The Surface of Prostate Carcinoma DU145 Cells Mediates the Inhibition of Urokinase-Type Plasminogen Activator by Maspin1

[CANCER RESEARCH 60, 4771–4778, September 1, 2000] The Surface of Prostate Carcinoma DU145 Cells Mediates the Inhibition of Urokinase-type Plasminogen Activator by Maspin1

Richard McGowen, Hector Biliran, Jr., Ruth Sager,2 and Shijie Sheng3 Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201 [R. M., H. B., S. S.], and Division of Cancer Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [R. S.]

ABSTRACT relation between maspin expression and the progression of breast cancer (1, 4). In addition, maspin expression is down-regulated in Maspin is a novel serine protease inhibitor (serpin) with tumor prostate carcinoma cells compared with that in immortalized normal suppressive potential in breast and prostate cancer, acting at the level of tumor invasion and metastasis. It was subsequently demonstrated prostate epithelial cells (5, 6). that maspin inhibits tumor invasion, at least in part, by inhibiting cell Biological studies demonstrate a tumor-suppressive role of maspin, motility. Interestingly, in cell-free solutions, maspin does not inhibit acting at the levels of tumor invasion and metastases. Mammary several serine proteases including tissue-type plasminogen activator carcinoma MDA-MB-435 cells transfected with maspin cDNA are and urokinase-type plasminogen activator (uPA). Despite the recent significantly inhibited in invasion and motility assays in vitro and are biochemical evidence that maspin specifically inhibits tissue-type plas- inhibited in tumor growth and metastasis in nude mice (1, 5, 7). It has minogen activator that is associated with fibrinogen or poly-L-lysine, been shown independently that induction of maspin expression in four the molecular mechanism underlying the tumor-suppressive effect of different breast tumor cell lines by ␥-linolenic acid dramatically maspin remains elusive. The goal of this study was to investigate the effect of maspin on cell surface-associated uPA. In our experimental reduces cell motility in vitro (8). Furthermore, purified recombinant system, we chose prostate carcinoma DU145 cells because these cells maspin proteins produced in three different expression systems inhibit mediate plasminogen activation primarily by uPA, as shown by two the invasion and motility of an array of breast cancer cell lines as well different colorimetric enzyme activity assays. Purified recombinant as three prostatic carcinoma cell lines in culture (5, 9). A study by maspin produced in baculovirus-infected Spodoptera frugiperda Sf9 Seftor et al. (10) shows that purified recombinant maspin provokes insect cells [rMaspin(i)] binds specifically to the surface of DU145 cells, changes of the integrin profiles on the MDA-MB-435 cell surface in inhibits the DU145 cell surface-bound uPA, and forms a stable complex favor of a more benign epithelial phenotype. Recently, Zhang et al. with the uPA in DU145 cell lysate. The inhibitory effect of rMaspin(i) (11) have shown that recombinant mouse maspin inhibits prostate on cell surface-bound uPA was similar to that of an uPA-neutralizing antibody and was reversed by a polyclonal antibody against the reac- tumor mitogenesis in vitro and inhibits human prostate tumor-induced neovascularization in a xenograft mouse model. Hence, maspin pro- tive site loop sequence of maspin. The Ki value for rMaspin(i) in cell surface-mediated plasminogen activation was 20 nM, which was com- tein, either reexpressed in carcinoma cells or as an exogenously added parable to the Ki values for plasminogen activator inhibitor 1 and purified reagent, may find novel therapeutic applications in the inter- plasminogen activator inhibitor 2, respectively. Furthermore, the pro- vention of both breast and prostate cancers. teolytic inhibitory effect of rMaspin(i) was quantitatively consistent The enthusiasm about the potential therapeutic value of maspin in with its inhibitory effect on the motility of DU145 cells in vitro. Our treating human malignancy is hampered by the lack of understanding data demonstrate an important role for the prostate carcinoma cell surface in mediating the inhibitory interaction between rMaspin(i) and of the molecular mechanism of maspin. Central to this issue, maspin uPA. Thus, future maspin-based therapeutic strategies may prove does not act as a classical serpin in cell-free conditions, i.e., it does not useful in blocking the invasion and metastasis of uPA-positive prostate inhibit a series of serine proteases including tPA and uPA (12–14), carcinoma. although the RSL sequence of maspin is critical for its biological activities (1, 5, 11). After intensive research, tPA preactivated by fibrinogen or poly-L-lysine has been identified as a target of maspin INTRODUCTION (12). Interestingly, detailed kinetic data suggest that recombinant maspin may use its two segregated domains to interact with the Maspin is a Mr 42,000 protein having an overall sequence homol- ogy with members of the serine protease inhibitor serpin superfamily catalytic and regulatory domains of tPA, respectively (12). The com- (1). The maspin gene has been mapped to chromosome 18q21.3–q23 plex interaction between recombinant maspin and tPA suggests that (2), within a cluster of serpins including PAI-24 and squamous cell the novel proteolytic inhibitory property of maspin depends not only carcinoma antigen (3). Accumulated evidence shows an inverse cor- on its intrinsic structural features but also on the microenvironment of the target enzyme.

