Membrane type-1matrix metalloproteinase (MT1-MMP)processing of pro-?v integrin regulates cross-talk between ?v ?3and ?2?1integrins
in breast carcinoma cells
Peter C.Baciu,a,*E.Aisha Suleiman,a Elena I.Deryugina,b and Alex Y.Strongin b
a
Allergan Inc.,2525Dupont Dr.,Irvine,CA 92612,USA
b
The Burnham Institute,10901North Torrey Pines Road,La Jolla,CA 92037,USA
Received 9December 2002,revised version received 9June 2003
Abstract
We have recently demonstrated that in breast carcinoma MCF7cells MT1-MMP processes the ?v ,?3,and ?5integrin precursors generating the respective mature S-S-linked heavy and light ?-chains.The precursor of ?2integrin subunit was found resistant to MT1-MMP proteolysis.The processing of the ?v subunit by MT1-MMP facilitated ?v ?3-dependent adhesion,activation of FAK signaling pathway,and migration of MCF7cells on vitronectin.To elucidate further the effects of MT1-MMP on cellular integrins,we examined the functional activity of ?5?1and ?2?1integrins in MCF7cells expressing MT1-MMP.Either expression of MT1-MMP alone or its coexpression with ?v ?3failed to affect the functionality of ?5?1integrin,and adhesion of cells to ?bronectin.MT1-MMP,however,profoundly affected the cross-talk involving ?v ?3and ?2?1integrins.In MT1-MMP-de?cient cells,integrin ?v ?3suppressed the functional activity of the collagen-binding ?2?1integrin receptor and diminished cell adhesion to type I collagen.Coexpression of MT1-MMP with integrin ?v ?3restored the functionality of ?2?1integrin and,consequently,the ability of MCF7cells to adhere ef?ciently to collagen.We conclude that the MT1-MMP-controlled cross-talk between ?v ?3and ?2?1integrins supports binding of aggressive,MT1-MMP-,and ?v ?3integrin-expressing malignant cells on type I collagen,the most common substratum of the extracellular matrix.?2003Elsevier Inc.All rights reserved.
Keywords:Adhesion;MT1-MMP;Integrin;Collagen;Breast carcinoma MCF7cells
Introduction
Membrane type matrix metalloproteinases (MT-MMPs)play a central role in locomotion of many cell types [1–3].MT1-MMP,the most common and thoroughly studied member of the MT-MMP subfamily,is widely expressed in tumors and is frequently associated with enhanced tumori-genicity of many cancer types.Prior studies have identi?ed the ability of MT1-MMP to modify the extracellular matrix and to initiate the activation pathway of the latent MMP zymogens [4–9].In addition,MT1-MMP proteolysis has been shown to play an important role in the processing of a variety of cell surface-associated molecules including tissue
transglutaminase,CD44,and the precursor of ?v integrin subunit (pro-?v )[9–13].
The MT1-MMP proteolysis of pro-?v represents a novel pathway of integrin maturation.It has been established that mature integrin subunits including ?3,?4,?5,?6,?7,?8,?9,?v ,?IIb,and ?E are generated by posttranslational endo-proteolytic cleavage of the respective precursors.The en-doproteolytic cleavage occurs at a highly conserved dibasic amino acids contained within one of two potential sequence motifs (RXR/KR or HXXXR/KR)present within integrin ?subunits.This cleavage results in the generation of an N-terminal heavy chain and a C-terminal light chain linked by a disul?de bridge [14].The cleavage at the highly conserved basic amino acid motif is a function of the furin-like pro-protein convertases.MT1-MMP,similar to furin-like pro-protein convertases,is able to speci?cally cleave pro-?chains containing the conserved basic amino acid motif to
*Corresponding author.Fax:?1-714-246-5578.
E-mail address:Baciu_Peter@https://www.doczj.com/doc/244110033.html, (P.C.
Baciu).
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generate the fully functional receptor.In addition to the initial cleavage adjacent to the dibasic amino acid motif at Leu892in the?v chain,a second cleavage event occurs localized within a loop between Cys852and Cys904involv-ing the disul?de-bonds linking heavy and light chains.In turn,?chains that do not exhibit the dibasic motif are resistant to furin cleavage(such as the?2integrin chain) and are also resistant to MT1-MMP.
