Induction of Plasminogen Activator Inhibitor-1 by Urokinase in Lung Epithelial Cells*

The plasminogen/plasmin system, urokinase-type plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1), influence extracellular proteolysis and cell migration in lung injury or neoplasia. In this study, we sought to determine whether tcuPA (two chain uPA) alters expression of its major inhibitor PAI-1 in lung epithelial cells. The expression of PAI-1 was evaluated at the protein and mRNA level by Western blot, immunoprecipitation, and Northern blot analyses. We found that tcuPA treatment enhanced PAI-1 protein and mRNA expression in Beas2B lung epithelial cells in a time- and concentration-dependent manner. The tcuPA-mediated induction of PAI-1 involves post-transcriptional control involving stabilization of PAI-1 mRNA. Inactivation of the catalytic activity of tcuPA had little effect on PAI-1 induction and the activity of the isolated amino-terminal fragment was comparable with full-length single- or two-chain uPA. In contrast, deletion of either the uPA receptor binding growth factor domain or kringle domain (^kringle) from full-length single chain uPA markedly attenuated the induction of PAI-1. Induction of PAI-1 by exposure of lung epithelial cells to uPA is a newly recognized pathway by which PAI-1 could regulate local fibrinolysis and urokinase-dependent cellular responses in the setting of lung inflammation or neoplasia.

gen activators. The former is mainly involved in extravascular proteolysis and is implicated in stromal remodeling and neoplasia. Plasminogen activator inhibitor type-1 (PAI-1), a member of the serpin family of serine protease inhibitors, binds and irreversibly inactivates both of these plasminogen activators (4), thereby regulating expression of plasminogen activator activity.
PAI-1 also modulates cell adhesion to extracellular matrix both by preventing cell detachment as a consequence of excess plasmin formation (5), but also through its interaction with vitronectin (1,6,7). PAI-1 binds to vitronectin exposed at sites of vascular interruption (8). Binding of PAI-1 to vitronectin stabilizes its activity (9) and alters its proteolytic specificity (10,11). In turn, PAI-1 exposes but transiently occludes the integrin binding site in vitronectin (12) and inhibits uPA-induced uPAR-mediated adhesion (13). Binding of uPA to PAI-1 lowers its affinity for vitronectin, restoring integrin binding, while promoting the affinity of uPA for the low density lipoprotein-related protein (14), which clears inactive complexes and recycles unoccupied uPAR to the cell surface (15,16). Thus, orderly cell migration along the provisional matrix requires a coordinated interaction between the expression and localization of uPA, PAI-1, and their (sub)cellular binding sites. Theoretically, both untoward or premature proteolysis, or excessive or ineffective development of adhesion forces, could retard cell migration along a provisional matrix.
It is then not surprising that pathologic overexpression of PAI-1 has been linked to a wide range of inflammatory and neoplastic lung diseases (4,17). PAI-1 is secreted by epithelial cells of many normal tissues, including the lung (18). 2 A defect of uPA-related fibrinolytic activity, in large part related to overexpression of PAI-1, has been associated with lung dysfunction in acute respiratory distress syndrome and interstitial lung diseases (20,21). There is also extensive and growing evidence for involvement of PAI-1 in cell migration, tumor invasion, and metastasis, where its mechanism of action is more complex (17). Growth of certain tumors is attenuated by PAI-1 (5,22). On the other hand, PAI-1 is required for tumorinduced angiogenesis in other experimental models and high levels of PAI-1, as well as uPA, and uPAR in lung tumor tissue correlate with poor prognosis (23,24). The mechanism underlying these seeming opposing roles for PAI-1 is unexplained, but points to the importance of factors that regulate the timing and level of PAI-1 expression as it relates to its dual antiproteolytic and anti-adhesive activities.
