B1+EGF-InitiatedInvasivePotentialinTransformedHuman ... · important mechanism in TGF-B1 +...

TGF-B1 + EGF-Initiated Invasive Potential in Transformed Human

Keratinocytes Is Coupled to a Plasmin/MMP-10/MMP-1–Dependent

Collagen Remodeling Axis: Role for PAI-1

Cynthia E. Wilkins-Port,1Qunhui Ye,

1Joseph E. Mazurkiewicz,

2and Paul J. Higgins

1

1Center for Cell Biology and Cancer Research and 2Center for Neuropharmacology and Neuroscience, Albany Medical College,Albany, New York

Abstract

The phenotypic switching called epithelial-to-mesenchymaltransition is frequently associated with epithelial tumor cellprogression from a comparatively benign to an aggressive,invasive malignancy. Coincident with the emergence of suchcellular plasticity is an altered response to transforminggrowth factor-B (TGF-B) as well as epidermal growth factor(EGF) receptor amplification. TGF-B in the tumor microen-vironment promotes invasive traits largely through reprog-ramming gene expression, which paradoxically supportsmatrix-disruptive as well as stabilizing processes. ras-trans-formed HaCaT II-4 keratinocytes undergo phenotypic changestypical of epithelial-to-mesenchymal transition, acquire acollagenolytic phenotype, and effectively invade collagen type1 gels as a consequence of TGF-B1 + EGF stimulation in athree-dimensional physiologically relevant model system thatmonitors collagen remodeling. Enhanced collagen degrada-tion was coupled to a significant increase in matrix metal-loproteinase (MMP)-10 expression and involved a proteolyticaxis composed of plasmin, MMP-10, and MMP-1. Neutraliza-tion of any one component in this cascade inhibited collagengel lysis. Similarly, addition of plasminogen activator inhi-bitor type 1 (SERPINE1) blocked collagen degradation as wellas the conversion of both proMMP-10 and proMMP-1 to theircatalytically active forms. This study therefore identifies animportant mechanism in TGF-B1 + EGF-initiated collagenremodeling by transformed human keratinocytes and pro-poses a crucial upstream role for plasminogen activatorinhibitor type 1–dependent regulation in this event. [CancerRes 2009;69(9):4081–91]

Introduction

Epithelial tumor progression, from a relatively indolent to amore aggressive phenotype, is frequently accompanied by acqui-sition of a plastic phenotype reminiscent of a developmentalprogram called epithelial-to-mesenchymal transition (EMT; ref. 1).This process is typified by loss of normal epithelial properties(e.g., cell polarity and junctional complexes) and a gain inmesenchymal traits (expression of vimentin and smooth muscleactin and enhanced cell motility; ref. 2). Although essential during

embryonic development, EMT is relatively limited in the adultorganism, occurring during wound repair or, more atypically, inadvanced pathologies largely in response to specific growth factorsassociated with tumor progression (3–5). Epidermal growth factor(EGF) receptor amplification and an altered cellular response totransforming growth factor-h (TGF-h), for example, accompanythe progression of epithelial tumor cells from a benign phenotypeto an aggressive, metastatic carcinoma (6–8). During thispathologic EMT, and despite increased autocrine/paracrine ex-pression of TGF-h, cells become refractory to the normally growth-suppressive effects of TGF-h family members. Mouse models ofmultistage skin carcinogenesis support the concept that TGF-hfunctions as a tumor suppressor in the early stages of benigngrowth; in late stage tumors, however, TGF-h accelerates malignantconversion (6). Down-regulation of TGF-h receptors, alterations inTGF-h-dependent Smad signaling components, or a combinationof both appear to contribute to this functional switch (8, 9).

TGF-h likely promotes tumor-invasive properties throughexpression of genes that encode stromal remodeling proteins,which paradoxically support matrix-disruptive as well as stabilizingprocesses. Structural extracellular matrix proteins such as fibro-nectin and collagen (10, 11) are up-regulated by TGF-h in conjunc-tion with their proteolytic regulators, including plasminogenactivator inhibitor type 1 (PAI-1; ref. 12) and matrix metallopro-teinase (MMP)-1, -3, -9, -10, -11, and -13 (13–15). Similar to TGF-h,EGF-dependent signaling contributes to up-regulation of severalMMPs (15–19) and is enhanced through increased receptor levelsin various cancers (7).

Stringent temporal and spatial controls on MMP activation areessential for maintaining tissue homeostasis. In addition totranscriptional regulation, MMP-dependent activities are alsomodulated through proteolytic activation (20). Proteolytic cascadeswithin the pericellular environment are largely initiated throughconversion of matrix plasminogen to the broad-spectrum proteaseplasmin, which, in turn, directly converts several proMMPs to theiractive form and triggers a positive feedback mechanism for MMPactivation (21). Indeed, regulation of plasminogen activation maysubstantially affect MMP-dependent remodeling processes andthereby cellular invasive traits. The ability of TGF-h and/or EGFstimulation to also up-regulate PAI-1 in several cell types (thisstudy; refs. 12, 22) provides a potential mechanism for upstreamnegative regulation or titration of the MMP cascade.