The processing of pro-?v by MT1-MMP and the subse-quent generation of the modi?ed?v?3integrin heterodimer affect outside-in signaling via the FAK pathway and corre-late with enhanced cell adhesion and migration on vitronec-tin[10,15,16].To better understand the functional attribute of MT1-MMP-mediated integrin processing,we analyzed the functional activity of?5?1and?2?1integrins in breast carcinoma MCF7cells coexpressing MT1-MMP with?v?3 integrin as well as in the MCF7cells expressing MT1-MMP and?v?3integrin alone.
Here,we report a novel,physiologically signi?cant path-way involving?2?1and?v?3integrins,which regulates adhesion of neoplastic cells to type I collagen,the main component of the extracellular matrix.This pathway,initi-ated by MT1-MMP,modulates a cross-talk between?2?1 and?v?3integrins.Our data also suggest that,in contrast to the enhancement of the activity of MT1-MMP-processed integrin?v?3,the similar processing of pro-?5by MT1-MMP did not affect the functionality of integrin?5?1in MCF7cells.We believe that the gain of functional activity of integrins resulting from MT1-MMP processing in MCF7 cells is largely restricted to the?v?3integrin and its cross-talk with other integrin types,especially,?2?1.
Materials and methods
Cell culture
Human breast carcinoma MCF7cells and their deriva-tion were described in a study by Deryugina et al.[15]. Cells were routinely cultured in Dulbecco’s modi?ed Ea-gle’s media(DMEM)(Invitrogen,Carlsbad,CA)containing 10%fetal bovine serum(FBS)and supplemented with250?g/ml neomycin and250?g/ml zeocin(Invitrogen).Prior to analysis,cells were replated in DMEM/10%FBS over-night followed by exchanging the culture media with serum-free AIM-V medium(Invitrogen)and cultured for an addi-tional48h in the presence or absence of inhibitor prior to analysis.Inhibitors used in these studies included the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk)(Bachem,King of Prussia,PA)and the hydroxamate inhibitor AG3340,which was synthesized in-ternally.Both inhibitors were used at a?nal concentration of30?M.cRGD(Bachem)was used at50?M.Cell adhesion
Cell adhesion studies were performed in96-well clear-bottom black well plates(Corning Costar,Acton,MA). Plates were coated overnight with matrix protein at4°C, then blocked for1h with1%heat denatured BSA in Hanks’balanced salt solution(HBSS)containing10mM HEPES, pH7.4.Prior to analysis,cells were labeled with?uorescent dye,10?M calcein AM(Molecular Probes,Eugene,OR), in HBSS for1h at37°C in a humidi?ed incubator.After labeling unincorporated dye was removed and cells de-tached using enzyme free dissociation buffer(Specialty Media,Phillipsburg,NJ).Detached cells were washed and resuspended in adhesion buffer(HBSS containing10mM HEPES,pH7.4,and1%heat denature BSA)at0.45?106 cells/ml.Suspended cells were then plated in triplicate,at 100?l/well,and incubated for1h at37°C in a humidi?ed incubator to allow cell attachment.At the end of the incu-bation period,unattached cells were removed by washing plates three times with PBS using an Embla96/384plate washer(Molecular Devices,Sunnyvale,CA).Total?uores-cence remaining after washing was determined using a Cyto?uor4000(Prospective Biosystems,Foster City,CA) with excitation and emission at485and530nm,respec-tively.Percent adhesion was determined by dividing total ?uorescence/well by the total initial?uorescence/well in unwashed control plates.To determine total initial?uores-cence,cells were plated in parallel onto96-well plates coated with poly-D-lysine and?uorescence determined without plate washing.Where indicated,anti-integrin anti-bodies JBS2,6S6,P1B5,BHA2.1,and LM609(Chemicon Int.,Temecula,CA)(20?g/ml each)were coincubated with cells for10min at37°C prior to plating.