Expression of PAI-1 is modulated by diverse stimuli including hormones, growth factors, endotoxin, glucocorticoids, and cytokines, acting at either the transcriptional or post-transcriptional levels (25)(26)(27)(28)(29)(30). However, to our knowledge, the possibil-* This work was supported by NHLBI National Institutes of Health Grants R01-HL71147-01, R01-HL-62453-01, and R01-45018-06, National Institutes of Health Grants HL60169, HL66442, and HL67381 (to D. B. C. and A. A. H.), and beginning Grant-in-aid 0060251U from the Mid-Atlantic Division of the American Heart Association (to K. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  ity that uPA itself regulates the expression of its inhibitor has not been studied. Overexpression of uPA, uPAR, or PAI-1 by tumor cells (31)(32)(33)(34) as well as autoregulatory feedback in normal epithelial cells (35,36) combined with the finding that uPA stimulates proliferation of varied cell types (37)(38)(39)(40)(41)(42) all suggested to us the hypothesis that uPA might regulate PAI-1 expression in lung epithelial cells. Also, the mechanism by which uPA mediates transcellular signaling remains unclear. uPA binds with high affinity through its growth factor domain to its cellular receptor (uPAR). However, the fact that uPAR is a glycolipid-anchored protein suggests that uPA may signal through other portions of the molecule directly or through conformational changes in uPAR via integrin ligands, integrins, or other transmembrane adapters (13,31). We, and others, have identified a signaltransducing region in the kringle of uPA that stimulates smooth muscle cell contraction and migration (43,44), but the effect of this domain on epithelial cells or on protein synthesis has not been explored. Moreover, uPA but not uPAR is required for smooth muscle cell migration and neointimal growth in vivo (45,46). In this paper, we describe a new paradigm through which PAI-1 expression by lung epithelial cells is regulated by uPA. This pathway could influence alveolar PAI-1 expression and thereby modulate uPA-mediated responses of lung epithelial cells in lung injury or neoplasia.
Cell Cultures-Human bronchial epithelial cells (Beas2B) were obtained from the ATCC. These cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 1% glutamine, and 1% antibiotics as previously described (42). Primary cultures of human small airway epithelial cells were obtained from Clonetics (San Diego, CA) and cultured in the same media, as previously described (48).
Total Protein Extraction and Western Blotting-Cells were grown to confluence and were serum starved overnight in RPMI 1640 media. The cells were then treated with or without recombinant human two-chain uPA or other agents for selected times in serum-free media supplemented with 0.5% BSA. Following these treatments, the cells were suspended in lysis buffer (10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, 15% glycerol, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 3-10 g of aprotinin per 100 ml). The cell lysates were prepared using three cycles of freezing and thawing. Proteins from Beas2B cell lysates (50 g) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 1% BSA in wash buffer for 1 h at room temperature followed by overnight hybridization with anti-PAI-1 monoclonal antibody in the same buffer at 4°C, washed, and PAI-1-immunoreactive proteins were detected by enhanced chemiluminescence. The membranes were stripped with ␤-mercaptoethanol and subjected to Western blotting with ␤-actin monoclonal antibody. Alternatively, we measured uPA-mediated PAI-1 expression by metabolic labeling using [ 35 S]methionine in combination with immunoprecipitation as we described earlier (41). In separate experiments, we also measured phosphotyrosine phosphatase 1C expression by PBS or uPAtreated cells in the presence or absence of sodium orthovanadate by Western blotting using an antiphotyrosine phosphatase 1C antibody.
Overexpression of uPA-Transfection of Beas2B Cells with uPA cDNA-uPA cDNA (49) was subcloned into the eukaryotic expression vector pRc/CMV2 (Invitrogen) containing the CMV promoter at HindIII/NotI sites. The orientations and sequences were confirmed by nucleotide sequencing. Beas2B cells were transfected with the prepared chimeric plasmid constructs using LipofectAMINE (Invitrogen). Stable cell lines were created by culturing Beas2B cells in neomycin-containing media for 3 months. Cells carrying plasmid DNA that survived neomycin treatment were scrapped from 6-well plates and grown in T75 flasks, and the presence of plasmid DNA was confirmed by PCR using specific primers. The overexpression of uPA by cDNA-transfected cells was confirmed by Western blotting of Beas2B cell lysates as well as conditioned media using a uPA monoclonal antibody. The effect of endogenous uPA overexpression on PAI-1 induction was then measured by Western blot and immunoprecipitation, as described above (35).
