2002). As a control, MK+/+ cells wereinfected at the same multiplicity of infection with anadenovirus that expresses GFP only. Expression of GFP andGFP-FRNK was confirmed by immunoblotting with anti-GFP(Fig. 5A). As shown previously for ventricular myocytes(Heidkamp et al., 2002), expression of GFP-FRNK in MK+/+

cells reduced FAK phosphorylation at Tyr-397 (data notshown). Infected cells were cultured on LN-5 ECM underserum-free conditions for 24 hours, and caspase-3 activationwas assayed by immunoblot. A higher proportion of caspase-3 was cleaved in GFP-FRNK-infected cells than in GFP-infected cells or in uninfected cells (Fig. 5B), indicating thatGFP-FRNK expression specifically induced apoptosis.

The apoptosis-inducing effects of GFP-FRNK expressionwere clearly evident upon microscopic analysis of infectedMK+/+ cells. Control GFP-infected cultures contained cellswith various levels of GFP expression, as determined byrelative fluorescence intensity (Fig. 5Ca). Although cultureunder serum-free conditions induced a rounded, apoptoticmorphology in a small proportion of these cells, the majorityof GFP-expressing cells maintained a spread morphologytypical of healthy MK cells on LN-5, including thoseexpressing high levels of GFP (Fig. 5Ca,b, arrowheads). Incontrast, cultures infected with GFP-FRNK showed a dramaticincrease in the number of cells with rounded, apoptoticmorphology (Fig. 5Cc,d). Many of these cells showedmembrane blebbing characteristic of apoptosis and haddetached from the substrate by 24 hours. Importantly, the vastmajority of GFP-FRNK expressing (i.e. fluorescent) cells wereapoptotic (Fig. 5Cc,d, arrowheads). A few uninfected (i.e. non-fluorescent) cells remained well spread over the course of theexperiment, providing an internal control (Fig. 5Cc,d, arrows).Taken together, results in Figs 4 and 5 suggest that α3β1-mediated FAK activation promotes keratinocyte survival.

α3β1 integrin mediates adhesion-dependent activationof MEK/ERK signalingA number of previous studies have implicated the MAP kinase


Fig. 4.α3β1 mediates adhesion-dependent activation of FAK/CASsignaling. Wild-type MK+/+ cells (+/+ lanes), α3-null MK–/– cells(–/– lanes), or α3-transfected MK–/– cells (α3 lanes) were kept insuspension (suspended) or adhered to LN-5 ECM under serum-freeconditions for the times indicated. (A,B) To assay levels of activatedFAK, cell lysates were immunoblotted with an antibody againstphospho-FAK (Tyr397) (pFAK, upper panel). Blots were strippedand reprobed for total FAK protein (FAK, lower panel). (C) To assaylevels of phosphorylated CAS, cell lysates were immunoprecipitatedwith anti-p130CAS monoclonal antibody, followed byimmunoblotting with monoclonal antibody 4G10 against phospho-Tyr (upper panel). Blots were stripped and reprobed for total CASprotein (lower panel).


ERK1/2 in the adhesion-dependent survival of keratinocytesand other epithelial cells (Jost et al., 2001; Gu et al., 2002;Frisch and Screaton, 2001). Although α3β1 has been shown toactivate ERK in some epithelial cells (Gonzales et al., 1999),a role for α3β1-mediated ERK activation in keratinocytesurvival has not been demonstrated. To determine whetherα3β1 regulates ERK activation in keratinocytes, MK+/+ cells,MK–/– cells, or α3-transfected MK–/– cells were kept insuspension or adhered to LN-5 ECM under serum-freeconditions. Cell lysates were then immunoblotted for theactivated forms of ERK with an antibody specific forphosphorylation on residues Thr202 and Tyr204 of p44/42ERK (Fig. 6A, upper panels), or for total ERK protein as acontrol (Fig. 6A, lower panels). In addition, ERK activity wasassayed using an in vitro kinase assay to detect ERK-mediatedphosphorylation of an Elk-1 substrate (Fig. 6B). Neitherphosphorylated ERK nor ERK activity was detected in MKcells that were held in suspension, despite high levels of ERKprotein in these cells (Fig. 6A,B, susp.). However, bothphosphorylated ERK and ERK activity were detected in wild-type MK+/+ cells 15 minutes after attachment to LN-5 ECM(Fig. 6A,B; LN-5 ECM, 15 min., +/+), demonstratingadhesion-dependent activation of ERK. In contrast, α3β1-deficient MK–/– cells contained considerably reduced levels ofphosphorylated ERK and ERK activity after adhesion to LN-5 ECM for 15 minutes (Fig. 6A,B; LN-5 ECM, 15 min., –/–).Stable transfection of MK–/– cells with α3 completely restoredboth ERK phosphorylation and ERK activity (Fig. 6A and B;LN-5 ECM, 15 min.,α3).