The immortalized adult human keratinocyte cell line HaCaT (23)harbors genetic changes similar to those that accompany prog-ression of a normal keratinocyte to an invasive squamous cell car-cinoma (24). Stimulation of activated ras-expressing HaCaT II-4cells with TGF-h alone or, more effectively, a combination of TGF-h1 and EGF promotes a highly plastic phenotype typified by lossof E-cadherin and de novo synthesis of N-cadherin and vimentin

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Paul J. Higgins, Center for Cell Biology and CancerResearch, Albany Medical College, MC-165, 47 New Scotland Avenue, Albany, NY12208. Phone: 518-262-5651; Fax: 518-262-5669; E-mail: [email protected].

I2009 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-09-0043

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(4, 25). The ability of TGF-h1 and/or EGF to elicit EMT-relatedresponses such as these in a more physiologically significantmodel, however, has not been explored. This article describes theuse of a three-dimensional collagen gel system to evaluateproteolytic events associated with TGF-h1 + EGF-stimulated EMTand collagen invasion by HaCaT II-4 keratinocytes. The invasivepotential of these keratinocytes was coupled to a plasmin/MMP-10/MMP-1–dependent collagen-remodeling axis, and a role for PAI-1as a critical upstream regulator of this remodeling process wasestablished.

Materials and Methods

Reagents. Vitrogen (Cohesion Technologies) or PureCol (Inamed;

Advanced BioMatrix) provided sources of bovine collagen type 1. Both

products yielded comparable results and were used interchangeably. Where

indicated, FITC-labeled collagen type 1 (Sigma-Aldrich) or DQ FITC-labeledcollagen type 1 (Molecular Probes/Invitrogen) were added to monitor gel

degradation. Recombinant human TGF-h1 (R&D Systems) was used at

1 ng/mL and recombinant human EGF (Upstate/Millipore) at 10 ng/mL.

Plasminogen, aprotinin, E-64, amiloride, o-phenylenediamine dihydrochlor-ide, hydrogen peroxide, and phosphate-citrate buffer were from Sigma-

Aldrich. Recombinant human PAI-1 protein and a2-antiplasmin were from

Calbiochem. GM6001 was obtained from Chemicon/Millipore and AG1478was obtained from Biosource International/Invitrogen. Immunofluores-

cence antibodies included tubulin (clone DM1A; Sigma), vimentin (LN6 Ab-

1; Calbiochem), E-cadherin (clone 36), and N-cadherin (clone 32) from BD

Biosciences; MMP-10 from Santa Cruz Biotechnology; and 4¶,6-diamidino-2-phenylindole stain, phalloidin-594, and Cell Tracker CMTPX from

Molecular Probes/Invitrogen. For Western blotting, antibodies to MMP-1

and MMP-10 or biotinylated antibodies to MMP-1 and MMP-10 were

obtained from R&D Systems; antibodies to actin or extracellular signal-regulated kinase 1/2 were from Santa Cruz Biotechnology. Antibodies

against human plasminogen and PAI-1 (neutralizing) were from American

Diagnostica. Neutralizing antibodies to MMP-1 and MMP-10 were obtainedfrom R&D Systems. A polyclonal antibody to PAI-1 was used for ELISA and

immunofluorescence. Unless otherwise indicated, Alexa Fluor 488 (green)

or 594 (red) conjugates (Molecular Probes/Invitrogen) were used for

immunocytochemistry detection; horseradish peroxidase conjugates (PierceBiotechnology) were used for Western blot and ELISA analyses.

Cell culture. HaCaT II-4 keratinocytes were maintained in low-glucose

DMEM supplemented with 10% fetal bovine serum (Life Technologies/

Invitrogen). Cells were harvested with trypsin/EDTA, washed with PBS,and seeded onto collagen in serum-free Advanced DMEM overnight before

stimulation with TGF-h1 and/or EGF. Phenol red-free medium (Life Tech-

nologies/Invitrogen) was used in fluorescence assays.