Plasmid Construction-Plasmid PAI-1/pGEM was obtained by polymerase chain reaction amplification of a human lung cDNA library. The cDNA corresponding to the coding region (0.5 kb) was subcloned to pGEM R -T vector (Promega) and the sequence of the clones was confirmed by nucleotide sequencing. The PAI-1 insert was released by NcoI and PstI, purified on 1% agarose gels, extracted with phenol/chloroform, and used as a cDNA probe for Northern blotting.
Random Priming of PAI-1 cDNA-The cDNA template of PAI-1 was released with NcoI/PstI, purified on 1% agarose gels, and labeled with [ 32 P]dCTP using a Rediprime labeling kit (Promega). Passage through a Sephadex G-25 column removed unincorporated radioactivity. The specific activity of the product was 6 -7 ϫ 10 8 cpm/g.
Nuclear Run-on Transcription Activation Assay-Confluent cells grown in two T182 flasks were serum-starved overnight in RPMI-BSA media. The cells were later treated with PBS or recombinant human two-chain uPA (1 g/ml) for 12 h at 37°C and analyzed using the transcription activation assay as described earlier (35).
PAI-1 mRNA Assessment by Northern Blotting-A Northern blotting assay was used to assess the steady-state level of PAI-1 mRNA. Confluent Beas2B cells were serum-starved overnight in RPMI-BSA media, and treated with two-chain human recombinant uPA for varying times (0 -24 h) in the same media. Total RNA was isolated using TRI reagent, RNA (20 g) was separated on agarose/formaldehyde gels. After electrophoresis, the RNA was transferred to Hybond N ϩ according to the instructions of the manufacturer. Prehybridization and hybridization were done at 65°C in NaCl (1 M), SDS (1%), and 100 g/ml salmon sperm DNA. Hybridization was performed with PAI-1 cDNA probes (1 ng/ml) labeled to ϳ6 -7 ϫ 10 8 cpm/g of DNA overnight. After hybridization, the filters were washed twice for 15 min at 65°C, with, respectively, 2 ϫ SSC, 1% SDS; 1 ϫ SSC, 1% SDS; and 0.1% SSC, 1% SDS. The membranes were exposed to x-ray film at Ϫ70°C overnight. The intensity of the bands was measured by densitometry and normalized against that of ␤-actin. uPA mRNA stability was assessed by transcription chase experiments. In these experiments, cells stimulated with or without uPA were then treated with 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole to inhibit ongoing transcription, after which total RNA was isolated at specific time points. PAI-1 mRNA was measured by Northern blot as described above.
In separate experiments, Beas2B cells were treated with PBS or uPA for 24 h and then with cycloheximide to inhibit ongoing translation. The stability of the PAI-1 protein expressed under these conditions was then measured. PAI-1 protein concentrations were determined at varying time periods (0 -24 h) by a combination of metabolic labeling and immunoprecipitation as described above.

RESULTS
Time-dependent Induction of PAI-1 by TcuPA-We previously found that lung carcinoma-derived cells differentially express PAI-1 in vitro (18). Based on this observation, we first sought to determine whether tcuPA induces PAI-1 expression in Beas2B cells, a non-malignant lung epithelial cell line. We treated the cells with the high molecular weight, two-chain form of uPA (tcuPA) for varying lengths (0 -24 h) of time. Total proteins from cell lysates were used for Western blotting using an anti-PAI-1 antibody. The data in Fig. 1a demonstrate that tcuPA induces PAI-1 expression in Beas2B cells in a time-dependent manner. The induction is detectable by 3 h after the addition of tcuPA and maintained for up to 24 h (Fig. 1b).
To see if contaminating lipopolysaccharides (LPS) in the high molecular weight uPA (tcuPA) preparation we used might be responsible for the observed activity, we first measured the LPS content by the Limulus Amebocyte lysate enzyme-linked immunosorbent assay method. We found that this tcuPA preparation contains negligible amounts (ϳ1 pg/ml) of LPS. To determine whether the LPS content of the preparation could account for the induction of PAI-1, we next treated Beas2B cells with this (1 pg/ml) as well as a 10-fold higher concentrations (10 pg/ml) of LPS for up to 12 h. We then measured PAI-1 expression by Western blotting as described above. We found that these concentrations of LPS failed to induce PAI-1 expression (data not shown) in this cell type, indicating that the induction of PAI-1 by the tcuPA preparation we used could not be attributed to LPS contamination.