Cell migration
Haptotactic cell migration assays were performed using 24-well Boyden chambers with8-?m-pore polycarbonate membranes(Corning Costar,Acton,MA).The undersur-face of the membranes were coated with either5?g/ml ?bronectin or1?g/ml type I collagen by placing chambers overnight at4°C in24-well plates containing300?l of matrix solution.After coating,membranes were washed with PBS and both sides of the membranes blocked for30 min with1%BSA in HBSS.Cells were harvested using EDTA,washed,and resuspended in AIM-V media.Sus-pended cells were then seeded in the upper chamber at 12,500cells/well in0.2ml of AIM-V medium.Each cham-ber was then placed in24-well plates containing0.5ml of AIM-V medium and incubated at37°C for6h in a humid-i?ed incubator containing5%CO2.After6h cells remain-ing in the upper chamber were removed with a cotton swab and cells that had migrated onto the lower membrane sur-face were?xed with3.5%paraformaldehyde for30min. Nuclei of the migrated cells were stained with Cyquant dye according to the manufacturer’s instructions(Molecular
168P.C.Baciu et al./Experimental Cell Research291(2003)167–175
Probes)and imaged using a Nikon E300microscope equipped with a Spot digital camera and a2?objective (Diagnostic Instruments,Sterling Heights,MN).The total number of nuclei within the central22mm2area of the membrane was determined using Image Pro analysis soft-ware(Media Cybernetics,Des Moines,IA).Data are ex-pressed as percentages of migrated cells relative to the total number of cells seeded in the upper chamber.
FACS analysis
Cells were cultured as described for the adhesion assay. After release from the plates with EDTA,cells were washed and resuspended at1?107cells/ml in staining buffer(PBS containing1%BSA and0.01%sodium azide at4°C);300?l of cell suspension was incubated with primary antibodies directed against either?1integrins,6S6or?2integrins,
monoclocal antibodies BHA2.1,and AK7(Chemicon)at a 1:100dilution for60min at4°C followed by washing. Bound primary antibodies were visualized by staining cells with a1:200dilution of FITC-conjugated goat anti-mouse IgG(Molecular Probes)for30min at4°C.Stained cells were then washed and the level of staining evaluated on a BD FACScalibar?ow cytometer.The data were analyzed with Cell Quest software(Becton Dickinson Immunocy-tometry Systems,San Jose,CA).
Immunoprecipitation and Western blotting
Cells were culture as indicated above.Treatment with inhibitors was done over48h prior to harvest.At the end of incubation period,cells were washed three times in HBSS and then surface-biotinylated using0.5mg/ml EZ-Link Sulfo-NHS-LC-Biotin(Pierce,Rockford,IL)in HBSS sup-plemented with10mM HEPES,pH7.4.Unincorporated Sulfo-NHS-LC-Biotin was removed by washing cells three times with PBS at4°https://www.doczj.com/doc/244110033.html,beled cells were lysed with modi?ed RIPA buffer(50mM Tris,pH7.4,0.1%SDS,1% Triton X-100,5mM EDTA,150mM NaCl,0.5mM sodium deoxycholate)containing Complete TM(Roche,Indianap-olis,IN)as protease inhibitor cocktail.Cell lysates were precleared on protein G beads(Pierce,Rockford,IL)for30 min at4°C.Precleared cell lysates(300?g of total protein) were incubated with2?g of polyclonal antibodies,AB1930 (Chemicon),for1h at4°C followed by absorption onto protein G-agarose beads for30min at4°C.Unbound ma-terial was removed by washing beads three times in the lysis buffer.Bound immune complexes were released by the addition of50?l of reducing2?SDS sample buffer fol-lowed by boiling for5min.Isolated immune complexes were separated on an8%SDS-PAGE gel and transferred onto an Immobilon P membrane(Millipore,Bedford,MA). Membranes were blocked overnight in PBS/0.2%Tween20 containing5%BSA.Bound proteins were visualized by HRP-conjugated aviding and ECL reagent(Pierce).Results
The expression of integrin?v?3and MT1-MMP alters cell adhesion to type I collagen
MT1-MMP is a key enzyme in cell locomotion[17,18]. Prior studies indicate that MT1-MMP accomplishes this,in part,through the endoproteolysis of the integrin?subunits [10,11,15,16].Speci?cally,enhanced migration on vitro-nectin correlated with MT1-MMP-mediated processing of pro-?v integrin in MCF7cells.To elucidate additional ac-tivities related to MT1-MMP,we examined the effects of MT1-MMP expression on cell adhesion to?bronectin and type I collagen.Cell lines used in this study were isolated in our earlier work and included cells stably transfected with empty vector(mock cells),the?3integrin subunit(?3 cells),the wild-type MT1-MMP(MT cells),and cotrans-fected with both?3and MT1-MMP(?3/MT cells).The status of MT1-MMP and integrin?v?3in the cells has been previously characterized by immunoprecipitation. Western blotting,?ow cytometry,gelatin zymography,?u-orescence substrate cleavage,and by the ability of MT1-MMP to induce activation of the latent MMP-2zymogen [10,15,16,19].
