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133 On the role of plasminogen activator inhibitor type-2(PAI-2) in cell survival
38
SESSION XI: Receptors, Proteases, Protease Inhibitors
133
134
ON THE ROLE OF PLASMINOGEN ACTIVATOR INHIBITOR TYPE-2 (PAI-2) IN CELL SURVIVAL
BINDING OF tPA TO HUMAN VASCULAR SMOOTH MUSCLE CELLS: EVIDENCE FOR A NOVEL, SPECIFIC RECEPTOR Vincent Ellis Tabir Razzaq, Finn Warner and Simon Whawell Thrombosis Research Institute, London, UK; Finsen Laboratory, Copenhagen, Denmark
Toni M. Antalis, May La Lin, Karen Donnan, Luis Maten, Joy Gardner, Joanne L. Dickinson, Kathy Buttigieg and Andreas Suhrbier. The Queensland Cancer Fund Experimental Ontology and EBV Units, The Queensland Institute of Medical Research, Brisbane, 4029, Australia Plasminogen activator inhibitor type -2 (PAI-2) is a serpin that is targeted to both extmcellular and intmcellular locations. Extmcellular PAI-2 is an efficient inhibitor of urokinase-type plasminogeu activator (uPA) and functions to regulate extracellniar uPA-initiated proteolytic events Involved in tissue remodelling, inflammation and tumor invasion and metastasis. We and others have demonstrated an inlracellular role for PAI-2 in the regulation of survival from tumour necrosis factor-a (TNF-a) mediated apoptusis. Here we show that PAI-2 also protects cells from the rapid eytopathic effects of alphavirus infection. Protection was associated with intracellular aad not extracellniar PAl2. In this case, protection was not associated with an effect on apoptosis but was mediated by a PAI-2 dependent induction of constitutive low level IFN a/13production. IFNcd[3 primed the cells for rapid induction of antiviral genes, resulting in the establishment of a persistent productive alphaviral infection in PAI-2 transfected cells. PAI-2 was also induced in macrophages in response to viral RNA suggesting that PAI-2 is a virus response gene. These observations and the ability of PAI-2 to inhibit TNF-c~ mediated apoptosis illustrate that PAI-2 has an additional and distinct function as an intracellular regulator of signal transduction pathway(s) and demonstrate a novel activity for a eukaryotie serpin.
Experiments in both normal and transgenic animals indicate that plasminogen activators play a role in the response of the vessel wall to injury, presumably by mediating the degradation of extracellular matrix by vascular smooth muscle cells (VSMC) which is necessary to facilitate their migration and proliferation. As these cells express both uPA and tPA in rive, we have studied whether they can assemble a tPA-catalysed plasminogen activation system on their surface in addition to the established uPA/uPAR system. tPA was found to bind to human VSMC, using a functional assay i.e. by plasminogeu activation. Analysis of the binding isotherms revealed 2 classes of binding site with Kcs of 25 and 660 nM. Enzyme kinetic analysis demonstrated that although both of these sites mediated plasminngan activation, the large enhancement of tPA activity observed (> 100-fold) involved the higher affinity site and required the cellular binding of plasminogen. The binding oftPA was not competed for either by antibodies to aanexin II, proposed as an endothelial tPA binding site, or by ligands competing the binding oftPA to a variety of other molecules such as LRP, mannose receptor or glycosaminoglycans.The speeificityoftPA binding was demonstrated, as Ser47S>AlatPA competed tPA binding by >85%. By contrast, PhePro-Arg-chloromethyl ketone (PPACK)-ina~vated tPA did not compete the binding of active tPA to VSMC, although it was effective at competing tPA binding to fibrin. As the domain deletion mutant K2P did not bind with high affinity, these data implicate both the serine pretense and N-terminal domains of tPA in the binding interaction. Preliminary aff~mtychromatography experiments have detected a tPAbinding protein of approx. 350 kD in detergent extracts of these cells. Comparison with the uPAR system reveals that tPA binding has the potential to mediate 10-25-fold greater plasmin generation on these cells. These data demonstrate the occurrence of a novel specific tPA receptor on human VSMC which may he important for the regulation of plasminogen activation and ECM degradation by these ceils in various vascular pathologies.
