Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia
Submitted 12 October 2004 ; accepted in final form 7 December 2004
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ABSTRACT |
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sphingosine 1-phosphate; suramin
Besides its increased expression, such as in human breast cancer (10), matriptase could play a role in cancer malignancy, such as in ovarian cancer, because of an imbalance of the protease relative to its cognate inhibitor, hepatocyte growth factor activator inhibitor 1 (HAI-1), in favor of proteolysis (23). Furthermore, deregulated activation and aberrant subcellular localization of the protease may also contribute to its role in breast cancer malignancy (2). In immortal mammary epithelial cells, activation of matriptase occurs at cell-cell junctions upon cellular stimulation with serum or sphingosine 1-phosphate (S1P) (1, 3, 7). However, breast cancer cells constitutively activate matriptase and concentrate the activated protease at membrane ruffles (2), a relocalization that may convert matriptase from a well-regulated cell junctional protease in mammary epithelial cells to a deregulated invasion protease at the leading edges of breast cancer cells. Furthermore, oligosaccharide modifications of matriptase by N-acetylglucosaminyltransferase V (GnT-V) in cancer cells could enhance the stability of matriptase, contributing to the prometastatic effect of GnT-V (8, 9).
Activation of matriptase requires cleavage at its canonical activation motif to convert the single-chain zymogen to a two-chain active protease, a common theme for most of the serine proteases (1). Interestingly, the activational cleavage of matriptase may not be carried out by other active proteases, because matriptase activation depends on its own active site triad and requires its noncatalytic domains and even its cognate inhibitor HAI-1 (25). Therefore, we have proposed that the cleavage at the activation motif of one matriptase zymogen molecule may be preceded by the weak proteolytic activity of another matriptase zymogen molecule as a process of transactivation (25). Dimerization or oligomerization of matriptase molecules thus may serve as a key step during matriptase autoproteolytic activation, a mechanism observed with C1r protease (5) and caspase 8 (33). The noncatalytic domains and posttranslational modifications of matriptase, as well as LDL receptor class A domain of HAI-1, could provide the structural basis for the oligomerization of these molecules during matriptase activation (25). In the present study, we sought to further understand the mechanisms that govern the process and regulation of matriptase autoproteolytic activation and the role of HAI-1 in matriptase functionality by characterizing and comparing the effects of two different exogenous inducers of matriptase activation in terms of the cellular events and signaling pathways involved in the activation of matriptase. Our data reveal that upon the stimulation of these two inducers, both matriptase and HAI-1 were translocated and accumulated at cell-cell junctions or vesicle-like structures where matriptase was activated. The close spatial association between matriptase and HAI-1 also allowed HAI-1 to inhibit active matriptase immediately after its activation.
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MATERIALS AND METHODS |
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Cell lines and culture conditions. Immortalized 184 A1N4 human mammary epithelial cells, a gift from Dr. Martha Stampfer (Lawrence Berkeley National Laboratory, Berkeley, CA), were maintained routinely as previously described (1). For time-kinetic and dose-response experiments, 184 A1N4 cells were seeded at a density of 1 x 105 cells per well in a six-well plate for 2 days. Cells were treated with different concentrations of suramin or for various durations with 50 µM suramin as indicated in the figure legends. Control cells were treated with DMSO alone. For immunofluorescent staining, 184 A1N4 cells were seeded in a well with a coverglass on the bottom of 12-well plates in improved minimum essential medium (IMEM) containing 0.5% FBS. Two days after being plated, cells were treated with 50 µM suramin or 50 ng/ml S1P in IMEM for 30 min. Control cells were treated with DMSO alone. After the treatment, cells were stained with fluorescent dye-labeled monoclonal antibodies (MAb) as described in the figure legends.
Monoclonal antibodies. Human matriptase protein was detected using MAb M32, which interacts with the third LDL receptor class A domain of matriptase (7, 18). The activated form of matriptase was detected using MAb M69, which recognizes an epitope present only in the activated (two-chain) form of the enzyme (1). Human HAI-1 was analyzed using MAb M19 (18).
Conjugation of MAb with fluorescent dyes. To conjugate MAb with fluorescent dyes, 1 mg of MAb, including M32, M69, and M19, were dissolved in 0.1 M NaHCO3 and then labeled with Alexa Fluor 488, 594, or 647 (Molecular Probes, Eugene, OR) according to the manufacturer's instructions.