Collagen gel-based studies. Collagen type 1 was neutralized accordingto the manufacturer’s instructions using 10� PBS and 0.1 N NaOH and then

diluted to 1.8 mg/mL (unless stated otherwise) with DMEM. Gels were

polymerized in 48-well tissue culture plates (150 AL), OptiCell Chambers(1.0 mL; USA Scientific), MatTek glass bottom dishes (200 AL; MatTek), or

onto cell culture inserts (20 AL, 700 Ag/mL) at 37jC for 2 to 3 h. For

OptiCell-based invasion assays, 2 � 105 cells were added in 1 mL Advanced

DMEM to gels polymerized vertically within the chamber. Cells were viewedon an inverted Olympus IX70 by laying the chamber on its side and images

were captured with Image-Pro Plus software. For Transwell invasion

assays, 1 � 105 cells were seeded onto thin collagen gels in Advanced

DMEM. Invading cells were visualized with 4¶,6-diamidino-2-phenylindoleand counted in four random fields. For collagen gel dissolution assays,

5 � 104 cells were seeded onto polymerized gels in Advanced DMEM;

plasminogen (5-20 Ag/mL) was added 24 to 48 h post-growth factorstimulation for up to 24 h. Cells were pretreated with inhibitors or neu-

tralizing antibodies as indicated. In experiments with anti-PAI-1 neutral-

izing antibody, time (not shown) and/or plasminogen concentration were

reduced to capture differences in the state of dissolution with increas-

ing antibody concentration. Cells on intact gels were fixed in 3% para-formaldehyde before viewing on an inverted Olympus IX70 microscope.

To quantify collagen degradation, FITC-labeled collagen (25 Ag/mL) was

incorporated into polymerized gels. Cells were stimulated in phenol red-free

DMEM and incubated with plasminogen for 7.5 h and 100 AL conditionedmedium was removed for fluorescence spectroscopy using Synergy HT

microplate reader equipped with KC4 software (BioTek Instruments).

Microscopy.HaCaT II-4 cells were seeded onto collagen-coated coverslips

(50 Ag/mL) or onto collagen gels polymerized in MatTek glass-bottomeddishes. Following treatment, cells were fixed in 3% paraformaldehyde,

permeabilized, blocked, and incubated with primary and secondary

antibodies for 1 h each. Coverslips were mounted using ProLong Gold with

4¶,6-diamidino-2-phenylindole and viewed on an Olympus BX61 microscopewith Image-Pro Lab software version 3.6.5. or an Olympus IX70 inverted scope

with Image-Pro Plus software. For visualization of collagen digestion, cells

were seeded onto coverslips coated with collagen type 1 (50 Ag/mL) + DQFITC-labeled collagen type 1 (25 Ag/mL).

Protein analysis. Collagen gels were digested with collagenase D; cells

were separated from digested collagen by centrifugation and lysed in a

50 mmol/L HEPES containing 150 mmol/L NaCl, 1% Triton X-100, 0.5%deoxycholate, 1% NP-40, 10 mmol/L NaF, 1 mmol/L orthovanadate,

and protease inhibitors; and extracts were probed for N-cadherin and

E-cadherin. Western blot analysis of MMP-1 and MMP-10 used conditioned

medium from cells stimulated with TGF-h1 + EGF followed by incubationwith plasminogen F inhibitors (as indicated). The Human MMP Antibody

Array 1.1 from RayBio (RayBiotech) was used to detect changes in MMP

protein levels in conditioned medium. For measurement of PAI-1 levels byELISA, 1.2 � 105 cells seeded on collagen type 1-coated, BSA-blocked wells

were maintained under serum-free conditions for 6 h, pretreated with

AG1478, as indicated, and then stimulated with TGF-h1 and/or EGF

overnight. Cells were fixed with 3% paraformaldehyde, permeabilized,blocked, and incubated with PAI-1 polyclonal antibodies for 1 h followed by

a horseradish peroxidase-conjugated secondary antibody. Cell layer PAI-1

was detected by colorimetric assay using an o-phenylenediamine dihydro-

chloride substrate and measured by spectrophotometer at 492 nm. Resultswere normalized to cell number by measuring the level of cell-associated

crystal violet staining.

Statistical analysis. The Student’s t test for two samples, assumingunequal variance, was used to compare conditions within a group. Two-

tailed values with P V 0.05 were considered significant.

Results

HaCaT II-4 keratinocytes stimulated with TGF-B1 + EGFundergo EMT and invade collagen gels in a MMP-dependentmanner. To recapitulate events associated with cutaneous EMT ina relevant context, p53 mutant, Ha-ras-expressing human kerati-nocytes (HaCaT II-4 cells; ref. 23) were cultured on a collagen coat(Fig. 1A) or onto a more physiologically related three-dimensionalcollagen gel (Fig. 1B and C) and simultaneously treated with TGF-h1 and EGF to mimic the increased TGF-h expression/EGFreceptor signaling characteristics of late-stage tumors. Under theseconditions, EGF stimulation was mitogenic, whereas TGF-h1maintained its growth-suppressive activity even in the presenceof EGF (Supplementary Fig. S1). Whereas HaCaT II-4 coloniescultured on a three-dimensional collagen gel appeared morecompact than cells cultured on a collagen coat, dually stimulatedcells displayed traits typical of an EMT (2) on both substratesincluding increased scattering (Fig. 1A and B ; tubulin), de novovimentin, and N-cadherin expression (Fig. 1A–C) as well as loss ofE-cadherin at cell-cell junctions (Fig. 1A–C). To our knowledge,these observations represent the first evidence that EMT-relatedevents take place in human keratinocytes cultured in a three-dimensional environment.