Basal levels of phosphorylated ERK were observed in MK–/–

cells adhered to LN-5 ECM, and these levels increasedsomewhat after overnight culture. However, phospho-ERKlevels remained considerably lower in MK–/– cells than in

α3β1-expressing MK cells even after 24 hours (Fig. 6A; LN-5 ECM, 24 hr.; also, see Fig. 7A). The delayed, low levels ofERK activation seen in MK–/– cells may occur in response togrowth factors or ECM ligands that are produced by thekeratinocytes during culture or that are present at low levels inthe LN-5 ECM preparation. Alternatively, basal levels of ERKactivation in MK–/– cells could be due to α6β4-mediatedadhesion, because this integrin has been reported to activateERK in keratinocytes (Mainiero et al., 1997). However, α6β4-mediated adhesion was not necessary to maintain high levelsof ERK activation in α3β1-expressing MK+/+ cells grown onLN-5 ECM for 24 hours, because blocking α6β4 function withGoH3 over this time course did not reduce the levels ofphosphorylated ERK (Fig. 6C). These results suggest thatα3β1, but not α6β4, is required for full activation of ERK inkeratinocytes adhered to LN-5.

Inhibition of MEK/ERK signaling reduces MK cellsurvival on LN-5 ECMBecause the absence of α3β1 from MK cells resulted in bothreduced ERK activation and increased apoptosis, we nextwanted to determine whether inhibition of ERK activationleads to increased apoptosis in MK cells. ERK isphosphorylated and activated by MAPK/ERK kinase (MEK),and ERK activation can be suppressed by treating cells withthe MEK-specific inhibitor U0126. MK+/+ cells or MK–/– cellswere cultured on LN-5 ECM in serum-free medium in thepresence of U0126, or DMSO as a control (Fig. 7). Treatmentwith 10 µM U0126 was sufficient to inhibit completely thehigh levels of ERK phosphorylation seen in MK+/+ cells, aswell as the basal levels of ERK phosphorylation seen in MK–/–

cells (Fig. 7A). Treatment with 10 µM U0126 also caused

Fig. 5.Exogenous FRNK expression induces keratinocyteapoptosis on LN-5 ECM. (A,B) Wild-type MK+/+ cells wereleft uninfected (uninfect. lanes) or infected with adenovirusexpressing either GFP-FRNK fusion protein (GFP-FRNKlanes) or GFP only as a control (GFP lanes). Cells were thensub-cultured on LN-5 ECM for 24 hours under serum-freeconditions. (A) Cell lysates were immunoblotted with anti-GFP antibody to confirm expression of exogenous proteins;the migratory positions of GFP (27-29 kDa) and GFP-FRNK(~68 kDa) are indicated. (B) Cells were assayed for activationof caspase-3 by immunoblotting as described in Fig. 2;migratory positions of pro-caspase-3 and cleaved caspase-3are indicated. A lower proportion of intact protein wasrecovered from GFP-FRNK expressing cells due to extensiveapoptosis (see panels Cc,d); therefore, a longer exposure ofthe GFP-FRNK lane is included to emphasize the proportionof caspase-3 that is cleaved. Filters were stripped and re-probed for keratin 14 (K14). (C) Fluorescence ofrepresentative fields of cells infected with either GFP (a) orGFP-FRNK (c). Corresponding phase images are shown in band d, respectively. Arrowheads point to examples of infected(i.e. fluorescent) cells. Note that most cells expressing GFPremained well spread with no signs of apoptosis (a,b), whilethe vast majority of GFP-FRNK expressing cells displayed anapoptotic phenotype (c,d). Arrows in panels c and d point touninfected cells, which do not show signs of apoptosis andserve as an internal control.