In our initial analysis,we examined the effects of MT1-MMP and?v?3integrin on MCF7cell adhesion to?bronec-tin.The expression of?v?3integrin and MT1-MMP indi-vidually or jointly in MCF7cells had no effect on cell adhesion to?bronectin(Fig.1A).In contrast,the?3cells that express?v?3integrin alone were less ef?cient than mock-transfected cells in adhering to type I collagen(Fig. 1B).The coexpression of MT1-MMP with?v?3integrin restored the adhesion ef?ciency to the levels observed in mock cells.The expression of MT1-MMP alone appeared to have no effect.These?ndings indicated that the expression of?v?3integrins alone affected the ef?cacy of the collagen-binding integrin receptors in MCF7cells while coexpres-sion of MT1-MMP with?v?3reversed this effect.These data indicate that MT1-MMP has no effect on?5?1-or ?2?1-mediated adhesion but does in?uence the cross-talk between collagen-binding integrin receptors and integrin ?v?3.
We suspected that the effect of?v?3integrin on the collagen-binding integrins represents an example of the trans-dominant inhibition observed earlier with integrins ?5?1and?iib?3in CHO cells.In the studies involving CHO cells the trans-dominant inhibition of the?5?1functionality by integrin?iib?3was reversed by increased levels of matrix protein[20].To investigate the matrix concentration depen-dence,we examined adhesion of cells over a wide range of type I collagen concentrations including those that exceeded by10-fold the values required for maximal adhesion.At all concentrations examined,?3cells were less adhesive rela-tive to mock or?3/MT cells(Fig.2).
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Integrin ?2?1is a target of inhibition by integrin ?v ?3Function-blocking anti-integrin antibodies were em-ployed to identify individual integrins involved in collagen binding by MCF7cells and susceptible to the inhibition by integrin ?v ?3.Adhesion of mock,?3,and ?3/MT cells to type I collagen were all inhibited by anti-?1and anti-?2inhibitory antibodies,indicating that ?2?1integrin is the primary collagen receptor in these cells (Fig.3A).No inhi-bition was observed with either control murine IgG or inhibitory antibodies speci ?c to integrin ?v ?3(LM609)or
to integrin ?3?1(P1B5).Because anti-?v ?3blocking LM609antibody had no effect on cell adhesion,it is clear that ?v ?3integrin is not involved in the binding of either ?3or ?3/MT cells to type I collagen.
Since our results indicate that the ?2?1integrin primarily mediates adhesion of MCF7cells to collagen we sought to determine if the levels of ?2?1are similar in ?3and ?3/MT cells.FACS analysis employing two ?2and one ?1mono-clonal antibodies demonstrate that mock,?3,and ?3/MT cells express similar cell surface levels of ?2and ?1inte-grins (Fig.3B).This observation indicates changes in the functional activity rather than expression levels of ?2?1integrin are a putative cause for decreased adhesion of ?3cells to collagen.To determine if ?3cells exhibit differing levels of activated forms of integrin ?2?1,we treated mock,?3,and ?3/MT cells with a stimulatory antibody speci ?c to ?2integrins or with 0.1mM MnCl 2to determine if further activation would increase adhesion relative to untreated cells.Pretreatment of ?3cells with either stimulatory anti-body or 0.1mM MnCl 2greatly increased cell adhesion in ?3cells while only modest effects were observed in mock or ?3/MT cells (Fig.3C).These results demonstrate that rela-tive to either mock or ?3/MT cells the functional activity of integrin ?2?1in ?3cells is suppressed as re ?ected in the higher level of adhesion induced with stimulatory antibod-ies or MnCl 2.
