Department of Oncology, Lombardi Cancer Center, Georgetown University Medical Center, Washington, District of Columbia 20057
Submitted 18 September 2003 ; accepted in final form 3 December 2003
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ABSTRACT |
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serine protease; phospholipid; actin
In addition to normal tissues, matriptase is also expressed in vivo by human carcinomas of various sites, including those of the breast, ovary, colon, endometrium, stomach, and prostate (20, 21, 25, 28). Because matriptase is able to activate urokinase-type plasminogen activator (uPA) and hepatocyte growth factor (HGF)/scatter factor in vitro, it has been proposed to play an important role in cancer invasion and metastasis, which require the degradation of extracellular matrix and enhanced cellular motility (14, 26). In addition, tight correlation between the expression of matriptase and its HGF substrate as well as the c-Met receptor was observed in a cohort of 330 node-negative breast carcinomas (9). More importantly, high-level expression of the c-Met receptor, matriptase, and matriptase cognate inhibitor HGF activator inhibitor 1 (HAI-1) (15) were associated with poor patient outcome in the same set of node-negative breast carcinomas (9).
Matriptase also may be deregulated at its activation in human breast cancer cells (3). Activation of matriptase, which requires cleavage at its activation motif to convert a single-chain zymogen to the two-chain active protease, is likely to be carried out by a transactivation mechanism (23), an alternative mechanism whereby latent matriptase zymogen molecules interact with each other, leading to the activational cleavage. Transactivation of serine proteases generally occurs for the serine proteases at the pinnacle of protease cascades to first generate active protease. The noncatalytic domains of matriptase, particularly its LDLR class A domains, and post-translational modifications, such as amino-terminal processing by cleavage at Gly149 within the SEA domain and glycosylation at the first CUB domain and at the serine protease domain, are required for matriptase activation (23). These structural requirements for matriptase activation could reflect the structural basis for protein-protein interactions among matriptase zymogens and other proteins required for transactivation of the protease (23). Once activated, HAI-1 acts to terminate active matriptase to avoid undesired proteolysis of matriptase. Subsequently, matriptase-HAI-1 complexes are shed from cell surfaces into the extracellular milieu (2). Although the details of how transactivation occurs remain unknown, deregulation of matriptase transactivation has been observed in breast cancer cells (3). In immortalized mammary epithelial cells, matriptase activation, its subsequent inhibition by HAI-1, and its clearance by shedding are all dependent on the presence of a blood-borne lipid mediator, sphingosine 1-phosphate (S1P) (2, 4). The S1P-dependent activation of matriptase is, however, lost in breast cancer cells (3).
S1P is a major lipid mediator in serum, and it possesses growth factor-like activities. S1P induces a wide spectrum of biological responses in a variety of cell types, including proliferation, survival, actin cytoskeletal rearrangement, cell shape changes, contraction, and cellular motility. These cellular responses are believed to involve family members of endothelial differentiation gene (EDG) receptors, a subfamily of G protein-coupled, heptahelical membrane receptors and their intracellular downstream effectors, including adenylate cyclase, Rho family GTPases, phospholipase C, protein kinase C, MAP kinase, and phosphatidylinositol 3-kinase (11, 13, 24, 29, 31). In our previous study (4), matriptase was identified as a unique extracellular effector of S1P. We also observed that S1P induces prominent actin cytoskeletal rearrangement and formation of subcortical actin belts, which are likely to be associated with assembly of adherens junctions, which are known to require extracellular Ca2+ and to be important for stable cell-cell adhesion. Because direct interaction of S1P with matriptase has been excluded in an extracellular Ca2+-dependent activation of matriptase (4), we now propose that matriptase activation could be an indirect, downstream consequence of S1P-induced cellular responses. In this study, we investigated the functional relationship between S1P-induced actin cytoskeletal rearrangement and matriptase activation. Our results suggest that in immortalized mammary epithelial cells, S1P-induced matriptase accumulation at cell-cell contacts depends on S1P-induced adherens junction assembly and subcortical actin belt formation, a mechanism that may ensure that matriptase functions only at cell-cell contacts.
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MATERIALS AND METHODS |
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Cell lines and culture conditions. Immortalized 184 A1N4 human mammary epithelial cells (provided by Martha Stampfer, Lawrence Berkeley National Laboratory, Berkeley, CA) were maintained as previously described (2). BT549 human breast cancer cells were maintained in culture by growth in Iscove's minimal essential medium (IMEM; Invitrogen, Rockville, MD) supplemented with 5% FBS in a humidified chamber at 37° C and 5% CO2.