Received 2/17/00; accepted 7/5/00. We report here that rMaspin(i) competitively inhibited plasminogen The costs of publication of this article were defrayed in part by the payment of page activation mediated by the surface of prostate carcinoma DU145 cells. charges. This article must therefore be hereby marked advertisement in accordance with In contrast, rMaspin(i) did not inhibit purified uPA, tPA, and plasmin 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Prostate Cancer Pilot Grant CA69845 from the National Cancer in cell-free conditions. Under our culture conditions, the DU145 cell Institute/Wayne State University (to S. S.), the Ruth Sager Memorial Fund established by surface used primarily uPA, but not tPA, to convert plasminogen to Dr. Arthur B. Pardee through Wayne State University (S. S.), and NIH Grant CA61253 (to R. S.). plasmin. We show that rMaspin(i) forms a stable complex with uPA 2 Deceased. in DU145 cell lysate. Furthermore, the maspin proteolytic inhibitory 3 To whom requests for reprints should be addressed, at Department of Pathology, effect correlated quantitatively with the inhibition of cell motility in Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201. Phone: (313) 997-8197; Fax: (313) 993-4112; E-mail: [email protected]. vitro. To our knowledge, this is the first evidence that maspin inhibits 4 The abbreviations used are: PAI, plasminogen activator inhibitor; rMaspin(i), recom- uPA. Moreover, our data demonstrate an important role of the prostate binant human maspin produced in baculovirus-infected Spodoptera frugiperda Sf9 insect cells; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; carcinoma cell surface in mediating the inhibitory interaction between uPAR, urokinase-type plasminogen activator receptor; RSL, reactive site loop; SRB, maspin and uPA. Thus, future maspin-based therapeutic strategies sulforhodamine B; CM, conditioned medium; SF, serum-free; CM, conditioned medium; CAA, colorimetric amidolytic activity assay; CCA-CSPA, coupled colorimetric assay for may prove useful in blocking the invasion and metastasis of uPA- cell surface-mediated plasminogen activation; MICS, membrane invasion culture system. positive prostate carcinomas. 4771

MATERIALS AND METHODS tration of 100 mM. The resulting mixtures were analyzed by Western blotting

with either the polyclonal antibody against the NH2-terminal variable sequence Cell Culture. Human prostate DU145 carcinoma cells (American Type of maspin [Abs3A (1)] or the monoclonal antibody against the A-chain of uPA Culture Collection) were maintained in RPMI 1640 (Life Technologies, Inc.) (American Diagnostica). medium supplemented with 5% fetal bovine serum (Hyclone) in a cell culture Immunofluorescent Staining of Maspin. Cells cultured in 8-well cham- incubator (constantly set at 37°C with 6.5% CO2). The CM was produced by ber slides (Nunc) in the maintenance medium were washed with PBS and incubating the cells (approximately 70% confluent) for 24 h in a SF keratino- incubated with PBS Ϯ 80 nM rMaspin(i) for 30 min. The cells were gently cyte growth medium (KGM; Life Technologies, Inc.) supplemented with washed twice with PBS, fixed with 4% freshly prepared paraformaldehyde/ epidermal growth factor (5 ng/ml). All CM thus obtained was concentrated by PBS for 10 min, blotted with Abs4A (5 ␮g/ml), and subsequently blotted with approximately 10-fold in Centricon-10 units (Amicon). R-phycoerythrin-antirabbit IgG (HϩL) (Zymed), as described previously (5). Chemicals and Reagents. Polyclonal antibodies against the RSL sequence The nuclear DNA was briefly counterstained with Hoechst 33528 at a final of maspin protein Abs4A were produced and purified as described previously concentration of 10 ␮g/ml. The stained cells were viewed and photographed (1). Purified recombinant PAI-1 was a generous gift from Dr. Thomas Reilly under a Leica fluorescent microscope. (Dupont Merck, Wilmington, DE). Reagents of molecular biology grade for ELISA Detection of rMaspin(i) Bound to DU145 Cells. Cells seeded at protein gel electrophoresis, protein staining, and protein concentration analysis 105 cells/well in a 96-well microplate were incubated in the maintenance were purchased from Bio-Rad. Trypan blue solution was purchased from Flow medium for 12 h. The cells were washed with PBS, incubated overnight at 4°C Laboratories (McLean, VA). Proteolytic enzymes, protease substrates, and in 100 ␮l/well SF keratinocyte growth medium containing rMaspin(i) at specific protease inhibitors purchased from American Diagnostica include various concentrations. The cells were washed gently, blocked with 1% BSA/ recombinant single-chain tPA, high molecular weight recombinant uPA, syn- PBS for 1 h, blotted with 15 ␮g/ml Abs4A for 1 h, and detected by the color thetic chromogenic peptide substrate of uPA (Spectrozyme UK), synthetic reaction of horseradish peroxidase-conjugated antirabbit IgG as described by chromogenic peptide substrate of tPA (Spectrozyme tPA), Glu-type plasmin- Harlow and Lane (15). ogen, purified plasmin, chromogenic plasmin substrate (Spectrozyme PL), In Vitro Motility Assays Using MICS. Briefly, the serum-starved cells (24 purified tPA-neutralizing monoclonal antibody (catalogue number 374B), h) were seeded onto the 8 ␮m polycarbonate membrane insert (precoated with purified uPA-neutralizing monoclonal antibody (catalogue number 394), and 50 ␮g/ml Matrigel) of the MICS chamber. rMaspin(i), Abs4A, or a preincu- purified recombinant human PAI-2. Unless otherwise specified, all other bated mixture of rMaspin(i) and Abs4A (37°C, 30 min) was added directly to chemicals and reagents were of the highest purity and obtained from Sigma. the cell cultures at the indicated final concentrations. The MICS chamber was