Processing of the alpha V chain correlates with extent of a cross-talk between ?v ?3and ?2?1integrin in MCF7cells
Previous studies have demonstrated that blocking ?chain maturation disrupts the functional activity of the het-erodimer [21,22].Furthermore,unconventional
processing
Fig.1.?3integrin suppresses adhesion of MCF7cells to type I collagen.MCF7cell adhesion to collagen and ?bronectin was examined by determining the number of cells bound to 96-well plates coated with either 3.0?g/ml ?bronectin (A)or 0.1?g/ml type I collagen (B).Percent adhesion represents the percentage of cells bound to the matrix after washing to remove nonadherent cells following a 1-h incubation period at 37°C in a humidi ?ed incubator.No change in cell adhesion to ?bronectin was observed among the cell lines examined.However,suppression of cell adhesion to type I collagen was observed in MCF7cells expressing ?3integrin.Mock,MCF7cells transfected with empty vector;?3,MCF7expressing ?v ?3integrin;?3/MT,MCF7cells expressing both ?v ?3integrin and MT1-MMP;MT,MCF7cells expressing MT1-MMP
alone.
Fig.2.?3-mediated suppression of MCF7cell adhesion to type I collagen is independent of matrix concentration.Ninety-six-well plates were coated with 1/2log serial dilution of type I collagen ranging from 1?g/ml to 0.003?g/ml and the ability of MCF7cells to adhere was examined as described in the materials and methods section.Percent adhesion represents the percentage of cells remaining after washing to remove nonadherent cells following a 1-h incubation period at 37°C in a humidi ?ed incubator.Maximal level of cell adhesion was obtained by all cell lines at 0.1?g/ml coatings of type I collagen with no further increase in adhesion observed at a 10-fold increase in matrix coatings (1?g/ml).Mock,MCF7cells trans-fected with empty vector;?3,MCF7expressing ?v ?3integrin;?3/MT,MCF7cells expressing both ?v ?3and MT1-MMP.
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of the pro-?v chain in the?v?3heterodimer by MT1-MMP results in enhanced functional activity of the integrin.These studies suggest that the processing of the pro-?v chain in MCF7cells may be responsible for the observed regulation of cell adhesion to type I collagen.To establish a link between pro-?v processing and cross-talk between?v?3and ?2?1integrins we disrupted pro-?v maturation by blocking the proprotein convertase pathway with the furin inhibitor dec-RVKR-cmk and the MT1-MMP pathway with the ma-trix metalloproteinase inhibitor AG3340.The effect of the inhibitors on integrin processing was analyzed by immuno-precipitating integrin?v?3from the lysates of surface-bi-otinylated cells followed by SDS-PAGE and Western blot-ting.The data demonstrate that dec-RVKR-cmk blocked pro-?v processing in?3cells but not in?3/MT cells(Fig. 4A).Alternatively,processing of pro-?v by MT1-MMP in ?3/MT cells was inhibited by AG3340(Fig.4A).Consistent with the requirement for proprotein convertase-mediated pro-?v processing for?v?3cross-talk with?2?1,treatment of?3cells with dec-RVKR-cmk increased cell adhesion to type I collagen to the levels seen in mock-transfected cells (Fig.4B).On the contrary,the furin inhibitor,which did not block pro-?v processing in?3/MT cells,failed to affect adhesion of?3/MT cells to collagen(not shown).Similary, pretreatment of?3/MT cells with AG3340,blocking MT1-MMP processing and shifting the balance to principally proprotein convertase-processed pro-?v,resulted in inhibi-tion of cell adhesion to the levels seen in?3cells(Fig.4C). These results are consistent with furin-processed pro-?v mediating the inhibition of?2?1by?v?3.Furthermore, exposure of?3/MT cells to AG3340during the adhesion assay did not alter?3/MT cell adhesion to collagen(Fig. 4D),indicating that MT1-MMP catalytic activity is not required during adhesion.However,MT1-MMP catalytic activity is required prior to adhesion under conditions al-lowing alternative processing of the pro-?v subunit by MT1-MMP to restored cell adhesion to collagen.This ob-servation agrees with previous studies demonstrating that short-term inhibition affects the cell surface-associated cat-alytic activity of MT1-MMP rather than the levels of the MT1-MMP-processed?v?3on the cell surface[23].Ac-cordingly,only by the extensive treatment of cells with the AG3340,a hydroxamate inhibitor,allowing replacement of MT1-MMP-processed pro-?v with that of proportein con-vertase-processed pro-?v,altered the functionality of inte-grin?v?3in?3/MT cells.