Monoclonal antibodies. Matriptase protein was detected with the M32 monoclonal antibody (MAb) that recognizes both the latent (1 chain) and activated (2 chain) forms of the protease or with the M69 monoclonal antibody that recognizes an epitope present only in the activated (2 chain) form of the enzyme (2, 4). MAb M32 recognizes a 70-kDa processed form and the 120-kDa matriptase-HAI-1 complex; M32 also recognizes the 95-kDa full-length matriptase, which was barely detectable in those cells endogenously expressing the protease but becomes prominent in those cells that were used for overexpression of matriptase (23). We have also found that the epitope against which MAb M32 is directed is destroyed by a point mutation in LDLR class A domain III (see Fig. 9). Therefore, MAb M84, which is directed against the serine protease domain of matriptase, was used in Fig. 9 to show the expression of matriptase mutants. Anti-E-cadherin MAb (clone 36) and FITC-labeled anti--catenin MAb (clone 14) were purchased from BD Bioscience.
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Labeling MAbs with fluorescent dyes. To label MAbs with fluorescent dyes, 1 mg each of MAbs M32 and M69 were solubilized in 0.1 M sodium bicarbonate and then labeled with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Eugene, OR), according to the manufacturer's instructions. The unbound Alexa Fluor dyes were removed by dialysis against PBS. The ratios of mole of dye per mole of IgG were determined to be 2.53.5 for Alexa Fluor 488 and 56 for Alexa Fluor 594.
Constructs and transfections. The cDNA clones for the HAI-1, wild-type matriptase, or matriptase mutants, bearing point mutations at the calcium cages of LDLR class A domains (23), in the vector pcDNA3.1 (Invitrogen, Carlsbad, CA) were used in transient transfections. Transient transfection of matriptase or HAI-1 constructs was accomplished by using Fugene 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol. When cotransfections were conducted, the amount of DNA used with the transfection reagent was kept constant for each individual transfection by including empty vector pcDNA3.1 DNA where appropriate.
Western blotting. Protein for Western blotting was prepared by the lysis of cells in 1% Triton X-100 in PBS or RIPA buffer (0.1% Nonidet-P40, 0.5% sodium deoxycholate, 0.1% SDS in PBS), after washing cells two times in PBS. Insoluble debris was removed by centrifugation, and the protein concentration was measured with bicinchoninic acid protein assay reagents (Pierce, Rockford, IL) according to the manufacturer's protocol. Lysates were resolved by SDS-PAGE under nonboiled and nonreduced conditions and then transferred to Protran nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The membranes were probed with MAbs. The binding of the primary antibody was followed by recognition with a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), and detected with the Western Lightening Chemiluminescence Reagent Plus (Perkin Elmer, Boston, MA). The MAbs recognizing matriptase were generated against conformation-dependent epitopes, and therefore samples were run under nonreducing SDS-PAGE conditions and were not boiled before electro-phoresis to preserve the formation of complexes between active matriptase and HAI-1.
Immunofluorescence microscopy. Cells plated onto microcover glasses were fixed in 3.7% formaldehyde in PBS for 20 min at room temperature. Cells were permeabilized by 0.05% Triton X-100 in PBS. Matriptase and activated matriptase were detected with Alexa Fluor dye-labeled M32 or M69. -Catenin was detected by FITC-labeled anti-
-catenin MAb (clone 14; BD Bioscience, San Jose, CA). E-cadherin was detected by anti-E-cadherin MAb (clone 36; BD Bioscience). For the double labeling of matriptase and E-cadherin, E-cadherin MAb was used, followed by FITC-labeled anti-mouse IgG and then Alexa Fluor 594-conjugated MAb M32 in PBS containing 250 µg/ml mouse IgG and 3% BSA. Actin was visualized with Texas red-conjugated phalloidin (Molecular Probes), and nuclei were visualized by DAPI staining. Cover glasses were mounted with Prolong Antifade (Molecular Probes) and observed on a Nikon Eclipse E600 digital fluorescence microscope, and images were captured with the Metavue software package for the Nikon digital microscope.
