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Tissue-Type Plasminogen Activator Is a Target of the Tumor Suppressor Gene Maspin

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Tissue-Type Plasminogen Activator Is a Target of the Tumor Suppressor Gene Maspin Proc. Natl. Acad. Sci. USA Vol. 95, pp. 499–504, January 1998 Biochemistry Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin SHIJIE SHENG*†,BINH TRUONG*, DEREK FREDRICKSON*, RUILIAN WU*, ARTHUR B. PARDEE‡, AND RUTH SAGER* *Division of Cancer Genetics and ‡Division of Cell Growth and Regulation, Dana–Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115 Contributed by Arthur B. Pardee, November 10, 1997§ ABSTRACT The maspin protein has tumor suppressor that PAI-1 also inhibits cell motility via an integrin-mediated activity in breast and prostate cancers. It inhibits cell motility pathway by its interaction with cell surface-bound uPA and and invasion in vitro and tumor growth and metastasis in nude vitronectin (8, 9). mice. Maspin is structurally a member of the serpin (serine The RSL of maspin is shorter than that of other serpins (1), protease inhibitors) superfamily but deviates somewhat from casting doubt on its ability to act as a classical inhibitory serpin. classical serpins. We find that single-chain tissue plasmino- Pemberton et al. (10) reported that purified rMaspin(i) is a gen activator (sctPA) specifically interacts with the maspin substrate instead of an inhibitor to an array of serine proteases, reactive site loop peptide and forms a stable complex with including uPA and tissue-type plasminogen activator (tPA). recombinant maspin [rMaspin(i)]. Major effects of rMas- However, our evidence that the RSL is crucial for the biolog- pin(i) are observed on plasminogen activation by sctPA. First, ical activity of maspin would be consistent with maspin acting rMaspin(i) activates free sctPA. Second, it inhibits sctPA as an inhibitory serpin. preactivated by poly-D-lysine. Third, rMaspin(i) exerts a Here we provide evidence that biologically active rMaspin(i) biphasic effect on the activity of sctPA preactivated by fibrin- specifically binds to purified single-chain tissue-type plasmin- y ogen gelatin, acting as a competitive inhibitor at low concen- ogen activator (sctPA) and has biphasic effects on sctPA, m trations (<0.5 M) and as a stimulator at higher concentra- activated to convert plasminogen to plasmin. rMaspin(i) had tions. Fourth, 38-kDa C-terminal truncated rMaspin(i) fur- y no effect on several other serine proteases, including uPA, ther stimulates fibrinogen gelatin-associated sctPA. under our assay conditions. Our model based on kinetic rMaspin(i) acts specifically; it does not inhibit urokinase-type analyses provides insight into the dual interactions between plasminogen activator, plasmin, chymotrypsin, trypsin, or rMaspin(i) and sctPA. elastase. Our kinetic data are quantitatively consistent with a model in which two segregated domains of maspin interact with the catalytic and activating domains of sctPA. These MATERIALS AND METHODS complex interactions between maspin and sctPA in vitro Cell Culture and Materials. Human mammary carcinoma suggest a mechanism by which maspin regulates plasminogen MDA-MB-435 cells (American Type Culture Collection) were activation by sctPA bound to the epithelial cell surface. cultured in aMEM (GIBCOyBRL) supplemented with 5% fetal calf serum (HyClone) (11). When the cells were approx- Since the isolation of the human maspin gene from mammary imately 70% confluent, the medium was switched to DFCI-1 epithelial cells (1), accumulating evidence supports its function medium (12). Following another 24 hr of continued cell as a tumor suppressor that inhibits motility, invasion, and culture, the conditioned medium was collected. The SulfoLink metastasis (2–4). To explore the diagnostic and therapeutic coupling gel was purchased from Pierce. Maspin RSL peptide potential of maspin, we have initiated investigations into the NH2-CIEVPGARILQHKDEL-COOH was synthesized and molecular mechanisms underlying its biological activities. was HPLC-purified at the Molecular Biology Core Facility of Identification of the maspin target was the first fundamental the Dana–Farber Cancer Institute. rMaspin(i) was produced objective. and purified as described (2). The 38-kDa N-terminal fragment Maspin is structurally a novel serine protease inhibitor of maspin resulting from limiting digestion by trypsin was a gift (serpin) that shares sequence homology with other inhibitory from LXR BioTechnology, Inc. (Richmond, CA) (10). sctPA, serpins, including plasminogen activator inhibitor type-1 and glutamate-type plasminogen (Glu-plasminogen), high molec- -2 (PAI-1 and PAI-2), as well as noninhibitory proteins like ular weight urokinase, chromogenic plasmin substrate Spec- ovalbumin (1). Functional studies demonstrate that recombi- trozyme PL, chromogenic urokinase substrate Spectrozyme nant maspin produced by baculovirus-infected insect cells [rMaspin(i)] acts at the