Active matrix metalloproteinase-2 promotes apoptosis of hepatic stellate cells via the cleavage of...

BAS IC STUDIES

Activematrixmetalloproteinase-2promotesapoptosisofhepatic stellatecells via the cleavage of cellularN-cadherinStephen N. Hartland1, Frank Murphy2, Rebecca L. Aucott1, Armand Abergel2, Xiaoying Zhou2, Julian Waung2,Nishit Patel2, Catherine Bradshaw1, Jane Collins3, Derek Mann3, R. Christopher Benyon2� and John P. Iredale1�

1 MRC/University of Edinburgh Centre for Inflammation Research, Edinburgh, UK

2 Liver Group and Mucosal Immunology Group, Division of Infection, Inflammation & Repair, Southampton University, Southampton, UK

3 Liver Group, Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK

Keywords

apoptosis – hepatic stellate cell – matrix

metalloproteinase – N-cadherin

Abbreviations

HSC, hepatic stellate cell; M2i, MMP-2

inhibitor I; MMP, matrix metalloproteinase;

PBS, phosphate-buffered saline; SF, serum-

free conditions; TBS, Tris buffer saline; TIMP,

tissue inhibitor of metalloproteinases;

ZVAD-fmk, benzyloxycarbonyl–

Val–Ala–Asp–fluoromethylketone.

Correspondence

J. P. Iredale, University of Edinburgh, MRC CIR,

47, Little France Crescent, Edinburgh EH16 4TJ,

UK

Tel: 144 0 131 242 6559

Fax: 144 0 131 242 6578

e-mail: [email protected]

Received 25 June 2008

Accepted 25 May 2009

DOI:10.1111/j.1478-3231.2009.02070.x

AbstractBackground and Aims: Hepatic stellate cells (HSC) are known to synthesiseexcess matrix that characterises liver fibrosis and cirrhosis. Activated HSCexpress the matrix-degrading matrix metalloproteinase enzymes (MMPs)and their tissue inhibitors (TIMPs). During spontaneous recovery fromexperimental liver fibrosis, the expression of TIMP-1 declines and hepaticcollagenolytic activity increases. This is accompanied by HSC apoptosis.In this study, we examine a potential mechanism whereby MMP activitymight induce HSC apoptosis by cleaving N-cadherin at the cell surface.Results: N-cadherin expression was upregulated in human HSC duringactivation in culture. Addition of function-blocking antibodies or a peptidetargeting the extracellular domain of N-cadherin, to cultured HSC, promotedapoptosis. During apoptosis, there was cleavage of N-cadherin into20–100 kDa fragments. MMP-2 became activated early during HSC apoptosisand directly cleaved N-cadherin in vitro. Addition of activated MMP-2 toHSCs in culture resulted in enhanced apoptosis and loss of N-cadherin.Conclusions: Together, these studies identify a role for both N-cadherin andMMP-2 in mediating HSC apoptosis, where N-cadherin works to provide acell survival stimulus and MMP-2 promotes HSC apoptosis concomitant withN-cadherin degradation.

The role of hepatic stellate cells (HSC) both in develop-ment and spontaneous resolution of liver fibrosis canbe considered a paradigm for solid organ fibrosis andwound healing in the body. Studies of experimentalfibrosis, including those in the liver, suggest that duringspontaneous resolution, there is a decrease in the fibro-genic cell population that is mediated by apoptosis(1–4). In recovery, HSC apoptosis accompanies matrixdegradation and is concomitant with increased matrix-degrading metalloproteinase activity. In contrast, duringprogressive fibrosis, the presence of excess tissue inhibi-tors of metalloproteinases (TIMPs) inhibit matrixmetalloproteinase (MMP) activity and prevents matrixdegradation, allowing the accumulation of matrixcomponents produced by HSCs.

The underlying stimulus for HSC apoptosis duringrecovery is unknown. However, a pro-apoptotic role of

MMPs is supported by a number of studies (5–7). Indeed,an investigation of transgenic mice expressing a range ofproteolytic enzymes demonstrated that mammary glandepithelial cells, which produced an auto-activated MMP-3(stromelysin-1), underwent unscheduled apoptosis dur-ing pregnancy. However, the same strain of mice, bred toalso express excess tissue inhibitor of metalloproteinase-1(TIMP-1), did not demonstrate an enhanced propensityfor apoptosis (8). One mechanism by which pericellularproteolysis may influence apoptosis is through cadherinmodification. Cadherins consist of a large family oftransmembrane glycoproteins that are responsible formediating Ca21-dependent, homophilic, cell–cell adhe-sion (9). Their primary role in adhesion renders cadher-ins essential for many processes such as cell growth,differentiation, proliferation and survival (10) as well astissue formation (11) and the maintenance of tissueintegrity in the adult organism (12–14). E-cadherin hasbeen shown to be targeted by MMP-3 in mammary�Contributed equally.

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epithelial cells (15–17) and is cleaved by metalloprotei-nases after induction of apoptosis (18). Furthermore,MMP inhibitors reduce shedding of both E- and VE-cadherin (19), ameliorate N-cadherin degradation (20),while increasing fibroblast adhesion through the stabi-lisation of focal adhesion contacts and increased cad-herin function (21). Together, these studies indicate thatMMPs modify adhesion molecules and cell–cell inter-action.