Induction of PAI-1 by Endogenous uPA-We also prepared uPA-overproducing Beas2B cells and vector-treated controls by transfecting these cells with the eukaryotic expression vector pRc/CMV 2 containing uPA cDNA or pRc/CMV 2 cDNA using lipofection. We analyzed the PAI-1 expression of the stable cell lines by Western blotting. As shown in Fig. 2a, Beas2B cells transfected with uPA cDNA expressed relatively large amounts of PAI-1 in comparison to vector-transfected or non-transfected control cells. We also found a comparable increase in PAI-1 protein expression by uPA cDNA-transfected cells compared with vector cDNA or non-transfected control cells when the cells were metabolically labeled with [ 35 S]methionine and the proteins immunoprecipitated using an anti-PAI-1 monoclonal antibody (Fig. 2b).
Induction of PAI-1 mRNA Expression by TcuPA in Lung Epithelial Cells-To evaluate the possibility that part of this increase is because of internalization of uPA-PAI-1 complexes by Beas2B cells, we examined the effect of tcuPA on expression of PAI-1 mRNA. We measured the steady-state levels of PAI-1 mRNA in tcuPA-treated Beas2B cells by Northern blotting using a PAI-1 cDNA probe. As shown in Fig. 3, tcuPA induces PAI-1 mRNA in Beas2B cells, with the induction observed as early as 3 h after treatment. Maximum accumulation of PAI-1 mRNA is achieved within 3 h. Whereas tcuPA induces both 3.2and 2.4-kb components of PAI-1 mRNA, induction of the 3.2-kb moiety was found to be greater. These data confirm that tcuPA increases PAI-1 mRNA expression and affects increased protein expression by Beas2B cells, as determined by Western blotting. The level of PAI-1 mRNA was quantified by densitometric scanning and normalized against ␤-actin loading controls. As shown by the composite data in Fig. 3b, resting Beas2B cells express a small amount of PAI-1 mRNA. Following addition of tcuPA, the level of PAI-1 mRNA was increased by 3 h and remained elevated over 24 h.
Evaluation of Transcriptional Activation of PAI-1 by TcuPA-Nuclear run-on experiments indicated that addition of tcuPA to Beas2B cells over 12 h did not induce PAI-1 mRNA (Fig. 4a).
Evaluation of the Effect of TcuPA on PAI-1 mRNA Stability-Because tcuPA did not enhance the rate of PAI-1 transcription, we next sought to determine whether tcuPA influenced the stability of PAI-1 mRNA. To address this possibility, we treated Beas2B cells with PBS or tcuPA for 12 h and then inhibited ongoing transcription with 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole for varying lengths of time. We analyzed PAI-1 mRNA by Northern blotting using 32 P-labeled PAI-1 cDNA as shown in Fig. 4b. PAI-1 mRNA of PBS-treated Beas2B cells has a very short half-life. However, tcuPA treatment stabilized PAI-1 mRNA over 6 h. We also sought to determine whether uPA increases PAI-1 protein stability. To address this possibility we inhibited translation of PAI-1 by PBS or uPA-treated cells with cycloheximde and then analyzed the stability of the PAI-1 protein over varying time periods. Our results (Fig. 4c) show that PAI-1 protein is detectable in the control PBStreated cells and that basal levels were not appreciably changed over 24 h. The low levels of basal PAI-1 expression are consistent with our findings in unstimulated cells as illustrated in Fig. 1. In the uPA-treated cells, PAI-1 protein expression was induced but then decreased to basal levels by 24 h, indicating that uPA treatment does not stabilize the induced PAI-1 protein over this interval.