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increased cleavage of caspase-3 in MK+/+ cells (Fig. 7B),indicating a requirement for MEK/ERK signaling in cellsurvival. U0126 treatment had no obvious effect on cellspreading before the development of apoptotic cellmorphology (data not shown). In some experiments, weobserved that caspase-3 activation was also increased slightlyin MK–/– cells treated with U0126 (Fig. 7B), suggesting thatthe basal levels of ERK activity observed in these cells maycontribute to survival. Taken together, results in Figs 6 and 7reveal an important role for MEK/ERK signaling inkeratinocyte survival, and indicate that α3β1 mediates themajority of ERK-dependent survival.

Oncogenic Ras rescues α3-null MK–/– cells from anoikisGrowth in serum dramatically reduced the amount of apoptosisthat occurred in α3-null MK–/– cells (Fig. 1G and Fig. 2A),consistent with the established importance of growth factorreceptor activation in keratinocyte survival (Rodeck et al.,1997; Jost et al., 2001; Sibilia et al., 2000). Many solublegrowth factors and some integrins promote cell survival

through activation of the small GTPase Ras (Giancotti andRuoslahti, 1999). Activating mutations in Ras occur frequentlyin epithelial cancers (Shields et al., 2000), and oncogenic formsof Ras can confer resistance to anoikis in keratinocytes andother epithelial cells (Frisch and Francis, 1994; Rosen et al.,2000; Zhu et al., 2002). To determine whether oncogenic Rascan rescue α3-null MK–/– cells from anoikis, we expressed theconstitutively active Ras-V12 mutant in MK–/– cells and testedits ability to inhibit caspase-3 activation under serum-freeconditions. MK–/– cells were infected with an adenovirusexpressing either HA-tagged Ras-V12 (Meadows et al., 2001)or β-galactosidase as a control, then cultured on LN-5 ECMunder serum-free conditions for 24 hours. Ras-V12 expressionwas confirmed by immunoblotting for HA-tag (Fig. 8A, HA-Ras-V12 blot, lanes 5 and 6). MK–/– cells infected with controladenovirus showed low basal levels of phospho-ERK, similarto those seen in uninfected cells (Fig. 8A, pERK blot, lanes 2and 3). In contrast, expression of Ras-V12 caused increasedlevels of phospho-ERK compared with control cells (Fig. 8A,pERK blot, lanes 3 and 5). Ras-V12-mediated ERKphosphorylation was inhibited completely by treatment withU0126 (Fig. 8A, pERK blot, lanes 5 and 6), indicating that Ras-V12 activates MEK/ERK signaling in MK cells.

Under serum-free conditions, MK–/– cells infected withcontrol adenovirus showed high levels of cleaved caspase-3that were comparable to those seen in uninfected MK–/– cells(Fig. 8B, lanes 2 and 3). In contrast, MK–/– cells infected withRas-V12 adenovirus showed reduced activation of caspase-3that was comparable to background levels seen in uninfectedMK–/– cells grown in the presence of serum (Fig. 8B, comparelanes 5 and 1). These results show that oncogenic activation ofRas can suppress apoptosis in α3β1-deficient keratinocytes.