A fate of programmed death has been proposed as adefault pathway for cells unless they receive continuoussurvival signals such as those from cell–cell and cell–matrix interactions (22, 23) as well as growth factors (24,25). Indeed, disruption of cadherin adhesion initiatesapoptosis both in normal and in cancerous cells (20, 26,27). Blockade of N-cadherin has been shown to promoteapoptosis of a number of cell types (23, 20, 28–30), whileeither administration of growth factor or transfection ofthe N-cadherin peptide can rescue cells from the inducedapoptotic fate (30, 23). In addition, intercellularN-cadherin adhesion has been shown to increase theexpression of anti-apoptotic Bcl-2 protein (31) withincreased PI-3 kinase phosphorylation of prosurvivalAkt and subsequent Bad phosphorylation (31, 23).Furthermore, studies investigating N-cadherin interac-tions at the cell surface and signal activation through thefibroblast growth factor receptor (FGFR) clearly illustratethe significance and diversity of N-cadherin signalling inthe maintenance of cell phenotype and fate (30–34).

During their activation to a myofibroblastic pheno-type by culture on plastic, HSC express abundant 72 kDaproMMP-2 and also activate this enzyme to its 66 and62 kDa forms (35). However, our studies show that underthese conditions, MMP-2 activity is highly suppresseddue to the co-expression of TIMPs by HSC (36). Duringexperimental induction of apoptosis, however, the activ-ity of MMP-2 in HSC-conditioned medium increases(37), implicating MMP-2 activity as a potential mediatorof HSC apoptosis. However, the pericellular substratesthat promote HSC survival, and that may be modified ordegraded by MMP-2, remain undetermined.

In this study, we demonstrate that disruption of N-cadherin function increases HSC apoptosis, an effectaugmented by MMP-2. We show evidence of N-cadherincleavage and degradation, following the induction ofapoptosis, and identify MMP-2 as a potential mediatorof this proteolysis.

Experimental procedures

Carbon tetrachloride-induced liver injury

An experimental model of liver fibrosis was established bya twice-weekly, intraperitoneal injection of carbon tetra-chloride (CCl4) (0.1ml/g) in cohorts of C57B6 micefor 4–8 weeks. Livers were harvested at peak fibrosis(24 h following the final injection of CCl4). Sex- and age-matched normal, untreated livers were also harvested foruse as controls in individual experiments.

Isolation of human and rodent hepatic stellate cells

Human HSC were extracted from normal human liverspecimens, removed from macroscopically normal marginsof liver resected from patients with colonic metastaticdisease, as described previously (38, 39). Extracted HSCwere activated to a myofibroblastic phenotype after 7–14days of culture on plastic. Primary human HSC were usedfor experimentation following activation. Cells were cul-tured in Dulbecco’s modified Eagle’s medium (Bio Whit-taker, Wokingham, UK) in the presence of 16% fetal calfserum, penicillin, streptomycin and gentamycin (Gibco,Paisley, UK). Rat and murine HSC were extracted andcultured as described previously (2) from normal and CCl4-treated fibrotic mouse liver. Local ethical approval wasobtained for all studies with human tissue, and UK homeoffice approval was obtained for studies using rodents.

Reverse transcriptase-polymerase chain reaction

N-cadherin expression was observed in normal and injuredmouse livers and HSC using a conventional reverse tran-scriptase-polymerase chain reaction (RT-PCR). One mi-crogram of total RNA was reverse transcribed in a reactionmix containing 4ml of 5� reaction buffer, 2ml dithiothrei-tol (DTT) (0.1 M), 1ml dNTPs (100mM), 2mg of randomhexamers, 1ml (200 U) superscript II reverse transcriptaseand 1ml (40 U) RNAse inhibitor (all Amersham, Buck-inghamshire, UK). Sense and antisense primers forN-cadherin (50-GGCGTCTGTGGAGGCTTCTGGTGAA-30 and 50-GTGATGACGGCTGTGGCTGTGTTTGA-30 re-spectively), following an amplification cycle of 901C for2 min, then 35 cycles of 941C for 30 s, 601 for 30 s and 681Cfor 1 min, followed by a final extension of 721C for 5 min,were used to create a 1.1 kb amplicon. A GAPDH controlwas produced using sense and antisense primers(50-TGTGAACGGATTTGGCCGTA-30, 50-AAGCAGTTGGTGGTGCAGGA-30 respectively) to create a 440 bp am-plicon. PCR products were run on a 2% agarose gel stainedwith ethidium bromide.