cell membrane (3) and that its activity UK, inhibitory monoclonal antibody against sctPA, and de- in inhibiting cell migration and invasion across a Matrigel fined DESAFIB-X des-AA-fibrinogen were purchased from matrix depends on an intact reactive site loop (RSL) (1–4). American Diagnostica (Greenwich, CT). Unless otherwise Based on this information, one may predict that maspin specified, all other chemicals and reagents were purchased inhibits a cell surface serine protease, which has dual activities from Sigma. in promoting cell motility and invasion. An example of such a protease is urokinase-type plasminogen activator (uPA) in the Abbreviations: PAI-1 and PAI-2, plasminogen activator inhibitor uPA receptoryuPA system (5). PAI-1 and PAI-2, inhibitors of type-1 and -2; RSL, reactive site loop; uPA, urokinase-type plasmin- ogen activator; tPA, tissue-type plasminogen activator; rMaspin(i), uPA, decrease cell invasion by inhibiting the proteolytic deg- recombinant maspin produced by baculovirus-infected insect cells; radation of extracellular matrix (6, 7). Recent studies suggest sctPA, single-chain tissue-type plasminogen activator; Glu- plasminogen, glutamate-type plasminogen. † The publication costs of this article were defrayed in part by page charge To whom reprint requests should be addressed at: Department of Pathology, Scott Hall, Room 9207, Wayne State University School of payment. This article must therefore be hereby marked ‘‘advertisement’’ in Medicine, 540 East Canfield Avenue, Detroit, MI 48201. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. © 1998 by The National Academy of Sciences 0027-8424y98y95499-6$2.00y0 §Contribution of this paper was initiated by Ruth Sager and, after her PNAS is available online at http:yywww.pnas.org. death (March 1997), completed by Arthur B. Pardee. 499 Downloaded by guest on October 2, 2021 500 Biochemistry: Sheng et al. Proc. Natl. Acad. Sci. USA 95 (1998) Purification of Maspin-Binding Proteins with an Affinity centrations of rMaspin(i) in 50 mM TriszHCl, pH 7.5y0.1% Column. The RSL affinity column was made according to Triton X-100 was added to the coated wells to start the instructions by the manufacturer. Briefly, 5 mg of the maspin reaction. Initial velocities for the production of plasmin were RSL peptide in PBS at pH 7.4 was mixed with 1 ml of SulfoLink calculated based on the changes in A405 by using Eq. 1. gel suspension. The gel mixture was incubated at room tem- sctPA Activity Assay in the Presence of FibrinogenyGelatin. perature for 1 hr and packed into a column (1 cm i.d.). The Spectrozyme PL at a final concentration of 0.2 mM was added column was washed thoroughly with PBS before loading 50 ml to a preincubation mixture of 0.4 NIH unit of sctPA, fibrinogen of conditioned DFCI-1 medium from the MDA-MB-435 cell (0.4 mM), and gelatin (133 mgyml) in 50 mM TriszHCl, pH culture. The nonspecific binding proteins were removed by 7.5y0.1% Triton X-100 (1 hr at 37°C). No sctPA was added in thorough washing with PBS until no protein was detected in the blank. Glu-plasminogen at a final concentration of 83 nM the washing (13). The protein specifically bound to the column and rMaspin(i) or trypsin-cleaved rMaspin(i) when indicated was eluted with 0.1 M glycine, pH 2.5, into 0.5-ml fractions that were added to start the assay. The final volume of each reaction were simultaneously neutralized with 0.1 vol of 1.0 M TriszHCl, was 150 ml. The absorbance of the reaction mixtures at 405 nm pH 9.5. was monitored at 37°C over the next 2-hr period. To determine Zymography. Zymogram gels with incorporated gelatin the dissociation constant of the sctPAyrMaspin(i) intermedi- substrate were prepared as described by Heussen and Dowdle ate, the concentrations of plasminogen and rMaspin(i) were (14). Plasminogen at a final concentration of 10 mgyml was varied under the optimized sctPA assay conditions. incorporated into the separating gel for the detection of Activity Assays of Other Serine Proteases. The amidolytic plasminogen activator activity. Protein samples were loaded at activities of uPA, sctPA, plasmin, elastase, chymotrypsin, and indicated amounts and subjected to electrophoretic separation trypsin were assayed by using specific synthetic chromogenic at 10 mA at 4°C. The gel was then washed twice with 2% Triton substrates Spectrozyme UK (0.2 mM in 50 mM TriszHCl, pH X-100 in double distilled H2O for 1 hr. The gel was subse- 8.0), Spectrozyme PL (0.2 mM in 50 mM TriszHCly0.1% 1 quently incubated at 37°C in 0.1 M glycine, pH 8.3, for 2 ⁄2 hr Triton X-100, pH 7.5), N-methoxysuccinyl-Ala-Ala-Pro-Val at 37°C, stained with 0.25% Coomassie brilliant blue R-250, p-nitroanilide (0.2 mM in 0.1 M Hepesy0.5 M NaCl, pH 7.5), y y and destained with a solution of methanol acetic acid water N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (0.2 mM in 0.2 M y (1:1:8, vol vol). phosphate buffer, pH 6.2), and N-succinyl-Ala-Ala-Pro-Phe Formation of sctPA–Maspin Complex.
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