Induction of Plasminogen Activator Inhibitor-1 by Urokinase in Lung Epithelial Cells
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m207445200
ISSN1083-351X
AutoresSreerama Shetty, Khalil Bdeir, Douglas B. Cines, Steven Idell,
Tópico(s)S100 Proteins and Annexins
ResumoThe 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. 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. urokinase-type plasminogen activator urokinase-type plasminogen activator receptor plasminogen activator inhibitor growth factor like domain bovine serum albumin amino-terminal fragment bronchial epithelial cells phenylmethylsulfonyl fluoride cytomegalovirus lipopolysaccharide phosphotyrosine phosphatase 1C Proteolytic enzymes, including urokinase (uPA)1 and metalloproteinases, have been implicated in the pathogenesis of lung inflammation and the growth of lung tumors. These proteases facilitate remodeling of the transitional stroma via the breakdown of basement membranes and extracellular matrix proteins, including fibrin (1Dano K. Andreasen P.A. Grondahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. 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Masson V. Gerard R.D. Schmitt P.M. Albert V. Praus M. Lund L.R. Frandsen T.L. Brunner N. Dano K. Fusenig N.E. Weidle U. Carmeliet G. Loskutoff D. Collen D. Carmeliet P. Foidart J.M. Noel A. J. Cell Biol. 2001; 152: 777-784Crossref PubMed Scopus (295) Google Scholar), but also through its interaction with vitronectin (1Dano K. Andreasen P.A. Grondahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2301) Google Scholar, 6Stefansson S. Haudenschild C.C. Lawrence D.A. Trends Cardiovasc. Med. 1998; 8: 175-180Crossref PubMed Scopus (32) Google Scholar, 7Stefansson S. Petitclerc E. Wong M.K. McMahon G.A. Brooks P.C. Lawrence D.A. J. Biol. Chem. 2001; 276: 8135-8141Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). PAI-1 binds to vitronectin exposed at sites of vascular interruption (8Seiffert D. Wagner N.N. Loskutoff D.J. J. Cell Biol. 1990; 111: 1283-1291Crossref PubMed Scopus (59) Google Scholar). Binding of PAI-1 to vitronectin stabilizes its activity (9Mimuro J. Loskutoff D.J. J. Biol. Chem. 1989; 264: 936-939Abstract Full Text PDF PubMed Google Scholar) and alters its proteolytic specificity (10Ehrlich H.J. Gebbink R.K. Preissner K.T. Keijer J. Esmon N.L. Merten K. Pannekoek H. J. Cell Biol. 1991; 115: 1773-1781Crossref PubMed Scopus (79) Google Scholar, 11Naski M.C. Lawrence D.A. Mosher D.F. Poder T.J. Ginsberg D. J. Biol. Chem. 1993; 268: 367-372Abstract Full Text PDF Google Scholar). In turn, PAI-1 exposes but transiently occludes the integrin binding site in vitronectin (12Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (607) Google Scholar) and inhibits uPA-induced uPAR-mediated adhesion (13Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1554Crossref PubMed Scopus (697) Google Scholar). 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 (14Nykjaer A. Conese M. Christensen E.I. Olson D. Cremona O. Gliemann J. Blasi F. EMBO J. 1997; 16: 2610-2620Crossref PubMed Scopus (293) Google Scholar), which clears inactive complexes and recycles unoccupied uPAR to the cell surface (15Li H. Kuo A. Kochan J. Strickland D. Kariko K. Barnathan E.S. Cines D.B. J. Biol. Chem. 1994; 269: 8153-8158Abstract Full Text PDF PubMed Google Scholar, 16Conese M. Olson D. Blasi F. J. Biol. Chem. 1994; 269: 17886-17892Abstract Full Text PDF PubMed Google Scholar). 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 (4Sprengers E.D. Kluft C. Blood. 1987; 69: 381-387Crossref PubMed Google Scholar, 17Mazar A.P. Henkin J. Goldfarb R.H. Angiogenesis. 2000; 3: 15-32Crossref Scopus (155) Google Scholar). PAI-1 is secreted by epithelial cells of many normal tissues, including the lung (18Shetty S. Idell S. Am. J. Physiol. 