Plasminogen Activator Inhibitor-1 and -3 Increase Cell Adhesion and Motility of MDA-MB-435 Breast Cancer Cells
2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês
10.1074/jbc.m202333200
ISSN1083-351X
AutoresDiane Palmieri, Sang Eun Lee, R. L. Juliano, Frank Church,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoPlasminogen activator inhibitor-1 (PAI-1), an inhibitor of urokinase plasminogen activator, is paradoxically associated with a poor prognosis in breast cancer. PAI-1 is linked to several processes in the metastatic cascade. However, the role of PAI-1 in metastatic processes, which may be independent of protease inhibitory activity, is not fully understood. We report herein that PAI-1, when added exogenously to or stably transfected in human MDA-MB-435 breast carcinoma cells, had disparate effects on adhesion to extracellular matrix proteins and motility in vitro. Specifically, exogenously added PAI-1 inhibited cell adhesion to vitronectin but not fibronectin, in agreement with the literature. By contrast, stably transfected PAI-1 stimulated adhesion to both proteins. Wild-type PAI-1 was required for this stimulation, because expression of a non-protease inhibitory P14 (T333R) PAI-1 mutant failed to enhance adhesion. Compared with non-inhibitory PAI-1, wild-type PAI-1 also increased cell motility in chemotaxic assays. Furthermore, stable transfection of a related serine protease inhibitor, plasminogen activator inhibitor-3 (PAI-3, or protein C inhibitor) gave results similar to wild-type PAI-1. The stimulatory activity of PAI-3 was not seen with a non-protease inhibitory P14 PAI-3 mutant (T341R). We show that a downstream effect of endogenous wild-type PAI-1 and PAI-3 overexpression, but not their non-inhibitory counterparts, was the altered expression of α2, α3, α4, α5, and β1 integrin subunits. Additionally, blocking antibodies to β1integrin inhibited PAI-1-induced adhesion. Our data provide experimental support for the stimulatory and inhibitory effects of PAI-1 in metastasis and introduce PAI-3 as another serpin potentially important in malignant disease. Plasminogen activator inhibitor-1 (PAI-1), an inhibitor of urokinase plasminogen activator, is paradoxically associated with a poor prognosis in breast cancer. PAI-1 is linked to several processes in the metastatic cascade. However, the role of PAI-1 in metastatic processes, which may be independent of protease inhibitory activity, is not fully understood. We report herein that PAI-1, when added exogenously to or stably transfected in human MDA-MB-435 breast carcinoma cells, had disparate effects on adhesion to extracellular matrix proteins and motility in vitro. Specifically, exogenously added PAI-1 inhibited cell adhesion to vitronectin but not fibronectin, in agreement with the literature. By contrast, stably transfected PAI-1 stimulated adhesion to both proteins. Wild-type PAI-1 was required for this stimulation, because expression of a non-protease inhibitory P14 (T333R) PAI-1 mutant failed to enhance adhesion. Compared with non-inhibitory PAI-1, wild-type PAI-1 also increased cell motility in chemotaxic assays. Furthermore, stable transfection of a related serine protease inhibitor, plasminogen activator inhibitor-3 (PAI-3, or protein C inhibitor) gave results similar to wild-type PAI-1. The stimulatory activity of PAI-3 was not seen with a non-protease inhibitory P14 PAI-3 mutant (T341R). We show that a downstream effect of endogenous wild-type PAI-1 and PAI-3 overexpression, but not their non-inhibitory counterparts, was the altered expression of α2, α3, α4, α5, and β1 integrin subunits. Additionally, blocking antibodies to β1integrin inhibited PAI-1-induced adhesion. Our data provide experimental support for the stimulatory and