Stimulation of hepatic stellate cell apoptosis andexamination of nuclear morphology, by acridine orange

Previous work by our group has used cycloheximide for theinduction of HSC apoptosis (40). Furthermore, the use ofgliotoxin in an animal model of liver fibrosis demonstratedits antifibrogenic potential (41). As a natural progression ofour previous work, we have cultured HSC in a serum-freemedium using gliotoxin (1.5mM) and cycloheximide(50mM) to induce apoptosis. Culture of cells in a mediumcontaining serum was used as a negative apoptotic control.The two different apoptogens were used to cross check thatthe observations made during apoptosis were independent.Examination of nuclear morphology, following stainingwith acridine orange, was used to determine the effect of amouse monoclonal, anti-N-cadherin blocking antibody(clone GC-4, Sigma, Gillingham, USA) and a blockingpeptide (HAVDI, Sigma Genesis, Haverhill, UK) on

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Hartland et al. Active MMP-2 promotes apoptosis of hepatic stellate cells

cultured human HSC. The optimum concentration of theHAVDI peptide, used in experiments (100–400mg/ml), wasdetermined following dose–response studies with acridineorange. Parallel experiments were also conducted usingcycloheximide (50mM), a known apoptotic stimulus forHSC. Also, the effect of the selective synthetic MMP-2inhibitor, cis-9-octadecenoyl-N-hydroxylamide (42)(MMP-2 inhibitor I, Calbiochem, Nottingham, UK) (con-centration range 0.17–17mM; KI = 1.7mM for MMP-2),was studied during HSC apoptosis induced by cyclohex-imide exposure (50mM). After a 4-h incubation at 371C,the nuclear morphology of the cells was examined underblue fluorescence, following the addition of acridine or-ange (final concentration 1mg/ml). The apoptotic indexwas calculated and expressed as a percentage, by dividingthe number of apoptotic cells with the total number of cellsobserved in the random high-power field.

Determination of caspase-3 activity

Caspase-3 activity of cell extracts was determined asdescribed previously (40). Apoptosis was quantified,according to the manufacturer’s instructions, using acolorimetric assay for caspase-3 activity (Calbiochem).Data are expressed as pmol pNA/min/mg protein extract.

Western blotting for N-cadherin

Western blot analysis of N-cadherin expression in humanHSC was undertaken using two different antibodies: (1)mouse monoclonal to an intracellular epitope of N-cadher-in (Clone 3B9, Zymed Laboratories Inc., San Francisco, CA,USA) and (2) mouse monoclonal to the N-terminal por-tion of the extracellular domain of N-cadherin (Clone GC-4, Sigma). Cellular protein extracts were prepared usinglysis buffer (50 mM HEPES, 1 mM DTT, 0.1 mM EDTAand 0.1%CHAPS, pH 7.4). The protein concentration ofextracts was measured using the Coomassie blue-bindingassay (BioRad, Hemel Hempstead, UK) and equal amounts(10mg of protein/well) were subjected to electrophoresis on12% sodium dodecyl sulphate-polyacrylamide gel electro-phoresis (SDS-PAGE) gel. Following electrophoresis, sam-ples were electrotransferred onto polyvinylidine difluoridemembranes. Membranes were blocked for 2 h in 5% non-fat dry milk in tris-buffered saline (TBS). Membranes wereincubated overnight at room temperature with the primaryantibody (1:100) or with non-immune IgG (as negativecontrol) in TBS. Membranes were then washed (3�) for15 min in TBS10.1% Tween (TBST) before the addition ofthe secondary antibody (rabbit anti-mouse IgG horseradishperoxidase, 1:2000 dilution) in TBS containing 0.5% non-fat dry milk for 1 h at room temperature. Followingincubation, membranes were washed in TBST (2�) for10 min, followed by distilled water for 10 min. Reactivebands were identified using enhanced chemiluminescence(ECLplus, Amersham, UK) and autoradiography accordingto the manufacturer’s protocol.

Assay of matrix metalloproteinase-2 degradation ofrecombinant human N-cadherin/Fc chimera

Recombinant human N-cadherin/Fc chimera (R&D sys-tem, Abingdon, UK) was used to study the effect of activeMMP-2 on the extracellular domain of N-cadherin. Thechimeric protein consists of the extracellular domain ofhuman N-cadherin (amino acid residues 1–724) fused tothe carboxy-terminal 6X histidine-tagged Fc region ofhuman IgG, via a polypeptide linker. The theoreticalmass of the chimeric protein is 89.4 kDa; however,glycosylation increases the mass and the protein migratesas an approximately 100–150 kDa protein on SDS-PAGE.The recombinant human N-cadherin/Fc chimera(100 ng) was used in reactions in MMP proteolysis buffer(50 mM Tris/HCl pH 7.8, 50 mM CaCl2 and 0.5 M NaCl),either with or without 10 or 100 nM of active human,recombinant MMP-2 (Calbiochem). The activity of theMMP-2 enzyme was within the range of 8.0–10 (DA412/h/mg protein) in standard thiopeptolide hydrolysis assays.Activity of the supplied MMP-2 was verified by gelatinzymography, demonstrating active 62 kDa MMP-2in addition to smaller autocatalytic-sized fragments ofactive MMP-2. These mixtures were incubated for 4 h at371C and the reactions were stopped by addition of2� SDS sample buffer. Samples were then immediatelyseparated on 12% SDS-PAGE gels and immunostained forthe Fc region of the recombinant N-cadherin/Fc proteinusing an Fc-specific anti-human IgG (Sigma Genesis).