Cancer Research

Cancer Res 2009; 69: (9). May 1, 2009 4082 www.aacrjournals.org




Figure 1. TGF-h1 + EGF costimulationinduces EMT-like plasticity in HaCaTII-4 cells cultured on a three-dimensional collagen matrix. A and B,immunocytochemical detection ofEMT-associated processes in TGF-h1and/or EGF stimulated HaCaT II-4 cellscultured on either a thin collagen coat (A )or a three-dimensional collagen gel (B ).Cells were stimulated 48 h (tubulin)and 60 h (vimentin, E-cadherin, andN-cadherin) with TGF-h1 and/or EGF.Green, vimentin, N-cadherin, andE-cadherin; red, cytoskeletal actin;yellow, colocalization between these twofluorochromes. Tubulin labeling (A and B )illustrates morphologic differences foreach condition. Bar , 100 Am (A) or50 Am (B). Bar , 50 Am for vimentin,N-cadherin, and E-cadherin (A and B).B, enlarged insets show the absenceor presence of vimentin, E-cadherin,or N-cadherin. C, Western blot analysisof N-cadherin and E-cadherin fromHaCaT II-4 cells cultured on collagengels and stimulated with TGF-h1 and/orEGF for 48 h. (0), nonstimulated cells.In all experiments, TGF-h1 is 1 ng/mLand EGF is 10 ng/mL.

PAI-1 Regulates MMP-10–Initiated Collagen Remodeling





Because the phenotypic plasticity characteristic of EMT maypromote tumor metastasis (5), it was important to evaluate theinvasive capacities of TGF-h1 + EGF-treated HaCaT II-4 cells.TGF-h1 + EGF enhanced cell invasion and migration into acollagen matrix, as assessed in both OptiCell and Transwell three-

dimensional systems (Fig. 2A and B), and was effectively attenu-ated by the broad-spectrum MMP inhibitor GM6001 (Fig. 2C).Confocal imagery of cells seeded onto thin collagen gels (Sup-plementary Fig. S2) also supported the observation that TGF-h1 +EGF promoted HaCaT II-4 collagen gel invasion. Enhanced

Figure 2. TGF-h1 + EGF stimulationenhances MMP-dependent collagen gelremodeling and invasion by HaCaT II-4cells. A, OptiCell tissue culture chamberswere used to visualize collagen gelinvasion 6 d post-stimulation withTGF-h1 and/or EGF. The diagramillustrates the chamber setup and servesas an orientation tool. Two magnificationlevels of the same chamber are presented.Bar, 100 Am. B, modified Boyden chamberassays were used to quantify invasionfollowing TGF-h1 + EGF stimulationovernight. Insets, representativequantitative fields. Bar, 100 Am. Mean FSE of multiple fields. *, P < 0.01. C,HaCaT II-4 cells seeded onto collagen gelsin the OptiCell system were stimulated withTGF-h1 + EGF 6 h before the addition ofGM6001 to inhibit MMP activity. Imageswere taken 2 d post-stimulation with TGF-h1 + EGF. Bar, 100 Am. D, evaluation ofcollagen type 1 degradation by TGF-h1and/or EGF-stimulated cells culturedon FITC-labeled DQ collagen-coatedcoverslips. Plg, plasminogen treatment(5 Ag/mL) F GM6001 (10 Amol/L) added30 min before the addition of plasminogen(17 h). Red, cytoskeletal actin; blue,nuclear staining with 4¶,6-diamidino-2-phenylindole; green, cleaved collagentype 1; grayscale, right, cleaved collagenalone. Bar , 10 Am. In all experiments,TGF-h1 is 1 ng/mL and EGF is 10 ng/mL.