inhibitory effects of PAI-1 in metastasis and introduce PAI-3 as another serpin potentially important in malignant disease. Metastasis is a multistep process that involves the coordinated events of proteolysis, adhesion, and migration. Plasminogen activator inhibitor-1 (PAI-1 1The abbreviations used are: PAI, plasminogen activator inhibitor; BSA, bovine serum albumin; CTX, chemotaxis; ECM, extracellular matrix; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; FN, fibronectin; HTX, haptotaxis; LN, laminin; MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; P14, non-inhibitory mutant for PAI-1 T333R and for PAI-3 T341R; PA, plasminogen activator; tPA, tissue-type plasminogen activator; uPA, urokinase plasminogen activator; serpin, serine protease inhibitor; uPAR, urokinase plasminogen activator receptor; VN, vitronectin; wt, wild-type; MEM, minimal essential medium; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. ;SERPINE1) and plasminogen activator inhibitor-3 (PAI-3; also known as protein C inhibitor; SERPINA5) are relatedserine proteaseinhibitors (serpins) of the plasminogen activator system (1Ginsburg D. Zehab R. Yang A.Y. J. Clin. Invest. 1986; 78: 1673-1680Google Scholar, 2Ny T. Sawyer M. Lawrence D.A. Millan J. Loskutoff D.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6776-6780Google Scholar, 3Suzuki K. Deyashiki Y. Nishioka J. Kurachi K. Akira M. Yamamoto S. Hashimoto S. J. Biol. Chem. 1987; 262: 611-616Google Scholar, 5Silverman G.A. Bird P.I. Carrell R.W. Church F.C. Coughlin P.B. Gettins P.G.W. Irving J.A. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. Remold-O'Donnell E. Salvesen G.S. Travis J. Whisstock J.C. J. Biol. Chem. 2001; 276: 33293-33296Google Scholar). PAI-1, the primary inhibitor of the plasminogen activator system, inactivates urokinase plasminogen activator (uPA), but it also has a role in cell adhesion and migration. Several studies have shown that PAI-1 expression in breast and other types of cancer is linked with a poor prognosis (6Andreasen P.A. Kjoller L. Christensen L. Duffy M.J. Int. J. Cancer. 1997; 72: 1-22Google Scholar, 7Andreasen P.A. Egelund R. Petersen H.H. Cell. Mol. Life Sci. 2000; 57: 25-40Google Scholar, 8Knoop A. Andreasen P.A. Andersen J.A. Hansen S. Laenkholm A.V. Simonsen A.C. Andersen J. Overgaard J. Br. J. Cancer. 1998; 77: 932-940Google Scholar). Identification of the biological function of PAI-1 in cancer is complicated by findings that PAI-1 can act independently of its protease inhibitory activity. PAI-1 inhibits in vitroadhesion of multiple cell lines to the extracellular matrix protein, vitronectin (VN) (9Deng G. Curriden S. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Google Scholar). PAI-1 binds VN (10Lawrence D.A. Berkenpas M.B. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 15223-15228Google Scholar), and this PAI-1-VN interaction blocks cell integrin (αvβ3 and αvβ5) adhesion to VN (11Stefansson S. Lawrence D. Nature. 1996; 383: 441-443Google Scholar). The uPA receptor (uPAR) can also bind VN and has been identified as an integrin-independent cell surface VN receptor (9Deng G. Curriden S. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Google Scholar). Although PAI-1 and uPAR both share N-terminal binding sites on VN (10Lawrence D.A. Berkenpas M.B. Palaniappan S. Ginsburg D. J. Biol. Chem. 1994; 269: 15223-15228Google Scholar), PAI-1 has a higher affinity for VN, and consequently, competitively inhibits cell uPAR-VN binding (12Waltz D. Natkin L. Fujita R. Wei Y. Chapman H. J. Clin. Invest. 1997; 100: 58-67Google Scholar). Deng et al. (9Deng G. Curriden S. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Google Scholar) proposed that increased PAI-1 could release cells bound to VN by uPAR and promote cell dissemination, possibly explaining the role of PAI-1 in a metastatic disease process. In addition to the adhesive interactions, there is increasing evidence that PAI-1 can mediate cell migration (12Waltz D. Natkin L. Fujita R. Wei Y. Chapman H. J. Clin. Invest. 1997; 100: 58-67Google Scholar, 13Kjoller L. Kanse S.M. Kirkegaard T. Rodenburg K.W. Ronne E. Goodman S.L. Preissner K.T. Ossowski L. Andreasen P.A. Exp. Cell Res. 1997; 232: 420-429Google Scholar, 14Degryse B. Sier C.F. Resnati M. Conese M. Blasi F. FEBS Lett. 2001; 505: 249-254Google Scholar, 15Stahl A. Mueller B.M. Int. J. Cancer. 1997; 71: 116-122Google Scholar, 16Chazaud B. Ricoux R. Christov C. Plonquet A. Gherardi R.K. Barlovatz-Meimon G. Am. J. Pathol. 2002; 160: 237-246Google Scholar). PAI-1 has been shown to either inhibit or stimulate cell migration on VN. Kjoller et al. (13Kjoller L. Kanse S.M. Kirkegaard T. Rodenburg K.W. Ronne E. Goodman S.L. Preissner K.T. Ossowski L. Andreasen P.A. Exp. Cell Res. 1997; 232: 420-429Google Scholar) added exogenous PAI-1 to a modified Boyden chamber migration assay in which the filters were coated with VN and found that active PAI-1 bound to VN, blocked uPAR and integrin binding, and subsequently blocked migration of human epidermoid carcinoma Hep-2 cells. By contrast, Stahl and Mueller (15Stahl A. Mueller B.M. Int. J. Cancer. 1997; 71: 116-122Google Scholar) found that exogenous PAI-1 stimulated melanoma cell migration on VN. It is unclear why exogenous PAI-1 had opposing effects in each of the studies, but the differences may be associated with differences in assay conditions, cell specificity, or protein conformation. PAI-3 is synthesized in the liver and in numerous steroid-responsive organs. PAI-3 antigen has been detected in saliva, cerebral spinal fluid, amniotic fluid, tears, and semen (4, 17Laurell M.A. Christensson A.P. Abrhamsson P.A. Stenflo J. Liljs H. J. Clin. Invest. 1992; 89: 1094-1101Google Scholar, 18Espana F. Gilabert J. Estelles A. Romue A. Aznar J. Cabo A. Thromb. Res. 1991; 64: 309-320Google Scholar, 19Espana F. Estelles A. Fernandez P. Gilabert J. Sanchez-Cuenca J. Griffin J. Thromb. Haemost. 1993; 70: 989-994Google Scholar). In contrast to PAI-1, PAI-3 inhibits a broad array of proteases, including uPA, tPA, activated protein C, thrombin (free and bound to thrombomodulin), and acrosin (19Espana F. Estelles A. Fernandez P. Gilabert J. Sanchez-Cuenca J. Griffin J. Thromb. Haemost. 1993; 70: 989-994Google Scholar, 20Suzuki K. Nishioka J. Hashimoto S. J. Biol. Chem. 1983; 258: 163-168Google Scholar, 21Jackson T.P. Cooper S.T. Church F.C. J. Protein Chem. 1997; 16: 819-828Google Scholar, 22Rezaie A.R. Cooper S.T. Church F.C. Esmon C.T. J. Biol. Chem. 1995; 270: 25336-25339Google Scholar, 23Elisen M.G.L.M. Bouma B.N. Church F.C. Meijers J.C.M. Fibr. Proteol. 1998; 12: 283-291Google Scholar). PAI-3, whose expression is increased in prostate cancer, also inhibits prostatic glandular kallikreins, suggesting that it may be linked to carcinogenesis in hormone-regulated tissues (17Laurell M.A. Christensson A.P. Abrhamsson P.A. Stenflo J. Liljs H. J. Clin. Invest. 1992; 89: 1094-1101Google Scholar,24Mikolajczyk S.D. Millar L.S. Kumar A. Saedi M.S. Int. J. Cancer. 1999; 81: 438-442Google Scholar). PAI-3 is found in many hormone-responsive tissues, is a uPA inhibitor, and can be localized to malignant breast tissue. The biological significance of a cancer expressing PAI-3 rather than other related serpins (PAI-1, PAI-2, or maspin) that have been implicated in various tumor cell biology processes is unknown (6Andreasen P.A. Kjoller L. Christensen L. Duffy M.J. Int. J. Cancer. 