JOURNAL OF CELLULAR PHYSIOLOGY 150:25%263 (1992)

Urokinase-Type Plasminogen Activator Mediates Basic Fibroblast Growth Factor-Induced Bovine Endothelial Cell Migration Independent of Its Proteolytic Activity LALE E. ODEKON,* Y A S U F U M I SATO, AND D A N I E L B. RlFKlN Department of Cell Biology and Kaplan Cancer Center, New York University Medical Center, and the Raymond and Beverly Sackler Foundation Laboratory, New York, New York 10076 The dependence of urokinase-type plasminogen activator (uPA) induction on endogenous basic fibroblast growth factor (bFGF) activity during endothelial cell migration was investigated utilizing a combination of wounded endothelial cell monolayers and substrate overlay techniques. Purified polyclonal rabbit immunoglobulin G (IgG) against bFGF blocked the appearance of uPA-dependent lytic activity normally observed at the edge of a wounded bovine aortic endothelial (BAE) cell monolayer. Additionally, the migration of cells into the denuded area was inhibited 30-50% by antibodies either to bFGF or to bovine uPA. Incubation of wounded monolayers with either purified bovine uPA or agents able to induce PA activity, such as phorbol myristate acetate (PMA), vanadate, or bFGF, resulted in enhanced migration of cells (28-50%). Anti-bovine uPA IgG blocked a significant fraction (25%) of BAE cell migration induced by exposure to exogenous bFGF. The role of uPA in migration of wounded BAE cells was not dependent on plasmin generation. Furthermore, the amino terminal fragment (ATF) of human recombinant (hr) uPA, which i s enzymatically inactive, stimulated BAE cell movement (36%) as well as intact uPA. ATF of hr uPA also stimulated endothelial cell movement in the presence of anti-bFGF IgG. These results suggest that BAE cell migration from the edge of a wounded monolayer i s dependent upon local increases of uPA mediated by endogenous bFGF. Moreover, the data support the conclusion that migration is stimulated via a signalling mechanism dependent upon occupancy of the uPA receptor but independent of uPA-mediated proteolysis

The process of neovascularization involves the proliferation and migration of endothelial cells. Basic fibroblast growth factor (bFGF) stimulates both of these processes (Klagsbrun and Shing, 1985; Moscatelli et al., 1985; Presta et al., 1986; Sat0 and Rifkin, 1988; Tsuboi et al., 1990). In addition bFGF increases the level of collagenase and plasminogen activator (PA) in endothelial cells (Moscatelli et al., 1985; Presta et al., 19861, proteases demonstrated to be necessary for bovine capillary endothelial (BCE) cell invasion (Mignatti et al., 1989). Addition of neutralizing anti-bFGF IgG to monolayers of bovine aortic endothelial (BAE) cells suppresses the basal level of PA and impedes the migration of BAE cells in a wound assay (Sato and Rifkin, 1988; Tsuboi et al., 1990). These results suggested that PA production may be a component of bFGF-induced BAE cell migration. Evidence for the involvement of PA in cell migration was first described by Ossowski et al. (1975) who demonstrated a requirement for plasminogen, which is converted into the active protease plasmin by PA, for the 0 1992 WILEY-LISS. INC

migration of several types of normal and transformed cells. The expression of uPA by migrating cells a t the edge of wounded monolayers and at focal adhesion plaques has been visualized in keratinocytes (Morioka

Received June 28, 1991; accepted August 29, 1991. Abbreviations used: aMEM, alpha minimal essential medium; ATF, amino terminal fragment; BAE, bovine aortic endothelial; BCE, bovine capillary endothelial; bFGF, basic fibroblast growth factor; EACA, t-amino-N-caproic acid; hr, human recombinant; IgG, immunoglobulin G; MDBK, Madin-Darby bovine kidney; PA, plasminogen activator; PBS, phosphate buffered saline; PMA, phorbol myristate acetate; SBTI, soybean trypsin inhibitor; tPA, tissue plasminogen activator; uPA, urokinase-type plasminogen activator. This work was presented a t the Second Congress of International Congresses on Inflammation (ICOI) in Rome, Italy, October 1991. *To whom reprint requestsicorrespondence should be addressed. Yasufumi Sato’s current address is First Department of Internal Medicine, Oita Medical School, Oita 879-56, Japan.

