The Role of Plasmhogen Activators in the Regulation of Connective Tissue Metalloproteinases” GILLIAN MURPHY,6 SUSAN ATKINSON, ROBIN WARD, JELENA GAVRILOVIC, AND JOHN J. REYNOLDS Stmqgemys Reseuxh Lubmtmy
W m cauretpay Gambn&e, United Kitghm CBl 4RN INTRODUCTION The conversion of plasminogen to plasmin is a key event in many physiological and pathologal processes requiring regulated extracellular proteolysis. Direct focal degradation of proteins involved in cell-cell and cell-matrix interactions by plasmin has been described,js as well as activation of other degradative enzymes, notably the matrix rnetall~proteinases~ (MMPs). Complex control of the plasminogen activator cascade has been shown to be required for the movement and re-organization of cells and matrix in events such as ovulation, trophoblast implantation, embryogenesis, and angiogenesis, as well as in the invasion and metastasis of tumors. A further level of regulation is thought to be effected by the release by plasmin of matrix-sequestered growth factors, such as basic fibroblast growth factor and trdnsbrming growth factor which are known to regulate the expression of both the plasminogen activatorinhibitor and the MMP-inhibitor systems2 /3,677
MATRIX METALLOPROTEINASES The MMPs play an important role in the degradation of connective tissue extracelMar matrices. Most connective tissue cells, including fibroblasts, chondrocytes, osteoblasts and endothelial cells, synthesize and secrete four types ofMMP. These have been purified tiom the conditioned medium of such cells and tissues in culture and characterized as having the combined ability to degrade all the components of the extracellular matrix.* The properties of each MMP type varies little with the species or with cell type. They are all Zn2+-and Ca2+-requiring proteinases and are secreted in a latent proform. Activation involves the loss of a propeptide, with an initial fill in the a This work was supported by the Arthritis and Rheumatism Council and the Medical Research Council, U.K. Author to whom correspondence should be addressed.
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Mr of about 1OOOO. The active enzymes are specifically inhibited by members of the fimily of tissue inhibitors of MMPs (TIMPs) which are secreted by most cells.9 The collagenases are the most specific of the MMPs, cleaving the native helix of the fib& collagens at a single locus. The gelatinases, or type IV co@nases, degrade types IV, V and VII collagens and may act synergistically with collagenases in the degradation of collagens, since they efficiently degrade denatured forms (gelatins). Two separate but homologous cDNAs encoding human gelatinases of Mr 95000 and 72 000 respectively have been identified to date and the corresponding proteins have been shown to be associated with many connective tissue cells as well as monocyte/ macrophages. The stromelysins are potentially a very important component of the degradative power of cells, having a broad pH optimum and substrate specificity and thus being able to degrade many extracellular matrix proteins; these enzymes also act as potentiators of cohgenase activity. Two separate but very homologous cDNAs encoding stromelysins have been identified. A more recently discovered MMP, punctuated metalloproteinase (pump), appears to be distantly related to the stromelysins, lacking the C-terminal domain and displaying some significant differences in specificity, notably the ability to degrade elastin.10
MEIlALLOPROTEINASE ACTIVATION Although transcriptionalregulation is a major control point in determining the extent of MMP expression, extracellular regulatory mechanisms may ultimately conml the level of enzyme activity in terms of matrix destruction. Consequently the nature of the MMP activation process and the hrther regulation of MMP activity by TIMPs has been the subject of intense investigation over the last frw years. Mechanisms by which secreted pro-MMPs may be activated extracellularly iff h are still not entirely clear. Studies on the biochemistry of the activation process have shown that the pro-MMPs can undergo a conformation change leading to self-cleavage of about 80 N-terminal amino acids. This process can be initiated by alteration of the chemical environment with ownomercurials, particularly 4aminophenylmercuric acetate. The conserved sequence PRCGVPD in the propeptide of MMPs may be involved by interaction of the cysteine thiol with the active site Zn2+.11J2 In the case of collagenase and stromelysin, proteolytic cleavage with either trypsin, plasmin, Mikrein or thermolysin can also cause activation, although a final self-cleavage event still occurs. 13.14 A role fbr plasmin as a potential physiological activator of procollagenase was first proposed by Eeckhout and Vaes.15 As part of a program to define such a role fbr plasmin we developed a model system for the analysis of matrix degradation. We have shown that cells such as synovial and gingival fibroblasts, chondrocytes, cap% endothelial cells, bone cells and VX2 tumor cells have the ability to degrade a 1%labeled type I collagen film in the presence of p I a ~ m i n 0 g e n . lThe ~ ~ ~collagenolytic activity was inhibited to greater than 90% by either the presence of TIMP, or a specific anti-collagenase IgG, implying a role fbr the enzyme collagenase in this process.17 Comparable data for tumor cell or endothelial cell invasion of the amnion have been reported by Mignatti et af. (reviewed in Moscatelli and Rifkinl). Using the collagen film model we have shown that the levels ofTIMP that are produced by the cells under study, as well as the amounts of available plasminogcn are crucial to the level of col-
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lagenolysis that is observed. Rabbit brain capillary endothelid cells stimulated with phorbol ester in the presence of low levels of plasminogen degraded the collagen to a limited extent (FIG. 1). Upon addition of a neutralizing anti-rabbit TIMP IgG, a marked increase in collagen degradation was observed as TIMP was removed fiom the system. Omission of plasminogen fiom the cultures prevented this stimulation (FIG. l), again suggesting that collagenase had been activated by way of plasmin generation.
STROMELYSIN ACTIVATION It is now well documented that urokinase-type plasminogen activator (uPA), and hence plasmin generation, is localized at cell adhesion sites by means of a specific receptor.2O-22 Plasminogen also binds to the cell sudice and to the extracellular maWe have demonstrated that stimulated cells of the type used in our collagen film degradation model (eg.,rabbit dermal fibroblasts) can activate exogenous human prostromelysin in the presence of plasminogen. Initially using a streptokinaseplasminogen system to generate plasmin at a concentration of 10 pg/ml, we found that stromelysin was activated within 3 h a t 37OC, with about 80% conversion from the Mr 57 000 profbrm to the Mr 46 000 active form (FIG. 2). The active stromelysin generated was identical in Mr to that generated by trypsin (FIG. 3). When plasminogen was incubated with stromelysin in the presence of monolayers of rabbit dermal fibroblasts we found that the cells were able to activate the stromelysin. Addition of streptokinase to the system, or the use of plasminogen-streptokinase in the absence of cell layers, did not modify the extent of stromelysin activation (TABLE 1). In further experiments the rate of stromelysin activation by cell layers and plasminogen was found to be similar with and without streptokinase (FIG. 4). Stromelysin was not activated by cell layers in the absence of plasminogen, nor by plasminogen in the absence of cells. It can therefbre be concluded that connective tissue cells can very rapidly activate stromelysin through a plasminogen-dependent mechanism. However, the presence of cell surfaces or their extracellular matrix did not appear to enhance this lOOr
FIGURE 1. The effect of TIMP or plasminogen depletion on the ability of capillaty endothelid cells to degrade collagen films.Rabbit brain capillary endothelial cells45 were cultivated on 1%labeled type I colhgcn films in medium containing 2% non-inhibitory rabbit serum as a source of plasminogen and 80 nM phorbol 12-myristate 13-acetate, PMA.17 The &, fragments of anti-rabbit TIMP IgG (60 j@ were also added to some wells to remove TIMP produced by the cells, markedly enhancing the rate of 14Ccollagen lysis. Prior rcmoval of plasminogen from the serum using lysine Sepharose and culture in the presence of PMA and anti-TIMP reduced the amount of collagen lysis. The mean of three wells f SEM are shown.
