Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA)

Action of purified human cathepsin B on recombinant single-chain urokinase-type plasminogen activator (pro-uPA) generated enzymatically active two-chain uPA (HMW-uPA), which was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot from plasmin-generated HMW-uPA and from elastase- or thrombin-generated inactive two-chain urokinase-type plasminogen activator. Preincubation of cathepsin B with E-64 (transepoxysuccinyl-L-leucylamino- (4-guanidino)butane, a potent inhibitor for cathepsin B) prior to the addition of pro-uPA prevented the activation of pro-uPA. The cleavage site within the cathepsin B-treated urokinase-type plasminogen activator (uPA) molecule, determined by N-terminal amino acid sequence analysis, is located between Lys158 and Ile159. Pro-uPA is cleaved by cathepsin B at the same peptide bond that is cleaved by plasmin or kallikrein. Binding of cathepsin B-activated pro-uPA to the uPA receptor on U937 cells did not differ from that of enzymatically inactive pro-uPA, indicating an intact receptor-binding region within the growth factor-like domain of the cathepsin B-treated uPA molecule. Not only soluble but also tumor cell receptor-bound pro-uPA could be efficiently cleaved by cathepsin B to generate enzymatically active two-chain uPA. Thus, cathepsin B can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. In contrast, no significant activation of pro-uPA by cathepsin D was observed. As tumor cells may produce both pro-uPA and cathepsin B, implications for the activation of tumor cell-derived pro-uPA by cellular proteases may be considered.     

Tumor-associated urokinase-type plasminogen activator: biological and clinical significance.

Evidence has accumulated that invasion and metastasis in solid tumors require the action of tumor-associated proteases, which promote the dissolution of the surrounding tumor matrix and the basement membranes. Receptor-bound urokinase-type plasminogen activator (uPA) appears to play a key role in these events. uPA converts plasminogen into plasmin and thus mediates pericellular proteolysis during cell migration and tissue remodeling under physiological and pathophysiological conditions. uPA is secreted as an enzymatically inactive proenzyme (pro-uPA) by tumor cells and stroma cells. uPA exerts its proteolytic function on normal cells and tumor cells as an ectoenzyme after having bound to a high-affinity cell surface receptor. After binding, pro-uPA is activated by serine proteases (e.g. plasmin, trypsin or plasma kallikrein) and by the cysteine proteases cathepsin B or L, resp. Receptor-bound enzymatically active uPA converts plasminogen to plasmin which is bound to a different low-affinity receptor on tumor cells. Plasmin then degrades components of the tumor stroma (e.g. fibrin, fibronectin, proteoglycans, laminin) and may activate procollagenase type IV which degrades collagen type IV, a major part of the basement membrane. Hence receptor-bound uPA will promote plasminogen activation and thus the dissolution of the tumor matrix and the basement membrane which is a prerequisite for invasion and metastasis. Tissues of primary cancer and/or metastases of the breast, ovary, prostate, cervix uteri, bladder, lung and of the gastrointestinal tract contain elevated levels of uPA compared to benign tissues. In breast cancer uPA and PAI-1 antigen in tumor tissue extracts are independent prognostic factors for relapse-free and overall survival.     

Pro-urokinase-type plasminogen activator is a substrate for hepsin

Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1') peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 x 10(5) m(-1) s(-1)) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 +/- 1.9 nm uPA h(-1), which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.     

Phorbol ester activation of a proteolytic cascade capable of activating latent transforming growth factor-betaL a process initiated by the exocytosis of cathepsin B

12-O-Tetradecanoylphorbol-13-acetate (TPA) suppresses the proliferation of the human breast epithelial cell line MCF10A-Neo by initiating proteolytic processes that activate latent transforming growth factor (TGF)-beta in the serum used to supplement culture medium. Within 1 h of treatment, cultures accumulated an extracellular activity capable of cleaving a substrate for urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA). This activity was inhibited by plasminogen activator inhibitor-1 or antibodies to uPA but not tPA. Pro-uPA activation was preceded by dramatic changes in lysosome trafficking and the extracellular appearance of cathepsin B and beta-hexosaminidase but not cathepsins D or L. Co-treatment of cultures with the cathepsin B inhibitors CA-074 or Z-FA-FMK suppressed the cytostatic effects of TPA and activation of pro-uPA. In the absence of TPA, exogenously added cathepsin B activated pro-uPA and suppressed MCF10A-Neo proliferation. The cytostatic effects of both TPA and cathepsin B were suppressed in cells cultured in medium depleted of plasminogen/plasmin or supplemented with neutralizing TGF-beta antibody. Pretreatment with cycloheximide did not suppress the exocytosis of cathepsin B or the activation of pro-uPA. Hence, TPA activates signaling processes that trigger the exocytosis of a subpopulation of lysosomes/endosomes containing cathepsin B. Subsequently, extracellular cathepsin B initiates a proteolytic cascade involving uPA, plasminogen, and plasmin that activates serum-derived latent TGF-beta.     

