Melanotransferrin stimulates t-PA-dependent activation of plasminogen in endothelial cells leading to cell detachment

Tissue plasminogen activator (t-PA) is an extracellular serine protease that converts the proenzyme plasminogen into the broad-spectrum substrate serine protease, plasmin. Plasmin, one of the most potent pro-angiogenic factors, is a key element in fibrinolysis, cell migration, tissue remodeling and tumor invasion. In the present investigation, we assessed the impact of the truncated form of soluble melanotransferrin (sMTf) on plasminogen activation by t-PA and subsequent endothelial cell detachment. Co-treatment of human endothelial microvessel cells with plasminogen, t-PA and sMTf significantly increased plasmin formation and activity in the culture medium. Plasmin generated in the presence of sMTf also led to a 30% reduction in fibronectin detection within cell lysates and to a 9-fold increase within the corresponding cell medium. Moreover, the presence of sMTf increases EC detachment by 6-fold compared to cells treated only with plasminogen and t-PA. Although the addition of alpha(2)-antiplasmin completely prevented plasmin formation and EC detachment, epigallocatechin gallate, GM6001 and a specific antibody directed against MMP-2 prevented cellular detachment without interfering with plasminogen activation. Overall, these data suggest that the anti-angiogenic properties of sMTf may result from local overstimulation of plasminogen activation by t-PA, thus leading to subsequent degradation of the Fn matrix and EC detachment.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: a key event in melanoma cell invasiveness in vitro.

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on MeWo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Activation of matrix metalloproteinase-9 (MMP-9) via a converging plasmin/stromelysin-1 cascade enhances tumor cell invasion

Matrix metalloproteinase-9 (MMP-9) may play a critical catalytic role in tissue remodeling in vivo, but it is secreted by cells as a stable, inactive zymogen, pro-MMP-9, and requires activation for catalytic function. A number of proteolytic enzymes activate pro-MMP-9 in vitro, but the natural activator(s) of MMP-9 is unknown. To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells (MDA-MB-231 breast carcinoma cells) that were induced to produce MMP-9 over a 200-fold concentration range (0.03-8.1 nM). The levels of tissue inhibitors of metalloproteinase (TIMPs) in the induced cultures remain relatively constant at 1-4 nM. Quantitation of the zymogen/active enzyme status of MMP-9 in the MDA-MB-231 cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether pro-MMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but through an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous urokinase-type plasminogen activator, is not an efficient activator of pro-MMP-9, neither the secreted pro-MMP-9 nor the very low levels of pro-MMP-9 associated with intact cells. Although plasmin can proteolytically process pro-MMP-9, this limited action does not yield an enzymatically active MMP-9, nor does it cause the MMP-9 to be more susceptible to activation. Plasmin, however, is very efficient at generating active MMP-3 (stromelysin-1) from exogenously added pro-MMP-3. The activated MMP-3 becomes a potent activator of the 92-kDa pro-MMP-9, yielding an 82-kDa species that is enzymatically active in solution and represents up to 50-75% conversion of the zymogen. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to both degrade extracellular matrix and transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodelling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9 blocking monoclonal antibody.     

Importance, localization and functional properties of the cell-associated form of plasminogen activator in mouse peritoneal macrophages

In inflammatory macrophages, plasminogen activator exists in two active forms, a soluble form released into the extracellular medium and a cell-associated form. This communication describes some properties of the cellular form of plasminogen activator, in intact macrophages and in cell lysates. Cellular plasminogen activator is a membrane protein, associated with the outer face of the plasma membrane; in intact macrophages, it participates in the activation of exogenous plasminogen and, thus, has to be considered as an ectoenzyme. A plasminogen activator activity can be detected in cell lysates (macrophage monolayers lysed in 0.1% Triton X-100) only when plasmin production is followed by the use of small synthetic substrates because a soluble inhibitor, released during extraction, blocks plasmin fibrinolytic activity. In these lysates, plasminogen activator molecules exist as high molecular weight unstable complexes exhibiting a high affinity for plasminogen.     