PMA anti-TIMP
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FIGURE 2. Activation of pmtmnelysin by plasmin. Purified ktcnt recombinant human p m tromclysin (2.5 was incubatedwith or without 10 @mI plasmin (generated from plasminogcn and streptokinase)at 37OC for up to 20 h. The activation reaction was stopped by the addition of 25 ccg/ml arpantiplasmin and cooling to O O C . Smmclysin activity was determined by percentage lysis of Wkaxin in the presence of 50 &ml soya bean trypsin inhibitor.
process in any way. The effect of inhibitors such as plasminogen activator inhibitors (PAIs) and TIMP, which may be produced by the cells, was minimized by the short term nature of the study. The mechanism of activation of prostmmelysin by proteinases has been nxently described by N a p e ct al.13 They showed that an initial attack by plasmin on the stromelysin propeptide cmtes an intermediate fbrm,which is then processed by an auto-cadytic cleavage at HissrPhes3.
COLLAGENASE ACI'IVmON Plasmin activation of pmcollagenase produced a marked fdl in Mr of the major form from 55000 to 44O00.5 The collagenase produced had an activity of only 200 unitsimg. Plasmin generated fmm plasminogen by fibroblast monolayers as described above was no more e&ctive. In the presence of stromelysin and plasmin, however, a hrther fdl in Mr to 43 OOO was observed and the activity of the collagenase rose up to 10-fold. A detailed analysis of the optimal conditions h r collagenase activation has shown that stmmelysin was most eflicient when the pre-activated form was present during w i n or plasmin activation of collagenase. Activation proceeded most rapidly at h@ concentrationsof collagenase even if the molar ratio of procollagenase: active stromelysin was 400:1 (TABLE 2). The high Mr (46OOO) fbrm of stromelysin and low Mr (28 OOO) brm were equally efficient on a molar basis in their ability to potentiate collagenase activau0n.u Pump, the truncated MMP and the Mr 22000 N-terminal portion of collagenase also acted as potentiators of collagenase activation,
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-997
FIGURE 3. Comparison of the activation of prostromelysin by plasmin and trypsin. Purified human prostrornelysinwas incubated: 1, alone, 37OC. 4 h; 2, with 5 p g h l trypsin, 25OC, 30 min; 3, with 10 &ml plasmin, 37%, 3 h. The products were subjected to polyacrylamide gel electrophoresis (1096 gel) and silver staining and stmmelysin activity was assessed in the 1% casein assay. The p m n zyme had no activity and the plasmin-activated material had 82% of the activity elicited by trypsin treatment.
-68 -55 -45
-35
-29 1
2
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TABLE 1. Activation of Prostromelysm by Fibroblast Monolayers Caseindegrading Activity Wml)
Prostromelysin and Prostromelysin and Prostromelysin and Prostromelysin and
plasminogcn plasminogen with celIs plasminogen with streptokinase plasrninogen with streptokinase and cells
0 0.041 0.033
0.033 Confluent monolayers of rabbit dermal fibroblasts stimulated with phorbol ester were washed and incubated for 2 h with 2.5 pg prostromelysin in the presence of 40 Icglml plasminogen. Stromelysin activity was assessed using 1% casein in the presence of a2 anti-plasmin and soya bean trypsin inhibitor. The extent of activation was compared with that elicited by plasminogen with or without streptokinase in the presence or absence of cells.
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FIGURE 4. Activation ofstromelysinby fibroblastmonolayers. Confluent monolayers of rabbit dermal fibroblasts stimulated with PMfi we= washed and incubaced for up to 6 h, with or without 2.5 pg of prosmmclysin in the pmence or absence of plasminogcnlsmptokinase. Stmmelysin activity was assesscd using 1Ccasciccascin in the p m n c e of az-antiplasmin and SBII. A, prostromelysin and plasminogcn/strcpmkinascin the presence of cells; A, ditto in the absence of cells; 0, prostromelysin and plasminogcn in the prescnce of cells; 0, ditto in the absence of cells; 0,plasminogcn/sncptokinase only in the p m n c e of cells; 0,prostromelysin in the pmence of cells.
apparently by the same mechanism2s (G. Murphy, unpublished observations). The ability ofstromelysin to potentiate trypsi activation of collagenase with a concomitant fall in Mr has been previously described (1987).MJ7Grant c t ~ 1 . described 2~ the initial trypsin c l e a w of the collagenase pmpeptide at +-Asn37, initiating subsequent self-cleavage along the pmpeptide. We have shown that fully activated collagenase has an N-terminal sequence generated from a cleavage at Glnso-Phee~.~~ The detailed
TABLE 2. The Effect of Collagenase Concentration on the Rate of Its Activation by Stromelysin in the Presence of Plasmin Rate of Collagcnase Activation (units/min)
Procollagenase 545 nM, plasmin Procollagcnase 27 nM, plasmin with 70 pM active smmelysin Procollagcnase 545 nM, plasmin with 1.4 nM active smmelysin
0.008
0.021 0.054
Procollagcnase (3 ccg) was incubated at 37OC for 1 h with active stromelysin at a molar ratio of collagcnase: stromelysin of approhatdy 400:1, in the presence of 10 &ml plasmin. The rate of collagcnase activation was ascsscd by assaying equivalent amounts of pmcollagcnasc in the 1% collagcn diffuse fibril assay. 1 unit of enzyme degrades 1 pg collagcn/min.