Although the presence of serum protected MK–/– cells fromapoptosis, as assayed by either TUNEL (Fig. 1B) or caspase-


Fig. 6.α3β1 is required for adhesion-dependent activation of ERK.(A) Wild-type MK+/+ cells (+/+ lanes), α3-null MK–/– cells (–/–lanes), or α3-transfected MK–/– cells (α3 lanes) were kept insuspension for 30 minutes or adhered to LN-5 ECM under serum-free conditions for 15 minutes or 24 hours, as indicated. To assayERK phosphorylation, cell lysates were immunoblotted withantibodies against phosphorylated ERK (pERK, upper panel) or totalERK (ERK, lower panel). (B) To assay ERK activity, lysates wereprepared from suspended cells or from cells adhered to LN-5 for 15minutes, then tested for in vitro phosphorylation of an Elk-1substrate (pELK), as described in the methods. (C) MK+/+ cells wereadhered to LN-5 ECM and cultured in serum-free medium for 24hours in the absence of antibody (no Ab), or in the presence of eitheranti-α6 blocking antibody (GoH3) or rat IgG2a isotype controlantibody (IgG), as described in Fig. 3. Cell lysates wereimmunoblotted with antibodies against phosphorylated ERK or totalERK, as described in A.

Fig. 7. MEK/ERK signaling is required for keratinocyte survival onLN-5 ECM. (A) Wild-type MK+/+ cells or α3-null MK–/– cells werecultured on LN-5 ECM in serum-free medium in the presence orabsence of the MEK inhibitor U0126 (10 µM), as indicated. Celllysates were assayed for phosphorylated ERK (pERK, upper panel)or total ERK (ERK, lower panel), as described in Fig. 6. Identicalresults were obtained in cells cultured for 24 or 48 hours. (B) Wild-type MK+/+ cells or α3-null MK–/– cells were cultured on LN-5 ECMin serum-free medium for 48 hours, in the presence or absence of 10µM U0126, as indicated. Cell lysates were assayed for caspase-3activation by western blot, as described in Fig. 2. Results arerepresentative of two independent experiments, one of which wasperformed in duplicate.


3 cleavage (Fig. 8B, lanes 1 and 2), only basal levels ofphospho-ERK were detected after 24 hours in the presence ofserum (Fig. 8A, lanes 1 and 2). These results suggest thatserum growth factors, in contrast with α3β1, can promote MKcell survival through pathways that do not require sustainedERK activity. Although Ras-V12 activated ERK in MK–/–

cells, Ras has numerous effectors and can initiate multipleintracellular signaling pathways (Marshall, 1996; Shields et al.,2000). Indeed, previous studies in MDCK epithelial cells haveshown that oncogenic Ras promotes survival through the PI3Kpathway, but not through the Raf/MEK/ERK pathway (Khwajaet al., 1997). Therefore, we wanted to determine the effects ofinhibiting MEK on the ability of Ras-V12 to suppressapoptosis in MK–/– cells. Treatment of infected cells with 10µM U0126 had no effect on the ability of Ras-V12 tocompletely inhibit caspase-3 activation (Fig. 8B, lanes 5 and6), even though this same treatment efficiently reduced ERK

phosphorylation to basal levels in the same cells (Fig. 8A, lanes5 and 6). These results indicate that sustained MEK/ERKsignaling was not required for Ras-V12-mediated survival, atleast over the time course of our assay, and suggest thatoncogenic Ras can stimulate keratinocyte survival throughpathways that are distinct from the MEK/ERK-dependentpathways induced by α3β1.

DiscussionPrevious studies have demonstrated that integrins can haveeither pro-survival or pro-apoptotic roles in the regulation ofcell survival (Stupack and Cheresh, 2002). Our findingsidentify a novel role for α3β1 integrin in promoting thesurvival of epidermal keratinocytes through a MEK/ERKsignaling pathway. This pro-survival role for α3β1 inkeratinocytes is clearly distinct from previously described rolesin cells of distinct origin or transformation status, where α3β1and/or its laminin ligands were shown either to promoteapoptosis (Seewaldt et al., 2001; Sato et al., 1999) or topromote cell survival in a MEK/ERK-independent manner (Guet al., 2002). Although it is known that LN-5 promoteskeratinocyte survival in the epidermis (Ryan et al., 1999;Nguyen et al., 2000), the relative roles of the LN-5-bindingintegrins α3β1 and α6β4 and the signaling pathways involvedare unclear. In the current study, we showed that while α3-nullkeratinocytes retained the ability to adhere to LN-5 throughintegrin α6β4, this adhesion did not fully protect cells fromapoptosis induced by serum deprivation. These observationsprovide a clear example of how integrin-mediated adhesion tolaminin per se may not be sufficient to inhibit anoikis ofepithelial cells completely, but that appropriate adhesionthrough a specific integrin receptor is necessary to protectepithelial cells fully from anoikis.