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RESULTS |
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Kinetics of matriptase activation at cell-cell contacts. We further examined the appearance and the localization of activated matriptase relative to total matriptase (Fig. 2) and to F-actin (Fig. 3) in 184 A1N4 cells at different time intervals after exposure to S1P. After growth in 0.5% serum for 2 days, 184 A1N4 cells do not express activated matriptase (24). Immunofluorescence using MAb M69 that recognizes two-chain matriptase showed only nonspecific staining, with weak, diffuse staining in the cytosol (Fig. 2A), as seen in the control with FITC-labeled secondary antibody alone (data not shown). Ten minutes after exposure to S1P, activated matriptase was detected mainly as tiny spots (Fig. 2D) and the total matriptase accumulated at cell-cell contacts (Fig. 2E). When both images were merged, activated matriptase was clearly colocalized with total matriptase at cell-cell contacts. At 20 and 30 min, activated matriptase was detected more brightly and in elongated patterns (Fig. 3, A and D). When the localization of activated matriptase was compared with the newly formed F-actin at the cell peripheries, activated matriptase closely coincided with F-actin at cell-cell contacts (Fig. 3, C and F). We also noted that subcortical actin belts were not homogeneously formed around the periphery of cells. More F-actin accumulated at the edges of cell-cell contacts (Fig. 3, B and E). Interestingly, activated matriptase was detected right at those areas where F-actin accumulated the most (Fig. 3, C and F). At 30 min, the staining of activated matriptase elongated along cell-cell contacts (Fig. 3D) and more F-actin accumulated at the cell peripheries (Fig. 3E). Additionally, activated matriptase, like total matriptase, was only detected at sites of cell-cell contacts, for example, in cell clusters (Figs. 2 and 3) or between two cells (Fig. 3G).
S1P induces assembly of adherens junctions in mammary epithelial cells. Because formation of subcortical actin belts at adherens junctions is induced by E-cadherin-mediated cell-cell contact formation (1), and because the role of S1P in adherens junction assembly has been well documented in endothelial cells (12), we further investigated whether S1P also can induce adherens junction assembly in mammary epithelial cells and whether the S1P-induced accumulation of matriptase at cell-cell contacts depends on adherens junction assembly. Given that extracellular Ca2+ is required for the homotypic binding between cadherin molecules on adjacent cells, we modulated the availability of extracellular Ca2+, in combination with S1P, to investigate whether S1P can induce assembly of adherens junctions. 184 A1N4 cells were first cultured in regular, calcium-containing (1.8 mM) medium supplemented with 0.5% serum for 2 days. After the cells were switched to calcium-free medium for 30 min, E-cadherin was detected as a diffuse pattern in cytosol, and the subcortical actin belt was not observed (Fig. 4A). When extracellular Ca2+ was increased to 1,000 µM, in the absence of S1P, the subcortical actin belt and E-cadherin began to appear at the sites of cell-cell contact, but at very low levels (Fig. 4A). We next exposed these cells to S1P for 30 min. In the absence of extracellular Ca2+, S1P caused some rounding of cells (Fig. 4B). Likewise, there was no translocation of E-cadherin to cell-cell contacts or formation of subcortical actin belts (Fig. 4B). When extracellular Ca2+ was increased to 100 and 1,000 µM, S1P induced to a greater extent translocation of E-cadherin to cell-cell contacts and the formation of subcortical actin belts (Fig. 4B). Although low levels of E-cadherin were seen at the cell-cell contacts when 184 A1N4 cells were grown in medium containing adequate extracellular Ca2+, in the absence of S1P, this cell-cell adhesion E-cadherin failed to resist the extraction of cells with a cytoskeletal stabilizing buffer containing 0.5% Triton X-100 (Fig. 5). In contrast, S1P induced assembly of strong adherens junctions, which were resistant to a wash with 0.5% Triton X-100 (Fig. 5). These data suggest that S1P can induce adherens junction assembly in mammary epithelial cells, as it does in endothelial cells. In contrast to E-cadherin, although matriptase accumulated at cell-cell contacts in response to S1P treatment, the protease was washed away by 0.5% Triton X-100 (Fig. 5). These observations suggest that although matriptase localization was coincident with that of E-cadherin, at cell-cell contacts the protease may not be incorporated into the tightly bound E-cadherin plaques.