1997; 72: 1-22Google Scholar, 7Andreasen P.A. Egelund R. Petersen H.H. Cell. Mol. Life Sci. 2000; 57: 25-40Google Scholar). We hypothesized that the role of PAI-1 in cell adhesion and motility would be independent of its ability to inhibit serine proteases. Because PAI-3 also inhibits uPA but does not bind VN, it was used to compare and contrast to the interactions of PAI-1. We established stably transfected MDA-MB-435 breast cancer cells expressing wild-type and non-inhibitory mutants of PAI-1 and PAI-3 and characterized their biological properties. In contrast to our initial hypothesis, what we report here is that changes in adhesion, integrin expression, and cell migration of MDA-MB-435 cells was dependent on expression of wild-type PAI-1 and PAI-3 and was not observed from their non-inhibitory serpin counterparts. These results suggest that expression of PAI-1 and PAI-3 may render a tumor cell better able to invade and may partly explain the association of PAI-1 with metastatic disease. MDA-MB-435 and MDA-MB-231 breast tumor cells and the hepatocellular carcinoma cell line Hep G2 were obtained from the University of North Carolina Tissue Culture Facility. Cells were maintained as monolayer culture in minimal essential media (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% sodium pyruvate in a humidified chamber with 5% CO2 at 37 °C. MDA-MB-435 cells were transfected with pcDNA3.1 vectors (Invitrogen) containing wt-PAI-1 (1Ginsburg D. Zehab R. Yang A.Y. J. Clin. Invest. 1986; 78: 1673-1680Google Scholar), P14-PAI-1 (T333R), wt-PAI-3 (25Phillips J.E. Cooper S.T. Potter E.E. Church F.C. J. Biol. Chem. 1994; 269: 16696-16700Google Scholar), or P14-PAI-3 (T341R) using Effectene (Qiagen) according to the manufacturer's recommendations. Stably transfected clones were selected for resistance to the neomycin analogue, G418 (Invitrogen). Total RNA was isolated from the MDA-MB-435 cells and HepG2 cells using RNeasy (Qiagen). Two micrograms of RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) per the manufacturer's recommendations. cDNA was amplified with each cycle consisting of a 1-min denaturation step at 94 °C, a 1-min annealing step at a primer-specific temperature (given in parentheses below for each primer set), and a 1-min elongation step at 72 °C. A pre-amplification denaturation at 94 °C for 5 min and a post-amplification elongation at 72 °C for 5 min were also included. The primer sequences were: PAI-1 (61 °C), sense 5′-AATCAGACGGCAGCACTGTC-3′, antisense 5′-CTGAACATGTCGGTCATTCC-3′; PAI-3 (55 °C), sense 5′-AGCAGGTGGAGAATGCACTGACTC-3′, antisense 5′-CCTGTTGAACACTAGCCTCTGAGAG-3′; uPA (55 °C), sense 5′GGCAGCAATGAACTTCATCAAGTTCC-3′, antisense 5′-TATTTCACAGTGCTGCCCTCCG-3′; tPA (60 °C), sense 5′-CCAGCAACATCAGTCATGGC-3′, antisense 5′-GCACTTCCCAGCAAATCCTTC-3′; uPAR (60 °C), sense 5′-ACAGGAGCTGCCCTCGCGAC-3′ and antisense 5′-GAGGGGGATTTCAGGTTTAGG-3′; annexin-II, sense 5′-TGCTTTGAACATTGAAACAGA-3′ and antisense 5′-TCTTGCTGGATATAATAGTAC-3′; and β-actin (55 °C), sense 5′-ATCATGTTTGAGACCTTCAA-3′ and antisense 5′-CATCTCTTGCTCGAAGTCCA-3′. Cells were plated at a density of 20,000/12 mm2 on sterile glass coverslips and allowed to adhere in normal growth media containing 10% FBS for 4 h, washed 1× with phosphate-buffered saline (PBS), and incubated overnight in serum-free MEM. Cells were fixed to the coverslips with 2% paraformaldehyde for 30 min at room temperature and permeabilized with a 0.5% Triton X-100 buffer containing 300 mm sucrose, 20 mm Hepes (pH 7.4), 50 mm NaCl, and 3 mm MgCl2 for 5 min on ice. Cells were blocked in 10% normal serum from the same species as the secondary antibody for 30 min. Staining was performed at room temperature for 1 h with the following primary antibodies: rabbit polyclonal anti-human PAI-1 (Molecular Innovations, Southfield, MI) and mouse monoclonal anti-human PAI-3 (G4–2, prepared in our laboratory). Biotinylated