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mechanism of activation of procollagenase has been elucidated recently by Suzuki et al." (1990). Plasmin cleavage of procollagenase gave intermediates that rapidly
underwent autacamlytic cleavage at Thw-Lem5. In the presence of stromelysin a cleavage at Glnso-Phesl generated fully active collagenase. Stromelysin alone was only able to e e c t this cleavage of procollagcnase at a very slow rate; the initial action of tissue or plasma proteinases was required b r rapid activation. A major question arising from these studies concerns the importance of stromelysin in the regulation of collagenase activity in pipa. Mignatti et al.,Jo (1989) showed that anti-stromelysin antibodies did not prevent invasion by bovine capillary endothelial cells, a collagenase-dependent process. However, the localized nature of plasmin production at the cell sudce and by inference, the collagenase activation cascade, may mean that the antibody cannot gain efficient access to activation sites. Since very low levels of stromelysin a~ required h r collagenase activation under optimal conditions, the lack of effect of anti-suomelysin antibody may not be significant. Using a human EJ bladder carcinoma cell line which constitutively secretes procollagenase in culture (2 units006 cells/24 h), but does not synthesize stromelysin, we have shown that these cells are unable to degrade 1% c o l l a p films in the presence of plasminogen. However, if purified prostromelysin is added to the culture medium, plasminogendependent collagenolysis proceeds (FIG.5). Active smmelysin alone had negligible activity on the collagen films.
ACTIVATION OF GELATINASES Plasmin fiiled to activate Mr 72 OOO fibroblast progclatinase even at a concentration of 40 %/ml (equimolar plasmin:gelatinase). After penads of 3-4 h at 37OC a limited cleavage of gelatinase to M r 70 OOO by plasmin was observable but this did not elicit any activity, nor could the active fbrm (Mr 64O00)be generated by gelatinase selfileavage on subsequent addition of 4-aminophenylmercuric acetate (data not shown). We bund that gelatinase is not activated by equimolar amounts of active collagenase and/or stromelysin. Other investigators have also bund that fibroblast gelat-
FIGURE 5. The degradation of collagen films by tumor cells: requirement hr plasminogcn and stromelysin. Human bladder cvcinoma cells, EJ.18..8.D (gdi of D. Porteous, Edinburgh), were seeded onto *4Glabckdcollagen films, cultured ovcmight and washed16 befbrc culturing fbr 96 h in medium containing 2% acid-mtcd rabbit Serum as a source of plasminogcn. Very low levels of film lysis, cxprcssed as a percentage of the total, could bc observed. In the presence of prosmmclysin (0.2 units) marked collagcnolysisoccurred in the cultures containing plasminogcn. Results arc the mean of three wells f SEM. Activated stromelysin alone did not degrade the films.
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inase is not activated by trypsin or plasmin.Jl.32 The elucidation of physiological mechanisms fbr the activation of this gelatinase remains crucial to our understanding of its regulation. We have recently demonstrated that this enzyme can be activated by a fibroblast membrane-mediated process which is insensitive to senne proteinase inh i b i t but ~ ~is~inhibited ~ by metalloproteinaseinhibitors. We do not yet know ifthis activation involves a membrane-proteinase. Mr 95 000 progelatinase, purified h m U937 cell conditioned medium,” can be activated by plasmin in a dosedependent manner WLE 3), although relatively large amounts of plasmin are required. A similar activation can be achieved when the progelatinase is incubated with active stromelysin WLE 3), but no synergy between the activity of plasmin and stromelysin was observed.
PERICELLULAR NATURE OF ACTIVATION CASCADES Recent studies have emphasized the heal nature of plasmin generation in relation cell adhesion plaques. Urokinase-type plasminogen activator receptors localized at these sites bind uPA and substantially mod@ its activity and susceptibilityto the action of inhibitors.22,35,J6The pericellular activation of plasminogen is thought to be an important prerequisite fbr cell migration, hence the expression of cellular receptors Plasmin generated at local sites may of uPA as a key event in regulating this itself become bound and resistant to inhibition.38 Consequently the generation of active MMPs may be an event that is closely cell-surfice associated. The essential role of plasmin in the activation of many metalloproteinasescan be demonstrated in model systems such as those described herein. We have noted that in rapidly resorbing systems in vim, active collagenase can be immunolocalized on collagen fibres,3941 but in vim studies have shown that procollagnase binds weakly to collagenq1 (J. Allan, unpublished data). However, stmmelysin binds to c o h p in both its pro and active forms and can sometimes be immunolocalizcd in tissues such as synovium and cartilage under diflkrent conditionsu (R. Hembry, unpublished data). Both hrms of gelatinase have a domain S i a r to that of the collagen (gelatin)binding region of fibronectin,*J and it might be predicted that they could bind colto
TABLE 3. Activation of Mr 95 000 Progelatinase by Plasmin and Stromelysi % of APMA-induced Activity
Incubation alone pg plasmin/ml: 25 100 250
&ml stromelysin: 20 100
7 34 73 95
83 106
Mr 95 OOO p’ogclatinasewas incubated at 37OC for 1 h either alone or in the presence of varying concentrationsof plasmin (generated from plasminogcn-streptokinase)or active suomelysin.The activated enzyme was assayed using 1% gelatin at 37OC and the results were compared with progelatinase activated with 4-arninophenylmermricacetate (APMA). f f i r e assay incubations containingplasmin were pretreated with a 10-fbld excess ofsoya bean trypsin inhibitor. Stromclysin had no activity on gelatin at these concentrations.
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INHIBITORS PAls a2AP TlMPs
FIGURE 6. Scheme depicting the pencellular activation cascade b r plasminogcn and matrix metalloprotcinases. Urokinase-type plasminogcn activator, uPA, bound to cell receptoa is functional in both the i t & chain (sc) and tm chain (tc) brms,activating cell or matrix bound plasminogen activator inhibitors (PAIs) which may only have limited access to rtceptor sites. Plasmin can activate prostromelysin and M,95 OOO progclatinase dirrctly, the pmccss being rrgulated by a2 anti-plasmin (azAP) and also by TIMPs, since the final s’;rgcs of activation arc autocatalytic. Plasmin and stromelysin can activate collagcnax by sequential deavagcs, as described in the text. by CYAP and TIMPs. The concept of localized and limited These events would also be +ted activation at cell-matrix adhesion sites is supported by the abundance of latent metalloprotcinases, TIMPs and PAIs observed in the culture medium of connective tissue cells.