In the absence of α3β1, α6β4-dependent adhesion appearedto compensate partially to promote keratinocyte survival,consistent with previous reports that α6β4 has pro-survivalfunctions in keratinocytes and other epithelial cells (Dowlinget al., 1996; Bachelder et al., 1999; Weaver et al., 2002). Insupport of this conclusion, the majority of α3-null MK–/– cellsthat were adhered to LN-5 ECM through α6β4 remainednegative for TUNEL-staining in our survival assays (Fig. 1).Furthermore, MK–/– cells that were adhered to LN-5 throughα6β4 showed lower levels of activated caspase-3 than didunattached MK–/– cells (Fig. 2). Importantly, however,blocking α6β4-mediated adhesion with GoH3 did not increaseMK cell apoptosis when α3β1-mediated adhesion was intact(Fig. 3), indicating that α3β1-mediated adhesion, but notα6β4-dependent adhesion, was sufficient for maintainingmaximal levels of cell survival in our assays.

We cannot exclude the possibility that other integrins alsocontribute to the increased survival that we observed inadherent α3-null MK–/– cells compared with non-adherent MKcells. Indeed, while α6β4 was clearly required for adhesion ofMK–/– cells to LN-5 ECM, it is possible that adherent MK–/–

cells subsequently interact with fibronectin or other ECMproteins that are present in the LN-5 ECM preparation or thatare deposited by the MK cells themselves followingattachment. This possibility is consistent with our earlierobservations in mice lacking both α3β1 and α6β4, whereincreased apoptosis was seen only in regions of epidermis that

Fig. 8.Expression of activated Ras-V12 promotes MEK/ERK-independent survival of α3-null keratinocytes. α3-null MK–/– cellswere infected with adenovirus expressing HA-tagged Ras-V12 (Ras-V12 lanes) or with a control adenovirus expressing β-galactosidase(β gal lanes) then sub-cultured on LN-5 ECM for 24 hours in serum-free medium in the presence or absence of 10 µM U0126, asindicated. As controls, uninfected MK–/– cells were cultured in thepresence of serum (uninfect., +serum) or in serum-free medium(uninfect., serum-free). (A) To confirm expression of Ras-V12, celllysates were immunoblotted with anti-HA tag (HA-Ras-V12). ERKactivation in Ras V12-infected cells was assayed by probing parallelblots with anti-phospho-ERK (pERK) or total ERK (ERK), asdescribed in Fig. 6. (B) Cells were assayed for activation of caspase-3 by western blot, as described in Fig. 2; migratory positions of pro-caspase-3 and cleaved caspase-3 are indicated. Filters were strippedand re-probed for keratin 14 (K14). Results are representative of fiveindependent experiments for adenoviral infections; inhibitortreatments were included in two experiments.

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had already detached from the basement membrane (DiPersioet al., 2000b).

Both α3β1 and α6β4 are expressed constitutively duringepidermal development and in adult epidermis (Watt, 2002).While our results indicate that both of these integrins cancontribute to keratinocyte survival in culture, the relativecontributions of these two integrins to cell survival in quiescentepidermis remain unclear. It is possible that α6β4, rather thanα3β1, is the primary LN-5 receptor for maintainingkeratinocyte survival in embryonic and adult epidermis,because it is clearly the major receptor for epidermal adhesionin vivo (van der Neut et al., 1996; Georges-Labouesse et al.,1996; Dowling et al., 1996). As mentioned above, interactionswith ECM ligands other than LN-5 are likely to contribute tokeratinocyte survival in normal epidermis, because TUNEL-positive keratinocytes in mutant mice that lack both α3β1 andα6β4 were restricted to detached regions of epidermis(DiPersio et al., 2000b).