135
136
TISSUE PLASMINOGEN ACTIVATOR BINDING TO THE TAIL DOMAIN OF ITS ENDOTHELIAL CELL RECEPTOR, ANNEXIN II: DIRECT MODULATION BY HOMOCYSTEINE. K.A. Haliar° , L. Mauri°, F. Zhong', U. tffu'za', B.T. Chait °'. Cornell University Medical College * and Rockefeller University", New York, NY. Both tis~a¢ plasmm'ogen activator (t-PA) and its substrate, plasminogeu (PLG), bind to the endothelial cell (EC) surface via the calcinm-regulated, phospholipidbinding protein annexin II (Ann II). Recent studies indicate that binding sites for both cell surface phosphulipid and the ligand PLG reside within the C-t~minal "core" region of Ann IL Binding oft-PA, but not PLG, is down-regulated upon exposure of EC to the thiol-euntainlng amino acid, homocysteine (HC, 1 raM, 18h). Here, to identify the t-PA binding domain of Ann II and to determine the mechanism of its modulation by HC, the chymotryptic "core" fragment recombinant human Ann II (r/am II) was isolated by ion exchange chromatography. Win3eexcess whole rAan ]I cempletely blocked specific binding of 125I-t-PA to mfive Ann II, the 33-kDa "core" fragment failed to do so, suggesting assignment of the t-PA-bindiag domain to the N-terminal "tail" region. Two overlapping "tail" pepfides (residues 2-13 and 7-18) each blocked 65-75% of mI-t-PA binding to native Ann H (I~ = 0.4 raM), while a third poptide (residues 14-25) blocked <10% of binding. A hexapeptide (LCKLSL) mimicking the residue 7-12 region of overlap, but not LGKLSL, completely inhibited binding of ~ - t - P A to both rAnn H and cultured EC (Is0= 0.2 and 1.1 raM), thus identifying a putative t-PA-binding domain- To further explore the role of C residues in t-PA binding, mutants containing all four possible C to G Ann H substitutions (C9G, C133G, C262G, and C335G) were tested. C133G, C262G, and C335G mutants expressed in 293 cells showed binding of mI-t-PA indistinguishable from that of the wild type construct, whereas =~sI-t-PAbinding to C9G-transfected cells was reduced by 61%. When rAnnlI was treated with HC (5 mM, 3h), electrospray ionization mass spectrometry (MS) revealed a 135 _+ 2 Da increase in mass, indicating a 1:1 stoichiometry of HC to Ann II adduct formation. Tandem MS of the N-termlnal tryptic peptide DPSTVHEILCK specifically localized this increment to C9. Treatment ofrAanlI with HC reduced its capacity to bind t-PA by 65% (I~ = 11 I~M). These data provide a molecular mechanism by which HC may impair the t-PA-binding capacity of Ann II, thereby inducing a prothrombotic effect in the blood vessel wall.
MODULATION OF PLASMINOGEN RECEPTOR EXPRESSION ON LEUKOCYTES Thomas Herren and Edward F. Plow Joseph J. Jacobs Center for Thrombosis and Vascular Biology, The Cleveland Clinic Foundation, Cleveland, OH 44195 A linkage between the plasminogen system and the inflammatory response has long been recognized. This association has been emphasized in studies of plasminogeudeficient mice which exhibit decreased leukocyte infiltration in response to inflammatory stimuli. Since the role of the plasminogeu system in cell migration depends upon intera~on of plasminogen with the cell surfaces, we have investigated the regulation of plasminogen receptors (PIgR) on neutrophils, Freshly isolated human peripheral blood neutrophils express low levels of PIgR (at an input concentration of I00 nM plasminogen, cells specifically bound 1.78 -~ 0.23 x 10~ moleodes/cell, n = 10). When cultured, either in the presence or absence of serum, the neutrophils extfibit a dramatic increase, up to 50-fold, in plasminogeu binding capacity. Although plasmin itself can increase the number of PIgR on cells by proteolytically generating new plasminogeu binding sites, depletion of the culture medium of plasminogen or addition of the plasmin inhibitor, aprotinin, affected upregulation of PlgR only modestly. However, addition of soybean trypsin inhibitor (SBTI) to culture medium suppressed up-regulation. Doses of SBTI as low as 0.05 mg/ml were suppressive and tmg/ml fully prevented up-regulation. These data implicate a proteolytic pathway for up-regulation of PIgR. Consistent with this interpretation, specific inhibitors of elastase and eathepsin G were also capable of suppressing the up-regulation of PlgR. Moreover, conditions known to increase the elastase binding capacity of neutrophils (LPS followed by flv1LP)increased PlgR on neotrophils. In a separate set of experiments, nentrophils were stimulated with PMA to induce cell adhesion. Unlike monoc~jtoidcells in which enhanced PlgR expression is restricted to the nonadherant cells, PlgR expression was increased in both the adherent and the non-adherent neutrophils. Under these circumstances, SBTI had only a modest effect on up-regulation. Taken together, these data suggest that neutrophils are capable of markedly altering their plasminogen binding capacity. Both proteolytic and non-proteolytic (or distinct proteinases) pathways may modulate PlgR receptor expression.