Effect of TcuPA Concentration on PAI-1 Protein Expression-We next treated Beas2B cells with varying amounts (0 -1 g/ml, 0 -18 nM) of tcuPA for 24 h and then measured PAI-1 expression by Western blotting. The data shown in expression was observed at concentrations of tcuPA between 250 and 1000 ng/ml. These data demonstrate that the induction of PAI-1 in Beas2B cells by tcuPA is a high affinity, concentration-dependent process with a K d comparable with that reported for binding to uPAR.
Effects of Phosphatase and Phosphotyrosine Kinase Inhibitors on tcuPA-mediated PAI-1 Induction-To determine whether tcuPA-mediated PAI-1 expression involves one or more of the pathways implicated in tcuPA signaling (31,32,35,36) are the same ones as responsible for the observed increase in mRNA stability, we pretreated Beas2B cells with herbimycin A and geneticin separately or in combination with tcuPA. As shown in Fig. 6a, herbimycin A and geneticin alone do not induce PAI-1 expression nor reverse tcuPA-mediated PAI-1 expression by Beas2B cells. Pretreatment of cells with tcuPA and sodium orthovanadate (a tyrosine phosphatase inhibitor), on the other hand, inhibited PAI-1 expression. We next used Western blot analysis of the cytosolic extracts of PBS or tcuPAtreated cells in the presence or absence of sodium orthovana-date to determine whether the induction of PAI-1 by tcuPA involved the expression of phosphotyrosine phosphatase (PTP) 1C. The data illustrated in Fig. 6b demonstrate that tcuPA induces PTP1C expression. However, pretreatment with sodium orthovanadate inhibited PTP1C expression in both PBSand tcuPA-treated cells.
Role of the Catalytic Domain of TcuPA on PAI-1 Expression-Experiments were then performed to determine whether the induction of PAI-1 by tcuPA requires retention of its catalytic activity. Induction of PAI-1 by tcuPA in Beas2B cells was partially inhibited by a urokinase-specific small molecule inhibitor, B428, by a monoclonal antibody directed at its catalytic site, and by the irreversible active site titrant, chloromethyl ketone (Fig. 7). ␣-Thrombin stimulated PAI-1 expression and appeared to augment PAI-1-inducing activity in the presence of tcuPA (Fig. 7), whereas plasmin or the plasmin inhibitor aprotinin had no effect (data not shown).
Role of the Non-catalytic Domains of tcuPA on PAI-1 Expression-These data strongly suggest that the capacity of tcuPA to  Fig. 1 were subjected to the transcription reaction in the presence of [ 32 P]UTP at 30°C for 30 min. 32 P-Labeled nuclear RNA was hybridized with uPA cDNA immobilized on nitrocellulose membrane. ␤-Actin and pcDNA3 cDNAs were used as positive and negative loading controls, respectively. b, effect of tcuPA on PAI-1 mRNA stability. Beas2B cells were treated with PBS or tcuPA for 12 h, after which 5,6-dichloro-1-␤-D-ribofuranosylbenzamidazole (10 g/ml) was added for various periods of time. PAI-1 mRNA was analyzed by Northern blotting. c, effect of tcuPA on PAI-1 protein stability. Beas2B cells were treated with PBS or tcuPA for 24 h, after which cycloheximde (10 g/ml) was added and the cycloheximide-treated samples were then assayed at different times over a 0 -24-h period. PAI-1 protein was measured by metabolic labeling using [ 35 S]methionine followed by immunoprecipitation with anti-PAI-1 monoclonal antibody (upper panel). The same samples were immunoprecipitated with a monoclonal antibody to ␤-actin as loading controls (lower panel).
induce PAI-1 resides in the non-proteolytic ATF. In accord with that hypothesis, the induction of PAI-1 by ATF was similar to that induced by scuPA or tcuPA (Fig. 8a). In contrast, the isolated low molecular weight catalytic domain had little activity, consistent with the amount of PAI-1 induced when the catalytic activity of tcuPA was inhibited (Figs. 7 and 8a). su-PAR totally blocked the activity of tcuPA (Fig. 8, b and c), excluding signaling through the uPA-uPAR complex (50,52) and eliminating the possibility of endotoxin or another mechanism of PAI-1 induction.