Cancer Research





collagen type 1 degradation following TGF-h1 + EGF treatment wasconfirmed by using collagen matrices prepared from a quenchedFITC-labeled collagen type 1 substrate that fluoresces on cleavage(Fig. 2D). Together, these data indicate that TGF-h1 and EGF playintegral roles in modulating HaCaT II-4-based collagenase activity,effectively supporting collagen gel invasion.TGF-B1 + EGF treatment enhances collagen degradation via

a plasmin/MMP dependent mechanism. Physiologic control ofpericellular proteolysis occurs primarily through the regulation ofplasminogen activation at the cell surface, which, in turn,contributes to downstream extracellular MMP activity (Fig. 3A).To explore the mechanisms associated with plasmin-basedproteolysis in a cutaneous model, exogenous plasminogen wasadded to HaCaT II-4 cultures stimulated with TGF-h1 and/or EGF,as HaCaT II-4 cells secrete only low levels of plasminogen (Fig. 3A).Dissolution of the supporting collagen matrix accompanied TGF-h1 + EGF + plasminogen treatment (Fig. 3B), a process significantlyreduced by the plasmin inhibitors aprotinin and a2-antiplasmin,but not with the cysteine protease inhibitor E-64 (Fig. 3C and D),which affects cellular cathepsins. Inhibition of MMP activity withGM6001 also blocked plasmin-initiated collagen degradation,confirming a role for MMPs in the remodeling process (Figs. 2Dand 3C and D). Control experiments revealed no evidence ofadverse effects arising from treatment with these inhibitors (datanot shown). Cells that were detached as a result of collagengel dissolution, in fact, reattached to tissue culture wells within24 h (Supplementary Fig. S3).

Plasmin-dependent collagen degradation was quantified throughthe release of digested FITC-labeled collagen type 1 (Fig. 3D).Stimulation with either TGF-h1 or EGF independently significantlyincreased collagen type 1 proteolysis within 7.5 h of plasminogenaddition. This by itself, however, was insufficient to trigger adistinguishable loss in the fibrillar network even at later timepoints (Fig. 3B). Stimulation with the combination of TGF-h1 +EGF clearly evoked a more substantial proteolytic response(Fig. 3D) that resulted in dissolution of the polymerized gel within20 h (Fig. 3B), suggesting that the collagenolytic activity promotedby combining these two growth factors in the presence of plas-minogen surpassed any threshold limitations.TGF-B1 + EGF-stimulated collagen gel dissolution occurs via

a plasmin/MMP-10/MMP-1–dependent axis. Because plasmin-dependent collagen degradation has been linked to MMP-13 up-regulation in mouse keratinocytes (26), it was necessary to assesswhether MMP-13 might be involved in TGF-h1 + EGF-initiatedcollagenolysis. Similar to what has been reported previouslyfor other HaCaT variants (13, 27), TGF-h1 stimulation signifi-cantly increased MMP-13 levels in HaCaT II-4 cells (Fig. 4A).Only a modest elevation in MMP-13 was evident, however, fol-lowing coincubation with TGF-h1 + EGF (Fig. 4A). MMP-10, incontrast, was substantially increased under these conditions(Fig. 4A and B).

ProMMP-10 is a plasmin substrate (21), and whereas activeMMP-10 does not cleave collagen type 1 directly, it does activatethe collagenase MMP-1 (20). Subsequent to TGF-h1 + EGFstimulation, MMP-10 activation was evident by 4 h post-plasmin-ogen addition and complete by 24 h (Fig. 5A, top), whereas thekinetics of MMP-1 activation closely followed the conversion ofMMP-10 to a catalytic form (Fig. 5A, bottom). To confirm the role ofMMP-10 in collagen matrix degradation, plasminogen was addedto TGF-h1 + EGF-stimulated HaCaT II-4 cultures in the presenceof increasing concentrations of neutralizing antibodies to either

MMP-1 or MMP-10. MMP-1 inhibition prevented plasminogen-dependent collagen dissolution (Fig. 5B, top). Importantly, neutral-ization of MMP-10 activity also blocked plasminogen-initiatedcollagen degradation (Fig. 5B, bottom), supporting a plasmin/MMP-10/MMP-1–dependent axis in matrix remodeling. A notable de-crease in the level of active MMP-1 was also consistently evidentfollowing MMP-10 neutralization (Fig. 5C). Despite residual levelsof active, likely plasmin-generated MMP-1, this activity by itselfwas insufficient, however, to trigger gel dissolution on MMP-10inhibition (Fig. 5B).PAI-1 functions as an upstream regulator of a MMP-10–

initiated collagenolytic phenotype. Similar to MMP-10, PAI-1expression in HaCaT II-4 cells is increased in response to TGF-h1stimulation (12), whereas the combination of TGF-h1 + EGFsynergistically enhanced PAI-1 protein levels (Fig. 6A ; Supplemen-tary Fig. S4A). Despite the inability of EGF alone to increase PAI-1levels in this system, enhanced PAI-1 synthesis resulting fromTGF-h1 + EGF stimulation, as well as from TGF-h1 alone, wasattenuated by inhibition of EGF receptor signaling with AG1478(Fig. 6A). Similar results were evident in human dermal fibroblasts(Supplementary Fig. S4B) and kidney epithelial cells (22), empha-sizing the generality of EGF receptor involvement in TGF-h1–dependent PAI-1 production.