secondary antibodies (Vector Laboratories) were used in conjunction with the Vectastain ABC Kit for detection. Nuclei were counterstained with hematoxylin. ELISAs were used to determine the concentration of PAI-1 or PAI-3 protein secreted by the transfected cells. A PAI-1 Imulyse kit (BioPool International) and PAI-3 Paired Antibody Sandwich ELISA kit (Affinity Biologicals) were used according to the manufacturer's instructions. Forty-eight well tissue culture plates were coated with VN (BD Bioscience), FN (BD Bioscience), or LN (BD Bioscience) at a concentration of 10 μg/well at 37 °C for 1 h. All plates were rinsed with PBS and blocked with 1% heat-inactivated bovine serum albumin (BSA) for 1 h at 37 °C. Plates were again rinsed with PBS and air-dried. Cells were seeded at 5 × 104 cells/well in 200 μl of serum-free medium and allowed to attach for 1 h at 37 °C. Non-adherent cells were removed with a multi-channel pipette, and adhered cells were gently washed twice with PBS. An MTT assay was performed to quantify the number of adhered cells (26Mosmann T. J. Immunol. Meth. 1983; 65: 55-63Google Scholar). Briefly, 200 μl of 5 μg/ml MTT in serum-free MEM was added to each well and incubated for 2 h at 37 °C. All MTT solution was removed, and 200 μl of dimethyl sulfoxide was added to solubilize the formazan crystals for 20 min at 37 °C. Samples were transferred to a 96-well plate, and the absorbance was measured at 600 nm in a V maxmicrotiter plate reader (Molecular Devices). In the competition studies for MDA-MB-435 cell adhesion to VN and FN surfaces, human wt-rPAI-1 was from Molecular Innovations, and human plasma PAI-3 was from Affinity Biologicals. For F-actin staining, cells were plated at a density of 20,000/12 mm2 on sterile glass coverslips previously coated with either VN or FN. The coverslips were coated according to the above-mentioned procedure for 48-well plates. Cells were allowed to adhere in the absence of serum for 1 h at 37 °C. After gently removing the non-adherent cells, coverslips were fixed as described above. Cells were stained with FITC-labeled phalloidin (Molecular Probes) at a 1:200 dilution in PBS for 30 min at room temperature. Nuclei were stained with Hoechst (1:40,000, Molecular Probes) for 1 min at room temperature. Coverslips were rinsed in PBS and mounted with 50% glycerol in PBS. Half a million cells were washed with PBS containing 1% BSA by centrifuging at 1000 rpm for 5 min at 4 °C. Pelleted cells were incubated with 100 μl of an anti-human integrin antibody solution diluted with PBS containing 1% BSA at a dilution of 1:50, on ice for 1 h. The monoclonal anti-human integrin antibody was either anti-α1 (mAB1973, Chemicon), anti-α2 (clone P1E6, Invitrogen), anti-α3 (clone P1B5, Invitrogen), anti-α4 (clone P4G9, Invitrogen), anti-α5(clone P1D6, Invitrogen), anti-α6 (clone GoH3, BD Pharmingen), anti-αv (clone VNR139 or VNR147, Invitrogen), anti-β1 (clone P4C10, Invitrogen), anti-β3 (clone 25E11, Chemicon), or anti-β4(clone 3E1, Invitrogen). Cells were then washed three times with PBS containing 1% BSA and incubated with 100 μl ofR-phycoerythrin-anti-mouse IgG in PBS containing 1% BSA at a dilution of 1:100 on ice for 45 min. The cells were washed as described above and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. To assess background staining, cells were labeled with only the secondary antibody, omitting the primary antibody. The threshold for an event in flow cytometric analysis was kept at 5%. A total of 1.5 × 104 events were counted for each sample. Listmode files were replayed for data analysis by using WinMDI 2.7 software. Production of uPA protein in the wt- and P14-PAI-1-expressing MDA-MB-435 cells was determined essentially as described by Ma et al. (27Ma