Immunohistochemistry

Five-micrometre-thick sections from paraffin-embedded12-week CCl4-injured mouse and rat livers were stainedwith a rabbit polyclonal antibody to N-cadherin (Abcamab12221, Cambridge, MA, USA). In brief, the sectionswere dewaxed in xylene and rehydrated in alcohol. Afterantigen retrieval was achieved by microwaving in 10 mMsodium citrate buffer for 15 min, slides were rinsed inphosphate-buffered saline (PBS) and endogenous perox-idase activity was blocked with 3% hydrogen peroxide for10 min. The slides were further blocked using the avidin/biotin kit (Vector Laboratories SP-2001, Peterborough,UK). Normal swine serum was added for 10 min to blocknon-specific protein binding. The sections were incu-bated overnight at 41C with the N-cadherin antibody at adilution of 1:200. Slides were rinsed in PBS and incu-bated with a swine anti-rabbit/biotin secondary antibody(Dako E0353, Cambridge, UK) at a 1:400 dilution for30 min at room temperature. Following further rinsingwith PBS, Vectastain Elite ABC reagent (Vector Labora-tories PK7100) was added for 30 min at room tempera-ture. Slides were 3,30-diaminobenzidine stained,counterstained, dehydrated in alcohol, cleared in xyleneand mounted. Primary HSC and the LX2 cell line werecultured on chamber slides and fixed in methanol for10 min. N-cadherin staining was repeated from afterthe antigen retrieval step (dilution 1:120 for rHSC).


Active MMP-2 promotes apoptosis of hepatic stellate cells Hartland et al.

Immunofluorescence confocal laser microscopy forN-cadherin

Human HSCs were grown on sterilised round glass cover-slips (Chance, Smethwick, UK). Cultured cells were sub-jected to a range of conditions including 16% serum,serum deprivation, exposure to gliotoxin (1.5mM) andco-incubation of gliotoxin with either MMP2-inhibitor I(17mM) (Calbiochem) or TIMP-1 (10 nM) (CN Bios-ciences, Nottingham, UK). Following a 4-h incubation at371C (in 5% CO2), the treatment medium was removedand the glass coverslips were washed in 0.01 M PBSsolution. The glass coverslips were fixed with acetone for10 min and washed three times for 5 min in 0.01 M PBSsolution. Primary antibodies for N-cadherin (Sigma Gen-esis) were prepared in 1% bovine serum albumin (BSA)PBS solution. Primary antibody was then placed onto eachglass coverslip and incubated at room temperature for 1 h.Following incubation, coverslips were washed three timesfor 5 min with 0.01 M PBS solution before subsequentincubation with mouse immunoglobulin–FITC-conju-gated secondary antibody (Dako) for 30 min, in 1% BSAPBS solution. Coverslips were then washed in 0.01 M PBSfor 5 min, before counter staining for 10 min with 7-aminoactinomycin D (Sigma) in 1% BSA PBS solution. The glasscoverslips were finally mounted on glass slides usingmoviol and observed under a Leica SP-2 confocal laserscanning microscope (Milton Keynes, UK).

Results

N-cadherin expression increases with activation ofhepatic stellate cells in culture

RNA and/or protein extracts from a murine liver homo-genate as well as cultured primary human and murineHSC were examined after both first and second passage. N-cadherin expression was observed in normal liver homo-genates (Fig. 1A). Furthermore, expression was associatedwith HSC activation after 7–17 days of culture (Fig. 1B andC), in both primary human and murine cells. Likewise, N-cadherin was detected by Western blotting in cells extractedfrom both untreated and CCl4-treated fibrotic murinelivers, following 9 days of culture [Fig. 1C(i) and (ii)].However, HSC extracted from CCl4-treated (4 weeks)fibrotic livers demonstrated an elevated expression relativeto cells extracted from normal livers.

In addition, both immunocytochemistry and immu-nohistochemistry staining clearly showed the presence ofN-cadherin in cultured primary human, mouse and ratHSC together with 12-week CCl4-treated mouse livers(Fig. 1D, F, H, J and L). Negative control slides exposed tosecondary antibody alone (Fig. 1E, G, I, K and M).

Disruption of N-cadherin binding promotes apoptosis ofhepatic stellate cells

To determine whether N-cadherin had a role in control-ling HSC survival, its function was disrupted by anN-cadherin blocking antibody (clone GC-4). Exposure

of HSC to this antibody caused a modest but significantincrease in apoptosis under serum-free conditions com-pared with non-immune controls (Fig. 2A). Comple-mentary experiments were undertaken using the HAVDIpeptide, which has been shown to block a site of N-cadherin involved in cell adhesion (43). Exposure ofhuman HSC to 400 mg/ml of peptide for 4 h significantlyincreased apoptosis (Fig. 2B). The effect of N-cadherinblockade by either method was more marked when HSCwere co-incubated with cycloheximide. Furthermore, theactivity of caspase-3 from HAVDI-treated HSC was alsoincreased (Fig. 2C). Together, these data indicate a rolefor N-cadherin binding in maintenance of HSC survival.

N-cadherin is degraded during hepatic stellate cellsapoptosis

Using an antibody directed against an intracellular epi-tope, a time-dependent upregulation of N-cadherin wasobserved following apoptosis, induced by gliotoxin ex-posure (Fig. 3). After 2 h of exposure, there was clearevidence of N-cadherin degradation, with the smallerdegraded fragments (60 and 35 kDa) detectable in whole-cell lysates following Western blotting. Furthermore,extracellular cleavage of N-cadherin to yield a 70 kDafragment was also evident, when analysing conditionedmedium from apoptotic cells, following 4 h of gliotoxinexposure (data not shown).