Expression of?3or MT1-MMP does not affect migration on type I collagen
We have previously established that the expression of
MT1-MMP enhanced the directional cell migration on vi-
tronectin[16].Accordingly,we tested whether the expres-
sion of?v?3integrin alone or its coexpression with MT1-MMP-altered cell migration on type I collagen.For these
purposes,we evaluated migration of?3and?3/MT cells on ?bronectin and type I collagen.Neither expression of the?3 integrin subunit alone or its coexpression with MT1-MMP affected cell migration on type I collagen(Fig.5A).The expression of MT1-MMP alone reduced the migration ef?-ciency probably because of the extensive degradation of the collagen matrix.In contrast,the expression of the?3inte-grin subunit alone and its coexpression with MT1-MMP facilitated migration of cells on?bronectin.The presence of ?v?3integrin in these cells evidently facilitated the recog-nition of?bronectin,a ligand of?v?3integrin.In agreement with this observation,cRGD reduced migration of?3and ?3/MT cells on?bronectin to levels seen in mock cells (Fig.5B).
Discussion
MMPs play a vital role in modulating cellular activity. MMPs,and speci?cally membrane-tethered MMPs from the membrane type(MT)-MMP subfamily,are directly associ-ated with the proteolysis of the extracellular matrix.Re-cently,MT-MMPs have been identi?ed as potential proteo-lytic regulators of the functionality of cell surface-associated receptors including transglutaminase,CD44,and certain individual members of the integrin receptor family [9–13].Proteolytic cleavage of integrin receptors by MT1-MMP occurs in a manner similar to that mediated by pro-protein convertases,suggesting that MT1-MMP functions as a prointegrin convertase in malignant cells and is directly involved in the processing of the single-chain pro-?v in breast carcinoma and glioma cells[16].Based on the amino acid sequence homology,it appears that at least several?
Fig.3.Changes in MCF7cell adhesion to collagen type I occurs through the?2?1integrin.(A)MCF7mock,?3,and?3/MT cells were incubated for10 min prior to and during the adhesion assay to collagen(0.1?g/ml coating)with inhibitory antibodies directed against either?1(6S6),?2(BHA2.1),?3 (P1B5),?v?3(LM609),or preimmune immunoglobulin(IgG).The level of cell adhesion was determined after a1-h incubation period and data plotted as a percentage of untreated control wells for each cell type.Data points represent the average of three separate experiments.Cell adhesion to type I collagen was blocked by?1(6S6)and?2(BHA2.1)inhibitory antibodies,indicating that cell adhesion to collagen type I occurs primarily through the?2?1integrin. Neither anti-?v?3(LM609),anti-?3(P1B5),nor preimmune(IgG)imunoglobulins inhibited adhesion to collagen.(B)Cell surface levels of?2and?1 integrins on mock,?3,and?3/MT-MCF7cells was determined by FACS analysis using AK7and BHA2.1monoclonal antibodies to?2integrins,and6S6 to?1integrins.FACS analysis indicates that the three cell lines express similar levels of?2?1integrin on the cell surface.(C)MCF7mock,?3,or?3/MT cells were either untreated(control),treated with?2integrin stimulatory monoclonals(JBS2,20?g/ml)or stimulatory levels of MnCl2(0.1mM)for10min prior to and during the adhesion assay to collagen(0.1?g/ml coating).Percent control represents the percentage of cells adhered relative to untreated control cells.Stimulation of?3cells with either JBS2or0.1mM MnCl2enhanced cell adhesion indicating that suppression of MCF7?3cell adhesion to collagen is due,in part,to suppression of?2?1integrin activity.