Z. Webb D.J., Jo, M. Gonias S.L. J. Cell Sci. 2001; 114: 3387-3396Google Scholar). MDA-MB-231 cells that constitutively express uPA were used as a positive control. From confluent cultures of wt- and P14-PAI-1-expressing MDA-MB-435 and MDA-MB-231 cells, 30 μg of total protein (by Bradford assay) from each cell type was subjected to SDS-PAGE, transferred to nitrocellulose, and probed with a uPA-specific monoclonal antibody (#390 from American Diagnostica, Inc.) followed by anti-mouse IgG-peroxidase conjugate. Secondary antibody was detected by enhanced chemiluminescence. Experiments were conducted using BioCoat® culture inserts (BD Bioscience) with an 8-μm diameter pore size membrane in a 24-well companion plate. For chemotaxis (CTX), VN or FN (50 μg/ml) was diluted in serum-free media with 0.1% BSA (750 μl) and added to each well of the plate. Cells were seeded at 5 × 104 cells (500 μl) in the culture insert in serum-free medium with 0.1% BSA and incubated for 4 h at 37 °C. For haptotaxis (HTX), the lower surfaces of the culture inserts were coated with VN or FN (50 μg/ml) in serum-free medium with 0.1% BSA for 2 h at 37 °C. Inserts were then rinsed with PBS and air-dried. Cells were seeded at 5 × 104 cells (500 μl) in the culture insert in serum-free media with 0.1% BSA, and the same medium was added to the plate. Cells were incubated for 5 h at 37 °C. After the incubation period for both the CTX and HTX experiments, the media was removed from the insert, and cells on the upper surface of the membrane were removed by "scrubbing" the membrane with a cotton-tipped applicator. Cells that migrated to the lower surface of the membranes were fixed to the membrane with 100% methanol for 5 min. Inserts were then washed with PBS and stained with Hoechst (Molecular Probes) diluted in PBS to a final concentration of 500 μg/ml for 2 min. The membranes were excised from the insert, inverted, and mounted on glass microscope slides. The total number of nuclei was counted in each of three fields at 40× magnification using UV fluorescence microscopy. Statistical analysis was performed using InStat, GraphPad Software, Inc. A one-way analysis of variance test was used followed by a Dunnett multiple comparison test or Tukey-Kramer multiple comparison analysis when appropriate. Exogenous PAI-1 can bind to VN and release cells bound to this substratum (9Deng G. Curriden S. Wang S. Rosenberg S. Loskutoff D.J. J. Cell Biol. 1996; 134: 1563-1571Google Scholar). We confirmed this interaction in adhesion experiments when exogenous wt-rPAI-1 and MDA-MB-435 were added to a VN coated plate at the same time, wt-rPAI-1 inhibited cell adhesion in a dose-dependent manner (Fig.1, upper panel). The addition of wt-rPAI-1 (50 nm) blocked MDA-MB-435 cell adhesion to VN by ∼50%. A PAI-1 blocking antibody added at the same time as the wt-rPAI-1 restored cell adhesion to approximately that of the untreated control cells, and exogenous wt-rPAI-1 did not block cell adhesion to FN, because PAI-1 does not bind FN (data not shown). Because PAI-3 lacks a VN binding site (28Neese L.L. Pratt C.W. Church F.C. Blood Coag. Fibrinol. 1994; 5: 737-746Google Scholar), exogenous PAI-3 should not affect cell adhesion to VN. Confirming this prediction, in adhesion experiments when exogenous wt-PAI-3 was added with MDA-MB-435 cells at the time of plating, there was no effect on cell adhesion either to VN (Fig. 1, lower panel) or to FN (data not shown). To test the effect of endogenously expressed plasminogen activator inhibitors on tumor cell adhesion and motility, MDA-MB-435 cells were stably transfected with PAI-1 and PAI-3. The parental MDA-MB-435 cells lack endogenous gene expression of PAI-1 and PAI-3 making them ideal candidates for evaluating a role for these