PAI-1, through its inhibition of urokinase-type plasminogenactivator, is critical for regulating the generation of pericellularplasmin. It was important, therefore, to assess the effect of PAI-1 oncollagen gel dissolution. Blocking urokinase-type plasminogenactivator activity with the inhibitor amiloride, or by adding astable recombinant form of PAI-1 protein (N150H, K154T, Q319L,and M345I; ref. 28), completely inhibited collagen gel dissolution(Fig. 6B). Addition of PAI-1 protein also effectively blocked con-version of MMP-10 and MMP-1 to their active forms (Fig. 6C).In contrast, neutralization of endogenous PAI-1 with function-blocking antibodies accelerated both collagenolysis (Fig. 6B)and activation of MMP-10 and MMP-1 (Fig. 6C). Collectively,these results indicate that, in the physiologically relevant settingof a complex three-dimensional collagen environment, PAI-1regulates MMP-10–initiated collagenolytic activity (Fig. 6D). Akey factor in this model is the ability of active MMP-10 tosuperactivate MMP-1, creating a plasmin/MMP-10/MMP-1 proteo-lytic axis that enhances collagen type 1 degradation and facilitatescollagen gel invasion.

Discussion

MMPs are integral components of a complex stromal remodel-ing program designed to modulate matrix integrity, releasebioactive fragments, growth factors, and cytokines from matrixconstituents, and enhance cell motility (29). Amplified MMP ex-pression appears linked to increased tumor aggressiveness, meta-stasis, and poor patient survival (30). Not surprisingly, recently,studies also implicate several MMPs, including MMP-3,-7, -9, and -28, in directly triggering EMT-related processes (30).The combination of TGF-h and EGF, which effectively promotesEMT, also up-regulates certain MMPs synergistically includingMMP-1, -3, -9, -10, and -14 (17, 18), posing some interestingquestions regarding potential mechanisms that support amplifica-tion of EMT-associated events.

An acute collagenolytic phenotype linked to plasmin-dependentactivation of stromelysin-2 (MMP-10) emerged in response tocostimulation of HaCaT II-4 keratinocytes with TGF-h1 and EGF






Figure 3. Plasmin inhibition blocks MMP-dependent collagen gel dissolution. A, Western blot of endogenous plasminogen from HaCaT II-4 and HepG2 cellscultured on collagen (serum-free) overnight and then stimulated F TGF-h1 + EGF for 8 h. Two samples of conditioned medium are presented for each. Purifiedhuman plasminogen was included as a control (Plg ). The diagram illustrates mechanisms associated with plasmin-based pericellular proteolysis and points ofinhibition; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor. B, HaCaT II-4 cells cultured on collagen type 1 gelswere stimulated with TGF-h1 and/or EGF overnight followed by treatment with plasminogen (20 Ag/mL; 20 h). Arrowheads, areas enlarged for insets, highlightingfibrillar characteristics of intact collagen gels versus conditions following TGF-h1 + EGF + plasminogen treatment. Bar , 100 Am. C, TGF-h1 + EGF-stimulatedHaCaT II-4 cells cultured on collagen gels and treated with plasminogen (20 Ag/mL) F serine protease inhibitors aprotinin or a2-antiplasmin (a2-AP ), cysteine proteaseinhibitor E-64, or the MMP inhibitor GM6001. Bars , 100 Am. D, quantification of collagen digestion by HaCaT II-4 cells stimulated with TGF-h1 and/or EGF(24 h) F plasminogen (20 Ag/mL) and inhibitors aprotinin (10 Amol/L) or GM6001 (20 Amol/L) was achieved by measuring the release of FITC-labeled collagentype 1 fragments into the medium 7.5 h post-plasminogen addition. Means F SE of duplicate wells, representative of multiple experiments. *, P < 0.05. In allexperiments, TGF-h1 is 1 ng/mL and EGF is 10 ng/mL.

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and was coincident with collagen gel invasion. MMP-10, which isgenerally limited to epithelial cells (15, 19), has broad substratespecificity, targeting proMMP-1, -7, -8, -9, and -13 as well as col-lagen types III, IV, and V, gelatin elastin, fibronectin, proteoglycans,and laminin (20, 21). Rigorous control over MMP-10 levels andactivation are likely critical, therefore, for normal cutaneoushomeostasis. MMP-10, in fact, is not evident in intact skin but isexpressed during cutaneous injury repair where it localizes tomigrating keratinocytes at the wound edge, suggesting that itspresence may facilitate invasive behavior (31).