Purification of Monomeric rMaspin(i). rMaspin(i) was first purified by a incubated for6hat37°C with 6.5% CO2. Cells attached to the bottom side of two-step chromatographic procedure as described previously (9). This rMas- the polycarbonate membrane insert were fixed, stained, and counted under the pin(i) preparation was further purified by a heparin column procedure using the microscope as described previously (5). medium-pressure BioLogic automated chromatographic system (Bio-Rad). Miscellaneous Procedures. Cell staining with SRB was performed as Briefly, 10 mg of the initially purified rMaspin(i) were loaded onto a heparin described by Garbin et al. (16). The trypan blue cell staining procedure was column. The unbound molecules were removed from the column by an performed as described by Broman et al. (17). Plasminogen-dependent gela- extensive washing (20 column volumes) with 20 mM Tris-HCl (pH 7.6). The tinolytic zymogram was performed as described previously (18). SDS-PAGE bound proteins were eluted by a NaCl gradient (0–0.5 M)in20mM Tris-HCl of protein was performed as described by Laemmli (19). Nondenaturing (pH 7.6). The fractions eluted at 0.1 M NaCl contained the purified monomeric protein gel electrophoresis was performed as described by Lomas et al. (20). rMaspin(i). The purified rMaspin(i) was sterilized by filtration through a 0.2 Western blotting of maspin was performed as described previously (1). Silver ␮m membrane for subsequent biochemical and biological analyses. nitrate staining of protein electrophoresis gels and protein concentration anal- Coupled Colorimetric Assay for Plasminogen Activation in Condi- yses using the Bio-Rad reagents were performed according to the manufac- tioned Culture Medium. Aliquots of 80 ␮l of reaction buffer [50 mM turer’s instructions. Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.2 ␮g/ml leupeptin, 0.32 ␮M Glu-plasminogen, 0.2 mM Spectrozyme PL, and 0.1% Triton X-100] were added to the RESULTS wells of a 96-well microplate. Glu-plasminogen was omitted in the blank reactions. The enzyme reaction was initiated by the addition of 20 ␮lofthe Purification of Monomeric rMaspin(i). To study the molecular concentrated CM of DU145 cells. The photometric absorbance of the reaction mechanism of maspin, a large quantity of pure and active maspin mixtures at 405 nm was monitored at 37°C over the next 20 min using a protein is essential. The rMaspin(i) preparation obtained previously Bio-Rad BenchMark microplate reader. from a two-step chromatographic procedure (9) was further purified in Detection of Cell Surface-associated Plasminogen Activators by CAA. this study by a heparin affinity chromatographic procedure using the Cells were seeded at 10,000 cells/well in 96-well plates in the maintenance medium and allowed to reach approximately 90% confluence. The cells were automated BioLogic system. Silver-stained SDS-PAGE (Fig. 1A) washed once with PBS and analyzed in a PBS-based reaction buffer (100 showed that the repurified rMaspin(i) was approximately 100% ho- ␮ ␮ l/well) containing 0.8 mM MgCl2 and 0.2 g/ml leupeptin. The reactions were initiated by the addition of the chromogenic amidolytic substrate specific for either uPA (Spectrozyme UK) or tPA (Spectrozyme tPA) to a final concentration of 0.2 mM. No amidolytic substrate was added in the blank reactions. The photometric absorbance of the reaction mixtures at 405 nm was monitored at 37°C over the next 30 min. CCA-CSPA. Cells were prepared as described for the CAA. A total of 100 ␮l of the reaction mixture (PBS, 0.2 mM Spectrozyme PL, 0.32 ␮M Glu- ␮ plasminogen, 0.8 mM MgCl2, 0.2 g/ml leupeptin, and various protein factors at the indicated final concentrations) was added to the cells in each well. The reaction was initiated by the addition of Glu-plasminogen, which was omitted in the blank reactions. The photometric absorbance of the reaction mixtures at Fig. 1. Purification of intact monomeric rMaspin(i) by heparin affinity chromatography using the BioLogic System (Fast Protein Liquid Chromatography). A, silver-stained 405 nm was monitored at 37°C over the next 20 min. ␮ ␮ SDS-PAGE gel. Lane 1, molecular weight standards; Lane 2, 0.75 g of the starting Chemical Cross-linking Assay. One hundred g of the total lysate of rMaspin(i) preparation; Lane 3, 0.75 ␮g of rMaspin(i) eluted with 0.1 M NaCl. B, DU145 cells were incubated in the presence or absence of 5 ␮g of rMaspin(i) silver-stained nondenaturing protein electrophoresis gel. Lane 1, molecular weight stand- at 37°C for 30 min. In parallel, 1 ␮g of purified uPA protein was incubated ards (phosphorylase b, Mr 94,000; BSA, Mr 66,000; carbonic anhydrase, Mr 31,000; ␤ ␮ with 5 ␮g of rMaspin(i) (or BSA as a negative control) at 37°C for 30 min. The -lactoglobulin, Mr 21,000). Lanes 2 and 3 each contain 0.75 g of protein from two different aliquots of the same elution fraction shown in Lane 3 in A. The aliquot of above-mentioned reaction mixtures were incubated with 0.1% glutaraldehyde rMaspin(i) in Lane 2 had been stored at Ϫ70°C, whereas the aliquot in Lane 3 had on ice for 30 min and then quenched by glycine (pH 8.0) at a final concen- undergone a series of freezing and thawing over a 1-month period. 4772