2000; 278: L148-L156PubMed Google Scholar). 2S. Shetty, unpublished results. 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 (20Idell S. Pueblitz S. Emri S. Gungen Y. Gray L. Kumar A. Holiday D. Koenig K.B. Johnson A.R. 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On the other hand, PAI-1 is required for tumor-induced 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 (23Pedersen H. Brunner N. Francis D. Osterlind K. Ronne E. Hansen H.H. Dano K. Grondahl-Hansen J. Cancer Res. 1994; 54: 4671-4675PubMed Google Scholar, 24Pedersen H. Grondahl-Hansen J. Francis D. Osterlind K. Hansen H.H. Dano K. Brunner N. Cancer Res. 1994; 54: 120-123PubMed Google Scholar). 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 anti-proteolytic 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 (25Colucci M. Paramo J.A. Collen D. J. Clin. 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Hansmann C. Stock J. Weidle U.H. Majdic O. Bartke I. Knapp W. Stockinger H. J. Exp. Med. 1995; 181: 1381-1390Crossref PubMed Scopus (355) Google Scholar, 32Dulmer I. Petri T. Schleuning W.D. FEBS Lett. 1993; 322: 37-40Crossref PubMed Scopus (92) Google Scholar, 33Nguyen G. Li X.-M. Peraldi M.-N. Zacharias U. Hagage J. Rondeau E. Srear J.D. Kidney Int. 1994; 46: 208-215Abstract Full Text PDF PubMed Scopus (15) Google Scholar, 34Konakova M. Hucho F. Schleuning W.-D. Eur. J. Biochem. 1998; 253: 421-429Crossref PubMed Scopus (94) Google Scholar) as well as autoregulatory feedback in normal epithelial cells (35Shetty S. Idell S. J. Biol. Chem. 2001; 276: 24549-24556Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 36Shetty S. Pendurthi U.R. Halady P.K. Azghani A.O. Idell S. Am. J. Physiol. 2002; 283: L319-L328Crossref PubMed Scopus (24) Google Scholar) combined with the finding that uPA stimulates proliferation of varied cell types (37Kirchheimer J.C. Wojta J. Christ G. Binder B.R. Eur. J. Biochem. 1989; 181: 103-107Crossref PubMed Scopus (44) Google Scholar, 38Kirchheimer J.C. Remold H.G. Blood. 1989; 74: 1396-1402Crossref PubMed Google Scholar, 39Rabbani S.A. Mazar A.P. Bernier S.M. Haq M. Bolivar I. Henkin J. Golzman D. J. Biol. Chem. 1992; 267: 14151-14156Abstract Full Text PDF PubMed Google Scholar, 40DePetro G. Copeta A. Barlati S. Exp. Cell Res. 1994; 213: 286-294Crossref PubMed Scopus (77) Google Scholar, 41Shetty S. Kumar A. Johnson A. Pueblitz S. Idell S. Am. J. Physiol. 1995; 268: L972-L982PubMed Google Scholar, 42Shetty S. Kumar A. Johnson A.R. Idell S. Antisense Res. Dev. 1995; 5: 307-314Crossref PubMed Scopus (33) Google Scholar) 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 (13Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1554Crossref PubMed Scopus (697) Google Scholar, 31Bahuslav J. Horejsi V. Hansmann C. Stock J. Weidle U.H. Majdic O. Bartke I. Knapp W. Stockinger H. J. Exp. Med. 1995; 181: 1381-1390Crossref PubMed Scopus (355) Google Scholar). We, and others, have identified a signal-transducing region in the kringle of uPA that stimulates smooth muscle cell contraction and migration (43Haj-Yehia A. Nassar T. Sachais B.S. Kuo A. Bdeir K. Al-Mehdi A.B. Mazar A. Cines D.B. Higazi A.A. FASEB J. 2000; 14: 1411-1422Crossref PubMed Google Scholar, 44Mukhina S. Stepanova V. Traktouev D. Poliakov A. Beabealashvilly R. Gursky Y. Minashkin M. Shevelev A. Tkachuk V. J. Biol. Chem. 2000; 275: 16450-16458Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), 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 (45Carmeliet P. Moons L. Herbert J.M. Crawley J. Lupu F. Lijnen R. Collen D. Circ. Res. 1997; 81: 829-839Crossref PubMed Scopus (191) Google Scholar, 46Carmeliet P. Moons L. Dewerchin M. Rosenberg S. Herbert J.M. Lupu F. Collen D. J. Cell Biol. 1998; 140: 233-245Crossref PubMed Scopus (117) Google Scholar). 