serpins in cancer cells (Fig. 2). MDA-MB-435 cells were transfected with pcDNA3.1 mammalian expression vectors containing either wt-PAI-1, P14-PAI-1, wt-PAI-3, or P14-PAI-3 genes. At least two clones were derived for each vector. Expression of the transfected genes and protein production was verified by rtPCR (Fig. 2,upper panel) and by immunoblot (data not shown). Protein expression was also detected by immunofluorescent staining for PAI-1 in clone 1 of wt-transfected and clone 2 of P14-PAI-1-transfected cells, and for clone 2 and clone 1 for wt and P14-PAI-3, respectively (Fig. 2,lower panel). There was no visible difference in protein localization between wt- and P14-expressing clones for PAI-1 or PAI-3, respectively. In non-permeabilized transfected MDA-MB-435 cells, immunofluorescent staining verified that both PAI-1 and PAI-3 were localized to the cell surface (data not shown). By ELISA, wt-PAI-1-expressing clone 1 and P14-PAI-1-expressing clone 2 secreted 12 and 8.0 μg/ml protein per 1 × 106cells, respectively. By ELISA, wt-PAI-3-expressing clone 2 and P14-PAI-3-expressing clone 1 secreted 6.1 and 8.4 μg/ml of protein per 1 × 106 cells, respectively. The P14-PAI-1- and P14-PAI-3-expressing MDA-MB-435 cells represent a population that was not only subjected to the same transfection method and antibiotic resistant selection as the wild-type population for both PAI-1- and PAI-3-expressing cells, but the P14 populations were transfected with an identical vector and gene sequence with the exception of a single nucleotide. Thus, changes in phenotype between the various PAI-1/PAI-3-expressing MDA-MB-435 clones should reflect biological differences between wild-type active and mutant non-inhibitory serpin effects on the MDA-MB-435 cells. Unexpectedly, we found that wt-PAI-1-expressing MDA-MB-435 cells adhered to both VN and FN 2- to 3-fold better than P14-PAI-1 and untransfected MDA-MB-435 cells (Fig.3, upper panel). Similarly, we found that adhesion of wt-PAI-3-expressing MDA-MB-435 cells increased 3- to 4-fold to both VN and FN compared with P14-PAI-3 and control MDA-MB-435 cells (Fig. 3, lower panel). Adhesion experiments were also performed with laminin (LN) and type I collagen as the substratum. Cell adhesion to LN (Fig. 3) and type I collagen (data not shown) was significantly increased for both the wt-PAI-1- and wt-PAI-3-expressing MDA-MB-435 cells but not for their non-inhibitory P14 counterparts. In control experiments, cell adhesion to tissue culture-treated plastic or to poly-l-lysine-coated plastic was similar between the wt-PAI-1, P14-PAI-1, wt-PAI-3, P14-PAI-3, or untransfected MDA-MB-435 cells. Additionally, six clones of wt-PAI-1-expressing cells isolated and cloned in a separate transfection experiment also adhered to both VN and FN 2- to 3-fold better than untransfected control MDA-MB-435 cells (data not shown). In adhesion experiments when exogenous wt-rPAI-1 (50 nm) was added to either PAI-1- or PAI-3-expressing MDA-MB-435 cell clones, adhesion was blocked by an average of 40% for wild-type and P14 populations (Fig. 4). Although wt-PAI-1- and wt-PAI-3-expressing cells showed 2- to 4-fold increased adhesion compared with untransfected MDA-MB-435 cells (Fig. 4), the addition of exogenous wt-rPAI-1 at time of plating can still bind VN and reduce cell adhesion compared with PAI-1- and PAI-3-expressing cells plated in the absence of exogenous wt-rPAI-1. The phenotype of the adhered cells was examined to understand why there was an increase in cell adhesion to VN and FN by MDA-MB-435 cells expressing wild-type PAI-1 and PAI-3. Cells were adhered to either VN- or FN-coated glass coverslips and stained with FITC-labeled phalloidin, which binds specifically to the cytoskeletal protein F-actin (Fig. 