uPA AND CELL MIGRATION

et al., 1987; Grgndhal-Hansen et al., 1988; McNeill and Jensen, 1990), fibroblasts and some tumor cells (Pollanen et al., 1987, 1988; Hebert and Baker, 19881, and endothelial cells (Pepper et al., 1987). However, the precise role of PA and plasmin in the migration of endothelial cells has been controversial. Schleef and Birdwell (1982) reported that depletion of plasminogen from the serum utilized in the culture medium or the inclusion of inhibitors of plasmin did not perturb the migration of wounded bovine endothelial cells. Therefore, these authors concluded that plasmin generation was not required for migration under their experimental conditions. Fibbi and co-workers (19881, however, observed that human uPA was chemotactic for BCE cells when assayed in blindwell chambers. This effect of uPA on BCE cell migration persisted in the presence of antibodies specific for the enzymatic site of uPA but was inhibited by monoclonal antibodies directed against the amino-terminal sequence of uPA, which binds to the plasma membrane uPA receptor (Stopelli et al., 1985). This observation strongly suggested that the interaction of uPA with its receptor may be a direct motility signal for bovine endothelial cells. In this study we investigated the potential role of uPA in BAE cell migration in response to bFGF. In addition we attempted to distinguish between a proteolytic contribution versus a signalling function for uPA. Our results indicate that induction of uPA activity and cell migration at the edge of a wounded BAE cell monolayer are both dependent on endogenous bFGF and that uPA mediates the bFGF-induced BAE cell migration in a manner independent of its proteolytic potential.

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2% non-fat dry milk, (Carnation Company, Los Angeles, CAI, 0.8% agar (Difco Laboratories, Detroit, MI) and 60 pg plasminogen. The cells were incubated a t 37°C until the caseinolytic zone could be clearly observed. The plates were photographed under dark-field illumination. In control experiments, plasminogen was omitted or amiloride (0.5mM) was added to the overlay mixture.

Wound assays for BAE cell migration Wound assays were performed a s previously described (Sato and Rifkin, 1988). Briefly, confluent monolayers of BAE cells in 35 mm dishes were wounded with a razor blade. After wounding, the cells were washed with PBS and further incubated in aMEM containing 0.1%gelatin for 20 h a t 37°C. The cells were fixed with absolute methanol following incubation and stained with Giemsa. Cells that had migrated from the edge of the wound were counted in successive (7) 125 pm increments at 100 x magnification using a light microscope with a n ocular grid. The cell numbers represent the mean determined from at least four different fields.