lagen in pipg, although there are no published descriptions of such a localization fbr gelatinases. It can be shown that similar effectors such as cytokines and growth factors regulate the extent of expression of uPA,PAIs,MMPs, and TIMPs (miwedin Murphy and Reynolds&) by cells. However, MMPs are produced in relatively small amounts relative to the plasminogen activators, with little constitutive expression. The overall picture that is emerging, as depicted in FIGURE6, is of plasminogen and M M P localization on the extracellular matrix and of plasminogen and uPA localization at sites of cell adhesion to the matrix. Thus the rate of plasmin activation and the extent of inhibitor activity at cell surfices could control the extent of MMP activity and hence matrix degradation. REIWRENCES D. & D. B. RXPKIN.1988. Membrane and matrix localization of proteinasex 1. MOSCATELLI, A common theme in tumor cell invasion and angiogcnesis. Biochim. Biophys. Acta 948: 67-85. 2. LIO-ITA, L. A., R H. GOLDPABB&
V. P. TJUANOVA. 1981. C l a w of laminin by thrombin and plasmin: Alpha thrombin sclcctively cleaves the beta chain of laminin. Thrombosis Rs.21: 663473. R BRUNDAGB, G. P. SIHOAL, V. THaaANova & S. GAR3. LIWA, L. A., R H. GOLDPABB, BISA. 1981. E&ct of plasminogcn activator (urokinase), plasmin, and thrombin on gly-
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coprotein and collagenous components of basement membrane. Cancer Res. 41: 4629436. 0.& D. B. RIFKIN. 1988.Cell-associated plasminogen activation: Regulation and 4. SAKSELA, physiological functions. Ann. Rev. Cell Biol. 4 93-126. 1991. Physiological mechanisms 5. MURPHY,G., R WARD,J. GAVRIMVIC& S. ATKINSON. for metalloproteinase activation. Matrix Suppl. 1: 224-230. 6. PEPPER,M. S.,D. BELIN,R MONTESANO,L. ORCI& J.-D. VASSALLI.1990.Transforming p w t h fictor-beta 1 modulates basic fibroblast growth fictor-induced proteolytic and angiogenic properties of endothelid cells in vitro. J. Cell Biol. llk 743-755. 7. SATO, Y., R TSUBOI, R LYONS,H . MOSES& D. B. RIFKIN.1990.Characterization of the activation of latent TGF-B by co-culturn of endotheli cells and pericytes or smooth muscle cells: A self-regulating system. J. Cell Biol. llk 757-763. 1988.Molecular studies on the connective tissue me8. MURPHY,G. & A . J. P. DOCHERTY. doproteinases and their inhibitor TIMP. In The Control of Tissue Damage. A. M. Glauert, Ed.: 223-241. Elxvier Science Publishers BV. Amsterdam. 9. DOCHERTY, A. J. P. & G. MURPHY.1990.The tissue metalloproteinase family and the inhibitor TIMP: A study using cDNAs and recombinant proteins. Ann. Rheum. Dis. 49: 469-479. 10. MURPHY,G., M. I. C ~ ~ K ER I TV. , WARD& A. J. P. DOCHERTY. 1991.Matrix metalloproteinase degradation ofelastin, type IV collagen and pmteoglycan. A quantitative comparison of the activities of 95 kDA and 72 kDA gelatinases, stromelysins-1 and -2and punctuated metalloproteinase (PUMP). Biochem. J. 277: 277-279. 11. SPRINGMAN, E. B., E. L. ANGLEION,H. BIRKEDAL-HANSON & H. E. VANWART. 1990. Multiple modes of activation of latent fibroblast collagenase: Evidence for the role of a cysteine active-site zinc complex in latency and a ”cysteine switch” mechanism for activation. Pmc.Natl. Acad. Sci. USA 87: 364-368. 12. DOCHERTY, A. J. P., J. O’CONNELL,T. CRABBE,S. ANGAL& G. MURPHY.1992.The matrix metalloproteinases and their natural inhibitors. Trends Biotechnol. 1 0 200-207. 13. NAGASE,H.,J.J. ENGHILD,K. Suzuw & G. SALVESEN. 1990.Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4aminophenyl)mercuric acetate. Biochemistry 29 5783-5789. & H. NAGASE.1990. Mecha14. SUZUKI,K.,J. J. ENGHILD,T. MOROWMI,G. SALVESEN nisms of activation of tissue pmcollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry 29: 10261-10270. 15. EECKHOUT,Y & G. VAES. 1977.Further studies on the activation of pmcollagenase, the latent precursor of bone collagenase. EfEcts of lysosomal cathepsin B, plasmin and Ml i i n , and spontaneous activation. Biochem. J. 166:21-31. 16. GAVRIMVIC,J., J. J. REYNOLDS& G. MURPHY.1985.Inhibition of type I collagen film degradation by cumour cells using a specific antibody to collagenase and the specific tissue inhibitor of metalloproteinases (TIMP). Cell Biol. Int. Rep. 9: 1W-1107. 17. GAVRIMVIC,J., R M. HEMBRY, J. J. REYNOLDS& G. MURPHY.1987.Tissue inhibitor of medoproteinases (TIMP) regulates extracellulartype I collagen degradation by chondrocytes and endothelial cells. J. Cell Sci. 87: 357-362. 18. GAVRIMVIC,J. & G. MURPHY.1989. The role of plasminogen in cell-mediated collagen degradation. Cell Biol. Int. Rep. 13: 367-375. 19. THOMPSON, B. M., S.J. ATKINSON, J. J. REYNOLDS& M. C. MEIKLE.1987.Degradation of type I collagen films by mouse osteoblasu is stimulated by 1,25 dihydroxyvitamin D3 and inhibited by human recombinant TIMP (tissue inhibitor of metalloproteinases). Biochem. Biophys. Res. Commun. 148: 596-602. J., 0.SAKSELA, E.-M. SALONEN,P. ANORBASEN,L. NIELSEN, K. DANO& A. 20. POLLANEN, VAHERI. 1987.Distinct localizations of umkinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells. J. Cell Biol. 104: 1085-1096. 21. KNUDSEN, B. S., R L. SILVERSTEIN, L. L. K. LEUNG,P. C. HARPEL& R L. NACHMAN. 1986.Binding of plasminogen to extracellularmatrix. J. Biol. Chem. 261: 10765-10771. 22. ELLIS,V.,N. BEHRENDT & K. DANO. 1991. Plasminogen activation by receptor-bound urokinase. J. Biol. Chem. 266: 12752-12758.
MURPHY ct d.:CONNECTIVE TISSUE MEIALIDPROTEINASE!3 23. 24.
25. 26. 27. 28. 29.
30. 31.
32.
33. 34. 35. 36.
37. 38. 39.
40. 41.