Apoptosis of human hepatic stellate cells in culture isassociated with promatrix metalloproteinase-2 activation

We found that cells exposed to gliotoxin developed anapoptotic morphology within 6 h, when assessed by acri-dine orange staining (Fig. 4A) and phase-contrast micro-scopy (data not shown), as had been described previously(41). Likewise, when defined by gelatin zymography con-ditioned medium from HSC was found to contain detect-able amounts of the 68 kDa proMMP-2, as has also beendescribed (35) (data not shown). Therefore, we sought toquantify active MMP-2 from conditioned medium, follow-ing induction of human HSC apoptosis by gliotoxin. Usingan MMP-2 activity assay that detects only the active matureenzyme, a progressive increase in MMP-2 activity, up to amaximum of three-fold at 6 h, was observed (Fig. 4B).While MMP-2 activity reached significance after 4 h ofgliotoxin exposure, cells maintained in serum-free mediumalone showed no significant increase in active MMP-2 overthe same period. Zymogram analysis of culture super-natants also demonstrated upregulation of the activeMMP-2 form in apoptotic HSC and whole liverundergoing spontaneous resolution of liver fibrosis follow-ing CCl4-intoxication (data not shown), a process char-acterised by HSC apoptosis (4, 35). These data supportthose of Preaux et al. (37), who demonstrated increasedpresence of active MMP-2 in response to cytochalasin andC2-ceramide-induced HSC apoptosis.



Active matrix metalloproteinase-2 promotes apoptosis ofhuman hepatic stellate cells in culture

In order to determine whether the increase in activeMMP-2 observed during the exposure of HSC to gliotox-in could itself act to induce apoptosis, we cultured

human HSC cells in the presence of either pro- or activerecombinant MMP-2 forms for 4 h (Fig. 5A). A signifi-cant increase in apoptosis, as assessed by caspase-3activity, was observed in cells cultured in the presence of10 nM of active MMP-2 compared with cells treated withMMP buffer alone. In addition, this effect was specific to

Fig. 1. Expression of N-cadherin increases during progressive activation of primary human hepatic stellate cells (HSC) cultured on plastic.Primary culture of both human and murine HSC was undertaken with cells harvested from both normal and carbon tetrachloride (CCl4)-treatedlivers for protein and RNA analysis. Human and murine protein samples were subjected to Western blotting for N-cadherin after 1–9 days ofculture; RNA was used for reverse transcriptase-polymerase chain reaction following 2 and 17 days of culture. (A) A 1.1 kb N-cadherin ampliconwas observed in normal murine liver homogenates and HSC RNA following 17 days of primary culture (B) but absent from control lanes (water).(C(i)) N-cadherin was observed in whole-cell lysates extracted from primary human HSC following 7 days of culture. HSC extracted from normal(C(i)) and CCl4-treated fibrotic murine liver (C(ii)) both demonstrated N-cadherin expression following 9 days of culture. However, expressionwas enhanced in cells extracted from CCl4-treated livers relative to that found in cells from normal murine livers. Cell and tissue staining for N-cadherin reveal expression in (D) human HSC, (F) mouse HSC, (H) rat HSC, (J) human LX2 hepatic stellate cell line and (L) 12-week CCl4-treatedmouse liver section; (E), (G), (I), (K) and (M) negative N-cadherin controls for (D), (F), (H), (J) and (L) respectively.



the active enzyme as treatment of cells with proMMP-2had little effect on caspase-3 activity or nuclear morphol-ogy (data not shown). Further assessment, using acridineorange, demonstrated a dose-dependent increase in thepercentage of HSC undergoing apoptosis with 2 or20 nM of active MMP-2 for 4 h (Fig. 5B). Moreover, thiseffect was considerably enhanced when cells were in-duced into apoptosis with the addition of cycloheximide.

Active matrix metalloproteinase-2 cleaves N-cadherin/Fc chimera recombinant protein

In order to determine whether the apoptotic effectobserved by the addition of active MMP-2 may occurvia cleavage of N-cadherin, it was necessary to identifywhether N-cadherin could act as a substrate for MMP-2.Recombinant N-cadherin/Fc protein was incubated with10 or 100 nM of active MMP-2 or with MMP bufferalone (control). Following separation by SDS-PAGE andWestern blotting for the Fc motif on the chimera protein,samples treated with MMP buffer alone appeared as fourbands 80–150 kDa in size (Fig. 6). However, treatment ofrecombinant N-cadherin with increasing quantities ofactive MMP-2 showed the dose-dependent increase inthe presence of cleavage products at approximately 60and 35 kDa, indicating the potential for in vitro cleavageof N-cadherin by MMP-2.

Active matrix metalloproteinase-2 promotes N-cadherincleavage in cultured hepatic stellate cells

After showing that N-cadherin was a substrate for MMP-2, we sought to determine whether the enzyme wouldpromote the cleavage of N-cadherin in cultured humanHSC. Therefore, activated HSC were exposed to activeMMP-2 with cycloheximide to promote HSC apoptosis.After 4 h, cells were harvested and analysed for N-cadherin by Western blotting. While cleavage products(60, 35 and 20 kDa) were visible under all conditions,indicating a low level of endogenous N-cadherin proces-sing by these cells, up to a nine-fold induction in thepresence of the 60 kDa-sized N-cadherin fragment waspresent from cells treated with active MMP-2 comparedwith untreated cells and a 3.6-fold induction over cellstreated with cycloheximide alone (Fig. 7).

