Soluble Urokinase-type Plasminogen Activator Receptor Inhibits Cancer Cell Growth and Invasion by Direct Urokinase-independent Effects on Cell Signaling
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m308808200
ISSN1083-351X
AutoresMinji Jo, Keena S. Thomas, Lihua Wu, Steven L. Gonias,
Tópico(s)Signaling Pathways in Disease
ResumoThe urokinase-type plasminogen activator receptor (uPAR) is released from human cancers and is readily detected in blood. In animal models, soluble uPAR (SuPAR) antagonizes cancer progression; however, the mechanism by which SuPAR functions in vivo remains unclear. It is generally thought that SuPAR scavenges uPA and prevents its interaction with membrane-anchored uPAR. In this study, we demonstrate a novel molecular mechanism by which SuPAR may inhibit cancer progression. We show that SuPAR has the potential to directly and in a uPA-independent manner block the signaling activity of membrane-anchored uPAR. Whether SuPAR inhibits signaling is cell type-specific, depending on the state of the endogenous uPA-uPAR signaling system. In uPAR-deficient cells that lack endogenous uPAR signaling, including uPAR–/–murine embryonic fibroblasts and human embryonal kidney 293 cells, SuPAR functions as a partial signaling agonist that activates ERK/mitogen-activated protein kinase. By contrast, in cells with potent autocrine uPA-uPAR signaling systems, including MDA-MB 231 breast cancer cells and low density lipoprotein receptor-related protein-1-deficient murine embryonic fibroblasts, SuPAR substantially decreases ERK activation. The mechanism probably involves competitive displacement of membrane-anchored uPAR-uPA complex from signaling adaptor proteins. As a result of its effects on cell signaling, SuPAR blocks cell growth and inhibits cellular invasion of Matrigel™. Cleavage of SuPAR by proteinases increases its signaling agonist activity and reverses its inhibitory effects on growth and invasion. Thus, proteolytic cleavage represents a molecular switch that neutralizes the anticancer activity of SuPAR. The urokinase-type plasminogen activator receptor (uPAR) is released from human cancers and is readily detected in blood. In animal models, soluble uPAR (SuPAR) antagonizes cancer progression; however, the mechanism by which SuPAR functions in vivo remains unclear. It is generally thought that SuPAR scavenges uPA and prevents its interaction with membrane-anchored uPAR. In this study, we demonstrate a novel molecular mechanism by which SuPAR may inhibit cancer progression. We show that SuPAR has the potential to directly and in a uPA-independent manner block the signaling activity of membrane-anchored uPAR. Whether SuPAR inhibits signaling is cell type-specific, depending on the state of the endogenous uPA-uPAR signaling system. In uPAR-deficient cells that lack endogenous uPAR signaling, including uPAR–/–murine embryonic fibroblasts and human embryonal kidney 293 cells, SuPAR functions as a partial signaling agonist that activates ERK/mitogen-activated protein kinase. By contrast, in cells with potent autocrine uPA-uPAR signaling systems, including MDA-MB 231 breast cancer cells and low density lipoprotein receptor-related protein-1-deficient murine embryonic fibroblasts, SuPAR substantially decreases ERK activation. The mechanism probably involves competitive displacement of membrane-anchored uPAR-uPA complex from signaling adaptor proteins. As a result of its effects on cell signaling, SuPAR blocks cell growth and inhibits cellular invasion of Matrigel™. Cleavage of SuPAR by proteinases increases its signaling agonist activity and reverses its inhibitory effects on growth and invasion. Thus, proteolytic cleavage represents a molecular switch that neutralizes the anticancer activity of SuPAR. The multifunctional, glycosylphosphatidylinositol-anchored membrane protein uPAR 1The abbreviations used are: uPAurokinase-type plasminogen activatoruPARuPA receptorERKextracellular signal-regulated kinaseSuPARsoluble uPARCSuPARcleaved soluble uPARLRP-1low density lipoprotein receptor-related proteinMEFmurine embryonic fibroblastHEKhuman embryonal kidneyPMSFphenylmethanesulfonyl fluorideMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.1The abbreviations used are: uPAurokinase-type plasminogen activatoruPARuPA receptorERKextracellular signal-regulated kinaseSuPARsoluble uPARCSuPARcleaved soluble uPARLRP-1low density lipoprotein receptor-related proteinMEFmurine embryonic fibroblastHEKhuman embryonal kidneyPMSFphenylmethanesulfonyl fluorideMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. is a receptor for both urokinase-type plasminogen activator (uPA) and vitronectin that regulates diverse aspects of cell physiology, including cell growth, apoptosis, cell adhesion, and migration (1Ossowski L. 