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P.J., C. A. DINARELM& T. B. STROM. 1986. Prostaglandinsposttranscriptionally inhibit monocyte exprrssion of interleukin 1 activity by increasing inuacellular cyclic adenosine monophosphate. J. Immunol. 137: 3189-3194. KOKLITIS,P.A., G. MURPHY,C. SU~TON& S. ANGAL.1991. Pudication of recombinant human pmtromelysin. Studies on heat activation to give high M,and low M, active forms, and a comparison of recombinant with natural smmelysin activities. Biochem. J. 276 217-221. QUA", B., G. MURPHY& R BRFATHNACH. 1989. Pump1 cDNA codes for a protein with characteristicssimilar to those of classical collagenax hnily members. Biochemistry 28: 5327-5334. MURPHY,G., M. I. C o c m , P. E. STEPHENS, B. J. S h i m i & A. J. P.DOCHERTY.1987. Stromelysin is an activator of procollagcnax. A study with natural and recombinant enzymes. Biochem. J. 248: 265-268. ITO, A. & H. NAGASE.1988. Evidence that human rheumatoid synovial matrix metalloproteinase 3 is an endogenous activator of pmllagenase. Arch. Biochem. Biophys. 267: 211-216. GRANT,G. A., A. Z. EISBN,B. L. Mmmx, W. T. Roswrr & G. I. GOLDBERG. 1987. The activation of human skin fibroblast pmollagenase. Sequence identification of the major conversion p d u c t s . J. Biol. Chem. 262: 58865889. WHITHAM,S . E., G. MURPHY,P. ANGEL, H . J . WMSDORF, B. J. S m , A. LYONS, T. J . R W s , J. J. REYNOLDS, P.HERRLICH & A. J. P. DOCHBRTY.1986. Comparison of human stromelysin and collagcrlase by cloning and sequence analysis. Biochem. J. 240: 913-916. MIGNATIT, P.,R TSUBOI, E. ROBBINS & D. B. RIpm. 1989. In vim angiogenesis on the human amniotic membrane: Requirement for basic fibroblast growth Ictor-induced pmteinases. J. Cell Biol. 108: 671682. COLLIER,I. E., S.M. WILHELM, A. Z. &SEN, B. L. hlARMBR, G. A. GRANT, J. L. Smnm, A. KRONBERGER, C. IIE, E. A. BAUER& G. I. GOLDBERGER. 1988. H-ras oncogenetransformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degradmg basement membrane collagen. J. Biol. Chem. 263: 6579-6587. OKADA,Y., T. MORODOMI,J. J. ENGHILD,K. Suzvm, A. YMU, I. NAICANISHI, G. SALVESEN & H. NAGASE. 1990. Matrix metalloproteinase2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. Eur. J. Biochem. 194: 721-730. P. M. SLOCOMBE, A. J. P. DOCHERTY,J. J. REYNOLDS& WARD,R V., S. J. ATKINSON, G. MURPHY.1991. Tissue inhibitor of metalloproteinase-2 inhibits the activation of 72 kDa progelatinax by fibroblast membranes. Biochim. Biophys. Acta 1079: 242-246. WARD,R V., R M. HF.MBRY, J. J. ~ Y N O L D S& G. MURPHY.1991. The purification of tissue inhibitor of metalloproteinases-2 from its 72 kDa progclatinase complex. Biochem. J. 278: 179-187. CUBELLIS, M. V.,T . 4 . WUN & F. BLASI. 1990. Receptor-mediated internalization and degradation ofurokinase is caused by its specific inhibitor PAI-1. EMBO J. 9: 1079-1085. ROLDAN, A. L., M. V.C U B ~ I SM. , T. m u c c ~N. , BEHRENDT, L. R LUND,K. DANO, E. APPELLA& F. BLASI.1990. Cloning and expmion of the receptor for human umkinase plasminogen activator, a central molecule in cell suhce, plasmin dependent pmteolysis. EMBO J. 9: 467-474. MANCHANDA, N. & B. S . Scmmn. 1991. Single chain urokinase. Augmentation of enzymatic activity upon binding to monocytes. J. Biol. Chem. 266: 14580-14584. HALL,S. W., J. E. HUMPI-IRIES & S. L. Go=. 1991. Inhibition of cell suhce receptor bound plasmin by cr2-macroglobulin. J. Biol. Chem. 266: 12329-12336. GAWLOVIC, J., R M. HEMBRY, J. J. ~ Y N O L D S& G. MURPHY.1989. Collagcnase is expressed by rabbit VX2 tumour cells in syngcneic and xenogeneic hosts. Matrix 9: 206-213. BROWN,C. C., R M. HEMBRY & J. J . R E ~ O L D S 1989. . Immunolocalization of metalloproteinam and their inhibitor in the rabbit growth plate. J. Bone Joint Surg. 7l-A: 580-593. HEMBRY, R M., G. MURPHY,T. E. CAWSTON, J. T. DJNGLE& J. J. ~ ~ O L D 1986. S . -EN,
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ANNALS NEW YORK ACADEMY OP SCIENCBS Characterization of a specific antiserum br mammalian collagcnase f h m several species: Immunolocalization of collagcnase in rabbit chondrocytcs and uterus. J. Cell Sci. 81:
105-123. 42. ALLAN, J. A., R M. HEMBRY, S. ANGAL,J. J. REYNOLDS & G. MURPHY. 1991. Binding of latent and hgh M,active brms of stromclysin to collagen is mediated by the Cterminal domain. J. Cell Sci. 99:789-795. S. M., I. E. COLLIER, B. L. MARMER, A. Z. EISEN,G. A. GRANT & G. I. GOLD43. WILHELM, BERG. 1989. SV4O-transbrmcd human lung fibroblasts secrete a 92-kDa type IV collag n a w which is identical to that secreted by normal human macrophap. J. Biol. Chem. 2&L: 17213-17221. 44. MURPHY,G. & J. J. REYNOLDS.1992. Extracellular matrix degradation. In Connective
Tissue and Its Heritable Disorders; Molecular, Genetic and Medical Aspects. P. RDyce & B. Steinman, Eds. Alan R. Lw.New Yo&. In p m . 45. BANDA,M. J., D. R KNIGIFIYIN,T. K. HUNT& Z. WEBB. 1982. Isolation of a nonmitognic angiogenesis .Factor f b m wound fluid. Proc. Natl. Acad. Sci. USA 79:7773-7777.