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. MASPIN INHIBITS uPA ON CELL SURFACE mogenous (Lane 3), whereas the starting material (Lane 2) had im- purities of higher and lower molecular weights. The yield of this highly purified rMaspin(i) was estimated to be at least 10% (w/w) of the total extractable proteins of the host insect cells. To test whether the purified rMaspin(i) was in a uniform conformation, an aliquot of rMaspin(i) shown in Lane 3 of Fig. 1A was analyzed by nondenaturing protein electrophoresis followed by silver staining. As shown in Fig. 1B, only one protein band was detected (Lane 2). Furthermore, the purified rMaspin(i) remained homogene- ous after being frozen and thawed several times over the next month (Lane 3). The identity of rMaspin(i) was further confirmed by West- ern blotting using the polypeptide-derived polyclonal antibody against the RSL sequence of maspin, Abs4A (Ref. 1; data not shown). Cell Surface-associate uPA as a Target of rMaspin(i). To address the possibility that maspin may inhibit cell surface-associated uPA, it was critical to use a homogenous cell population that produces uPA as the predominant plasminogen activator. For this reason, human prostate carcinoma cell line DU145 was chosen for our study. Fig. 2A shows the zymographic profile of the plasminogen-dependent gelatinolytic activities of both the total cell lysate and the SF-CM of DU145 cells. As compared with the purified tPA and uPA standards, both the lysate and the SF-CM of DU145 cells contained primarily uPA, although a trace amount of tPA was also detected. To further identify the plasminogen activator(s) produced by DU145 cells, the SF-CM of DU145 cells was first tested by the coupled assay for plasminogen activation. As shown in Fig. 2B, the SF-CM of DU145 cells was active in converting Glu-plasminogen to plasmin, which in turn led to the cleavage of Spectrozyme PL, as shown by an increased photometric absorbance at 405 nm (12). The uPA-neutralizing antibody blocked this plasminogen activation activity in a dose-dependent manner. In contrast, the tPA-neutralizing antibody had no significant effect. CAA was also performed. When the chromogenic amidolytic substrate specific for uPA or tPA was added to the monolayer of DU145 cells, only the uPA substrate, Fig. 2. DU145 cells produce primarily uPA. A, gelatinolytic zymographic profiles of Spectrozyme UK, was cleaved (Fig. 2C). The uPA-neutralizing anti- plasminogen activators in the total cell lysate and CM of DU145 cells. A total of 20 ␮g body, but not a tPA-neutralizing antibody, blocked the cleavage of of the lysate protein or 20 ␮l of the concentrated CM was loaded on the zymogram gel containing both plasminogen and gelatin as the protease substrates. Purified single-chain Spectrozyme UK. In parallel experiments, the tPA-specific substrate, tPA (left lane; 0.4 NIH unit) and high molecular weight uPA (second lane from the left; Spectrozyme tPA, was not cleaved. 0.3 NIH unit) were used as standards. B, plasminogen activation mediated by the CM of There appeared to be a specific interaction between rMaspin(i) and DU145 cells was analyzed by the coupled colorimetric assay in the presence of the uPA-neutralizing antibody (f) or the tPA-neutralizing antibody (F) at the indicated the DU145 cell surface. DU145 cells do not produce endogenous concentrations. The data represent an average result of quadruplicate repeats, and the maspin (Refs. 5 and 6; Fig. 3A). However, after the cells were treated error bars stand for the SDs. C, detection of the amidolytic activity of the plasminogen with rMaspin(i) and washed thoroughly, cell-bound rMaspin(i) was activators on the DU145 cell surface using Spectrozyme UK (●), Spectrozyme UK plus 2.5 ␮g/ml uPA-neutralizing monoclonal antibody (f), Spectrozyme tPA (), and Spec- detected by immunofluorescent staining under a nonpermeablizing trozyme tPA plus 2.5 ␮g/ml tPA-neutralizing monoclonal antibody (Œ). Changes of condition (Fig. 3A). In addition, when added to the SF culture me- photometric absorbance at 405 nm for each reaction were normalized by the number of remaining cells quantified by SRB staining (A ). The data represent an average of Ͻ M 550 nm dium, rMaspin(i) at low concentrations ( 100 n ) bound to DU145 quadruplicate repeats. The SEs are not shown for clarity. cells in a dose-dependent manner (Fig. 3B). As the concentration of rMaspin(i) exceeded 100 nM, the amount of cell surface-bound rMas- pin(i) was not further increased, suggesting that the binding of rMas- activator on the surface of DU145 cells, cells were preincubated with pin(i) had reached a level of saturation. specific neutralizing antibody of uPA or tPA and analyzed by CCA- To examine the effect of rMaspin(i) on DU145 cell surface-medi- CSPA. As shown in Fig. 4B, the uPA-neutralizing antibody inhibited ated plasminogen activation, the CCA-CSPA was used. As shown in DU145 cell-mediated plasminogen activation to a similar extent as Fig. 4A, rMaspin(i) at a final concentration of 90 nM exhibited a rMaspin(i). Cells preincubated with the uPA-neutralizing antibody significant inhibition of DU145 cell surface-mediated plasminogen were not further inhibited by rMaspin(i) in this assay. In contrast, the activation. In addition, purified Abs4A blocked the inhibitory activity tPA-neutralizing antibody neither affected the basal level cell-medi- of rMaspin(i) in a dose-dependent manner, whereas the preimmune ated plasminogen activation nor altered the proteolytic inhibitory rabbit IgG had no significant effect. It is worth noting that throughout effect of rMaspin(i). These data further support a specific inhibitory this biochemical assay, cells remained intact, showing little or no activity of rMaspin(i) toward the cell surface-associated uPA. plasma membrane permeability by trypan blue and a full recovery of rMaspin(i) did not seem to act as a protease substrate. Fig. 4C proliferation in the subsequent cell growth assay (data not shown). shows the Western blotting of rMaspin(i) before (Lane 2) and after The number of remaining cells in each reaction, as quantified by the (Lane 3) the CCA-CSPA, respectively. rMaspin(i) was not degraded SRB staining procedure (16), was used to normalize the initial veloc- during CCA-CSPA. Under our assay conditions, the cleavage of the ities of plasminogen activation. plasmin substrate Spectrozyme PL was dependent on the exogenous To further test whether rMaspin(i) inhibits a specific plasminogen supply of either Glu-plasminogen (data not shown) or plasmin. The 4773