Purification of bovine urokinase-type plasminogen activator (uPA) from MDBK cell conditioned medium Serum-free conditioned medium from MDBK cells incubated with lo-' M PMA was processed as follows. Conditioned medium (10 1) was collected and centriMATERIALS AND METHODS fuged (2,OOOg)for 20 min a t 4°C. Tween-80 was added to Materials the supernatant at a final concentration of 0.01%, the Recombinant human prouPA and rhATF were gener- pH adjusted to 5.25, and the solution applied to a SPous gifts from Dr. Jack Henkins (Abbot Laboratories, Sephadex column. The column was washed first with Chicago, IL) and a,-plasmin inhibitor was graciously 0.05 M ammonium acetate (pH 5.251, then with 0.1 M donated by Dr. P. Harpel (Mount Sinai Medical School, ammonium sulfate in 0.05 M ammonium acetate (pH New York, NY). Aprotinin, amiloride, E-amino-N-ca- 5.25), and eluted with 1 M ammonium sulfate at 4°C. proic acid (EACA), phorbol myristate acetate (PMA), PA activity of the fractions was determined using the and soybean trypsin inhibitor (SBTI) were purchased fibrin plate assay (Gross et al., 1982). Active fractions from Sigma (St. Louis, MO). Sodium orthovanadate were pooled and concentrated by ultrafiltration using was purchased from Fisher Scientific (Springfield, NJ). a n Amicon filter (10,000 mol wt cutoff3 and dialysed against 0.05 M ammonium acetate (pH 5.25) a t 4°C. The retentate was applied to a Mono-S FPLC column, Cell cultures BAE cells were isolated as described previously and the column was eluted with a gradient of NaCl(0-2 (Gross et al., 1982) and were cultured on gelatin coated M). The active fractions were pooled and dialysed dishes in alpha minimal essential medium (aMEM) against 1 mM acetic acid (pH 3.0), adjusted to 0.02 M (Flow Laboratories, McLean, VA) containing 10% calf Tris (pH 6.2), and applied to a Mono Q column. The serum (Flow Laboratories), 2 mM L-glutamine (Gibco column was eluted with a gradient of NaCl (0-2 M). Laboratories), 0.14 mgiml streptomycin, and 500 pgiml The PA activity was identified by zymography (Tsuboi penicillin. Madin-Darby bovine kidney (MDBK) cells et al., 1990). were purchased from the American Type Culture Collection (Rockville, MD). Preparation of antibodies Casein zymography of wounded BAE Female New Zealand white rabbits were immunized cell monolayers as previously described (Joseph-Silverstein e t al., Confluent monolayers of BAE cells in 35 mm dishes 1988). Briefly, human recombinant bFGF (Synergen were wounded with a rubber policeman; the cultures Inc., Boulder, CO) or bovine uPA was diluted in comwere washed twice with aMEM, and incubated in plete Freund's adjuvant and injected into popliteal aMEM containing 0.1% gelatin. After 20 h incubation lymph nodes. The rabbits were boosted at three week a t 37"C, the cells were washed twice with phosphate intervals with incomplete Freund's adjuvant and bled buffered saline (PBS) and overlaid with PBS containing 7-14 days after boosts. IgG was purified with protein

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Fig. 1. Caseinolysis by wounded endothelial cell monolayers in the presence and absence of anti-bFGF IgG. Monolayers of BAE cells were wounded and, after a 20 h incubation in aMEM containing 1%gelatin, overlaid with casein-agar as described in Methods. A Control culture. B Culture in which plasminogen was omitted from the casein-agar

A-Sepharose (Pharmacia, Piscataway, NJ) according to the manufacturer's instructions. The anti-uPA antibodies recognized bovine uPA but not human uPA or tissue plasminogen activator in both blotting and activity experiments (data not shown).

RESULTS Previously, we showed that both the spontaneous movement of BAE cells into a denuded area and the level of PA activity of confluent BAE cells were dependent upon endogenous bFGF a s antibodies to bFGF suppressed cell migration and PA levels (Sato and Rifkin, 1988). Since Pepper et al. (1987) demonstrated that increased synthesis of uPA could be observed specifically a t the edge of a wounded BAE cell monolayer, we examined whether this induction of uPA activity at the wound edge was dependent on endogenous bFGF and attempted to clarify the potential role of uPA in bFGFinduced endothelial cell migration. When confluent monolayers of BAE cells were overlaid with caseinagar substrate 20 h after wounding, a lytic zone, seen as a clearing of the opalescent background, appeared along the edge of the wound when plasminogen was a component of the overlay (Fig. 1A). In the absence of plasminogen or in the presence of amiloride, a specific inhibitor of uPA (Vassalli and Belin, 19871, caseinolysis was dramatically reduced (Fig. lB,C) demonstrating that the lytic activity was derived from uPA as originally reported by Pepper et al. (1987). To determine whether the increase in uPA at the wound edge depended upon endogenous bFGF, wounded cultures were overlaid with substrate containing plasminogen and either anti-bFGF or non-immune IgG. Whereas exposure of the cultures to non-immune IgG had little effect on the appearance of the lytic zones (Fig. lD), the presence of anti-bFGF antibodies in the overlay gel significantly decreased caseinolysis (Fig. 1E).These ob-

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mixture. C: Culture in which the casein-agar mixture contained amiloride (0.5 mM). D: Culture overlaid with casein-agar containing non-immune IgG (100 pgiml). E: Culture overlaid with casein-agar containing anti-bFGF IgG (100 pgiml).