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. Culture media, penicillin, streptomycin, and fetal calf serum were purchased from Invitrogen; tissue culture plastics were from BD Bioscience. α-Thrombin, herbimycin A, genestein, bovine serum albumin (BSA), ovalbumin, Tris base, aprotinin, dithiothreitol, phenylmethylsulfonyl fluoride, silver nitrate, ammonium persulfate, and phorbol myristate acetate were from Sigma. Acrylamide, bisacrylamide, and nitrocellulose were from Bio-Rad. Recombinant high molecular weight two-chain uPA was a generous gift from Drs. Jack Henkin and Andrew Mazar from Abbott Laboratories (Abbott Park, IL). Anti-PAI-1 and anti-uPA antibodies were obtained from American Diagnostics (Greenwich, CT). Antiphosphotyrosine phosphatase 1C antibody was from Upstate Pharmaceuticals (Lake Placid, NY). The uPA antagonist B428 was the generous gift of Dr. Andrew Mazar (Angstrom Pharmaceuticals, San Diego, CA). XAR x-ray film was purchased from Eastman Kodak. uPA deletion mutants were cloned and the recombinant proteins were expressed in S2 cells and purified, including the amino-terminal fragment (ATF) (amino acids 1–135), low molecular weight uPA fragments (amino acids 136–411), and the deletion mutants GFD-scuPA (amino acids 4–43) and kringle-scuPA (amino acids 47–135), as previously described (47Bdeir K. Kuo A. Mazar A. Sachais B.S. Xiao W. Gawlak S. Harris S. Higazi A.A. Cines D.B. J. Biol. Chem. 2000; 275: 28532-28538Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). 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 (42Shetty S. Kumar A. Johnson A.R. Idell S. Antisense Res. Dev. 1995; 5: 307-314Crossref PubMed Scopus (33) Google Scholar). Primary cultures of human small airway epithelial cells were obtained from Clonetics (San Diego, CA) and cultured in the same media, as previously described (48Shetty S. Idell S. J. Biol. Chem. 2000; 275: 13771-13779Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 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 μg of aprotinin per 100 ml). The cell lysates were prepared using three cycles of freezing and thawing. Proteins from Beas2B cell lysates (50 μg) 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 β-mercaptoethanol and subjected to Western blotting with β-actin monoclonal antibody. Alternatively, we measured uPA-mediated PAI-1 expression by metabolic labeling using [35S]methionine in combination with immunoprecipitation as we described earlier (41Shetty S. Kumar A. Johnson A. Pueblitz S. Idell S. Am. J. Physiol. 1995; 268: L972-L982PubMed Google Scholar). In separate experiments, we also measured phosphotyrosine phosphatase 1C expression by PBS or uPA-treated cells in the presence or absence of sodium orthovanadate by Western blotting using an antiphotyrosine phosphatase 1C antibody. uPA cDNA (49Axelrod J.H. Reich R. Miskin R. Mol. Cell. Biol. 1987; 9: 2133-2141Crossref Scopus (126) Google Scholar) was subcloned into the eukaryotic expression vector pRc/CMV2 (Invitrogen) containing the CMV promoter atHindIII/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 (35Shetty S. Idell S. J. Biol. Chem. 2001; 276: 24549-24556Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). 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 pGEMR-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. The cDNA template of PAI-1 was released with NcoI/PstI, purified on 1% agarose gels, and labeled with [32P]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 × 108 cpm/μg. 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 μg/ml) for 12 h at 37 °C and analyzed using the transcription activation assay as described earlier (35Shetty S. Idell S. J. Biol. Chem. 2001; 276: 24549-24556Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). 