Similar to what has been observed in other systems (15, 18,19, 27), MMP-10 and MMP-1 were up-regulated in HaCaT II-4 cellsseeded onto collagen gels and stimulated with TGF-h1 and/or EGF.Both proenzymes are plasmin substrates; however, following MMP-10 inhibition, the residual level of active, likely plasmin-generatedMMP-1 (Fig. 5C) was insufficient by itself to trigger collagengel dissolution (Fig. 5B). These data likely reflect the establishedability of MMP-10 to ‘‘superactivate’’ MMP-1 and enhance its col-lagenase activity 7- to 10-fold over that observed with plasminalone (19). Given these parameters, neutralization of MMP-10activity would have quenched this hypercollagenase activity and,as observed, impeded collagen degradation. Similar results regard-ing MMP-10–dependent superactivation of MMP-1, -8, and -13 inan arthritis-based model have been reported but not linked toplasminogen activation (32). Consequently, this article is the first toshow a plasmin/MMP-10/MMP-1–dependent collagen remodelingaxis and establish its relevance in a keratinocyte-based three-dimensional model.

Clearly, MMP-10 activity can have significant stromal con-sequences, particularly in a cutaneous environment, irrespectiveof its level of up-regulation. Amplified MMP-10 expression does,

however, accompany the progression of several epithelial cancers,including squamous cell carcinomas of the head and neck andesophagus (33, 34). Increased MMP-10 expression also occurs incolorectal carcinoma (35), breast cancer (36), prostate cancer(37), and lymphoma (38). It localizes to cells at the invasive frontof renal cell carcinomas and signals a lower survival ratecompared with patients with MMP-10–negative tumors (39).Similarly, tumor MMP-10 levels predict poor survival in non-smallcell lung cancer (40).

Like MMPs, lysosomal proteinase cathepsins are also associatedwith tumor cell invasion, particularly the cysteine proteinasescathepsins L and B, which degrade collagen type 1 and activateMMP-1, respectively (41). Inhibition of cysteine cathepsins had noeffect, however, on collagen gel dissolution in the HaCaT II-4model, whereas serine proteinase blockade effectively attenuatedcollagen degradation. These data emphasize a critical role foractive plasmin, and not cathepsins, in the initiation of collagendegradation by TGF-h1 + EGF-stimulated cells and are consistentwith observations regarding the ability of TGF-h to down-regulatecathepsins (42).

Previously, intermediates other than MMP-10, including MMP-13, have been associated with linking keratinocyte-based collagentype 1 dissolution and plasminogen activation (26, 43). Studiessuggest, however, that contrary to observations in primary humankeratinocytes, MMP-13 expression is, in fact, correlated withtransformation of human keratinocytes and enhanced in thesecells, including HaCaT derivatives, following stimulation with TGF-h1 (13, 27). Our data in a three-dimensional system also indicatethat TGF-h1 stimulation alone increases the level of MMP-13;however, the combination of TGF-h1 + EGF did not produce thiseffect. Costimulated cells instead exhibited a robust induction of

Figure 4. TGF-h1 + EGF stimulation increases MMP-10 levels in HaCaT II-4 cells cultured on collagen. A, evaluation of downstream MMP targets by proteinmicroarray analysis of conditioned medium from HaCaT II-4 cells cultured on collagen gels and stimulated with TGF-h1 + EGF for 30 h. B, immunofluorescencemicroscopy of HaCaT II-4 cells cultured on collagen-coated coverslips (50 Ag/mL) and stimulated with TGF-h1 and/or EGF (60 h). Green, MMP-10; cytoskeletal actin(red) was utilized for cell morphology. Bar , 10 Am. In all experiments, TGF-h1 is 1 ng/mL and EGF is 10 ng/mL.






MMP-10. This disparity may be due, in part, to differences amongthe ras-HaCaT variants in MMP expression programs (44), TGF-h/EGF-related receptor cross-talk promoting EGF-dependent down-regulation of TGF-h1–enhanced MMP-13 levels (18, 45), or culturein two-dimensional versus a more complex three-dimensionalstromal-equivalent system. The potential for EGF to counteract aTGF-h1–dependent increase in MMP-13 production reinforces thecomplexity of this process and presents some intriguing questionsfor future investigation regarding the regulation of these matrix-modifying enzymes in cancer progression.

Consistent with recent observations regarding receptor crosstalkand synergy (17, 18), PAI-1 levels increased synergistically followingTGF-h1 + EGF treatment of HaCaT II-4 cells on a collagensubstrate (Fig. 6A). During tumor progression, synergistic ampli-fication of PAI-1 would, in effect, inhibit a disproportionate level of

stromal degradation and, in doing so, facilitate cell migration bypreserving stromal architecture as well as by interacting with thelow-density lipoprotein-related receptor (46). Up-regulation of bothMMP-10 and PAI-1 in conjunction with a TGF-h1 + EGF-stimulatedEMT, therefore, promotes an environment conducive to the acqui-sition of an invasive phenotype. Indeed, like MMP-10, PAI-1expression is up-regulated in various cancers where its presenceis associated with poor patient outcome (47, 48). The incidenceof stromal PAI-1 is an important factor in determining tumorprogression, reflecting its capacity to stabilize the microenviron-ment and promote tumor vascularization (49, 50).