SESSION XI: Receptors, Proteases, Protease Inhibitors
133
134
ON THE ROLE OF PLASMINOGEN ACTIVATOR INHIBITOR TYPE-2 (PAI-2) IN CELL SURVIVAL
BINDING OF tPA TO HUMAN VASCULAR SMOOTH MUSCLE CELLS: EVIDENCE FOR A NOVEL, SPECIFIC RECEPTOR Vincent Ellis Tabir Razzaq, Finn Warner and Simon Whawell Thrombosis Research Institute, London, UK; Finsen Laboratory, Copenhagen, Denmark
Toni M. Antalis, May La Lin, Karen Donnan, Luis Maten, Joy Gardner, Joanne L. Dickinson, Kathy Buttigieg and Andreas Suhrbier. The Queensland Cancer Fund Experimental Ontology and EBV Units, The Queensland Institute of Medical Research, Brisbane, 4029, Australia Plasminogen activator inhibitor type -2 (PAI-2) is a serpin that is targeted to both extmcellular and intmcellular locations. Extmcellular PAI-2 is an efficient inhibitor of urokinase-type plasminogeu activator (uPA) and functions to regulate extracellniar uPA-initiated proteolytic events Involved in tissue remodelling, inflammation and tumor invasion and metastasis. We and others have demonstrated an inlracellular role for PAI-2 in the regulation of survival from tumour necrosis factor-a (TNF-a) mediated apoptusis. Here we show that PAI-2 also protects cells from the rapid eytopathic effects of alphavirus infection. Protection was associated with intracellular aad not extracellniar PAl2. In this case, protection was not associated with an effect on apoptosis but was mediated by a PAI-2 dependent induction of constitutive low level IFN a/13production. IFNcd[3 primed the cells for rapid induction of antiviral genes, resulting in the establishment of a persistent productive alphaviral infection in PAI-2 transfected cells. PAI-2 was also induced in macrophages in response to viral RNA suggesting that PAI-2 is a virus response gene. These observations and the ability of PAI-2 to inhibit TNF-c~ mediated apoptosis illustrate that PAI-2 has an additional and distinct function as an intracellular regulator of signal transduction pathway(s) and demonstrate a novel activity for a eukaryotie serpin.
Experiments in both normal and transgenic animals indicate that plasminogen activators play a role in the response of the vessel wall to injury, presumably by mediating the degradation of extracellular matrix by vascular smooth muscle cells (VSMC) which is necessary to facilitate their migration and proliferation. As these cells express both uPA and tPA in rive, we have studied whether they can assemble a tPA-catalysed plasminogen activation system on their surface in addition to the established uPA/uPAR system. tPA was found to bind to human VSMC, using a functional assay i.e. by plasminogeu activation. Analysis of the binding isotherms revealed 2 classes of binding site with Kcs of 25 and 660 nM. Enzyme kinetic analysis demonstrated that although both of these sites mediated plasminngan activation, the large enhancement of tPA activity observed (> 100-fold) involved the higher affinity site and required the cellular binding of plasminogen. The binding oftPA was not competed for either by antibodies to aanexin II, proposed as an endothelial tPA binding site, or by ligands competing the binding oftPA to a variety of other molecules such as LRP, mannose receptor or glycosaminoglycans.The speeificityoftPA binding was demonstrated, as Ser47S>AlatPA competed tPA binding by >85%. By contrast, PhePro-Arg-chloromethyl ketone (PPACK)-ina~vated tPA did not compete the binding of active tPA to VSMC, although it was effective at competing tPA binding to fibrin. As the domain deletion mutant K2P did not bind with high affinity, these data implicate both the serine pretense and N-terminal domains of tPA in the binding interaction. Preliminary aff~mtychromatography experiments have detected a tPAbinding protein of approx. 350 kD in detergent extracts of these cells. Comparison with the uPAR system reveals that tPA binding has the potential to mediate 10-25-fold greater plasmin generation on these cells. These data demonstrate the occurrence of a novel specific tPA receptor on human VSMC which may he important for the regulation of plasminogen activation and ECM degradation by these ceils in various vascular pathologies.