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Fig. 2. Effect of anti-bFGF and anti-bovine uPA IgG on BAE cell migration. Confluent monolayers of BAE cells were wounded as described in Methods. After wounding, medium alone (1);anti-bFGF IgG (200 pgiml) (2); increasing concentrations of anti-bovine uPA (25, 50, 100, 200 pgiml) IgG (3, 4, 5, 6, respectively); or non-immune (200 pgiml) IgG (7)were added to the cultures. The cells were fixed after an overnight incubation, and the number of cells which had migrated from the edge of the wound determined as described in Methods.

servations indicate that induction of uPA activity in migrating BAE cells is dependent on endogenous bFGF. We next asked whether bFGF-dependent uPA expression was a required component of bFGF-induced BAE cell migration. As seen in Figure 2, anti-bovine uPA IgG inhibited cell migration in a dose-dependent manner and as effectively a s anti-bFGF IgG, while nonimmune IgG had no inhibitory effect on BAE cell migration. The degree of observed inhibition was rarely more than 50%;the failure to inhibit migration by more than this amount is not understood. The results. how-

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tumor cell migration. However, we were unable to inhibit BAE cell movement into denuded areas by including the inhibitors of plasminogen activation or plasmin aprotinin (100 pg/ml), EACA (50 pglrnl), SBTI (100 150pg/ml), or u,-plasmin inhibitor (2 pglml) in the culture medium. These results are in agreement with those published by Schleef and Birdwell (1982). These two apparently contradictory observations, a 7 requirement for uPA in migration and a failure of plasmin inhibitors to block migration, could be resolved by assuming that the receptor binding but not the proteolytic activity of uPA was necessary for its ability to stimulate migration. To test this, we utilized the ATF of human recombinant uPA which consists of the first 135 amino acids of uPA including the sequence necessary for binding to the uPA receptor (Stopelli et al., 1985). While uPA-receptor interaction is species specific when murine and human systems are considered 1 2 3 4 (Appella et al., 19871, human uPA binds in a specific manner to bovine capillary endothelial cells (Fibbi Fig. 3. Effects of anti-bovine uPA IgG on BAE cell migration induced et al., 1988) and to bovine aortic endothelial cells (L. by exogenous bFGF. Confluent monolayers of BAE cells were Odekon, unpublished results). We, therefore, expected wounded and incubated with medium (11, medium and anti-bovine uPA IgG (200 pgiml) (2), bFGF (10 ngiml) (31,bFGF (10 ngiml) + anti- that the ATF of h r uPA may function a s a ligand for the bovine uPA IgG (200 pgiml) (4), and processed as described in uPA receptors on bovine endothelial cells. As seen in Methods. Figure 4A, h r ATF potentiated the migration of BAE cells from the wound edge in a dose-dependent manner with maximal stimulation a t 1 nM ATF (36%). The ever, suggested that uPA expression induced by bFGF decrease in stimulation with higher concentrations of was necessary for bFGF stimulation of BAE cell migra- ATF may reflect desensitization of the response (Schifftion. Thus, the effects on BAE cell migration of other mann, 1982; Devreotes and Zigmond, 1988). The extent reagents which elevate BAE cell PA levels were deter- of stimulation of