The potential importance of α3β1 in keratinocyte survivalbecomes more obvious when one considers the changes in cell-ECM interactions and the dramatic shifts in integrin functionthat occur during cutaneous wound healing. Activatedkeratinocytes at the wound edge show a redistribution of α6β4from the basal cell surface to the baso-lateral surface,presumably reflecting a requirement to disassemblehemidesmosomes and reduce stable adhesion in migratingkeratinocytes (Nguyen et al., 2000). Concurrently, α3β1redistributes mainly to the basal surface of keratinocytes in theleading edge of the wound, where it can bind to newlydeposited LN-5 and promote cell migration (Lampe et al.,1998; Nguyen et al., 2000). This switch from α6β4-LN-5adhesion to α3β1-LN-5 adhesion in leading edge keratinocytesalso occurs in in vitro scrape wounds of breast epithelial cells(Goldfinger et al., 1999) or keratinocytes (D. Choma and C. M.DiPersio, unpublished). We propose a model in whichincreased binding of α3β1 to LN-5 in activated keratinocytesat the wound edge becomes important for cell survivalfollowing the dissolution of α6β4-LN-5 adhesions (Fig. 9).This function may be most critical during the initial phase ofkeratinocyte activation, when leading edge cells have not yetengaged other ECM ligands present in the wound bed. In thismodel, α3β1-mediated adhesion to LN-5 would facilitate atleast two processes essential to re-epithelialization of thewound: (1) keratinocyte migration into the wound bed and (2)maintenance of keratinocyte survival during migration andECM remodeling. Importantly, our findings do not rule out pro-migratory or pro-survival roles for other integrin-ECMinteractions that occur during wound healing.

Activation of ERK signaling is important for adhesion-dependent survival in a number of cell types (Frisch andScreaton, 2001). In keratinocytes, a MEK/ERK-dependentsurvival pathway can be induced by EGF receptor (EGFR)activation (Jost et al., 2001). In the current study, we show thatα3β1 integrin can also stimulate MEK/ERK-dependentsurvival. Two major mechanisms whereby integrins activatethe Ras-ERK signaling cascade are through FAK activation andthrough the Fyn/Shc pathway (Giancotti and Ruoslahti, 1999).FAK can be activated by most integrins, including α3β1. Incontrast, only a subset of integrins can activating the tyrosinekinase Fyn, which activates Ras pathways through recruitmentof the Shc and Grb2 adaptor proteins (Wary et al., 1996;

Giancotti and Ruoslahti, 1999). Interestingly, α3β1 was amongthose integrins that did not activate the Fyn/Shc pathway (Waryet al., 1996), suggesting that α3β1-dependent ERK activationprobably occurs through a FAK-mediated pathway. Indeed, ithas been well established that integrin-mediated FAK signalingcan lead to ERK activation (Schlaepfer et al., 1997). Consistentwith this idea, we showed that α3β1 stimulates both FAKactivation and ERK activation in keratinocytes, and thatinhibition of either FAK function through FRNK over-expression or ERK signaling through MEK inhibition leads toincreased apoptosis.

As we did not assay FAK or ERK activation withinindividual cells, we were unable to determine whether reducedFAK or ERK signaling occurred in all cells of α3-null MK–/–

cultures, or whether a subpopulation of cells retainedFAK/ERK signaling despite the absence of α3β1. Indeed, alarge fraction of MK–/– cells remained TUNEL-negative for upto 48 hours (Fig. 1), suggesting that there is heterogeneityamong α3-null keratinocytes regarding their sensitivity toapoptosis. Future experiments using in situ approaches toassess the phosphorylation or sub-cellular localization of FAKand ERK within individual cells should help to distinguishbetween these possibilities.