5, figure shows representative fields). There were striking differences in the phenotype of the wt-PAI-1- and wt-PAI-3-expressing cells when adhered to VN and FN. Within 1 h of adhesion to VN and FN, there was an increase in the number of wt-PAI-1-expressing MDA-MB-435 cells adhered compared with the P14-PAI-1-expressing and MDA-MB-435 cell controls. Moreover, these cells flattened and formed focal contacts when adhered to VN and FN (Fig. 5, A and B, respectively). Although comparable numbers of wt-PAI-3- and wt-PAI-1-expressing MDA-MB-435 cells adhered to VN, the wt-PAI-3-expressing MDA-MB-435 cells did not flatten or form focal contacts on VN in 1 h (Fig.5 G). However, wt-PAI-3 cells did flatten and form focal contacts when adhered to FN (Fig. 5 H), resembling the wt-PAI-1-expressing MDA-MB-435 cells (Fig. 5, compare B andH). There was no apparent difference in the numbers or the phenotype of adhered cells between the control MDA-MB-435 cells and the P14-PAI-1 and P14-PAI-3-expressing MDA-MB-435 cells on either VN or FN.Panels C, F, I, L, andO of Fig. 5 show actin staining of the control, wt-, and P14-PAI-1-expressing MDA-MB-435 cells, and wt- and P14-PAI-3-expressing MDA-MB-435 cells, respectively, adhered to uncoated, native glass coverslips. The phenotype of all cells appears similar with approximately the same number of cells adhering per condition. The expression profiles of various integrin subunits on the surface of the wt-PAI-1- and wt-PAI-3-expressing MDA-MB-435 cells were determined to begin to investigate a mechanism for the increased cell adhesion observed. The levels of α1, α2, α3, α4, α5, α6, αv, β1, β3, and β4 integrins were assessed by flow cytometry. Theleft side of Fig. 6 shows the integrin profiles for α2, α3, α4, α5, and β1 in the control, wt-PAI-1- and P14-PAI-1-expressing MDA-MB-435 cells, respectively. The levels of these integrin subunits were increased on the surface of wt-PAI-1-expressing MDA-MB-435 cells compared with the P14-PAI-1-expressing cells and the control MDA-MB-435 cells. Similar results were observed for wt-PAI-3 compared with P14-PAI-3-expressing MDA-MB-435 cells (Fig. 6, right side). There were also increased levels of the α1 and αv integrin subunits detected on the wt-PAI-3-expressing MDA-MB-435 cells (data not shown). We detected no change in the integrin profiles for α1, α 6, αv, β3, or β4 subunits on wt-PAI-1-, P14-PAI-1-, or P14-PAI-3-expressing MDA-MB-435 cells compared with control MDA-MB-435 cells. Because the prototypic VN receptor is αvβ3, we were surprised that we did not detect increased levels of these integrin subunits on the surface of the wt-PAI-1- and wt-PAI-3-expressing cells. Further flow cytometry analysis with an antibody that detected the αvβ5 heterodimer was performed. There was no detectable difference in the expression of αv, β3, or αvβ5subunits on the surface of the wt-PAI-1-expressing cells, or on the P14-PAI-1-expressing and control populations. To investigate whether the increased expression of the β1integrin subunit was at least partially responsible for the increased adhesion of the wild-type-expressing cells, we conducted a series of adhesion experiments with a β1-blocking antibody to substrates that require integrin adhesion through β1subunits, namely FN and LN, but not VN. Addition of an anti-human β1 antibody significantly decreased wt-PAI-1-expressing MBA-MB-435 cell adhesion to both FN and LN, but the antibody had no effect on adhesion to VN (Fig. 7). In control experiments, a nonspecific IgG did not significantly alter wt-PAI-1-expressing MBA-MB-435 cell adhesion to any of the protein substrates (data not shown). Because the phenotype of the MDA-MB-435 cells expressing either w
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