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 μg) was separated on agarose/formaldehyde gels. After electrophoresis, the RNA was transferred to Hybond N+according to the instructions of the manufacturer. Prehybridization and hybridization were done at 65 °C in NaCl (1 m), SDS (1%), and 100 μg/ml salmon sperm DNA. Hybridization was performed with PAI-1 cDNA probes (1 ng/ml) labeled to ∼6–7 × 108 cpm/μg of DNA overnight. After hybridization, the filters were washed twice for 15 min at 65 °C, with, respectively, 2 × SSC, 1% SDS; 1 × SSC, 1% SDS; and 0.1% SSC, 1% SDS. The membranes were exposed to x-ray film at −70 °C overnight. The intensity of the bands was measured by densitometry and normalized against that of β-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-β-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. We previously found that lung carcinoma-derived cells differentially express PAI-1 in vitro (18Shetty S. Idell S. Am. J. Physiol. 2000; 278: L148-L156PubMed Google Scholar). 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). Identical tcuPA treatment also induced PAI-1 expression in primary small airway epithelial cells (Fig.1c). 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 (∼1 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. We also prepared uPA-overproducing Beas2B cells and vector-treated controls by transfecting these cells with the eukaryotic expression vector pRc/CMV2 containing uPA cDNA or pRc/CMV2cDNA 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 [35S]methionine and the proteins immunoprecipitated using an anti-PAI-1 monoclonal antibody (Fig. 2b). 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 3.2- and 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 β-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. Nuclear run-on experiments indicated that addition of tcuPA to Beas2B cells over 12 h did not induce PAI-1 mRNA (Fig.4a). 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-β-d-ribofuranosylbenzimidazole for varying lengths of time. We analyzed PAI-1 mRNA by Northern blotting using32P-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 PBS-treated 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. We next treated Beas2B cells with varying amounts (0–1 μg/ml, 0–18 nm) of tcuPA for 24 h and then measured PAI-1 expression by Western blotting. The data shown in Fig.5 indicate that tcuPA induced PAI-1 expression by Beas2B cells in a concentration-dependent manner. The effect is apparent at concentrations as low as 10 ng/ml (0.18 nm). Maximum PAI-1 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 aKd comparable with that reported for binding to uPAR. To determine whether tcuPA-mediated PAI-1 expression involves one or more of the pathways implicated in tcuPA signaling (31Bahuslav J. Horejsi V. Hansmann C. Stock J. Weidle U.H. Majdic O. Bartke I. Knapp W. Stockinger H. J. Exp. Med. 1995; 181: 1381-1390Crossref PubMed Scopus (355) Google Scholar, 32Dulmer I. Petri T. Schleuning W.D. FEBS Lett. 1993; 322: 37-40Crossref PubMed Scopus (92) Google Scholar, 35Shetty S. Idell S. J. Biol. Chem. 2001; 276: 24549-24556Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 36Shetty S. Pendurthi U.R. Halady P.K. Azghani A.O. Idell S. Am. J. Physiol. 2002; 283: L319-L328Crossref PubMed Scopus (24) Google Scholar) 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 tcuPA-treated cells in the presence or absence of sodium orthovanadate 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 PBS- and tcuPA-treated cells. Experiments were then performed to determine whether the induction of PAI-1 by tcuPA requires retention of i
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