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Cleaved SuPAR (CSuPAR) activates the Src family member hck and induces changes in the cytoskeleton and relocation of integrins to the leading edge of cellular migration, mimicking the responses observed when cells are treated with uPA (30Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (302) Google Scholar, 31Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar, 32Degryse B. Resnati M. Rabbani S.A. Villa A. Fazioli F. Blasi F. Blood. 1999; 94: 649-662Crossref PubMed Google Scholar). We reported that SuPAR promotes MCF-7 cell migration, again mimicking the response observed with uPA (33Nguyen D.H. Webb D.J. Catling A.D. Song Q. Dhakephalkar A. Weber M.J. Ravichandran K.S. Gonias S.L. J. Biol. Chem. 2000; 275: 19382-19388Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Similarly, Aguire Ghiso et al. 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The purpose of the present investigation was to test the hypothesis that SuPAR inhibits cancer progression by directly regulating cell signaling, as opposed to "uPA scavenging." To test this hypothesis, we studied the effects of human SuPAR on murine embryonic fibroblasts (MEFs). Human SuPAR does not bind murine uPA, eliminating uPA scavenging as a possible mechanism (47Estreicher A. Wohlwend A. Belin D. Schleuning W. Vassalli J. J. Biol. Chem. 1989; 264: 1180-1189Abstract Full Text PDF PubMed Google Scholar). Our results demonstrate that SuPAR may activate or inhibit ERK phosphorylation, depending on the state of the autocrine uPA-uPAR signaling system. In cells that have a highly activated autocrine signaling system, including low density lipoprotein receptor-related protein (LRP-1)-deficient MEFs and MDA-MB 231 breast cancer cells, SuPAR inhibits ERK activation, cell growth, and Matrigel™ invasion. The most likely mechanism involves competition with membrane-anchored uPAR-uPA complex for signaling adaptor proteins. Proteinase cleavage serves as a molecular switch, converting SuPAR from a partial agonist into a full agonist and thereby eliminating its function as an anticancer agent. Reagents and Antibodies—Recombinant human SuPAR was from R & D Systems, Inc. (Minneapolis, MN). CSuPAR was prepared by treating SuPAR with 2.0 nm chymotrypsin for 7 h at 37 °C. The chymotrypsin was inactivated with 1.0 mm PMSF, as described (22Behrendt N. Ploug M. Patthy L. Houen G. Blasi F. Dano K. J. Biol. Chem. 1991; 266: 7842-7847Abstract Full Text PDF PubMed Google Scholar). Chymotrypsin, poly-l-lysine, sodium orthovanadate, and protease inhibitor mixture were from Sigma-Aldrich. Antibody that specifically detects phosphorylated ERK1 and ERK2 was from Cell Signaling Technologies (Beverly, MA). Polyclonal antibody that recognizes total ERK was from Zymed Laboratories Inc. (San Francisco, CA). Horseradish peroxidase-conjugated antibodies specific for rabbit IgG was from Amersham Biosciences. Cell Culture—LRP-1-deficient MEF-2 cells were originally prepared by Willnow and Herz (48Willnow T.E. Herz J. J. Cell Sci. 1994; 107: 719-726Crossref PubMed Google Scholar). uPAR–/– MEFs (A1 cells) were prepared as previously described (13Ma Z. Thomas K.S. Webb D.J. Moravec R. Salicioni A.M. Mars W.M. Gonias S.L. J. Cell Biol. 2002; 159: 1061-1070Crossref PubMed Scopus (92) Google Scholar). Both cell lines and human embryonal kidney (HEK) 293 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). MDA-MB 231 cells (ATCC) were maintained in L-15 medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). The cells were plated in tissue culture wells that were precoated with vitronectin (5 μg/ml) purified as previously described (49Yatohgo T. Izumi M. Kashiwagi H. Hayashi M. Cell Struct. Funct. 1988; 13: 281-292Crossref PubMed Scopus (437) Google Scholar), fibronectin (10 μg/ml), type-1 collagen (25 μg/ml) (BD biosciences), or poly-l-lysine (25 μg/ml). ERK Phosphorylation—MEF-2, A1 MEFs, and MDA-MB 231 cells were plated in 60-mm dishes and cultured in serum-free medium. After 18 h, the cultures were washed and then treated with recombinant human SuPAR or CSuPAR, as indicated. The cells were extracted in 1% (v/v) Nonidet P-40, 10 mm HEPES, 150 mm NaCl, 2 mm EDTA, pH 7.5, containing protease inhibitor mixture and sodium orthovanadate (1 mm). The protein concentration in each extract was determined by bicinchoninic acid assay (Sigma-Aldrich). Equal amounts of cell extracts were subjected to SDS-PAGE on 10% slabs, electrotransferred to polyvinylidene difluoride membranes, and probed with specific antibodies for phosphorylated ERK and total ERK. Control experiments were