W m cauretpay Gambn&e, United Kitghm CBl 4RN INTRODUCTION The conversion of plasminogen to plasmin is a key event in many physiological and pathologal processes requiring regulated extracellular proteolysis. Direct focal degradation of proteins involved in cell-cell and cell-matrix interactions by plasmin has been described,js as well as activation of other degradative enzymes, notably the matrix rnetall~proteinases~ (MMPs). Complex control of the plasminogen activator cascade has been shown to be required for the movement and re-organization of cells and matrix in events such as ovulation, trophoblast implantation, embryogenesis, and angiogenesis, as well as in the invasion and metastasis of tumors. A further level of regulation is thought to be effected by the release by plasmin of matrix-sequestered growth factors, such as basic fibroblast growth factor and trdnsbrming growth factor which are known to regulate the expression of both the plasminogen activatorinhibitor and the MMP-inhibitor systems2 /3,677
MATRIX METALLOPROTEINASES The MMPs play an important role in the degradation of connective tissue extracelMar matrices. Most connective tissue cells, including fibroblasts, chondrocytes, osteoblasts and endothelial cells, synthesize and secrete four types ofMMP. These have been purified tiom the conditioned medium of such cells and tissues in culture and characterized as having the combined ability to degrade all the components of the extracellular matrix.* The properties of each MMP type varies little with the species or with cell type. They are all Zn2+-and Ca2+-requiring proteinases and are secreted in a latent proform. Activation involves the loss of a propeptide, with an initial fill in the a This work was supported by the Arthritis and Rheumatism Council and the Medical Research Council, U.K. Author to whom correspondence should be addressed.
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Mr of about 1OOOO. The active enzymes are specifically inhibited by members of the fimily of tissue inhibitors of MMPs (TIMPs) which are secreted by most cells.9 The collagenases are the most specific of the MMPs, cleaving the native helix of the fib& collagens at a single locus. The gelatinases, or type IV co@nases, degrade types IV, V and VII collagens and may act synergistically with collagenases in the degradation of collagens, since they efficiently degrade denatured forms (gelatins). Two separate but homologous cDNAs encoding human gelatinases of Mr 95000 and 72 000 respectively have been identified to date and the corresponding proteins have been shown to be associated with many connective tissue cells as well as monocyte/ macrophages. The stromelysins are potentially a very important component of the degradative power of cells, having a broad pH optimum and substrate specificity and thus being able to degrade many extracellular matrix proteins; these enzymes also act as potentiators of cohgenase activity. Two separate but very homologous cDNAs encoding stromelysins have been identified. A more recently discovered MMP, punctuated metalloproteinase (pump), appears to be distantly related to the stromelysins, lacking the C-terminal domain and displaying some significant differences in specificity, notably the ability to degrade elastin.10
MEIlALLOPROTEINASE ACTIVATION Although transcriptionalregulation is a major control point in determining the extent of MMP expression, extracellular regulatory mechanisms may ultimately conml the level of enzyme activity in terms of matrix destruction. Consequently the nature of the MMP activation process and the hrther regulation of MMP activity by TIMPs has been the subject of intense investigation over the last frw years. Mechanisms by which secreted pro-MMPs may be activated extracellularly iff h are still not entirely clear. Studies on the biochemistry of the activation process have shown that the pro-MMPs can undergo a conformation change leading to self-cleavage of about 80 N-terminal amino acids. This process can be initiated by alteration of the chemical environment with ownomercurials, particularly 4aminophenylmercuric acetate. The conserved sequence PRCGVPD in the propeptide of MMPs may be involved by interaction of the cysteine thiol with the active site Zn2+.11J2 In the case of collagenase and stromelysin, proteolytic cleavage with either trypsin, plasmin, Mikrein or thermolysin can also cause activation, although a final self-cleavage event still occurs. 13.14 A role fbr plasmin as a potential physiological activator of procollagenase was first proposed by Eeckhout and Vaes.15 As part of a program to define such a role fbr plasmin we developed a model system for the analysis of matrix degradation. We have shown that cells such as synovial and gingival fibroblasts, chondrocytes, cap% endothelial cells, bone cells and VX2 tumor cells have the ability to degrade a 1%labeled type I collagen film in the presence of p I a ~ m i n 0 g e n . lThe ~ ~ ~collagenolytic activity was inhibited to greater than 90% by either the presence of TIMP, or a specific anti-collagenase IgG, implying a role fbr the enzyme collagenase in this process.17 Comparable data for tumor cell or endothelial cell invasion of the amnion have been reported by Mignatti et af. (reviewed in Moscatelli and Rifkinl). Using the collagen film model we have shown that the levels ofTIMP that are produced by the cells under study, as well as the amounts of available plasminogcn are crucial to the level of col-
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lagenolysis that is observed. Rabbit brain capillary endothelid cells stimulated with phorbol ester in the presence of low levels of plasminogen degraded the collagen to a limited extent (FIG. 1). Upon addition of a neutralizing anti-rabbit TIMP IgG, a marked increase in collagen degradation was observed as TIMP was removed fiom the system. Omission of plasminogen fiom the cultures prevented this stimulation (FIG. l), again suggesting that collagenase had been activated by way of plasmin generation.
STROMELYSIN ACTIVATION It is now well documented that urokinase-type plasminogen activator (uPA), and hence plasmin generation, is localized at cell adhesion sites by means of a specific receptor.2O-22 Plasminogen also binds to the cell sudice and to the extracellular maWe have demonstrated that stimulated cells of the type used in our collagen film degradation model (eg.,rabbit dermal fibroblasts) can activate exogenous human prostromelysin in the presence of plasminogen. Initially using a streptokinaseplasminogen system to generate plasmin at a concentration of 10 pg/ml, we found that stromelysin was activated within 3 h a t 37OC, with about 80% conversion from the Mr 57 000 profbrm to the Mr 46 000 active form (FIG. 2). The active stromelysin generated was identical in Mr to that generated by trypsin (FIG. 3). When plasminogen was incubated with stromelysin in the presence of monolayers of rabbit dermal fibroblasts we found that the cells were able to activate the stromelysin. Addition of streptokinase to the system, or the use of plasminogen-streptokinase in the absence of cell layers, did not modify the extent of stromelysin activation (TABLE 1). In further experiments the rate of stromelysin activation by cell layers and plasminogen was found to be similar with and without streptokinase (FIG. 4). Stromelysin was not activated by cell layers in the absence of plasminogen, nor by plasminogen in the absence of cells. It can therefbre be concluded that connective tissue cells can very rapidly activate stromelysin through a plasminogen-dependent mechanism. However, the presence of cell surfaces or their extracellular matrix did not appear to enhance this lOOr
FIGURE 1. The effect of TIMP or plasminogen depletion on the ability of capillaty endothelid cells to degrade collagen films.Rabbit brain capillary endothelial cells45 were cultivated on 1%labeled type I colhgcn films in medium containing 2% non-inhibitory rabbit serum as a source of plasminogen and 80 nM phorbol 12-myristate 13-acetate, PMA.17 The &, fragments of anti-rabbit TIMP IgG (60 j@ were also added to some wells to remove TIMP produced by the cells, markedly enhancing the rate of 14Ccollagen lysis. Prior rcmoval of plasminogen from the serum using lysine Sepharose and culture in the presence of PMA and anti-TIMP reduced the amount of collagen lysis. The mean of three wells f SEM are shown.