migration by h r ATF (36%)is compamined. The addition of PMA (20 ngiml) or vanadate (10 rable to that provided by bovine uPA (28%) and PMA pM), both of which increase PA levels (Gross et al., (37%). However, the degree of stimulation is less than 1982; Montesano et al., 1988), to cultures of wounded that provided by maximal stimulatory concentrations BAE cells stimulated migration 28% and 50%, respec- of bFGF (60%), supporting the interpretation that tively. The addition of purified bovine uPA (10 IU/ml) bFGF stimulation may involve additional mechanisms. When wounded BAE monolayers are treated with also stimulated migration by 37%. These observations are in agreement with previous studies by Fibbi et al. anti-bFGF IgG, uPA induction is abolished (Fig. 1)and (1988) demonstrating that addition of human uPA to migration impeded (Fig. 2). As the data from Figure 3 bovine capillary cells stimulated chemotaxis. Treat- and the experiments with inhibitors of plasminogen ment of wounded BAE cell cultures with bFGF (10 ngi activator and plasmin indicate that the role of uPA in ml) provided a greater stimulation of cell migration this system is independent of its proteolytic activity, (60%) than that observed with purified bovine uPA, supplementation of wounded cultures with anti-bFGF PMA, or vanadate. This suggests t h a t additional mech- IgG and ATF should reverse the inhibition of migration anisms besides induction of uPA may be involved in normally observed with anti-bFGF IgG. Indeed, as seen in Figure 4B, addition of 1nM ATF together with antibFGF-stimulated cell motility. The requisite role of bFGF-induced uPA expression bFGF antibody abrogated the 30% inhibition due to in BAE cell migration is further defined by the results anti-bFGF IgG (column 2) and restored migration to of the experiment illustrated in Figure 3. Since anti- 98% (column 4) of control but not to the level seen in the bodies to bovine uPA appeared to block BAE cell migra- absence of neutralizing antibodies to bFGF (130%, coltion, we compared the highest effective dose of anti- umn 2). These results are consistent with the interprebovine uPA IgG on endogenous (Sato and Rifkin, 1988) tation that uPA is a component of BAE cell migration and exogenous bFGF-mediated cell migration. The ap- mediated by bFGF and that the engagement of the uPA plication of antibodies to uPA to control and bFGF- receptor is sufficient for promotion of migration indetreated cultures resulted in a comparable degree of in- pendent of any proteolytic activity. hibition (29% and 26%, respectively) of cell migration DISCUSSION in both cases indicating a requirement for uPA in both We previously reported that both spontaneous migrasituations. We cannot, however, completely exclude the possibility that in the bFGF-treated cultures some non- tion of BAE cells into a denuded area and basal PA activity of BAE cells are dependent on endogenous neutralized uPA remained. As uPA is a potent activator of plasminogen, it was bFGF (Sato and Rifkin, 1988; Tsuboi et al., 1990). The necessary to determine if uPA mediated migration in- results presented here illustrate that the induction of directly via the generation of plasmin which Ossowski uPA activity observed in migrating endothelial cells et al. (1975) described a s necessary and sufficient for after wounding is also dependent on endogenous bFGF