Cell surface complex of cathepsin B/annexin II tetramer in malignant progression

The cysteine protease cathepsin B is upregulated in a variety of tumors, particularly at the invasive edges. Cathepsin B can degrade extracellular matrix proteins, such as collagen IV and laminin, and can activate the precursor form of urokinase plasminogen activator (uPA), perhaps thereby initiating an extracellular proteolytic cascade. Recently, we demonstrated that procathepsin B interacts with the annexin II heterotetramer (AIIt) on the surface of tumor cells. AIIt had previously been shown to interact with the serine proteases: plasminogen/plasmin and tissue-type plasminogen activator (tPA). The AIIt binding site for cathepsin B differs from that for either plasminogen/plasmin or tPA. AIIt also interacts with extracellular matrix proteins, e.g., collagen I and tenascin-C, forming a structural link between the tumor cell surface and the extracellular matrix. Interestingly, cathepsin B, plasminogen/plasmin, t-PA and tenascin-C have all been linked to tumor development. We speculate that colocalization through AIIt of proteases and their substrates on the tumor cell surface may facilitate: (1) activation of precursor forms of proteases and initiation of proteolytic cascades; and (2) selective degradation of extracellular matrix proteins. The recruitment of proteases to specific regions on the cell surface, regions where potential substrates are also bound, could well function as a 'proteolytic center' to enhance tumor cell detachment, invasion and motility.     

Activation of the zymogen to urokinase-type plasminogen activator is associated with increased interdomain flexibility

A key regulatory step for serine proteases of the trypsin clan is activation of the initially secreted zymogens, leading to an increase in activity by orders of magnitude. Zymogen activation occurs by cleavage of a single peptide bond near the N-terminus of the catalytic domain. Besides the catalytic domain, most serine proteases have N-terminal A-chains with independently folded domains. Little is known about how zymogen activation affects the interplay between domains. This question is investigated with urokinase-type plasminogen activator (uPA), which has an epidermal growth factor domain and a kringle domain, connected to the catalytic domain by a 15-residue linker. uPA has been implicated under several pathological conditions, and one possibility for pharmacological control is targeting the conversion of the zymogen pro-uPA to active uPA. Therefore, a small-angle X-ray scattering study of the conformations of pro-uPA and uPA in solution was performed. Structural models for the proteins were derived using available atomic-resolution structures for the various domains. Active uPA was found to be flexible with a random conformation of the amino-terminal fragment domain with respect to the serine protease domain. In contrast, pro-uPA was observed to be rigid, with the amino-terminal fragment domain in a fixed position with respect to the serine protease domain. Analytical ultracentrifugation analysis supported the observed difference between pro-uPA and uPA in overall shape and size seen with small-angle X-ray scattering. Upon association of either of two monoclonal Fab (fragment antigen-binding) fragments that are directed against the catalytic domain of, respectively, pro-uPA and uPA, rigid structures were formed.     

Interplay of human tissue kallikrein 4 (hK4) with the plasminogen activation system: hK4 regulates the structure and functions of the urokinase-type plasminogen activator receptor (uPAR)

The plasminogen activation system is involved in cancer progression and metastasis. Among other proteolytic factors, it includes the serine protease urokinase-type plasminogen activator (uPA) and its three-domain (D1D2D3) receptor uPAR (CD87), which focuses plasminogen activation to the cell surface. The function of uPAR is regulated in part through shedding of domain D1 by proteases, e.g., uPA itself or plasmin. Human tissue kallikrein 4 (hK4), which is highly expressed in prostate and ovarian tumor tissue, was previously shown to cleave and activate the pro-enzyme forms of prostate-specific antigen (PSA, tissue kallikrein hK3) and uPA. Here we demonstrate that uPAR is also a target for hK4, being cleaved in the D1-D2 linker sequence and, to a lesser extent, in its D3 juxtamembrane domain. hK4 may thus modulate the tumor-associated uPA/uPAR-system activity by either activating the pro-enzyme form of uPA or cleaving the cell surface-associated uPA receptor.     

Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo. Activation of pro-urokinase by glandular kallikrein (mGK-6) in plasminogen-deficient mice

The plasminogen activation (PA) system is involved in the degradation of fibrin and various extracellular matrix proteins, taking part in a number of physiological and pathological tissue remodeling processes including cancer invasion. This system is organized as a classical proteolytic cascade, and as for other cascade systems, understanding the physiological initiation mechanism is of central importance. The attempts to identify initiation routes for activation of the proform of the key enzyme urokinase-type plasminogen activator (pro-uPA) in vivo have been hampered by the strong activator potency of the plasmin, that is generated during the progress of the cascade. Using gene-targeted mice deficient in plasminogen (Plg -/- mice) [Bugge, T. H., Flick, M. J., Daugherty, C. C., and Degen, J. L. (1995) Genes Dev. 9, 794-807], we have now demonstrated and identified a component capable of initiating the cascade by activating pro-uPA. The urine from Plg -/- mice contained active two-chain uPA as well as a proteinase capable of activating exogenously added pro-uPA. The active component was purified and identified by mass spectrometry-based peptide mapping as mouse glandular kallikrein mGK-6 (true tissue kallikrein). The pro-uPA converting activity of the mGK-6 enzyme, as well as its ability to cleave a synthetic substrate for glandular kallikrein, was inhibited by the serine proteinase inhibitor leupeptin but not by other serine proteinase inhibitors such as aprotinin, antithrombin III, or alpha(1)-antitrypsin. We suggest that mouse glandular kallikrein mGK-6 is an activator of pro-uPA in the mouse urinary tract in vivo. Since this kallikrein is expressed in a number of tissues and also occurs in plasma, it can also be considered a candidate for a physiological pro-uPA activator in other locations.     

Inhibition of tumor invasion by genomic down-regulation of matriptase through suppression of activation of receptor-bound pro-urokinase

Urokinase-type plasminogen activator (uPA) degrades the extracellular matrix and plays critical roles in tumor invasion and metastasis. Matriptase, a membrane-bound serine protease, was shown to activate uPA in a uPA receptor-free, solution-based study. We now investigate whether matriptase affects activation of receptor-bound uPA and contributes to the invasiveness of HRA human ovarian cancer cells in vitro and tumor behavior in nude mice. Here we show the following. 1) uPA expression was effectively stimulated by TGF-beta1 in HRA cells. 2) Antisense (AS)-matriptase transfection achieved a marked inhibition of receptor-bound pro-uPA activation without altering expression of uPA and uPA receptor mRNA and proteins, irrespective of whether cells were stimulated with TGF-beta1. 3) Tumor cell receptor-bound pro-uPA could be efficiently cleaved by matriptase to generate enzymatically active two-chain uPA. Thus, matriptase can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active uPA. 4) The AS-matriptase-treated cells had a decreased ability to invade an extracellular matrix layer, as compared with control cells. 5) When the AS-matriptase-treated cells were injected intraperitoneally into nude mice, the mice developed smaller tumors. Our data identify a novel role for matriptase for activation of receptor-bound uPA.     

Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin

Urokinase plasminogen activator receptor (uPAR) binds pro-urokinase plasminogen activator (pro-uPA) and thereby localizes it near plasminogen, causing the generation of active uPA and plasmin on the cell surface. uPAR and uPA are overexpressed in a variety of human tumors and tumor cell lines, and expression of uPAR and uPA is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed mutated anthrax toxin-protective antigen (PrAg) proteins in which the furin cleavage site is replaced by sequences cleaved specifically by uPA. These uPA-targeted PrAg proteins were activated selectively on the surface of uPAR-expressing tumor cells in the presence of pro-uPA and plasminogen. The activated PrAg proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A, thereby killing the uPAR-expressing tumor cells. The activation and cytotoxicity of these uPA-targeted PrAg proteins were strictly dependent on the integrity of the tumor cell surface-associated plasminogen activation system. We also constructed a mutated PrAg protein that selectively killed tissue plasminogen activator-expressing cells. These mutated PrAg proteins may be useful as new therapeutic agents for cancer treatment.     

Receptor-mediated regulation of plasminogen activator function: plasminogen activation by two directly membrane-anchored forms of urokinase.