Matrix metalloproteinase-2 inhibition prevents cleavageof N-cadherin during apoptosis

Following identification of N-cadherin cleavage by exo-genous MMP-2, further studies examined whether

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Fig. 2. Blockade of homophilic N-cadherin binding promotesapoptosis of hepatic stellate cells (HSC). (A) HSC incubated for fourhours with an N-cadherin blocking antibody (clone GC-4) (40mg/ml)to an extracellular epitope on N-cadherin and assessed by acridineorange. Blockade was compared with non-immune IgG control atthe same concentration. Morphology was assessed with andwithout the presence of cycloheximide. Data are expressed as meanof the apoptotic index (SEM, ��Po0.005 by Student’s t-test, n = 3,SF denotes serum-free conditions). (B) The blocking peptide, HAVDI(400mg/ml), was used as an alternative means to block N-cadherinbinding with a 4-h exposure in serum-free medium. Apoptosis wasquantified by acridine orange staining and cell counting. Data areexpressed as mean of the apoptotic index (SEM, ��Po 0.005 byStudent’s t-test, n = 3). (C) The pro-apoptotic effect of the HAVDIpeptide as measured by caspase-3 activity assay (SEM, �Po 0.05 byStudent’s t-test, n = 3).



endogenous MMP-2, secreted by HSC, had similareffects. Cultured human HSC were induced into apopto-sis to promote cleavage of N-cadherin. Cells grown inserum-containing medium had intact N-cadherin135 kDa in size. In contrast, cells induced into apoptosisby cycloheximide exposure (50 mM) showed evidence ofN-cadherin degradation with an abundant immunoreac-tive species at 60 kDa (Fig. 8). However, when cells wereco-incubated with cycloheximide and the MMP-2 inhi-bitor (17 mM), degradation to the 60 kDa form wasinhibited with N-cadherin remaining intact, similar tocontrol conditions. Fragmented bands observed at 35and 20 kDa in all lanes are thought to represent a lowlevel of constitutive N-cadherin processing by these cells.These data suggest that MMP-2 may mediate autocrineN-cadherin degradation during apoptosis and thatMMP-2 inhibition would protect N-cadherin from de-gradation during this process.

Matrix metalloproteinase-2 inhibitor maintained N-cadherin at the cell surface after exposure to gliotoxin

Once the association between apoptosis and MMP-2cleavage of N-cadherin had been established by Westernblotting, confocal microscopy was undertaken to provideconfirmatory evidence. Staining of cultured human HSCfor N-cadherin (intracellular epitope) showed that itslocation was consistent with cell surface expression (Fig.9B and C). To further study the effect of MMP-2inhibition, human HSC were exposed to gliotoxin alone(Fig. 9D) or gliotoxin with either MMP-2 inhibitor (Fig.9E) or TIMP-1 (Fig. 9E) for 4 h. Following exposure togliotoxin there was reduced expression of N-cadherincompared with cells left untreated in either serum-

containing medium or serum-free conditions (Fig. 9Dvs B and C respectively). In contrast, MMP-2 inhibitor(Fig. 9E) and TIMP-1 (Fig. 9F) countered the effect ofgliotoxin and maintained the cellular expression of N-cadherin.

Discussion

In these studies, we have defined cleavage of N-cadherinas a possible mechanism, whereby the resurgence ofhepatic MMP activity, during resolution of liver fibrosis,might trigger or amplify apoptosis of HSC. Furthermore,we demonstrate this process to be MMP dependent andhighlight the potential of MMP-2 as a mediator of N-cadherin cleavage and subsequent apoptosis.

The interaction of cells with the extracellular matrix(ECM) is crucial for the normal development and func-tion of multicellular organisms. Disruption of theseinteractions can significantly alter tissue function andcell fate. MMPs are a major group of enzymes thatparticipate in ECM turnover. There is, however, a grow-ing awareness that the substrate repertoire of MMPs iswide and includes many non-matrix proteins such as cellsurface molecules (19), growth factors (44) and theirreceptors (45). Indeed, our studies (46) and those ofothers (47) indicate that both proliferation and activa-tion of HSC are regulated by ECM. Equally, intercellularinteractions also play a key role in supporting cellsurvival (17). N-cadherin is involved in cell–cell bindingand our studies show its expression by activated HSC.Utilising two independent methods of assessment, dis-ruption of N-cadherin-binding interactions, with a spe-cific neutralising antibody or HAVDI peptide, promotedHSC apoptosis, demonstrating its role as a prosurvivalstimulus. Although novel in HSC, these findings are inaccordance with earlier work, utilising similar methodsof functional disruption, in granulosa, endothelial andepithelial cells (28, 30, 48, 20).