tively (Lane 3). This polymerization pattern resembles that seen with

the trypsin-cleaved maspin, a Mr 38,000 NH2-terminal fragment of maspin (13). These data suggest that rMaspin(i) did not form a complex with soluble uPA but rather was significantly degraded. When rMaspin(i) was incubated with DU145 lysate, however, a

new protein band of approximately Mr 100,000 was detected by Western blotting of maspin (Fig. 6, Lane 5). When the same membrane containing Lanes 4 and 5 was reblotted with the uPA antibody,

a uPA-containing protein of approximately Mr 100,000 was detected in the mixture of DU145 cell lysate and rMaspin(i) (Fig. 6, Lane 7).

In the absence of rMaspin(i), only the monomeric uPA of Mr 55,000 was detected in DU145 cell lysate (Fig. 6, Lane 6). Given that the combined molecular weight of rMaspin(i) and uPA is 97,000, it is

likely that the new band of approximately Mr 100,000 detected by both Abs3A and the monoclonal antibody against uPA was the complex between rMaspin(i) and uPA in DU145 cells. The Biologically Active rMaspin(i) Acts as a Competitive In- hibitor of the Cell Surface-mediated Plasminogen Activation. Ki- netic analyses were performed to further investigate the mode of

Fig. 3. rMaspin(i) binds specifically to the DU145 cell surface. A, immunofluorescent staining of maspin (red) on the DU145 cell surface. The blue centroids indicate cell nuclei (ϫ630). B, ELISA detection of rMaspin(i) bound to DU145 cells after the cells were preincubated with purified rMaspin(i) at the concentrations indicated on the horizontal axis. The data represent an average of triplicate repeats. Error bars, SDs.