Identification of the human plasma protein which inhibits fibrinolysis associated with malignant cells

Mixed cultures of mouse fibroblasts and mouse fibroblasts transformed with Kirsten murine sarcoma virus were grown in petri dishes and overlayed with casein. The appearance of focal lysis zones required the presence of transformed cells in the culture and plasminogen in the overlay, indicating that caseinolysis was due to plasminogen activator released by the malignant cells. Caseinolysis was inhibited by addition of human plasma or bovine pancreatic trypsin inhibitor to the overlay, 1 ml of plasma being equivalent to 67 ± 18 (mean ± S.E.) kallikrein inhibitor (KI) units of trypsin inhibitor.The culture fluid of a human melanoma line induced lysis of a fibrin clot, 1 ml of culture fluid being equivalent to 250 CTA units of urokinase (EC 3.4.99.26). Fibrinolysis was inhibited by addition of human plasma or trypsin inhibitor, 1 ml of plasma being equivalent to 94 ± 34 KI units of trypsin inhibitor.Specific removal of antiplasmin, the fast-reacting plasmin inhibitor (Collen, D. (1976) Eur. J. Biochem. 69, 209), from plasma by immunoabsorption completely abolished its inhibitory activity, both in the caseinolytic and fibrinolytic assays. It is therefore concluded that antiplasmin is the only protein in human plasma capable of inhibiting the fibrinolytic activity associated with oncogenic transformation or neoplasia. Whether this effect is exclusively due to inhibition of formed plasmin or also to interference with plasminogen activvtion remains unsettled.     

Inhibition of human MDA-MB-231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen.

Matrix metalloproteinase (MMP)-3 inhibited human MDA-MB-231 breast cancer cell invasion through reconstituted basement membrane in vitro. Inhibition of invasion was dependent upon plasminogen and MMP-3 activation, was impaired by the peptide MMP-3 inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH2 and was associated with: rapid MMP-3-mediated plasminogen degradation to microplasminogen and angiostatin-like fragments; the removal of single-chain urokinase plasminogen activator from MDA-MB-231 cell membranes; impaired membrane plasminogen association; reduced rate of tissue plasminogen activator (t-PA) and membrane-mediated plasminogen activation; and reduced laminin-degrading capacity. Purified human plasminogen lysine binding site-1 (kringles 1-3) exhibited a similar capacity to inhibit MDA-MB-231 invasion, impair t-PA and cell membrane-mediated plasminogen activation and impair laminin degradation by plasmin. Our data provide evidence that MMP-3 can inhibit breast tumour cell invasion in vitro by a mechanism involving plasminogen degradation to fragments that limit plasminogen activation and the degradation of laminin. This supports the hypothesis that MMP-3, under certain conditions, may protect against tumour invasion, which would help to explain why MMP-3 expression, associated with benign and early stage breast tumours, is frequently lost in advanced stage, aggressive, breast disease.     

Fibroblast remodeling of adsorbed collagen type IV is altered in contact with cancer cells

A series of co-culture experiments between fibroblasts and H-460 human lung carcinoma cells were performed to learn more about the fate of adsorbed type IV collagen (Coll IV). Fibroblasts were able to spatially rearrange Coll IV in a specific linear pattern, similar but not identical to the fibronectin (FN) fibrils. Coll IV partly co-aligns with fibroblast actin cytoskeleton and transiently co-localize with FN, as well as with beta1 and alpha2 integrin clusters, suggesting a cell-dependent process. We further found that this Coll IV reorganization is suppressed in contact with H460 cells. Zymography revealed strongly elevated MMP-2 activity in supernatants of co-cultures, but no activity when fibroblasts or cancer cells were cultured alone. Thus, we provide evidence that reorganization of substrate associated Coll IV is a useful morphological approach for in vitro studies on matrix remodeling activity during tumorigenesis.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: A key event in melanoma cell invasiveness in vitro