The ATF is composed of the uPAR-binding GFD and a kringle. To determine whether either or both domains are required for the inductive effect, we studied scuPA variants lacking one or the other domain. Deletion of the uPAR-binding GFD almost totally abolished the inductive effect, as did deletion of the kringle (Fig. 8, b and c). These data suggest that uPAR or, less likely, another cellular receptor, must bind to the uPA GFD for induction of PAI-1 to occur, but that the kringle is needed either because it contains the signaling element or is required to induce the active conformation of the GFD⅐uPAR complex. In addition, treatment of Beas2B cells with growth factor domain (ˆGFD) or kringle (ˆkringle) uPA deletion mutants inhibited PTP1C expression. These observations suggest that tcuPAmediated induction of PAI-1 at least in part involves phosphotyrosine phosphatases, and that the induction of PTP1C by uPA involves both the GFD and kringle regions (Fig. 8d). DISCUSSION uPA-dependent proteolysis is critical for cellular migration and tissue remodeling in inflammation, tumor growth, and the development of metastasis (53,54). The tcuPA-uPAR interaction can promote cellular movement and regulate cellular attachment to surrounding ground substance, processes that may contribute to remodeling of the lung in the acute respiratory distress syndrome or in the interstitial lung diseases (21,55). The interaction between uPA and uPAR at the cancer cell surface also appears to be a critical event in the pathogenesis of neoplastic growth and metastasis, mediating tissue remodeling, tumor cell invasion, adhesion, and proliferation (1,39). Binding of uPA to uPAR mediates cell proliferation in several cell types including nonmalignant lung epithelial cells, lung carcinoma-derived cells, and mesothelioma (41,42). Tumor cell invasion is also facilitated by occupancy of uPAR with host-or tumor-derived uPA (56,57). Oligomerization of uPAR may facilitate vitronectin-mediated cell adhesion and migration (58). Pathways that regulate the uPA-uPAR-PAI-1 system are, therefore, germane to the pathogenesis of lung injury, its repair and neoplasia.
Plasminogen activation is regulated in part by two specific, fast-acting plasminogen inhibitors, PAI-1 and PAI-2. These inhibitors belong to the serpin family and are products of different genes. These inhibitors bind and inactivate both receptor-bound and fluid-phase uPA. PAI-1 is the major PAI in FIG. 5. Effect of tcuPA concentration on PAI-1 protein expression. a, the cells grown to confluence were treated with varying amounts of tcuPA (0 -1 g/ ml) for 24 h at 37°C in basal medium containing 0.5% BSA. The total proteins from cell lysates were subjected to immunoblotting as described in the legend to Fig. 1. b, the figure shown illustrates the mean band densities of four independent experiments.
FIG. 6. a, effect of tyrosine kinase and phosphatase inhibitors on tcuPA-mediated PAI-1 expression. Cells grown to confluence were treated with or without herbimycin A (Herb), geneticin (Gen), and sodium orthovanadate (Naor) for 2 h followed by two-chain (tcuPA, 1 g/ml) for 24 h at 37°C and subjected to immunoblotting with anti-PAI-1 antibody as described in the legend to Fig. 1. The bar graph illustrates the mean band densities of three independent experiments. b, effect of tyrosine phosphatase inhibitors on phosphotyrosine phosphatase 1C expression. Cells grown to confluence were treated with PBS or tcuPA in the presence or absence of sodium orthovanadate as described above. The cytosolic extracts were subjected to Western blotting using an anti-phosphotyrosine phosphatase antibody.
plasma and in most tissues, and a wide diversity of hormones, cytokines, and growth factors regulate its cellular expression. In the lung, PAI-1 is expressed at the surface of nonmalignant lung epithelial cells, which also elaborate uPA and thereby regulates the delicate PA-PAI balance that determines expression of fibrinolytic activity in the alveolar compartment (55,59).