increased cleavage of Spectrozyme PL by exogenously added pure plasmin, however, was not affected by rMaspin(i) (Fig. 4B). Thus, it seemed unlikely that rMaspin(i) directly inhibited plasmin. rMaspin(i) Exerts Differential Effects on Cell Surface-associated and Free Plasminogen Activators. Because DU145 cells secreted both tPA and uPA into the culture media (Fig. 2A), it was important to investigate whether rMaspin(i) inhibited the unbound plasminogen activators. As shown in Fig. 5A, rMaspin(i) added at a final concentration of 90 nM did not inhibit the basal level activity of plasminogen activation in the SF-CM of DU145. The exogenously added tPA and uPA both significantly stimulated the plasminogen activation in the CM. However, in each case, rMaspin(i) failed to inhibit the accelerated plasminogen activation. Interestingly, the preincubation of DU145 cells with purified uPA did not lead to an elevated plasminogen activation on the cell surface, nor did this treatment change the inhibitory activity of rMaspin(i) (Fig. 5B). It is possible that the surface of DU145 cells was saturated with the endogenous uPA and thus could not accommodate additional plasminogen activators added exogenously. It has been reported that, under an acidic condition, the cell surface-associated uPA may be stripped and subsequently replaced by purified uPA (21). However, treatment of DU145 cells by this method caused significant damage to Fig. 4. rMaspin(i) inhibits DU145 cell surface-mediated uPA. A, the RSL sequence of the cell membrane, as indicated by an increased trypan blue perme- rMaspin(i) is critical for its proteolytic inhibitory activity. The CCA-CSPA was performed ability (data not shown), thus preventing an accurate assessment of the in the presence of no additional factor (F), 90 nM rMaspin(i) (f), 90 nM rMaspin(i) plus binding as well as the activity of uPA on DU145 cell surface. 5 ␮g/ml Abs4A (Œ), 90 nM rMaspin(i) plus 10 ␮g/ml Abs4A (), or 90 nM rMaspin(i) plus 10 ␮g/ml preimmune rabbit IgG (ࡗ). B, rMaspin(i) specifically inhibits uPA on the To investigate whether rMaspin(i) interacts directly with uPA, DU145 cell surface. CCA-CSPA was performed in the presence of Glu-plasminogen (0.32 chemical cross-linking experiments were performed. As shown in Fig. ␮M), plasmin (0.1 nM), rMaspin(i) (90 nM), uPA-neutralizing antibody (2.5 ␮g/ml), and ␮ 6, the polymerization of rMaspin(i) in cell-free solution resulted in a tPA-neutralizing antibody (2.5 g/ml) in the indicated combinations. C, Western blotting of maspin. Lane 1 shows 0.5 ␮g of purified rMaspin(i) control with a molecular weight Ͼ ␮ f high molecular weight rMaspin(i) oligomer (Mr 125,000; Fig. 6, of Mr 42,000. Lanes 2 and 3 are 35 l of postassay supernatants from reaction ( )inA Lane 1). When rMaspin (i) was incubated with purified uPA, three and its corresponding blank (without Glu-plasminogen), respectively. In A and B, change major polymer forms of rMaspin(i) were detected with molecular of photometric absorbance at 405 nm for each reaction was normalized by the number of remaining cells (A550 nm). The data represent an average of quadruplicate repeats. The SEs weights of approximately 80,000, 120,000, and Ͼ125,000, respec- are not shown for clarity in A, whereas the error bars in B represent the SDs. 4774

bation with Abs4A, but not with the preimmune rabbit IgG, abolished the inhibitory activity of rMaspin(i). Thus, the RSL of rMaspin(i) was critical for both its proteolytic inhibitory activity (Fig. 4A) and its biological activity in inhibiting tumor cell motility.