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Adherens junction assembly and subcortical actin belt formation are required for matriptase accumulation and activation at cell-cell contacts. The concurrence of matriptase accumulation and activation at cell-cell contacts, adherens junction assembly, and subcortical actin belt formation suggests a potential functional relationship between these S1P-induced events. Therefore, we investigated further whether prevention of subcortical actin belt formation by disruption of F-actin polymerization and prevention of adherens junction assembly by prevention of homotypic binding of E-cadherin each affect the accumulation and activation of matriptase. The pharmacological agents latrunculin B and cytochalasin D (which can inhibit actin polymerization and disrupt microfilament organization) abolished S1P-induced activation of matriptase (Fig. 6). Both latrunculin B (Fig. 6, lanes 2 and 3) and cytochalasin D (Fig. 6, lane 7), but not nocodazole (Fig. 6, lane 5), an inhibitor of micro-tubule polymerization, completely inhibited S1P-induced matriptase activation. Immunofluorescent staining further revealed that cytochalasin D (Fig. 7) and latrunculin B (data not shown) prevented not only formation of subcortical actin belt and assembly of adherens junction but also accumulation of matriptase to cell peripheries.
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We further modulated the availability of extracellular Ca2+, in combination with S1P, to examine the effects of adherens junction assembly on matriptase accumulation at cell-cell contacts. When 184 A1N4 cells were cultured in medium containing 10 µM Ca2+, S1P failed to induce translocation and accumulation of matriptase at cell-cell contacts or -catenin, an adherens junction marker protein (Fig. 8). When extracellular Ca2+ to was increased 1,000 µM, in the absence of S1P, low levels of matriptase and
-catenin were observed at cell-cell contacts. When these cells were exposed to S1P, both matriptase and
-catenin significantly accumulated at cell-cell contacts (Fig. 8). In addition to preventing the accumulation of matriptase at cell-cell contacts, removal of extracellular Ca2+ was shown to abrogate S1P-induced matriptase activation (4). These data suggest that S1P-induced assembly of adherens junctions is required for S1P-induced accumulation and activation of matriptase at cell-cell contacts.
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MAb M32, which is directed against matriptase LDLR class A domain III, inhibits S1P-induced matriptase activation but not S1P-induced actin cytoskeletal rearrangement. In our previous study (23), the intact LDLR class A domains of matriptase were shown to be required for matriptase activation in breast cancer cells. This observation is consistent with the hypothesis that matriptase activation is carried out by transactivation, which could require complex protein-protein interactions among matriptase zymogens and other unidentified molecules. Alterations at the calcium cages in LDLR class A domains of matriptase by site-directed point mutation abolished matriptase activation in breast cancer cells (23). Interestingly, this alteration in LDLR class A domain III also destroyed the epitope recognized by the anti-matriptase MAb M32 (Fig. 9). Therefore, we tested further whether this anti-matriptase MAb could inhibit S1P-induced matriptase activation and S1P-induced actin cytoskeletal rearrangement in immortalized mammary epithelial cells. Pretreatment of 184 A1N4 cells with MAb M32 for 1 h clearly showed inhibition of S1P-induced matriptase activation (Fig. 10G). This inhibition of S1P-induced matriptase activation by MAb M32 could result from the potential inhibitory activity of matriptase transactivation. Alternatively, pretreatment of MAb M32, which could bind to the matriptase on the cell surfaces before S1P treatment (Figs. 1A, 2B, and 8), could cause internalization of matriptase and thus may interfere with its later accumulation induced by S1P treatment, because matriptase was seen as punctate after S1P treatment (Fig. 10D). In contrast to the inhibition of matriptase activation, anti-matriptase MAb M32 did not affect the formation of subcortical actin belts (Fig. 10, E and H). These results suggest that although actin cytoskeletal rearrangement is required for S1P-induced matriptase activation, matriptase activity is not important for S1P-induced actin cytoskeletal rearrangement.
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DISCUSSION |
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The identification of cell-cell contacts as the functional location of matriptase in immortalized mammary epithelial cells is in contrast to its localization in breast cancer cells. In T-47D breast cancer cells, matriptase was detected at cell peripheries (17), as in 184 A1N4 mammary epithelial cells. However, in T-47D cells the protease was detected on cell surfaces in isolated breast cancer cells as well as on outer cell surfaces in cell clusters (17). Notably, in cancer cells, the protease is concentrated on membrane ruffles, suggesting that T-47D breast cancer cells could convert a physiological "cell junction protease" to a potential cancer "invasion protease." Matriptase, expressed in cancer cells, could recruit and activate uPA, an important extracellular matrix-degrading protease, and HGF, a prominent cell motility factor, to invasive cell edges (14, 26). Furthermore, the accumulation of activated matriptase on membrane ruffles was significantly increased by epidermal growth factor (EGF) (3). Besides its role in growth regulation, EGF has been implicated in cell motility and potentially in cancer invasion and metastasis (30). In MCF-7 breast cancer cells, the activated matriptase was also detected within cells, suggesting an intracellular activation or an active internalization of the protease. Furthermore, in contrast to immortalized mammary epithelial cells, which depend on S1P for matriptase activation, breast cancer cells have developed an autonomous mechanism to constitutively activate matriptase regardless of the presence of S1P (3). Therefore, breast cancer cells could deregulate matriptase, with respect to both its subcellular localization and its activation.