The balance between proteolytic enzymes and their inhibitors is also critical in the regulation of tissue remodeling and normal angiogenesis (60). Cell migration along provisional matrix involves sequential and topographically directed adhesion/ disadhesion. The traction forces involved must fall within a critical range. The fact that both excessive and inadequate cell contacts preclude coordinate assembly/disassembly of focal contacts helps to explain the otherwise seemingly paradoxical requirement for PAI-1 in cell migration and its negative correlation with prognosis in a variety of human tumors. The possibility that this balance is achieved through an autoregulatory process, in this case initiated by uPA, has received little attention. Therefore, we sought to determine whether uPA contributed to the regulation of PAI-1 expression by lung epithelial cells.
In this study, we confirm that this is the case and demonstrate that uPA induces expression of PAI-1 in cultured Beas2B as well as primary small airway epithelial cells. This pathway provides a versatile regulatory system through which the uPA concentration of the ambient microenvironment could regulate PA activity and pericellular proteolysis by up-regulating expression of PAI-1. This molecular mechanism may be a crucial determinant of cellular invasiveness of lung carcinomas, in which excessive uPA-dependent pericellular proteolysis increases cellular invasiveness (17), an effect that can be regulated by local expression of PAI-1.
Regulation of PAI-1 expression involves both transcriptional and post-transcriptional mechanisms. In previous studies, we found that a post-transcriptional pathway influences levels of PAI-1 mRNA in lung cancer-derived cell lines and nonmalignant lung epithelial cells (18). Similar findings were previously reported in phorbol myristate acetate, insulin, insulin-like growth factor, and cyclic nucleotide analogue-treated cells (61)(62)(63)(64). Cytokines expressed in the setting of acute lung injury or in the tumor microenvironment increased PAI-1 expression (65). The identification of a newly identified post-transcriptional mechanism by which the lung epithelium regulates PAI-1 suggested the possibility that other novel pathways could likewise influence expression of this major PA inhibitor. Based upon our previous observations, we therefore hypothe- a, cells grown to confluence were treated with either the aminoterminal (1 g/ml) or low molecular weight (LMW, 1 g/ml) fragments of uPA, two-chain (tcuPA, 1 g/ml) or single chain (scuPA) for 24 h at 37°C in basal medium containing 0.5% BSA. Cellular proteins were immunoblotted as described above with anti-PAI-1 or anti-␤-actin antibody. The data illustrated are representative of the findings of four independent experiments. b, Beas2B cells were treated with PBS, growth factor domain deletion (ˆGFD), kringle domain deletion (ˆkringle) mutant, active uPA (tcuPA), chloromethyl ketone-inactived uPA (Chl. uPA), or uPA in the presence of excess soluble uPAR (suPAR). All proteins were used in a concentration of 1 g/ml and suPAR was used in a 10-fold molar excess. PAI-1 expression was measured by Western blotting, as described above. The data are representative of four independent experiments. c, composite densitometric analyses of the effect of deletion fragments of uPA on PAI-1 induction in Beas2B cells. The cells grown to confluence were treated with the same concentrations (1 g/ml) of scuPA, ATF, growth factor domain deletion (ˆGFD), or kringle domain (ˆkringle) mutants, low molecular weight uPA, tcuPA alone, or in the presence of excess amounts of soluble uPAR (suPAR, 10-fold excess versus tcuPA) for 24 h at 37°C. The cellular proteins were subjected to immunoblotting using anti-PAI-1 and anti-␤-actin monoclonal antibodies. d, effect of growth factor domain or kringle domain deletions from single chain uPA on PTP1C expression. Cells grown to confluence were treated with PBS, single chain uPA (scuPA), two-chain uPA, or single chain uPA lacking either the GFD (ˆGFD) or kringle domain (ˆkringle). The cytosolic extracts were subjected to Western blotting using an anti-PTP1C antibody, as described above. The figure represents the findings of two independent experiments. sized that the induction of PAI-1 in lung epithelial cells by uPA could involve post-transcriptional regulation. We now confirm that PAI-1 mRNA is stabilized by tcuPA treatment. We have previously shown that tcuPA-mediated induction of its own expression as well as that of uPAR, and that the processes also involve post-transcriptional regulation (35,36). However, tcuPA treatment did not stabilize the induced PAI-1 protein itself. Studies are in progress to study the inter-relationship between these pathways and to identify the responsible regulatory factors. Differences in cell receptors, transcription factors, or other components of the signaling pathway may participate in the characteristic untoward migration of tumor cells compared with the coordinated regulation of normal repair processes including angiogenesis and wound healing.