Functional N-cadherin is increasingly recognised toaffect multiple signalling cascades. However, it is theassociation of N-cadherin with FGFR on the cell surfacethat appears to mediate much of its observed effects (28,30, 32–34, 49). The N-cadherin–FGFR coclustering (49,50) is thought to stabilise FGFR, preventing internalisa-tion and facilitating sustained activation (34). Studies byTrolice et al. (28) and Erez et al. (30) both demonstratereduced FGFR activation and increased apoptosisthrough the disruption of N-cadherin-mediated con-tacts. In both the studies the addition of basic FGFreduced apoptosis. However, while Trolice induced apop-tosis using antibodies to both N-cadherin and FGFR,Erez did so using an HAV-containing peptide but ob-served no increase in apoptosis when using antibodies toN-cadherin. Thus, although the specific nature of theinteraction remains to be defined, these studies suggestthat the activation of prosurvival signals occurs via theN-cadherin–FGFR complex. In the current study, wefound that both HAV-containing peptide as well as

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Fig. 3. Gliotoxin-induced apoptosis leads to cleavage of cellular N-cadherin. Western blot using an antibody raised against anintracellular epitope for N-cadherin. Cell extracts were made over atime course of 0–4 h of gliotoxin exposure (1.5mM) under serum-free conditions. Following quantification of total protein byCoomassie blue-binding assay (BioRad), equal quantities of protein(10mg/well) were loaded onto PAGE gels for analysis. Theemergence of smaller immunoreactive bands around 60 and 35 kDain size was associated with human HSC apoptosis. Data arerepresentative of four separate experiments.



antibodies to N-cadherin promoted apoptosis of HSC.Future work to determine how these methods of apopto-tic induction affect the activation status of the FGFR andthe subsequent activation of specific intracellular signal-ling cascades may help to define the nature of the anti-apoptotic function of N-cadherin in HSC.

Here we have shown, using a variety of different ap-proaches, that N-cadherin degradation occurs during HSCapoptosis and that this process appears to be, at least in part,MMP-2 mediated. Sequence analysis of human N-cadherinshows the extracellular domain to contain multiple (ap-proximately 20) potential MMP cleavage sites, each contain-ing the consensus MMP cleavage motif Pxx/Hy (P is proline,x is any amino acid and Hy is any hydrophobic amino acid)(51). Furthermore, using previously published motifs foractive MMP-2 (LIxx/Hy, HySx/L and Hxx/Hy) (52), fivepotential cleavage sites were identified in the N-cadherinamino acid sequence (data not shown). Indeed, our studies

using the N-cadherin/Fc chimera protein provide evidenceof direct MMP-2-mediated cleavage and suggest that withinthe extracellular domain of N-cadherin, there are a numberof cleavage sites yielding fragments ranging between 20 and60 kDa. Cells undergoing apoptosis demonstrated increasedN-cadherin processing that was blocked by MMP-2 inhibi-tor, in accordance with published data (20), and enhancedby exogenous MMP-2. The presence of N-cadherin cleavageproducts under all conditions suggests a low level ofconstitutive processing by these cells.

Using tissue culture models, we have previously de-monstrated TIMP-1 to inhibit HSC apoptosis via itsinhibition of MMP activity (40). While MMP-2 isproduced by activated HSC during fibrogenesis its ex-pression and activity persist during spontaneous resolu-tion of liver fibrosis (35). Our current study and that ofothers (37) showing an increase in active MMP-2production by apoptotic HSCs supports a potential

Gliotoxin exposure

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Fig. 4. Apoptosis of human HSC in culture is associated with proMMP-2 activation. (A) Acridine orange staining under blue fluorescencebefore and after exposure to gliotoxin (1.5mM) (left and right panel respectively). Gliotoxin-induced apoptosis is highlighted by arounded cell morphology with evidence of nuclear condensation and fragmentation (red arrow). (B) Increasing quantity of endogenousactive MMP-2 during gliotoxin-induced apoptosis in serum-free, conditioned medium from human HSC, as measured by MMP-2 activityassay (Biotrak, GE HealthCare, Little Chalfont, UK). Data are expressed as mean and SEM pM of active MMP-2, (�Po 0.05; NS, not significantby ANOVA with the Bonferroni test, n = 3).



role for this protease in resolving disease via the degrada-tion of cell surface N-cadherin. Indeed, gliotoxin-induced HSC apoptosis correlated directly with increas-ing levels of endogenous active-MMP-2 [confirmingprevious reports (37)], and addition of MMP-2 tocultured human HSC induced apoptosis but only whenin its active form. Crucially, we have shown a clear rolefor N-cadherin in supporting HSC survival, demon-strating its cleavage and degradation as a possiblemechanism through which MMP-2 may mediate itspro-apoptotic effect.

The susceptibility of N-cadherin to MMP-2-mediateddegradation suggests that its anti-apoptotic function may

be dependent on the local TIMP/MMP balance. Indeed,N-cadherin degradation may not be specific to MMP-2alone. While investigation of the role played by otherMMPs is beyond the scope of the present study, theseenzymes are known to have a broad, overlapping speci-ficity and a number of MMPs have now been linked toapoptosis (53, 54), although their specific mechanism ofaction may differ. Likewise, this is not the first study toimplicate a pro-apoptotic role for either MMP-2 orcadherin modification. However, this is the first study,to our knowledge, to specifically link HSC apoptosisthrough MMP-2-mediated N-cadherin degradation inresolving liver fibrosis.