Fig. 9.A model for α3β1-mediated keratinocyte survival at thecutaneous wound edge. (A) In quiescent epidermis, α6β4 (blackintegrin) is polarized to the basal cell surface in hemidesmosomes,where it mediates stable adhesion of keratinocytes to LN-5 in thebasement membrane. α6β4 also contributes to maintenance ofkeratinocyte survival, along with other ECM receptors (see text fordiscussion); α3β1 may contribute to survival, as well. (B) Duringepidermal wound healing, activated keratinocytes at the wound edgedisassemble hemidesmomes, and α3β1 (grey integrin) redistributesto the basal surface where it binds to newly deposited LN-5. Inaddition to promoting cell migration (Nguyen et al., 2000), α3β1-LN-5 interactions at the wound edge may contribute to keratinocytesurvival following the loss of α6β4-mediated adhesion.


There are several potential mechanisms whereby α3β1-mediated activation of FAK could lead to MEK/ERK-dependent keratinocyte survival. For example, FAK/CASinteractions may play a role in cell survival, because CAS/Crkcoupling and ERK activation have been linked to suppressionof apoptosis in some cells (Cho and Klemke, 2000). Inaddition, FAK interaction with the Grb2-mSOS complex leadsto activation of the Ras/Raf/MEK/ERK cascade (Schlaepfer etal., 1994). Another possibility is that activation of FAK leadsto activation of PI3K, which in turn can activate PAK. ActivePAK can then enable a Ras/Raf/MEK/ERK signaling cascadeby phosphorylating Raf-1 at a site that is necessary for itsactivation by Ras (King et al., 1998; Giancotti and Ruoslahti,1999; Eblen et al., 2002). Consistent with a role for PI3K inkeratinocyte survival, treatment of α3β1-expressing MK cellswith the PI3K inhibitor LY294002 also induced caspase-3activation (data not shown). However, PI3K also promotesERK-independent cell survival through activation of the kinaseAKT (Frisch and Screaton, 2001), and further experimentationis required to determine whether PI3K and ERK promote MKcell survival through overlapping or distinct pathways.

Integrin-dependent changes in cell shape and cytoskeletalintegrity can also play a key role in regulating cell survival(Giancotti and Ruoslahti, 1999; Frisch and Screaton, 2001).For example, the nuclear localization of ERK can be regulatedby integrin-ECM interactions (Aplin et al., 2001) and may playa role in survival of some cell types (Lai et al., 2002). Indeed,adhesion-dependent changes in the actin cytoskeleton thatreduce ERK nuclear localization can lead to decreasedtranscription of genes that promote cell survival (Schulze et al.,2001). Keratinocytes that lack integrin α3β1 adhere to LN-5efficiently through integrin α6β4, but they spread poorly anddisplay defects in the actin cytoskeleton that are evident bothin cultured cells and in vivo (DiPersio et al., 1997; Hodivala-Dilke et al., 1998; DiPersio et al., 2000a). α3-null kidneycollecting duct cells display similar defects in cytoskeletalorganization (Wang et al., 1999). Therefore, the ability of α3β1to activate ERK and promote cell survival may be due, at leastin part, to the ability of this integrin to promote cell spreadingon LN-5 and organize the actin cytoskeleton, a function whichα6β4 cannot fulfill. Future experiments will directly test theimportance of cell spreading for activation of MEK/ERKsurvival pathways in keratinocytes.

We thank Andrew Aplin for critical reading of the manuscript, andLivingston Van De Water for helpful discussions. We also thank AllenSamarel for providing adenovirus encoding GFP-FRNK fusionprotein, and Patrick Bryant for preparation of adenovirus. Thisresearch was supported by a grant from the National Institutes ofHealth to C.M.D. (R01CA84238), and a grant from the NationalInstitutes of Health to K.P. (R01CA081419). S. G. Shome wassupported by a post-doctoral training grant from the National Instituteof General Medical Sciences (NIH-T32-GM-07033). J. Lamar and V.Iyer were supported by a pre-doctoral training grant from the NationalHeart, Lung, and Blood Institute (NIH-T32-HL-07194).

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β1 integrin promotes keratinocyte cell survival through ... · through a mek/erk signaling...

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