The generation of the broad specificity serine protease plasmin in the pericellular environment is regulated by binding of the urokinase-type plasminogen activator (uPA) to its specific glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor, uPAR. This interaction potentiates the reciprocal activation of the cell-associated zymogens pro-uPA and plasminogen. To further study the role of uPAR in this mechanism, we have expressed two directly membrane-anchored chimeric forms of uPA, one anchored by a C-terminal GPI-moiety (GPI-uPA), the other with a C-terminal transmembrane peptide (TM-uPA). These were expressed in the monocyte-like cell lines U937 and THP-1, which are excellent models for kinetic and mechanistic studies of cell-surface plasminogen activation. In both cell-lines, GPI-uPA activated cell-associated plasminogen with characteristics both qualitatively and quantitatively indistinguishable from those of uPAR-bound uPA. By contrast, TM-uPA activated cell-associated plasminogen less efficiently. This was due to effects on the K, for plasminogen activation (which was increased up to five-fold) and the efficiency of pro-uPA activation (which was decreased approximately four-fold). These observations suggest that uPAR serves two essential roles in mediating efficient cell-surface plasminogen activation. In addition to confining uPA to the cell-surface, the GPI-anchor plays an important role by increasing accessibility to substrate plasminogen and, thus, enhancing catalysis. However, the data also demonstrate that, in the presence of an alternative mechanism for uPA localization, uPAR is dispensable and, therefore, unlikely to participate in any additional interactions that may be necessary for the efficiency of this proteolytic system. In these experiments zymogen pro-uPA was unexpectedly found to be constitutively activated when expressed in THP-1 cells, suggesting the presence of an alternative plasmin-independent proteolytic activation mechanism in these cells.     

A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction. Cystatin with a urokinase receptor binding site

Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19-31). The inhibition constants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K(i), papain (pm), 1.9-2.4; K(i), cathepsin B (nm), 1.0-1.7; K(i), cathepsin L (pm), 0.12-0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. The measured K(D) value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 17 nm (K(D) value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors.     

High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.     

Binding of single-chain prourokinase to the urokinase receptor of human U937 cells

The single-chain form of human urokinase plasminogen activator (uPA) is the major form of the enzyme found in cells, tissues, and extracellular fluids. The protein, called pro-uPA, has high (Kd = 0.5 nM) affinity for the specific uPA receptor of U937 human monocyte-like cells. Its conversion to two-chain uPA by plasmin does not appreciably change the binding parameters. In addition, conversion of pro-uPA to uPA occurs with receptor-bound pro-uPA and does not lead to dissociation from the membrane. These data show that secreted pro-uPA can find its way to the specific surface receptor without previous conversion to the two-chain form and that, once bound, can be activated by plasmin.     

Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.     

Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation.     

The human fibrinolytic system is a target for the staphylococcal metalloprotease aureolysin

The major opportunistic pathogen Staphylococcus aureus utilizes the human fibrinolytic system for invasion and spread via plasmin(ogen) binding and non-proteolytic activation. Because S. aureus secretes several proteases recently proposed as virulence factors, we explored whether these enzymes could add to the activation of the host's fibrinolytic system. Exposure of human pro-urokinase [pro-uPA (where uPA is urokinase-type plasminogen activator)] to conditioned growth media from staphylococcal reference strains results in an EDTA-sensitive conversion of the single-chain zymogen into its two-chain active form, an activity not observed in an aureolysin-deficient strain. Using purified aureolysin, we verified the capacity of this thermolysin-like metalloprotease to activate pro-uPA, with a 2.6 x 10(3) M(-1) x s(-1) catalytic efficiency. Moreover, activation also occurs in the presence of human plasma, as well as in conditioned growth media from clinical isolates. Finally, we establish that aureolysin (i) converts plasminogen into angiostatin and mini-plasminogen, the latter retaining its capacity to be activated by uPA and to hydrolyse fibrin, (ii) degrades the plasminogen activator inhibitor-1, and (iii) abrogates the inhibitory activity of alpha(2)-antiplasmin. Altogether, we propose that, in parallel with the staphylokinase-dependent activation of plasminogen, aureolysin may contribute significantly to the activation of the fibrinolytic system by S. aureus, and thus may promote bacterial spread and invasion.     