performed in which PMSF alone or PMSF-treated chymotrypsin was added to cultures. Changes in ERK phosphorylation were not observed. Cell Growth—Cell growth was measured by MTT assay, using the Cell Proliferation Kit I (Roche Applied Sciences). The cells were plated in 96-well plates and cultured for 18 h in serum-containing medium. The cultures were then washed with serum-free medium and treated with SuPAR, CSuPAR, or vehicle in serum-free medium for 48 h. MTT hydrolysis was determined, as directed by the manufacturer and detected on the basis of the absorbance at 570 nm. In each experiment, parallel cultures were analyzed prior to the 48-h incubation with SuPAR, CSuPAR, or vehicle. MTT hydrolysis is proportional to the number of viable cells in a culture. Matrigel™ Invasion—Matrigel™ invasion chambers with polymerized growth factor-reduced Matrigel™ were purchased from BD Biosciences. MDA-MB 231 cells were plated in vitronectin-coated wells and cultured in serum-free L-15 medium for 18 h. The cells were then treated with SuPAR, CSuPAR, or vehicle as indicated, released from monolayer culture, and transferred to the upper wells of the Invasion Chambers. SuPAR or CSuPAR were added to both chambers when the cells had been pretreated with either reagent. The lower chamber also contained 10% fetal bovine serum. The cells were allowed to invade for 24 h, at which time the Matrigel™ and cells that were associated with the top surfaces of the membranes were removed using cotton swabs. Cells that penetrated through the Matrigel™ to the underside surfaces of the membranes were fixed and stained with 0.1% crystal violet, as previously described (50Webb D.J. Nguyen D.H. Gonias S.L. J. Cell Sci. 2000; 113: 123-134Crossref PubMed Google Scholar). The dye was eluted with 10% acetic acid, and the absorbance at 570 nm was determined. Regulation of ERK Activation by SuPAR—There is considerable evidence that SuPAR interacts directly with transmembrane adaptor proteins to elicit cellular responses that are equivalent to those caused by membrane-associated uPAR-uPA complex (17Resnati M. Pallavicini I. Wang J.M. Oppenheim J. Serhan C.N. Romano M. Blasi F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1359-1364Crossref PubMed Scopus (320) Google Scholar, 30Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (302) Google Scholar, 51Mizukami I.F. Todd III, R.F. J. Leukocyte Biol. 1998; 64: 203-213Crossref PubMed Scopus (35) Google Scholar). To eliminate the possibility that SuPAR scavenges endogenously produced uPA and thereby indirectly affects cell signaling, we took advantage of the strict species specificity in uPA action (47Estreicher A. Wohlwend A. Belin D. Schleuning W. Vassalli J. J. Biol. Chem. 1989; 264: 1180-1189Abstract Full Text PDF PubMed Google Scholar) and studied the response of MEFs to human SuPAR. A1 MEFs are uPAR-deficient murine cells, prepared from uPAR gene knock-out embryos (13Ma Z. Thomas K.S. Webb D.J. Moravec R. Salicioni A.M. Mars W.M. Gonias S.L. J. Cell Biol. 2002; 159: 1061-1070Crossref PubMed Scopus (92) Google Scholar). Because uPAR is absent, these cells cannot establish autocrine uPA-uPAR signaling. When A1 cells were cultured in serum-free medium for 18 h, the basal level of ERK phosphorylation was very low, irrespective of whether the cells were plated on vitronectin, fibronectin, type-1 collagen, or poly-l-lysine (Fig. 1). Treatment with 10 nm human SuPAR for 2 min activated ERK in quiescent A1 cells, and the extent of activation was similar under each of the culture conditions. MEF-2 cells are murine and LRP-1-deficient. These cells have increased levels of uPA and uPAR and establish autocrine signaling in serum-free medium so that the basal level of activated ERK is elevated (13Ma Z. Thomas K.S. Webb D.J. Moravec R. Salicioni A.M. Mars W.M. Gonias S.L. J. Cell Biol. 2002; 159: 1061-1070Crossref PubMed Scopus (92) Google Scholar, 52Weaver A.M. Hussaini I.M. Mazar A. Henkin J. Gonias S.L. J. Biol. Chem. 1997; 272: 14372-14379Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Similarly, MDA-MB 231 breast cancer cells express large amounts of uPA and uPAR, which form an autocrine signaling loop to maintain an elevated level of activated ERK under serum-free conditions (53Ma Z. Webb D.J. Jo M. Gonias S.L. J. Cell Sci. 2001; 114: 3387-3396Crossref PubMed Google Scholar). In both of these cells lines, SuPAR (10 nm, 2 min) actually decreased ERK phosphorylation, and this result was extracellular matrix protein-independent. One explanation for these results is that SuPAR scavenges uPA and prevents uPA binding to membrane-anchored uPAR. However, in experiments with MEF-2 cells, human SuPAR cannot scavenge murine