ODEKON ET AL.

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Fig. 4. Effect of human recombinant ATF on BAE cell migration under basal and inhibitory conditions. A Confluent monolayers of BAE cells were wounded and incubated with medium (1)or increasing concentrations of hr ATF (.25, .5,1,5 nM) ( 2 , 3 , 4 , 5 ,respectively) and processed as described in Methods. B Confluent monolayers of BAE

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cells were wounded and incubated with medium (11,medium containing hr ATF (1.0 nM, final concentration) (21, medium containing antibFGF IgG (100 Fgiml) (3), or anti-bFGF (100 I*.g/ml)+ hr ATF (1.0 nM) (4)and processed as described in Methods.

and that this uPA is a direct contributing factor to BAE teraction of the receptor-binding domain of uPA is sufficient to promote migration. These observations are in cell migration. Several observations in this study indicate that the agreement with the results of Fibbi et al. (1988) who induction of uPA at the wound edge is a mechanistic reported that human uPA stimulates chemotaxis of component of BAE cell migration due to bFGF. First, BCE cells by a mechanism independent of its proteothe induction of uPA at the wound edge and the basal lytic activity. Three other studies have suggested a posmigration of BAE cells are both blocked by inclusion of sible function for the binding of the ATF to the uPA antibodies to bFGF in the assay. Secondly, antibodies to receptor. Kirchheimer et al. (1987) have indicated that bovine uPA block basal migration a s effectively a s do uPA-receptor interactions may induce intracellular the antibodies to bFGF. Third, cell movement in re- signals which initiate mitosis. Additionally, the ATF of sponse to exogenous bFGF is also impeded by treatment uPA derived from a tumor was found to be mitogenic for with anti-bovine uPA antibodies. Fourth, the ATF por- osteoblast-like cells (Rabbani et al., 1990). Recently tion of uPA reverses the inhibition of BAE cell migra- Nusrat and Chapman (1991) demonstrated that recombinant human ATF mediates a required step in PMAtion by anti-bFGF antibodies. The precise role of uPA in cell movement has been induced differentiation of myeloid cell lines. The eviunclear. Initial studies by Ossowski et al. (1975) indi- dence for the transmission of a signal by the interaction cated that PA participated in the migratory process by of uPA with its receptors is only suggestive a t this time. generating plasmin. More recently, other studies have The conclusive demonstration of a signal mediated by shown spatial and/or temporal changes in uPA in mi- occupancy of the uPA receptor awaits a more direct grating cells, supporting the hypothesis that this en- analysis of signal transmission. The requirement for PA in invasion by both tumor zyme participates in cell migration (Morioka et al., 1987; Pepper et al., 1987; Pollanen et al., 1988; Hebert and normal cells (Ossowski and Reich, 1983; Mignatti and Baker, 1988; Grqjndhal-Hansen et al., 1988; Erick- et al., 1986, 1989; Ossowski, 1988; Yagel et al., 1988) son and Isseroff, 1989). Consistent with the proposed has been clearly demonstrated. Plasmin, the product of role for uPA in migration, we observed that agents PA activation of plasminogen, participates in the degwhich elevated the endogenous level of PA production radation of basement membrane and matrix compoor the addition of purified bovine uPA enhanced cell nents (Liotta e t al., 1981) a s well as the activation of migration. However, several plasmin inhibitors were metalloprotease zymogens required for the lysis of colunable to block BAE cell migration, supporting the ear- lagens (Paranjpe e t al., 1980). It is possible that the lier suggestion by SchIeef and Birdwell (1982) that migration of cells requires the lysis of specific matrix or plasmin was not necessary for the migration of BAE adhesive proteins that may normally impede migration cells. The effectiveness of polyclonal IgG against bovine or detachment. In this regard uPA may be well uPA in impeding BAE cell migration when considered equipped to promote cell motility. While one domain of in the context of the results with plasmin inhibitors the molecule may impart a motility signal, another suggested that the antibodies may function by interfer- domain may trigger a cascade of proteolytic events faing with domains on uPA other than its enzymatic size. cilitating the progression of cells through tissue archiThe stimulation of BAE cell migration by the ATF of tecture. uPA or pro uPA in the presence of aprotinin (not In conclusion the results presented here support the shown) lent further support to the hypothesis that in- argument that uPA is a mechanistic component of

uPA AND CELL MIGRATION

bFGF-induced endothelial cell migration. ATF is necessary and sufficient for uPA-mediated effects in this system and the effectiveness of ATF in promoting cell migration is a function of the availability of receptors. An understanding of the mechanism of endothelial cell migration due to bFGF may provide in the future more specific control of this phenomenon.