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FIGURE 2. Activation of pmtmnelysin by plasmin. Purified ktcnt recombinant human p m tromclysin (2.5 was incubatedwith or without 10 @mI plasmin (generated from plasminogcn and streptokinase)at 37OC for up to 20 h. The activation reaction was stopped by the addition of 25 ccg/ml arpantiplasmin and cooling to O O C . Smmclysin activity was determined by percentage lysis of Wkaxin in the presence of 50 &ml soya bean trypsin inhibitor.
process in any way. The effect of inhibitors such as plasminogen activator inhibitors (PAIs) and TIMP, which may be produced by the cells, was minimized by the short term nature of the study. The mechanism of activation of prostmmelysin by proteinases has been nxently described by N a p e ct al.13 They showed that an initial attack by plasmin on the stromelysin propeptide cmtes an intermediate fbrm,which is then processed by an auto-cadytic cleavage at HissrPhes3.
COLLAGENASE ACI'IVmON Plasmin activation of pmcollagenase produced a marked fdl in Mr of the major form from 55000 to 44O00.5 The collagenase produced had an activity of only 200 unitsimg. Plasmin generated fmm plasminogen by fibroblast monolayers as described above was no more e&ctive. In the presence of stromelysin and plasmin, however, a hrther fdl in Mr to 43 OOO was observed and the activity of the collagenase rose up to 10-fold. A detailed analysis of the optimal conditions h r collagenase activation has shown that stmmelysin was most eflicient when the pre-activated form was present during w i n or plasmin activation of collagenase. Activation proceeded most rapidly at h@ concentrationsof collagenase even if the molar ratio of procollagenase: active stromelysin was 400:1 (TABLE 2). The high Mr (46OOO) fbrm of stromelysin and low Mr (28 OOO) brm were equally efficient on a molar basis in their ability to potentiate collagenase activau0n.u Pump, the truncated MMP and the Mr 22000 N-terminal portion of collagenase also acted as potentiators of collagenase activation,
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-997
FIGURE 3. Comparison of the activation of prostromelysin by plasmin and trypsin. Purified human prostrornelysinwas incubated: 1, alone, 37OC. 4 h; 2, with 5 p g h l trypsin, 25OC, 30 min; 3, with 10 &ml plasmin, 37%, 3 h. The products were subjected to polyacrylamide gel electrophoresis (1096 gel) and silver staining and stmmelysin activity was assessed in the 1% casein assay. The p m n zyme had no activity and the plasmin-activated material had 82% of the activity elicited by trypsin treatment.
-68 -55 -45
-35
-29 1
2
3
TABLE 1. Activation of Prostromelysm by Fibroblast Monolayers Caseindegrading Activity Wml)
Prostromelysin and Prostromelysin and Prostromelysin and Prostromelysin and
plasminogcn plasminogen with celIs plasminogen with streptokinase plasrninogen with streptokinase and cells
0 0.041 0.033
0.033 Confluent monolayers of rabbit dermal fibroblasts stimulated with phorbol ester were washed and incubated for 2 h with 2.5 pg prostromelysin in the presence of 40 Icglml plasminogen. Stromelysin activity was assessed using 1% casein in the presence of a2 anti-plasmin and soya bean trypsin inhibitor. The extent of activation was compared with that elicited by plasminogen with or without streptokinase in the presence or absence of cells.
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FIGURE 4. Activation ofstromelysinby fibroblastmonolayers. Confluent monolayers of rabbit dermal fibroblasts stimulated with PMfi we= washed and incubaced for up to 6 h, with or without 2.5 pg of prosmmclysin in the pmence or absence of plasminogcnlsmptokinase. Stmmelysin activity was assesscd using 1Ccasciccascin in the p m n c e of az-antiplasmin and SBII. A, prostromelysin and plasminogcn/strcpmkinascin the presence of cells; A, ditto in the absence of cells; 0, prostromelysin and plasminogcn in the prescnce of cells; 0, ditto in the absence of cells; 0,plasminogcn/sncptokinase only in the p m n c e of cells; 0,prostromelysin in the pmence of cells.
apparently by the same mechanism2s (G. Murphy, unpublished observations). The ability ofstromelysin to potentiate trypsi activation of collagenase with a concomitant fall in Mr has been previously described (1987).MJ7Grant c t ~ 1 . described 2~ the initial trypsin c l e a w of the collagenase pmpeptide at +-Asn37, initiating subsequent self-cleavage along the pmpeptide. We have shown that fully activated collagenase has an N-terminal sequence generated from a cleavage at Glnso-Phee~.~~ The detailed
TABLE 2. The Effect of Collagenase Concentration on the Rate of Its Activation by Stromelysin in the Presence of Plasmin Rate of Collagcnase Activation (units/min)
Procollagenase 545 nM, plasmin Procollagcnase 27 nM, plasmin with 70 pM active smmelysin Procollagcnase 545 nM, plasmin with 1.4 nM active smmelysin
0.008
0.021 0.054
Procollagcnase (3 ccg) was incubated at 37OC for 1 h with active stromelysin at a molar ratio of collagcnase: stromelysin of approhatdy 400:1, in the presence of 10 &ml plasmin. The rate of collagcnase activation was ascsscd by assaying equivalent amounts of pmcollagcnasc in the 1% collagcn diffuse fibril assay. 1 unit of enzyme degrades 1 pg collagcn/min.
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mechanism of activation of procollagenase has been elucidated recently by Suzuki et al." (1990). Plasmin cleavage of procollagenase gave intermediates that rapidly
underwent autacamlytic cleavage at Thw-Lem5. In the presence of stromelysin a cleavage at Glnso-Phesl generated fully active collagenase. Stromelysin alone was only able to e e c t this cleavage of procollagcnase at a very slow rate; the initial action of tissue or plasma proteinases was required b r rapid activation. A major question arising from these studies concerns the importance of stromelysin in the regulation of collagenase activity in pipa. Mignatti et al.,Jo (1989) showed that anti-stromelysin antibodies did not prevent invasion by bovine capillary endothelial cells, a collagenase-dependent process. However, the localized nature of plasmin production at the cell sudce and by inference, the collagenase activation cascade, may mean that the antibody cannot gain efficient access to activation sites. Since very low levels of stromelysin a~ required h r collagenase activation under optimal conditions, the lack of effect of anti-suomelysin antibody may not be significant. Using a human EJ bladder carcinoma cell line which constitutively secretes procollagenase in culture (2 units006 cells/24 h), but does not synthesize stromelysin, we have shown that these cells are unable to degrade 1% c o l l a p films in the presence of plasminogen. However, if purified prostromelysin is added to the culture medium, plasminogendependent collagenolysis proceeds (FIG.5). Active smmelysin alone had negligible activity on the collagen films.