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on Me Wo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Identification of cells responsible for urokinase-type plasminogen activator synthesis and secretion in human diploid kidney cell cultures

Human urokinase-type plasminogen activator (uPA) is a serine protease that converts plasminogen to plasmin. It is produced and secreted by a variety of different human cells in vivo and in vitro. We have studied human diploid kidney cell (HKC) cultures prepared from neonatal kidney tissue and cultures of purified populations of HKC to determine which cells synthesize and secrete uPA into the culture medium. Antibodies against cell specific antigens and uPA were used to correlate specific kidney cell types with uPA synthesis. In addition, secretion of uPA activity into growth and uPA production media was determined for each cell type and cultures containing a mixture of cell types. The results of these studies demonstrated that glomerular visceral epithelial and kidney tubular epithelial cells synthesize and secrete uPA into the culture medium.     

Production of a fibronectin-associated lymphokine by cloned mouse T cells

Azobenzenearsonate-specific cloned mouse T cells able to transfer delayed hypersensitivity reactions in vivo produced macrophage agglutination factor (MaggF) after stimulation with mitogen or antigen in vitro. Mitogen (Con A) elicited MAggF production directly from T cells. Responses to Ag were Ag-specific, required syngeneic accessory cells in addition to T cells, and were independent of T cell fine specificity for azobenzenearsonate. Mouse MAggF shared a number of biochemical and immunochemical properties with the fibronectins (FN): 1) high Mr similar to that of plasma FN; 2) binding to gelatin, heparin, and polyclonal antibodies and mAb specific for cellular and plasma FN; 3) inhibition of activity in solution by monoclonal anti-human FN directed against plasma FN gelatin-binding domain; and 4) action on peritoneal exudate macrophages mediated through a FN-receptor cross reactive with one on human monocytes. MAggF production required active protein synthesis and was associated with significant increases in gelatin-binding immunoreactive FN (Mr 440 kDa on immunoblotting) in culture supernatants and T cell lysates. Metabolically labeled peptides could be precipitated by anti-FN from culture supernatants of activated T cells. Stimulated cultures contained significantly more cells with immunohistologically demonstrable cytoplasmic FN than unstimulated control cultures. We suggest that T cell FN is a distinct species of cellular FN which may play an important role in mediating delayed hypersensitivity inflammatory reactions in vivo.     

Impact of plasminogen on an in vitro wound healing model based on a perfusion cell culture system

Vascular reorganization in wound healing is a complex process, which involves coagulation, endothelial cell proliferation and migration, basement membrane regeneration, and fibrinolysis. During this healing process, the hemostatic system and the angiogenic system are intimately interconnected. To elucidate the contribution of plasminogen in the process of wound healing, we have established a perfusion cell culture system. Using this novel cell culture system, we found that addition of plasminogen in the perfusion medium allowed the "scratch-wounded" endothelial cells to recover completely, while mini-plasminogen only affected the migration but not the proliferation of the endothelial cells. In the process of recovery with the addition of plasminogen, significant plasmin activity could only be detected when the growth of the endothelial cells have almost reached confluence. This finding indicates that wound healing is triggered and promoted during the absence of the proteolytic activity of plasmin. In addition, we could not detect any matrix metalloproteinase activity in the perfusion culture medium throughout the whole culture period. However, we did found that the circulating medium collected from the perfusion system at the early phase of the healing process has stimulatory activity on the growth of endothelial cells, but the proliferative activity decreased back to the basal level when the cells reached confluence. Thus, by using the perfusion cell culture system, we found that proliferation of endothelial cells is regulated by plasminogen and the wound healing process is controlled by a temporal interaction between the endothelial cells and plasminogen.     