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Mignatti, P., Tsuboi, R., Robbins, E., and Rifkin, D.B. (1989) In vitro angiogenesis on the human amniotic membrane: Requirement for basic fibroblast growth factor-induced proteinases. J. Cell Biol., 10857 1-682. Montesano, R., Pepper, M.S., Belin, D., Vassalli, J.-D., and Orci, L. (1988) Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases. J . Cell. Physiol., 134t460-466. Morioka, S., Lazarus, G.S., Baird, J.L., and Jensen, P.J. (1987)Migrating keratinocytes express urokinase-type plasminogen activator. J. Invest. Dermatol., 88t418-423. ACKNOWLEDGMENTS Moscatelli, D., Presta, M., and Rifkin, D.B. (1985) Purification of a factor from human placenta that stimulates capillary endothelial We are grateful to Dr. J. Henkins for his gift of r h pro cell protease production, DNA synthesis, and migration. Proc. Natl. uPA and ATF, and to Dr. P. Harpel for his gift of aZ- Acad. Sci. U.S.A., 83t2091-2095. plasmin inhibitor. We would like to thank Dr. D.A. Nusrat, A.R., and Chapman, H.A., J r . (1991) An autocrine role for Moscatelli for critical review of the manuscript, and Dr. urokinase in phorbol ester-mediated differentiation of myeloid cell lines. J. Clin. Invest., 87t1091-1097. R. Manejias for the preparation of the antiserum to L. (1988) Plasminogen activator dependent pathways in the bovine uPA. This work was supported by grants from Ossowski, dissemination of human tumor cells in the chick embryo. Cell, the National Institutes of Health (D.B.R.), the Ameri52:321-328. can Cancer Society (D.B.R.), and the New York Heart Ossowski, L., Quigley, J.P., and Reich, E. (1975) Plasminogen, a necessary factor for cell migration in vitro. In: Proteases and Biological Association (L.E.O.). Lale E. Odekon is the recipient of Control. E. Reich, D.B. Rifkin, and E. Shaw, eds. Cold Spring Harthe Jack Murphy Memorial Fellowship for Research bor Laboratory, Cold Spring Harbor, New York, pp. 901-903. (AHA, New York City affiliate). Ossowski, L., and Reich, E. (1983) Antibodies to plasminogen activator inhibit human tumor metastasis. Cell, 35r611-619. LITERATURE CITED Paranjpe, M., Engel, L., Young, N., and Liotta, L.A. (1980) Activation of human breast carcinoma collagenase through plasminogen actiAppella, E., Robinson, E.A., Ullrich, J.S., Stopelli, M.P., Corti, A., vator. Life Sci., 26t1223-1231. Cassani, G., and Blasi, F. (1987) The receptor-binding sequence of Pepper, M.S., Vassali, J.-D., Montesano, R., and Orci, L. (1987)Urokiurokinase. J . Biol. Chem., 262:44374440. nase-type plasminogen activator is induced in migrating capillary Devreotes, P.N., and Zigmond, S.H. (1988) Chemotaxis in eukaryotic endothelial cells. J . Cell Biol., 105t2535-2541. cells: A focus on leukocytes and dictyostelium. Annu. Rev. Cell. Pollanen, J., Hedman, K., Nielsen, L.S., Dano, K., and Vaheri, A. Biol., 4t649-686. (1988) Ultrastructural localization of plasma membrane associated Erickson, C.A., and Isseroff, R.R. (1989) Plasminogen activator activurokinase-type plasminogen activator a t focal contacts. J. Cell. ity is associated with neural crest cell motility in tissue culture. J . Biol., 106r87-95. Exp. Zool., 25t123-133. Pollanen, J., Saksela, O., Salonen, E.-M., Anderson, P., Nielsen, L., Fibbi, G., Ziche, M., Morbidelli, L., Magnelli, L., and Del Rosso, M. Dano, K., and Vaheri, A. (1987) Distinct localizations of urokinase(1988) Interaction of urokinase with specific receptors stimulates type plasminogen activator and its type-1 inhibitor under cultured mobilization of bovine adrenal capillary endothelial cells. Exp. Cell human fibroblasts and sarcoma cells. J . Cell Biol., 104t1085-1096. Res., 179:385-395. Presta, M., Moscatelli, D., Joseph-Silverstein, J., and Rifkin, D.B. Gr0ndhal-Hansen, J., Lund, L.R., Ralfkiar, E., Otteranger, V., and (1986) Purification from a human hepatoma cell line of a basic Dano, K. (1988) Urokinase and tissue-type plasminogen activators fibroblast growth factor-like molecule that stimulates capillary enin keratinocytes during wound reepithelialization in vitro. J . Invest. dothelial cell plasminogen activator production, DNA synthesis, Dermatol., 90:790-795. and migration. Mol. Cell. Biol., 6:4060-4066. Gross, J.L., Moscatelli, D., Jaffe, E.A., and Rifkin, D.B. (1982) Plasminogen activator and collagenase production by cultured capillary Rabbani, S.A., Desjardins, J., Bell, A.W., Danville, D., Mazar, A., Henkin, J., and Golzmann, D. (1990) An amino-terminal fragment endothelial cells. J . Cell Biol., 95t974-981. of urokinase isolated from a prostate cancer cell line (PC-3) is mitoHBbert, C.A., and Baker, J.B. (1988) Linkage of extracellular plasgenic for osteoblast-like cells. Biochem. Biophys. Res. Comm., minogen activator to the fibroblast skeleton: Colocalization of cell 137: 1058-1064. surface urokinase with vinculin. J . Cell Biol., 106:1241-1247. 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Urokinase-type plasminogen activator mediates basic fibroblast growth factor-induced bovine endothelial cell migration independent of its proteolytic activity.

The dependence of urokinase-type plasminogen activator (uPA) induction on endogenous basic fibroblast growth factor (bFGF) activity during endothelial...
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