Temporal and spatial balance between extracellular componentsthat reorganize tissue architecture is a significant aspect oftumor progression. This study has identified an importantproteolytic axis for regulating collagen type 1 degradation in a

Figure 5. MMP-10 and MMP-1 directplasmin-dependent collagen geldissolution. A, Western blot analysisto detect levels of active MMP-10 orMMP-1 (bottom band of each doublet)in conditioned medium from HaCaTII-4 cells cultured on collagen FTGF-h1 + EGF for 24 h and incubated Fplasminogen (5 Ag/mL) for 2-24 h.B, HaCaT II-4 cells cultured on collagengels and stimulated with TGF-h1 + EGFfor 24 h were treated F plasminogen(20 Ag/mL) F neutralizing antibodies toMMP-1 or MMP-10 for 8 h. IgG servesas control for nonspecific antibody-basedeffects. Bar , 100 Am. C, Western blotanalysis of conditioned medium fromHaCaT II-4 cells cultured on collagen,stimulated with TGF-h1 + EGF (24 h),followed by plasminogen treatment(5 Ag/mL) for the indicated times F aneutralizing antibody to MMP-10(30 Ag/mL). Extracellular signal-regulatedkinase (ERK ) represents a loadingcontrol for each blot. Representative ofmultiple experiments. In all experiments,TGF-h1 is 1 ng/mL and EGF is 10 ng/mL.

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Figure 6. PAI-1 regulates MMP-10/MMP-1–dependent collagen gel dissolution by HaCaT II-4 cells. A, cell-based ELISA for PAI-1 in TGF-h1 and/or EGF-stimulatedHaCaT II-4 cells cultured on collagen-coated tissue culture plastic (50 Ag/mL). AG1478 was added 30 min before stimulation with TGF-h1 and/or EGF (15 h). PAI-1 wasdetected by colorimetric assay (see Materials and Methods). *, P < 0.05. B, effects of urokinase-type plasminogen activator inhibitors amiloride and PAI-1 or aneutralizing antibody to PAI-1 on collagen gel dissolution by TGF-h1 + EGF-stimulated HaCaT II-4 cells incubated with plasminogen (20 Ag/mL). Plasminogenwas reduced (10 Ag/mL) to capture differences in dissolution levels with increasing concentrations of anti-PAI-1 antibody. Bar , 100 Am. C, Western blot analysis forMMP-10 and MMP-1 activation in conditioned medium from TGF-h1 + EGF-stimulated HaCaT II-4 cells cultured on collagen and incubated with plasminogen(5 Ag/mL) F a PAI-1–neutralizing antibody or PAI-1 protein for 2 to 24 h. D, proposed mechanistic model for TGF-h1 + EGF-enhanced plasmin-dependent collagenmatrix remodeling and its contribution to the evolution of an invasive phenotype (see text for discussion). In all experiments, TGF-h1 is 1 ng/mL and EGF is 10 ng/mL.






three-dimensional environment. The data provided are consistentwith a model (Fig. 6D) in which transformed keratinocytes, inresponse to TGF-h1 + EGF in the microenvironment, up-regulateproMMP-10, which is converted to its active form in the presenceof plasmin and can subsequently superactivate the catalytic activityof MMP-1. This process results in a plasmin/MMP-10/MMP-1–dependent proteolytic axis that effectively enhances collagen type 1degradation and facilitates collagen gel invasion. PAI-1 plays acrucial role in this paradigm through its ability to counter excessivecollagen degradation and maintain stromal integrity for cellmigration. Identification of critical components involved inmanaging the rate and level of collagen type 1 degradation may

have far reaching implications for therapeutic targeting ofcutaneous pathologies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Received 1/7/09; revised 3/3/09; accepted 3/6/09; published OnlineFirst 4/21/09.Grant support: NIH grant GM57242 (P.J. Higgins) and NIH training grant T32-

HL07194 (C.E. Wilkins-Port).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

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2009;69:4081-4091. Published OnlineFirst April 21, 2009.Cancer Res Cynthia E. Wilkins-Port, Qunhui Ye, Joseph E. Mazurkiewicz, et al. Axis: Role for PAI-1

Dependent Collagen Remodeling−Plasmin/MMP-10/MMP-1 Human Keratinocytes Is Coupled to a

1 + EGF-Initiated Invasive Potential in TransformedβTGF-

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