The mechanism by which tcuPA induces PAI-1 in Beas2B cells appears to be largely independent of its proteolytic activity. Inhibitors of the catalytic site or proteolytic inactivation by thrombin caused little loss of PAI-1-inducing activity. Moreover, essentially identical amounts of PAI-1 were induced by ATF and full-length single-and two-chain forms of uPA. In contrast, little or no PAI-1 was induced when either the growth factor domain or the kringle domain were deleted from the full-length molecule. Binding of GFD to uPAR may be required to approximate a signaling element in the kringle with a yet to be identified transmembrane adapter. Signaling mediated through the uPA kringle domain has previously been reported in vascular smooth muscle cells (43,44). The alternative explanation, i.e. that the kringle is required to generate a productive interaction of the GFD⅐uPAR complex with an adapter, such as an integrin, cannot be excluded. However, the fact that suPAR inhibited PAI-1 induction, although suPAR⅐uPA complexes bind to vitronectin (50), makes this latter explanation somewhat less likely. The regulatory mechanism may also involve the participation of other factors. We previously reported that tumor necrosis factor-␣ induces uPA in Beas2B cells (67). This mechanism could contribute to increased expression of uPA, which could in turn increase uPA-mediated expression of PAI-1 by these cells. Multiple other such interactions could likewise influence the expression of PAI-1 and require further study.
The process also involves cellular signaling mainly through activation of tyrosine phosphatases. The inhibitory effect of tyrosine phosphatase inhibitors on PAI-1 expression indicates that tyrosine phosphorylation is involved in the signaling process and that the signaling mechanism could involve PTP1C. This possibility is supported by the induction of PTP1C by the uPA GFD and kringle, regions that are otherwise implicated in expression of PAI-1. uPA may also induce synthesis of growth factors or cytokines, which in turn may induce increases in the level of PAI-1 mRNA. The elucidation of the mechanisms responsible for the prolonged effect of uPA on the PAI-1 mRNA level remain to be determined. The induction of PAI-1 by tcuPA and thrombin was additive whereas plasmin did not influence the process (data not shown), indicating that the induction mechanism was not subject to inhibitory cross-talk involving these proteases.
Studies performed with other cell types have demonstrated the involvement of uPAR in uPA-mediated signaling. Along this line, activation of focal adhesion kinase and mitogen-activated protein kinases in cultured endothelial cells has been reported (68). Similarly, activation of a 38-kDa tyrosine-phosphorylated uPAR-associated protein has been identified in U937 cells (32). In both cases, signaling by uPA was abolished when the cells were treated with phosphatidylinositol-phospholipase C and other studies also supported the association of uPAR with protein kinase C and cytokeratin (66). In vascular smooth muscle cells, uPAR has been associated with JAK1, Tyk2, and Src kinases (33), whereas in kidney epithelial tumor cells uPAR associates with gp130 and JAK1 (19). We now show that uPA mediates induction of PAI-1 and that the signaling mechanism involves phosphotyrosine phosphatases, including PTP1C.
In summary, we demonstrate that uPA stimulates expression of PAI-1 by lung epithelial cells in culture. If operative in vivo, this pathway could contribute to the relative local overexpression of PAI-1 and the paucity of alveolar fibrinolytic capacity associated with inflammatory lung disease (55). On the other hand, failure to counterbalance the exuberant production of uPA characteristic of tumor cells by the timely production of sufficient PAI-1 by host cells or the tumor cells themselves may contribute to neoplastic proliferation and formation of metastasis. Identification of the intracellular mediators of this process may provide a new handle on the regulation of impaired or untoward cell migration. This newly identified pathway is, to our knowledge, the first description of the ability of uPA to regulate the expression of its major inhibitor, PAI-1, in any cell type. The physiological and pathological implications of uPA-mediated PAI-1 expression will require further study.