DISCUSSION We show here that purified rMaspin(i) acts as a competitive inhib-

itor of DU145 cell surface-associated uPA, having an apparent Ki value of 20 nM. The proteolytic inhibitory activity of rMaspin(i) correlates with its inhibition of the motility of DU145 cells in vitro. These data support the hypothesis that maspin blocks cell motility and invasion, at least in part, by inhibiting the tumor cell-mediated plasminogen activation. The biological activity of rMaspin(i) in inhibiting cell motility and invasion has been initially localized on the cell membrane (5). Data from the current study demonstrated an important role for the epithelial cell surface in regulating the inhibitory interaction between rMas- pin(i) and the localized plasminogen activation system. First, rMas- Fig. 5. The differential effects of rMaspin(i) toward cell surface-associated and free plasminogen activators. DU145 cells cultured in a 96-well microplate were incubated for pin(i) bound to intact DU145 cells in a saturable fashion (Fig. 3), 30 min at 37°C with 50 ␮l of SF medium (1 and 2), or SF medium plus 8 NIH units/ml suggesting that it may interact with a specific cell surface-associated tPA (3 and 4), or SF medium plus 8 NIH units/ml uPA (5 and 6). The resulting CM were molecule. Second, rMaspin(i) exhibited a strong inhibitory effect on collected and analyzed by the coupled plasminogen activation assay in the presence (2, 4, and 6) or absence (1, 3, and 5) of rMaspin(i) at a final concentration of 90 nM (shown in DU145 cell surface-mediated plasminogen activation, comparable A). In parallel, the remaining cells were analyzed by CCA-CSPA in the presence (2, 4, and with that of PAI-1 and PAI-2 in parallel experiments (Fig. 7). In 6) or absence (1, 3, and 5) of rMaspin(i) at a final concentration of 90 nM (shown in B). The changes of photometric absorbance at 405 nm for each reaction were normalized by contrast, rMaspin(i) did not inhibit free uPA (12–14) in solution, nor the number of remaining cells (A550 nm). In both A and B, the data represent an average did it inhibit the plasminogen activators secreted by DU145 cells to of quadruplicate repeats, and the error bars represent the SDs. the CM (Fig. 5). Moreover, we showed that rMaspin(i) forms a complex with the DU145 cell-associated uPA but acted as a substrate of purified uPA in solution (Fig. 6). molecular interaction between rMaspin(i) and the DU145 cell surface- The DU145 cell surface may conceivably enhance the inhibitory associated uPA. The initial velocities of plasminogen activation are interaction between rMaspin(i) and its target plasminogen activators presented in the Lineweaver-Burk plots as shown in Fig. 7A.Inthe via two mechanisms that are not mutually exclusive. On one hand, presence of subsaturating concentrations of plasminogen (0–30 nM), specific interactions between uPA and its associated proteins on the the rate of the plasminogen activation mediated by DU145 cell surface cell surface may render uPA prone to inhibition by rMaspin(i). It is was dependent on the concentration of Glu-plasminogen, [s]. The proposed that the activation of uPA on the cell surface is mediated by slope of the Lineweaver-Burk plot was initially increased as the a specific receptor, uPAR, which not only controls the localized concentration of rMaspin(i) increased from 0 to 90 nM, demonstrating plasminogen activation (22–24) but also regulates cell-matrix inter- a dose-dependent inhibition by rMaspin(i). Furthermore, these plots intersect close to the 1/v axis, suggesting a constant maximum velocity (Vmax). Thus, rMaspin(i) appeared to inhibit the cell surface- mediated plasminogen activation as a competitive inhibitor. As shown in Fig. 7B, the replot of the slopes derived from Fig. 7A versus the concentrations of rMaspin(i) was linear. Based on Fig. 7B, an apparent

Ki value of approximately 20 nM was deduced. Interestingly, rMas- pin(i) became less inhibitory as its concentration was further increased to 240 nM (Fig. 7A). In parallel, both recombinant PAI-1 and PAI-2 exhibited competitive inhibition, to slightly different extents, on DU145 cell-mediated plasminogen activation. The Lineweaver-Burk plots with PAI-1 and PAI-2 were obtained (data not shown). The slopes of these plots were replotted against the concentration of the corresponding inhibitor as shown in Fig. 7C. The apparent Ki values for PAI-1 and PAI-2 deduced from these replots were approximately 10 and 25 nM, respectively. We have shown previously that rMaspin(i) inhibits the invasion and motility of three prostatic carcinoma cell lines including DU145 (5). To test whether repurified monomeric rMaspin(i) retained biological activity, we performed the MICS motility assay as described previously (5). As shown in Fig. 8, the purified monomeric rMaspin(i) exhibited a biphasic dose-dependent inhibition on the motility of Fig. 6. The molecular interaction between rMaspin(i) and uPA. Mixtures of rMaspin(i), DU145 cells with a nadir at 125 nM. This biphasic dose-response purified single-chain uPA, DU145 cell lysate, and BSA in the indicated combinations were cross-linked using a glutaraldehyde procedure and immunoblotted with Abs3A curve coincides with the biphasic effects of rMaspin(i) on DU145 against maspin (Lanes 1–5) or with the monoclonal antibody against uPA (Lanes 6 and 7; cell-mediated plasminogen activation (Fig. 7A). In addition, preincu- see “Materials and Methods”). 4775

Fig. 7. rMaspin(i) as a competitive inhibitor of the plasminogen activation mediated by DU145 cell surface. A, the Lineweaver-Burk plots (1/v versus 1/s) for DU145 cell surface-mediated plasminogen activation in the presence of rMaspin(i) at 0(F), 30 (Œ), 60 (ࡗ), 90 (), and 240 nM (f), 2 respectively. v in the unit of A405 nm/A550 nm/min is the initial velocity normalized by the number of remaining cells (A550 nm). [s] is the concentration of Glu-plasminogen. Dashed lines represent the linear regressions of the plots by the least square method using Sigma Plot 2.0 software. The data represent an average of quadruplicate repeats, and SE bars are not shown for clarity. B, replot of the slopes of the Lineweaver-Burk plots in Fig. 6A versus the concentration of rMaspin(i). The intercept on the horizontal axis indicates the apparent Ki value as 20 nM. C, replot of the slopes of the Lineweaver-Burk plots versus the concentration of PAI-1 (Œ) and PAI-2 (), respectively. The intercepts on the horizontal axis indicate an apparent Ki value of 10 nM for PAI-1 and 25 nM for PAI-2.

action through its close association with integrins, the transmembrane required for its inhibitory effect on the motility of DU145 cells (Fig. receptors of extracellular matrix proteins (25). The elegant study of 8) and further supports the role of maspin as an inhibitory serpin.