Serine phosphorylation of biosynthetic pro-urokinase from human tumor cells

Phosphorylation is a potent mechanism regulating the activity of many intracellular enzymes. We have discovered that the product of the human urokinase plasminogen activator gene, pro-uPA, is phosphorylated in serine in at least two human cell lines. Phosphorylation occurs within the cell during biosynthesis, and phosphorylated intracellular pro-uPA is secreted into the medium. Of the secreted pro-uPA molecules, 20-50% are phosphorylated in serine, thus representing a meaningful fraction of the total biosynthetic pro-uPA. Although the sites of phosphorylation have not yet been determined, at least two such sites must exist; in fact plasmin cleavage of phosphorylated single chain pro-uPA yields a two chain uPA in which both chains are phosphorylated. A specific function for pro-uPA phosphorylation has not yet been identified; however, it is tempting to speculate that, as in many other cases, phosphorylation may affect the activity of the enzyme, its response to inhibitors or the conversion of pro-uPA zymogen to active two-chain uPA. This would represent an additional way of regulating extracellular proteolysis, an important pathway involved in both intra- and extravascular phenomena like fibrinolysis, cell migration and invasiveness.     

Activation of hepatocyte growth factor activator zymogen (pro-HGFA) by human kallikrein 1-related peptidases

Hepatocyte growth factor activator (HGFA) is a serine protease and a potent activator of prohepatocyte growth factor/scatter factor (pro-HGF/SF), a multifunctional growth factor that is critically involved in tissue morphogenesis, regeneration, and tumor progression. HGFA circulates as a zymogen (pro-HGFA) and is activated in response to tissue injury. Although thrombin is considered to be an activator of pro-HGFA, alternative pro-HGFA activation pathways in tumor microenvironments remain to be identified. In this study, we examined the effects of kallikrein 1-related peptidases (KLKs), a family of extracellular serine proteases, on the activation of pro-HGFA. Among the KLKs examined (KLK2, KLK3, KLK4 and KLK5), we identified KLK4 and KLK5 as novel activators of pro-HGFA. Using N-terminal sequencing, the cleavage site was identified as the normal processing site, Arg407-Ile408. The activation of pro-HGFA by KLK5 required a negatively charged substance such as dextran sulfate, whereas KLK4 could process pro-HGFA without dextran sulfate. KLK5 showed more efficient pro-HGFA processing than KLK4, and was expressed in 50% (13/25) of the tumor cell lines examined. HGFA processed by these KLKs efficiently activated pro-HGF/SF, and led to cellular scattering and invasion in vitro. The activities of both KLK4 and KLK5 were strongly inhibited by HGFA inhibitor type 1, an integral membrane Kunitz-type serine protease inhibitor that inhibits HGFA and other pro-HGF/SF-activating proteases. These data suggest that KLK4 and KLK5 mediate HGFA-induced activation of pro-HGF/SF within tumor tissue, which may thereafter trigger a series of events leading to tumor progression via the MET receptor.     

Sulfated Glycosaminoglycans Enhance Tumor Cell Invasionin Vitroby Stimulating Plasminogen Activation

Metastasizing tumor cells invade host tissues by degrading extracellular matrix constituents. We report here that the highly sulfated glycosaminoglycans, heparin and heparan sulfate, as well as the sulfated polysaccharide, fucoidan, significantly enhanced tumor cell invasionin vitrointo fibrin, the basement membrane extract, Matrigel, or through a basement membrane-like extracellular matrix. The enhancement of tumor cell invasion was due to a stimulation of the proteolytic cascade of plasminogen activation since the effect required plasminogen activation and was abolished by inhibitors of urokinase-type plasminogen activator (uPA) or plasmin. Sulfated polysaccharides enhanced five reactions of tumor-cell initiated plasminogen activation in a dose-dependent manner. They amplified plasminogen activation in culture supernatants up to 70-fold by stimulating (i) pro-uPA activation by plasmin and (ii) plasminogen activation by uPA. (iii) In addition, sulfated polysaccharides partially protected plasmin from inactivation by α₂-antiplasmin. Sulfated polysaccharides also stimulated tumor-cell associated plasminogen activation, e.g., (iv) cell surface pro-uPA activation by plasmin and (v) plasminogen activation by cell surface uPA. These results suggest that sulfated glycosaminoglycans liberated by tumor-cell mediated extracellular matrix degradationin vivomight amplify pericellular plasminogen activation and locally enhance tumor cell invasion in a positive feedback manner.