uPA, eliminating this mechanism in favor of a model in which SuPAR suppresses ERK activation by direct interaction with the cells. The Effects of SuPAR on ERK Activation Are Sustained— ERK activation by uPA may be highly transient. In MCF-7 breast cancer cells, the response is sustained for 5 min or less; however, this duration of response is sufficient to induce long-lived effects on cell migration (10Nguyen D.H. Hussaini I.M. Gonias S.L. J. Biol. Chem. 1998; 273: 8502-8507Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Cell growth and the rate of apoptosis are not affected by uPA in MCF-7 cells, consistent with the work of others demonstrating the requirement for sustained ERK phosphorylation for cells to enter the S phase (54Weber J.D. Raben D.M. Phillips P.J. Baldassare J.J. Biochem. J. 1997; 326: 61-68Crossref PubMed Scopus (374) Google Scholar, 55Balmanno K. Cook S.J. Oncogene. 1999; 18: 3085-3097Crossref PubMed Scopus (200) Google Scholar). Fig. 2A shows that ERK activation by SuPAR in uPAR-deficient A1 MEFs is sustained through 60 min. As a second cell culture model system in which the cells do not express uPAR, we studied HEK 293 cells (17Resnati M. Pallavicini I. Wang J.M. Oppenheim J. Serhan C.N. Romano M. Blasi F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1359-1364Crossref PubMed Scopus (320) Google Scholar). Again, SuPAR activated ERK, and the response was sustained. Suppression of ERK activation by SuPAR, in LRP-1-deficient MEF-2 cells, was also sustained, without apparent change through 60 min (the time course of the complete experiment). We also explored the SuPAR concentration dependence of ERK activation and deactivation. SuPAR, at a concentration of 10 pm, consistently activated ERK in A1 MEFs (Fig. 2B). Maximum ERK activation was observed with SuPAR at concentrations from 0.1 to 10 nm. Similarly, in experiments with MEF-2 cells, inhibition of ERK activation was consistently observed with 10 pm SuPAR. These results indicate that fairly low concentrations of SuPAR are sufficient to directly regulate cell signaling. Because SuPAR induced sustained changes in ERK activation, we undertook experiments to examine the potential of SuPAR to regulate cell growth. We measured cell growth using the MTT assay, which determines the total number of viable cells. As shown in Fig. 3, SuPAR increased A1 MEF proliferation by 2.2-fold in 48 h (p < 0.05, n = 8). By contrast, in MEF-2 and MDA-MB 231 cells, SuPAR essentially blocked cell growth over the same time period. Thus, the effects of SuPAR on cell growth correlate with its effects on the level of ERK activation. SuPAR may also have promoted apoptosis in MEF-2 and MDA-MB 231 cells. This effect would have been indistinguishable from growth inhibition by MTT assay. SuPAR Cleavage by Proteinase Reverses Its Inhibitory Activity—Chymotrypsin cleaves SuPAR in the linker region between D1 and D2 + D3, revealing the sequence, SRSRY, as the new N terminus of D2. Because of this activity, chymotrypsin serves as a model of proteinases that activate SuPAR for cell signaling (30Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (302) Google Scholar, 31Fazioli F. Resnati M. Sidenius N. Higashimoto Y. Appella E. Blasi F. EMBO J. 1997; 16: 7279-7286Crossref PubMed Scopus (230) Google Scholar). We performed experiments to determine whether cleavage of SuPAR by chymotrypsin reverses its activity as an inhibitor of ERK activation and cell growth. Fig. 4A shows that chymotrypsin completely cleaved SuPAR, generating new bands with the anticipated mobilities of D1 and D2 + D3. CSuPAR and SuPAR activated ERK equivalently in uPAR–/– A1 MEFs, indicating that the proteinase-cleaved product was active (Fig. 4B). In control experiments, the chymotrypsin, which was used to cleave SuPAR, had no effect on ERK activation in A1 MEFs, when added alone (data not shown). The chymotrypsin was treated with PMSF before incubation with the cells, following the protocol used to prepare CSuPAR. In MEF-2 cells and MDA-MB 231 cells, SuPAR and CSuPAR yielded different results. After incubation with cells for 2 min, SuPAR decreased ERK phosphorylation as already described; however, in multiple experiments, CSuPAR either had no effect on or slightly increased ERK phosphorylation. Equivalent results were obtained when the time of incubation with CSuPAR was extended (data not shown). Because CSuPAR did not inhibit ERK activation, in MEF-2 and MDA-MB 231 cells, we tested its effects on cell growth. As anticipated, CSuPAR did not inhibit growth of the MEF-2 and MDA-MB 231 cells (Fig. 5). Thus, SuPAR cleavage neutralizes its ability to function as an inhibitor of ERK
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