Schwartz et al. (26) demonstrates that uPAR-bound uPA and free uPA Furthermore, given the earlier in vitro evidence that the Mr 38,000 are inhibited by serpin PAI-3 via different mechanisms. NH2-terminal fragment of rMaspin(i) specifically interacts with the On the other hand, the cell surface microenvironment may play a regulatory domain of tPA (12), it is likely that the functional deter- critical role in mediating the transition of rMaspin(i) from a latent minants of maspin are not restricted to its RSL. It is worth noting that conformation to an active conformation. Based on a current paradigm, PAI-1 uses its NH2-terminal domain to interact with heparin and the initial docking of the RSL of an inhibitory serpin into the catalytic vitronectin (31–33). The latter is an extracellular matrix component site of the target enzyme induces a massive ␤ sheet rearrangement of and regulates cellular functions via its integrin receptor and uPAR the serpin molecule, leading to a stabilized enzyme/inhibitor complex (25). An X-ray crystallographic study of cleaved PAI-1 revealed two (27, 28). Accordingly, based on protein sequence alignments, serpins additional potential regulatory sites on ␤ strand 3A and 5A, respec- such as chick ovalbumin and maspin, whose RSL may not undergo tively, that may render PAI-1 inactive on additional intermolecular such conformational rearrangement without substantial energy com- interaction (34). Future study is needed to address what other func- pensation, were predicted to be noninhibitory (29). However, a study tional domains of rMaspin(i) are involved in its specific interaction by Mellet et al. (30) showed that chick ovalbumin could be converted with the cell surface-bound plasminogen activators. In addition, be- to a potent competitive serine protease inhibitor by heat denaturation. cause rMaspin(i) stimulates the adhesion of mammary carcinoma In fact, the activity of many inhibitory serpins such as PAI-1 can be MDA-MB-435 cells to fibronectin and alters the integrin profiles on greatly enhanced by serpin cofactors (31, 32). Central to the molecular the cell surface (10), it is of particular importance to investigate mechanism of maspin, we show here that the RSL of rMaspin(i) was whether fibronectin binds directly to maspin and regulates its proteolytic inhibitory activity. Regarding the biphasic dose effect of rMaspin(i) on DU145 cell surface-mediated plasminogen activation (Fig. 7A) and on cell motility (Fig. 8), our chemical cross-linking evidence (Fig. 6) supports an earlier notion that purified recombinant maspin may undergo a concentration-dependent polymerization (35). Although the intact monomeric rMaspin(i) used in this study did not undergo fast spontaneous polymerization and was not degraded during storage (Fig. 1), at higher local concentrations, rMaspin(i) may polymerize and lose its proteolytic inhibitory potency. Meanwhile, current evidence does not ex- clude the possibility that the reduced biochemical and biological activities of rMaspin(i) at higher concentrations are caused by an increased nonspecific interaction between rMaspin(i) and the plasminogen activation system because rMaspin(i) has been shown to use its NH -terminal domain to interact with the regulatory domain of tPA Fig. 8. The dose-dependent effect of the intact monomer form of rMaspin(i) on the 2 motility of DU145 cells. The motility data with untreated DU145 cells (zz) were normal- (12). Furthermore, because the specific receptors and other molecules ized as 100%, which represent approximately 12% of the starting cell population. The associated with the plasminogen activation system on the cell surface motility data from cells treated with rMaspin(i) (Ⅺ), rMaspin(i) plus Abs4A (f), and rMaspin(i) plus the preimmune rabbit IgG (oo) are expressed as a percentage of this are of fundamental importance in cell biology (22–26, 31–33, 36–39), control. The data represent the average of three parallel experiments. Error bars, SEs. an inhibitor of plasminogen activators such as rMaspin(i), when added 4776

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. MASPIN INHIBITS uPA ON CELL SURFACE at high doses, may not only shift the balance against the enzymatic 7. Sager, R., Sheng, S., Pemberton, P., and Hendrix, M. J. C. Maspin, a tumor sup- activity of its target but may also trigger further compensating cellular pressing serpin. Adv. Exp. Med. Biol., 425: 77–88, 1997. 8. Jiang, W. G., Hiscox, S., Horrobin, D. F., Bryce, R. P., and Mansel, R. E. ␥-Linolenic responses. acid regulates expression of maspin and the motility of cancer cells. Biochem. It was advantageous to use DU145 cells to investigate the effect of Biophys. Res. Commun., 237: 639–644, 1997. 9. Sheng, S., Pemberton, P., and Sager, R. Production, purification and characterization maspin on cell-associated uPA because these cells do not produce of recombinant maspin proteins. J. Biol. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. The Surface of Prostate Carcinoma DU145 Cells Mediates the Inhibition of Urokinase-type Plasminogen Activator by Maspin

Richard McGowen, Hector Biliran, Jr., Ruth Sager, et al.

Cancer Res 2000;60:4771-4778.

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