In our previous study (23), we showed that activation of matriptase is likely to be carried out by transactivation, an unconventional mechanism for serine protease activation whereby a matriptase zymogen molecule could interact with and cleave (activate) another matriptase zymogen molecule. We have proposed that transactivation could occur in an activation complex that contains matriptase zymogen molecules and other yet unidentified proteins. Because the LDL receptor class A domains and N-glycosylation of the protease at its CUB domain and serine protease domain are required for its activation, these structural requirements for matriptase activation could result from the protein-protein interactions in this proposed activation complex (23). However, how the transactivation is regulated in immortalized mammary epithelial cells and why breast cancer cells constitutively activate matriptase remain largely unknown. In this study, we have shown that S1P induces accumulation of matriptase at cell-cell contacts (Fig. 1) and that activation of matriptase may begin at tiny areas of cell-cell contact, presumably signifying activation centers (Fig. 2). Activation then spreads along cell-cell contacts (Fig. 3). It is likely that S1P could also translocate other required components for matriptase transactivation to those activation centers, for the onset of matriptase activation. After initial activation of matriptase, there may be two possible mechanisms for its further activation: transactivation and activation of matriptase by newly active matriptase along the cell-cell contacts, where matriptase zymogen has accumulated.
Among the S1P downstream effectors, matriptase may be unique. The functional locations of other S1P downstream effectors are either in the cytosol, as for adenylate cyclase and ERK2 MAP kinase, or on the cytoplasmic face of the plasma membrane, as for Rho family GTPases. The bulk of matriptase molecules, including their serine protease domains, are located at the exoplasmic face of the plasma membrane (17). Therefore, matriptase could act on its substrates at the extracellular face of cell-cell junctions. Three protein substrates of matriptase in vitro have been identified to date. These are HGF, uPA, and the protease-activated receptor-2 (PAR-2) (14, 26). List et al. (18) suggested that the defects in epidermal barrier function, hair follicle development, and thymic homeostasis in matriptase-deficient mice are not likely to result from the lack of activation of uPA. Although matriptase has been proposed to activate uPA in human breast cancer, where uPA is overexpressed, it remains to be further determined whether matriptase could activate uPA at cell-cell contacts in physiological settings. HGF, a secreted cell growth/motility factor, has pleiotropic functions, including direct control of cell-cell and cell-substrate adhesion in epithelia. The HGF membrane receptor c-Met is expressed at cell-cell contacts of epithelial cells (6). Thus matriptase could serve as an activator for HGF at the cell-cell contacts where the c-Met receptor is located. PAR-2, a seven-transmembrane G protein-coupled receptor, has been identified in a number of epithelial tissues and is likely to be located at the basolateral surface of cells (5, 7). Matriptase could cleave within the extracellular amino terminus of PAR-2 to release a tethered ligand that binds to and activates the cleaved receptor. Therefore, matriptase could serve as a unique S1P effector that can transduce signals from G protein-coupled receptors to growth factor receptors or other G protein-coupled receptors.
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ACKNOWLEDGMENTS |
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Present addresses: R.-J. Hung, I.-W. J. Hsu, and M.-J. Lee, Dept. of Life Science, National Tsing-Hua University, 101, Section 2 Kuang Fu Road, Hsinchu 300, Taiwan; M. D. Oberst, National Cancer Institute, National Institutes of Health, Building 10 Room 3B47, 9000 Rockville Pike, Bethesda, MD 20892.
GRANTS
This study was supported by a grant from Corvas International, Inc., National Cancer Institute (NCI) Grant P50-CA058185, and US Department of Defense (DOD) Grant DAMD 17-02-1-0391. Salary support for C.-Y. Lin was also provided by US DOD Grant DAMD 17-01-1-252 and by Susan G. Komen Breast Cancer Foundation Grant BCTR0100345. This work was supported in part by the Lombardi Cancer Center Microscopy and Imaging Shared Resource, NCI Grant 2P30-CA-51008.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* R.-J. Hung and I.-W. J. Hsu contributed equally to this work.
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