By degrading N-cadherin, endogenous HSC-derivedMMP-2 would promote and amplify apoptosis whilesimultaneously promoting matrix degradation and scarresolution. MMP-2 and its biological activator MMP-14(or MT1 MMP) are highly expressed by activated HSCboth in vivo and in vitro, while expression and activitypersist during recovery (35). During progressive liverinjury, the activity of MMP-2 is likely to be held in checkby the simultaneously produced TIMPs. Our previouswork indicates that TIMP-1, in the quantities producedby activated HSC, highly suppressed HSC-derived MMP-2 (36). Recent studies have also positively correlatedMMP-2 activity with HSC apoptosis, demonstratingMMP-2 activation to occur in association with cyto-

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active

Fig. 5. Active matrix metalloproteinase (MMP)-2 induces apoptosisof human hepatic stellate cells (HSC) in culture. (A) Caspase-3activity from extracts of cultured HSC. Under serum-free conditionsa significant induction of caspase-3 activity was demonstrated incells treated with 10 nM of active MMP-2 for 4 h, compared withcells treated with pro-MMP-2 or MMP buffer alone (data areexpressed as mean� SEM, n = 3, �Po 0.05 by Student’s t-test).(B) Human HSC cultured under serum-free conditions were exposedto 2 or 20 nM of recombinant active MMP-2 for 4 h, either with orwithout 50 mM cycloheximide, to promote apoptosis. The apoptoticindex was calculated following acridine orange staining as describedin the methods (data are expressed as mean� SEM of the apoptoticindex, n = 3, ��Po 0.01, ��Po 0.005).

160k

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Fig. 6. Active matrix metalloproteinase (MMP)-2 degrades N-cadherin/Fc chimera. Active MMP-2 (10 or 100 nM) or MMP bufferalone (control) was incubated with recombinant N-cadherin/Fcprotein for four hours. Following protein quantification equalquantities (10mg/well) were loaded onto polyacrylamide gelelectrophoresis (PAGE) gels for analysis. Western blotting reveals theappearance of N-cadherin fragments at 60 and 35 kDa (arrows)when the protein is exposed to active MMP-2. Fragmented bandsare absent from control. Data are representative of three separateexperiments.



chalasin D- or C2 ceramide-induced apoptosis (37), afinding we have confirmed with gliotoxin-inducedapoptosis. Furthermore, the gene promoter for MMP-2has been shown to contain a functioning p53 motif(55), further supporting its potential role as a regulatorof apoptosis.

Pharmacological approaches to promote MMP-2 ac-tivity in the fibrotic liver may be a potential method ofeither inducing or accelerating resolution of liver fibrosisin humans. Indeed, resolution of experimental liverfibrosis in rats was achieved through the genetic deliveryof a urokinase-type plasminogen activator (56), a processthought to be mediated through the subsequent down-stream activation of MMPs. Gliotoxin has been shown topromote apoptosis of HSC in culture and has beenshown to promote recovery of experimental liver fibrosisin rats (41). The current study shows that gliotoxin-

induced apoptosis of human HSC is associated withMMP-2 activation. Although the natural trigger forHSC apoptosis in resolving fibrosis needs to be defined,this study supports the intriguing concept that MMP-2may enhance the resolution of fibrosis by encouragingHSC apoptosis involving the cleavage of N-cadherin onthe cell surface.

Overall, these data provide cogent evidence thatHSC survival is influenced by the stabilization or degra-dation of N-cadherin and active MMP-2 is able to dis-rupt these survival signals directly by degrading thismolecule.

Acknowledgements

The authors wish to acknowledge the help of ProfessorJohn Primrose of Southampton University and MrMyrrdin Rees of North Hampshire Hospital NHS Trustfor supplying human liver resection specimens.

Grant support: F. M. was an MRC clinical trainingfellow. J. P. I., D. M. and R. C. B. were members of anMRC co-operative group (G9900297) and J. P. I. was anMRC senior clinical fellow (Grant G116/84) during partof the time this work was undertaken. J. P. I. currentlyholds an MRC Programme grant that supports S. N. H.

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Fig. 7. Active matrix metalloproteinase (MMP)-2 directly cleavesN-cadherin on cultured hepatic stellate cells (HSC). Cultured HSCwere exposed to active MMP-2 (20 nM) for 4 h. Following proteinquantification of the conditioned medium, equal quantities (10 mg/well) were loaded onto polyacrylamide gel electrophoresis gels foranalysis. An increase in the 60 kDa-sized N-cadherin fragment waspresent from cells treated with active MMP-2 (20 nM MMP-2 withcycloheximide in serum-free medium) compared with cells treatedwith cycloheximide alone (0, baseline, cycloheximide alone in serum-free medium). The bar graph shows fold induction of the 60 kDaband over control (serum-free medium alone) levels.

Cycloheximide(50µM)

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Fig. 8. Matrix metalloproteinase (MMP)-2 inhibitor I inhibitsN-cadherin cleavage during hepatic stellate cells (HSC) apoptosis.The selective MMP-2 inhibitor was used to test whether inhibition ofendogenous MMP-2 would affect N-cadherin cleavage in culture.Following protein quantification of cell lysates, equal quantities(10mg/well) were loaded onto polyacrylamide gel electrophoresisgels for analysis [Serum, serum-containing medium; 0,cycloheximide only in serum-free medium; M2i, cycloheximide andMMP-2 inhibitor (17mM) in serum-free medium]. Data arerepresentative of three separate experiments.



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