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Fig.4.Modulation of MCF7cell adhesion to collagen through ?2?1integrin correlates with alternative processing of the ?v subunit.(A –C)?3and ?3/MT cells were treated with either 30?M dec-RVKR-cmk or 30?M AG3340for 48h prior to analysis of pro-?v processing by Western analysis (A)or alterations in cell adhesion (B and C).(D)MCF7?3/MT cells were treated with AG3340during the adhesion assay alone to determine if MT1-MMP catalytic activity is required during adhesion to promote attachment to type I collagen (0.1?g/ml).(A)MCF7?3cells treated with 30?M dec-RVKR-cmk inhibited processing of pro-?v but this inhibitor had no effect on MT1-MMP processed pro-?v in ?3/MT cells.However,AG3340blocked MT1-MMP processing of pro-?v in ?3/MT cells,although pro-?v maturation still occurs through the proprotein convertase-dependent pathway.(B)?3cells were cultured in the presence or absence of 30?M dec-RVKR-cmk for 48h followed by analysis of cell adhesion to type I collagen.As shown,?3cell adhesion to type I collagen was restored to levels seen in mock cells when treated with the proprotein convertase inhibitor dec-RVKR-cmk.(C)Treatment of ?3/MT cells with AG3340for 48h results in suppression of cell adhesion to type I collagen to the levels similar to those observed in ?3cells.(D)?3/MT cell adhesion to type I collagen was determined in the presence or absence of AG3340during the adhesion assay.Blocking MT1-MMP activity in the cell adhesion assays had no effect on ?3/MT cell adhesion to type I collagen.
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integrin subunit types requiring proprotein convertase cleavage for maturation are also susceptible to MT1-MMP.Consistent with this hypothesis,MT1-MMP is capable of processing pro-?5and pro-?3.In turn,pro-?2,which is resistant to proprotein convertase cleavage and does not exhibit the cleavage motif,was found to be resistant to MT1-MMP cleavage [16].
The processing of pro-?v by MT1-MMP in breast carci-noma MCF7cells and the generation of the ?v ?3integrin heterodimer increased cell migration and adhesion on vitro-nectin through the upregulation of the FAK signal transduc-tion pathway.In this report,we demonstrate the profound in ?uence of MT1-MMP on the cross-talk imposed by ?v ?3integrin on ?2?1integrin,which itself is resistant to MT1-MMP.Our adhesion ?ow cytometry,and immunoprecipi-tation studies employing function-blocking integrin anti-bodies and synthetic inhibitors of furin and MMPs demonstrate that the expression of ?v ?3integrin alone re-presses the functional activity of ?2?1in breast carcinoma cells and,accordingly,diminishes the ef ?ciency of cell adhesion to type I collagen.In turn,the coexpression of MT1-MMP with integrin ?v ?3reverses this undesirable effect and restores the adhesion ef ?ciency of cells on type I collagen to the levels observed with control cells.The cur-rent studies indicate that pro-?v processing by MT1-MMP plays an essential role in this regulatory pathway.In agree-ment,the regulation of ?2?1integrin via the MT1-MMP/?v ?3axis was not observed in the cells expressing MT1-MMP alone.
MT1-MMP-dependent processing of the pro-integrin ?chains was observed,not only in breast carcinoma cells,but also in the other cell types including Chinese hamster ovary CHOK1cells,glioma U251cells,and embryonic kidney HEK293cells (data not shown).The mechanism by which
MT1-MMP affects the cross-talk between ?v ?3and ?2?1integrins is not precisely understood.Potential hypotheses include downstream regulation of the FAK signaling path-way [24]and alterations in the pool of cellular proteins associated with the cytoplasmic domain of the ?3integrin subunit [25,26].We suggest that the MT1-MMP-controlled cross-talk between ?v ?3and ?2?1integrins provides an alternative mechanism employed by malignant cells to reg-ulate adhesion to the extracellular matrix.This mechanism overrules the repression of ?2?1integrin,imposed by con-ventionally processed ?v ?3integrin,and supports the adhe-sive activity of aggressive,MT1-MMP,and ?v ?3integrin-expressing malignant cells on type I collagen,the most common substratum of the extracellular matrix.Collec-tively,our data further emphasize the complex functional relationship that exists between the integrin adhesion recep-tors and MT1-MMP and suggest a mechanism by which aggressive neoplastic cells downregulate counterproductive negative cooperativity between cell surface-associated inte-grin adhesion receptors.
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