Vascular smooth muscle cells efficiently activate a new proteinase cascade involving plasminogen and fibronectin

The plasminogen/plasmin system is involved in vascular wall remodeling after injury, through extracellular matrix (ECM) degradation and proteinase activation. Vascular smooth muscle cells (VSMCs) synthesize various components of the plasminogen/plasmin system. We investigated the conversion of plasminogen into plasmin in primary cultured rat VSMCs. VSMCs efficiently converted exogenous plasminogen into plasmin in a time- and dose-dependent manner. We measured plasmin activity by monitoring the hydrolysis of Tosyl-G-P-R-Mca, a fluorogenic substrate of plasmin. Cell-mediated plasmin activation was associated with the degradation of ECM, as revealed by fibronectin proteolysis. Plasmin also activated a proteinase able to hydrolyze Mca-P-L-G-L-Dpa-A-R-NH(2), a fluorogenic substrate of matrix metalloproteinases (MMPs). However, this proteinase was not inhibited by an MMP inhibitor. Furthermore, this proteinase displayed similar biochemical and pharmacological properties to fibronectin-proteinase, a recently identified zinc-dependent metalloproteinase located in the gelatin-binding domain of fibronectin. These results show that VSMCs convert exogenous plasminogen into plasmin in their pericellular environment. By hydrolyzing matrix protein plasmin activates a latent metalloproteinase that differs from MMP, fibronectin-proteinase. This metalloproteinase may participate to vascular wall remodeling, in concert with other proteinases.     

Lipoprotein (a) inhibits the generation of transforming growth factor beta: an endogenous inhibitor of smooth muscle cell migration

Conditioned medium (CM) derived from co-cultures of bovine aortic endothelial cells (BAECs) and bovine smooth muscle cells (BSMCs) contains transforming growth factor-beta (TGF-beta) formed via a plasmin-dependent activation of latent TGF-beta (LTGF beta), which occurs in heterotypic but not in homotypic cultures (Sato, Y., and D. B. Rifkin. 1989. J. Cell Biol. 107: 1199-1205). The TGF-beta formed is able to block the migration of BSMCs or BAECs. We have found that the simultaneous addition to heterotypic culture medium of plasminogen and the atherogenic lipoprotein, lipoprotein (a) (Lp(a)), which contains plasminogen-like kringles, inhibits the activation of LTGF-beta in a dose-dependent manner. The inclusion of LDL in the culture medium did not show such an effect. Control experiments indicated that Lp(a) does not interfere with the basal level of cell migration, the activity of exogenous added TGF-beta, the release of LTGF-beta from cells, the activation of LTGF-beta either by plasmin or by transient acidification, or the activity of plasminogen activator. The addition of Lp(a) to the culture medium decreased the amount of plasmin found in BAECs/BSMCs cultures. Similar results were obtained using CM derived from cocultures of human umbilical vein endothelial cells and human foreskin fibroblasts. These results suggest that Lp(a) can inhibit the activation of LTGF-beta by competing with the binding of plasminogen to cell or matrix surfaces. Therefore, high plasma levels of Lp(a) might enhance smooth muscle cell migration by decreasing the levels of the migration inhibitor TGF-beta thus contributing to generation of the atheromatous lesions.     

Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity

Cultured bovine capillary endothelial (BCE) cells synthesize heparan sulfate proteoglycans (HSPG), which are both secreted into the culture medium and deposited in the cell layer. The nonsoluble HSPGs can be isolated as two predominant species: a larger 800-kD HSPG, which is recovered from preparations of extracellular matrix, and a 250-kD HSPG, which is solubilized by nonionic detergent extraction of the cells. Both HSPG species bind bFGF. 125I-bFGF bound to BCE cell cultures is readily released by either heparinase or plasmin. When released by plasmin, the growth factor is recovered from the incubation medium as a complex with the partly degraded high molecular mass HSPG. Endogenous bFGF activity is released by a proteolytic treatment of cultured BCE cells. The bFGF-binding HSPGs are also released when cultures are incubated with the inactive proenzyme plasminogen. Under such experimental conditions, the release of the extracellular proteoglycans can be enhanced by treating the cells either with bFGF, which increases the plasminogen activating activity expressed by the cells, or decreased by treating the cells with transforming growth factor beta, which decreases the plasminogen activating activity of the cells. Specific immune antibodies raised against bovine urokinase also block the release of HSPG from BCE cell cultures. We propose that this plasminogen activator-mediated proteolysis provides a mechanism for the release of biologically active bFGF-HSPG complexes from the extracellular matrix and that bFGF release can be regulated by the balance between factors affecting the pericellular proteolytic activity.     