135
136
TISSUE PLASMINOGEN ACTIVATOR BINDING TO THE TAIL DOMAIN OF ITS ENDOTHELIAL CELL RECEPTOR, ANNEXIN II: DIRECT MODULATION BY HOMOCYSTEINE. K.A. Haliar° , L. Mauri°, F. Zhong', U. tffu'za', B.T. Chait °'. Cornell University Medical College * and Rockefeller University", New York, NY. Both tis~a¢ plasmm'ogen activator (t-PA) and its substrate, plasminogeu (PLG), bind to the endothelial cell (EC) surface via the calcinm-regulated, phospholipidbinding protein annexin II (Ann II). Recent studies indicate that binding sites for both cell surface phosphulipid and the ligand PLG reside within the C-t~minal "core" region of Ann IL Binding oft-PA, but not PLG, is down-regulated upon exposure of EC to the thiol-euntainlng amino acid, homocysteine (HC, 1 raM, 18h). Here, to identify the t-PA binding domain of Ann II and to determine the mechanism of its modulation by HC, the chymotryptic "core" fragment recombinant human Ann II (r/am II) was isolated by ion exchange chromatography. Win3eexcess whole rAan ]I cempletely blocked specific binding of 125I-t-PA to mfive Ann II, the 33-kDa "core" fragment failed to do so, suggesting assignment of the t-PA-bindiag domain to the N-terminal "tail" region. Two overlapping "tail" pepfides (residues 2-13 and 7-18) each blocked 65-75% of mI-t-PA binding to native Ann H (I~ = 0.4 raM), while a third poptide (residues 14-25) blocked <10% of binding. A hexapeptide (LCKLSL) mimicking the residue 7-12 region of overlap, but not LGKLSL, completely inhibited binding of ~ - t - P A to both rAnn H and cultured EC (Is0= 0.2 and 1.1 raM), thus identifying a putative t-PA-binding domain- To further explore the role of C residues in t-PA binding, mutants containing all four possible C to G Ann H substitutions (C9G, C133G, C262G, and C335G) were tested. C133G, C262G, and C335G mutants expressed in 293 cells showed binding of mI-t-PA indistinguishable from that of the wild type construct, whereas =~sI-t-PAbinding to C9G-transfected cells was reduced by 61%. When rAnnlI was treated with HC (5 mM, 3h), electrospray ionization mass spectrometry (MS) revealed a 135 _+ 2 Da increase in mass, indicating a 1:1 stoichiometry of HC to Ann II adduct formation. Tandem MS of the N-termlnal tryptic peptide DPSTVHEILCK specifically localized this increment to C9. Treatment ofrAanlI with HC reduced its capacity to bind t-PA by 65% (I~ = 11 I~M). These data provide a molecular mechanism by which HC may impair the t-PA-binding capacity of Ann II, thereby inducing a prothrombotic effect in the blood vessel wall.
MODULATION OF PLASMINOGEN RECEPTOR EXPRESSION ON LEUKOCYTES Thomas Herren and Edward F. Plow Joseph J. Jacobs Center for Thrombosis and Vascular Biology, The Cleveland Clinic Foundation, Cleveland, OH 44195 A linkage between the plasminogen system and the inflammatory response has long been recognized. This association has been emphasized in studies of plasminogeudeficient mice which exhibit decreased leukocyte infiltration in response to inflammatory stimuli. Since the role of the plasminogeu system in cell migration depends upon intera~on of plasminogen with the cell surfaces, we have investigated the regulation of plasminogen receptors (PIgR) on neutrophils, Freshly isolated human peripheral blood neutrophils express low levels of PIgR (at an input concentration of I00 nM plasminogen, cells specifically bound 1.78 -~ 0.23 x 10~ moleodes/cell, n = 10). When cultured, either in the presence or absence of serum, the neutrophils extfibit a dramatic increase, up to 50-fold, in plasminogeu binding capacity. Although plasmin itself can increase the number of PIgR on cells by proteolytically generating new plasminogeu binding sites, depletion of the culture medium of plasminogen or addition of the plasmin inhibitor, aprotinin, affected upregulation of PlgR only modestly. However, addition of soybean trypsin inhibitor (SBTI) to culture medium suppressed up-regulation. Doses of SBTI as low as 0.05 mg/ml were suppressive and tmg/ml fully prevented up-regulation. These data implicate a proteolytic pathway for up-regulation of PIgR. Consistent with this interpretation, specific inhibitors of elastase and eathepsin G were also capable of suppressing the up-regulation of PlgR. Moreover, conditions known to increase the elastase binding capacity of neutrophils (LPS followed by flv1LP)increased PlgR on neotrophils. In a separate set of experiments, nentrophils were stimulated with PMA to induce cell adhesion. Unlike monoc~jtoidcells in which enhanced PlgR expression is restricted to the nonadherant cells, PlgR expression was increased in both the adherent and the non-adherent neutrophils. Under these circumstances, SBTI had only a modest effect on up-regulation. Taken together, these data suggest that neutrophils are capable of markedly altering their plasminogen binding capacity. Both proteolytic and non-proteolytic (or distinct proteinases) pathways may modulate PlgR receptor expression.