ACTIVATION OF GELATINASES Plasmin fiiled to activate Mr 72 OOO fibroblast progclatinase even at a concentration of 40 %/ml (equimolar plasmin:gelatinase). After penads of 3-4 h at 37OC a limited cleavage of gelatinase to M r 70 OOO by plasmin was observable but this did not elicit any activity, nor could the active fbrm (Mr 64O00)be generated by gelatinase selfileavage on subsequent addition of 4-aminophenylmercuric acetate (data not shown). We bund that gelatinase is not activated by equimolar amounts of active collagenase and/or stromelysin. Other investigators have also bund that fibroblast gelat-
FIGURE 5. The degradation of collagen films by tumor cells: requirement hr plasminogcn and stromelysin. Human bladder cvcinoma cells, EJ.18..8.D (gdi of D. Porteous, Edinburgh), were seeded onto *4Glabckdcollagen films, cultured ovcmight and washed16 befbrc culturing fbr 96 h in medium containing 2% acid-mtcd rabbit Serum as a source of plasminogcn. Very low levels of film lysis, cxprcssed as a percentage of the total, could bc observed. In the presence of prosmmclysin (0.2 units) marked collagcnolysisoccurred in the cultures containing plasminogcn. Results arc the mean of three wells f SEM. Activated stromelysin alone did not degrade the films.
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inase is not activated by trypsin or plasmin.Jl.32 The elucidation of physiological mechanisms fbr the activation of this gelatinase remains crucial to our understanding of its regulation. We have recently demonstrated that this enzyme can be activated by a fibroblast membrane-mediated process which is insensitive to senne proteinase inh i b i t but ~ ~is~inhibited ~ by metalloproteinaseinhibitors. We do not yet know ifthis activation involves a membrane-proteinase. Mr 95 000 progelatinase, purified h m U937 cell conditioned medium,” can be activated by plasmin in a dosedependent manner WLE 3), although relatively large amounts of plasmin are required. A similar activation can be achieved when the progelatinase is incubated with active stromelysin WLE 3), but no synergy between the activity of plasmin and stromelysin was observed.
PERICELLULAR NATURE OF ACTIVATION CASCADES Recent studies have emphasized the heal nature of plasmin generation in relation cell adhesion plaques. Urokinase-type plasminogen activator receptors localized at these sites bind uPA and substantially mod@ its activity and susceptibilityto the action of inhibitors.22,35,J6The pericellular activation of plasminogen is thought to be an important prerequisite fbr cell migration, hence the expression of cellular receptors Plasmin generated at local sites may of uPA as a key event in regulating this itself become bound and resistant to inhibition.38 Consequently the generation of active MMPs may be an event that is closely cell-surfice associated. The essential role of plasmin in the activation of many metalloproteinasescan be demonstrated in model systems such as those described herein. We have noted that in rapidly resorbing systems in vim, active collagenase can be immunolocalized on collagen fibres,3941 but in vim studies have shown that procollagnase binds weakly to collagenq1 (J. Allan, unpublished data). However, stmmelysin binds to c o h p in both its pro and active forms and can sometimes be immunolocalizcd in tissues such as synovium and cartilage under diflkrent conditionsu (R. Hembry, unpublished data). Both hrms of gelatinase have a domain S i a r to that of the collagen (gelatin)binding region of fibronectin,*J and it might be predicted that they could bind colto
TABLE 3. Activation of Mr 95 000 Progelatinase by Plasmin and Stromelysi % of APMA-induced Activity
Incubation alone pg plasmin/ml: 25 100 250
&ml stromelysin: 20 100
7 34 73 95
83 106
Mr 95 OOO p’ogclatinasewas incubated at 37OC for 1 h either alone or in the presence of varying concentrationsof plasmin (generated from plasminogcn-streptokinase)or active suomelysin.The activated enzyme was assayed using 1% gelatin at 37OC and the results were compared with progelatinase activated with 4-arninophenylmermricacetate (APMA). f f i r e assay incubations containingplasmin were pretreated with a 10-fbld excess ofsoya bean trypsin inhibitor. Stromclysin had no activity on gelatin at these concentrations.
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INHIBITORS PAls a2AP TlMPs
FIGURE 6. Scheme depicting the pencellular activation cascade b r plasminogcn and matrix metalloprotcinases. Urokinase-type plasminogcn activator, uPA, bound to cell receptoa is functional in both the i t & chain (sc) and tm chain (tc) brms,activating cell or matrix bound plasminogen activator inhibitors (PAIs) which may only have limited access to rtceptor sites. Plasmin can activate prostromelysin and M,95 OOO progclatinase dirrctly, the pmccss being rrgulated by a2 anti-plasmin (azAP) and also by TIMPs, since the final s’;rgcs of activation arc autocatalytic. Plasmin and stromelysin can activate collagcnax by sequential deavagcs, as described in the text. by CYAP and TIMPs. The concept of localized and limited These events would also be +ted activation at cell-matrix adhesion sites is supported by the abundance of latent metalloprotcinases, TIMPs and PAIs observed in the culture medium of connective tissue cells.
lagen in pipg, although there are no published descriptions of such a localization fbr gelatinases. It can be shown that similar effectors such as cytokines and growth factors regulate the extent of expression of uPA,PAIs,MMPs, and TIMPs (miwedin Murphy and Reynolds&) by cells. However, MMPs are produced in relatively small amounts relative to the plasminogen activators, with little constitutive expression. The overall picture that is emerging, as depicted in FIGURE6, is of plasminogen and M M P localization on the extracellular matrix and of plasminogen and uPA localization at sites of cell adhesion to the matrix. Thus the rate of plasmin activation and the extent of inhibitor activity at cell surfices could control the extent of MMP activity and hence matrix degradation. REIWRENCES D. & D. B. RXPKIN.1988. Membrane and matrix localization of proteinasex 1. MOSCATELLI, A common theme in tumor cell invasion and angiogcnesis. Biochim. Biophys. Acta 948: 67-85. 2. LIO-ITA, L. A., R H. GOLDPABB&
V. P. TJUANOVA. 1981. C l a w of laminin by thrombin and plasmin: Alpha thrombin sclcctively cleaves the beta chain of laminin. Thrombosis Rs.21: 663473. R BRUNDAGB, G. P. SIHOAL, V. THaaANova & S. GAR3. LIWA, L. A., R H. GOLDPABB, BISA. 1981. E&ct of plasminogcn activator (urokinase), plasmin, and thrombin on gly-
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