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.     

Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants.

The urokinase-type plasminogen activator (uPA) and the matrix-degrading metalloproteinases MMP-2 and MMP-9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP-2 and MMP-9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA-plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo- or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose- and time-dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA-plasmin system may represent a physiological mechanism for the control of gelatinase activity.     

Collagenolysis-dependent angiogenesis mediated by matrix metalloproteinase-13 (collagenase-3)

We have demonstrated previously that new blood vessel formation induced by angiogenic growth factors in onplants placed on the chorioallantoic membrane (CAM) of the chick embryos is critically dependent on the cleavage of fibrillar collagen by a previously unidentified interstitial collagenase. In the present study we have used a quantitative CAM angiogenesis system to search for and functionally characterize host avian collagenases responsible for the collagen remodeling associated with angiogenesis. Among the matrix metalloproteinases (MMPs) identified in the CAM onplant tissue, the chicken MMP-13 (chMMP-13) was the only enzyme whose induction and expression coincided with the onset of angiogenesis and blood vessel formation. The chMMP-13 cDNA has been cloned and recombinantly expressed. The chMMP-13 protein has been purified, characterized in vitro, and examined in situ in the CAM. MMP-13-positive cells appear in the CAM shortly after angiogenic stimulation and then accumulate in the collagen onplant tissue. Morphologically, the chMMP-13-containing cells appear as hematopoietic cells of monocyte/macrophage lineage. In vitro, the chMMP-13 proenzyme is rapidly and efficiently activated through the urokinase plasminogen activator/plasminogen/plasmin cascade into a collagenase capable of cleaving native but not the (r/r) mutant collagenase-resistant collagen. Surprisingly, nanogram levels of purified chMMP-13 elicit an angiogenic response in the CAM onplants comparable with that induced by the angiogenic growth factors. The chMMP-13-mediated response was efficiently blocked by select protease inhibitors indicating that plasmin-activated chMMP-13 can function as an angiogenic factor in vivo. Altogether, the results of this study extend the physiological role of MMP-13, previously associated with cartilage/bone resorption, to the collagen remodeling involved in the angiogenic cascade.     

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.     

uPA/plasmin system-mediated MMP-9 activation is implicated in bronchial epithelial cell migration

To examine the effects of the uPA/plasmin system on cell migration in relation to the activation of MMP-9, we used ex vivo and in vitro wound-repair models of human bronchial epithelial cells and videomicroscopy techniques that make possible cell tracking and quantification of cell migration speeds. We observed that uPA was only detected in migrating cells at the wound edges and located at crucial sites for cell/extracellular matrix interactions. The implication of uPA in human bronchial epithelial cell migration was studied by incubating cultures with a monoclonal antibody raised against uPA and these experiments led to a 70% reduction in cell velocity. To examine the effects of the plasmin system on cell migration, we incubated cultures with increasing concentrations of plasmin or activated MMP-9. We observed a significant dose-dependent increase in cell migration velocity with plasmin (P < 0.001) and MMP-9 (P < 0.001). Moreover, addition of exogenous plasmin led to a twofold increase of activated MMP-9 in migrating cells. We also demonstrated that the addition of anti-uPA IgG led to an inhibition of 43% of activated MMP-9. In conclusion, these results show that uPA is involved in human bronchial epithelial cells migration. This action is mediated by the generation of plasmin, which in turn activates MMP-9, thus making possible cell migration.