Western blot analysis. Cell lysates for Western blotting were prepared by lysing cells in 1% Triton X-100 in PBS after the cells were washed twice in PBS. The nuclei and cell debris were removed by centrifugation at 10,000 g for 10 min at 4°C. The protein concentration was determined using bicinchoninic acid protein assay reagents (Pierce, Rockford, IL) according to the manufacturer's protocol. For immunoblotting, an aliquot of the total lysate protein was electrophoresed in a 7.5% SDS gel under nonboiled and nonreduced conditions and transferred onto Protran nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The membranes were incubated with MAb as indicated, and the proteins were visualized using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Boston, MA).
Immunodepletion. An aliquot of 400 µl of total cell lysates from suramin-stimulated 184 A1N4 cells was incubated with 30 µl of MAb M19-conjugated Sepharose 4B (5 mg of M19 per ml of Sepharose 4B beads) at 4°C for 2 h. The supernatant was separated from M19-Sepharose 4B by centrifugation.
Immunofluorescence microscopy. Cells were plated onto microcoverglasses and grown for 2 days. Subconfluent cells were subjected to the treatments indicated in each figure legend. Cells were then fixed in 3.7% formaldehyde and permeabilized within 0.05% Triton X-100 in PBS for 20 min at room temperature. Cells were washed with PBS three times. Matriptase, activated matriptase, and HAI-1 were detected with Alexa Fluor dye-conjugated M32, M69, and M19, respectively. F-actin was visualized using Texas red-labeled phalloidin (Molecular Probes), and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). FITC-labeled anti-mouse IgG was used for the staining controls, which resulted in a weak, diffuse background (data not shown). After fluorescent staining, coverglasses were mounted with Prolong Antifade (Molecular Probes) and the fluorescent images were captured using the MetaVue software package (Universal Imaging, Downingtown, PA) in conjunction with a Nikon Eclipse E600 digital fluorescence microscope.
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RESULTS |
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Suramin-induced matriptase activation in immortal human mammary epithelial cells. To analyze the effect of suramin on the activation of matriptase, we first determined the optimal concentration (Fig. 1A) and time course (Fig. 1B) of cellular treatments using this compound. Matriptase activation was determined by the levels of activated matriptase using immunoblot analysis of cell lysates and immunofluorescent staining of fixed cells. For these studies, we used MAb M69, which specifically recognizes the two-chain activated matriptase but not the single-chain zymogen (1). Activation of matriptase induced by suramin occurred in a dose-responsive manner, with a minimal required concentration of 25 µM (Fig. 1A). The activated matriptase was detected in complexes with its cognate inhibitor, HAI-1, at 120 and 85 kDa (Figs. 1 and 2). We also used another anti-matriptase MAb, M32, to determine the levels of matriptase. M32 MAb recognizes the third LDL receptor class A domain of matriptase (7) and thus can react with the single-chain latent protease of 70 kDa and with two-chain activated matriptase in the 120-kDa HAI-1 complex, but not with the 85-kDa HAI-1 complex, which likely contains the serine protease domain of matriptase and the full-length HAI-1 (Fig. 2). M32 reactivity provided data indicating the extent to which latent matriptase is converted to its activated form by comparing the levels of the 70-kDa form in controls with those in treated samples. For example, the majority of matriptase was converted to its activated form with 50 and 100 µM suramin. Matriptase-HAI-1 complexes induced by suramin also were observed to appear in a dose-responsive manner in association with anti-HAI-1 MAb M19. M19 recognizes the unbound, full-length HAI-1 at 55 kDa and both the 120- and 85-kDa matriptase-HAI-1 complexes (Fig. 1A). In contrast to matriptase, however, the majority of HAI-1 was detected in its unbound form, even after treatment with 100 µM suramin. These data suggest that the complexed HAI-1 represents only a small portion of total HAI-1, even under conditions in which the majority of matriptase is activated and bound to HAI-1. Therefore, 184 A1N4 cells express HAI-1 in much higher ratios than matriptase. Matriptase activation was detected within 5 min of suramin treatment and reached to the maximal activation after 30-min treatment (Fig. 1B). This rapid response of matriptase activation is similar to that observed with S1P (3).
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It is possible that the 85-kDa complex is derived from the 120-kDa complex. Indeed, the 120-kDa complex was converted to the 85-kDa complex by incubating the cell lysate at 37°C (Fig. 2B). Because MAb M32 did not recognize the 85-kDa complex, the conversion of the 120-kDa complex to a 85-kDa complex could result from the loss of the noncatalytic domains of matriptase, which contain its third LDL receptor class A domain, where the epitope recognized by M32 resides. Furthermore, the addition of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) into the cell lysis buffer inhibited this conversion even for the samples incubated at 37°C. Because the loss of noncatalytic domains of matriptase occurred only for activated matriptase and not for latent matriptase, and because DTNB can inhibit protein disulfide isomerase (15), it is very likely that, after cell lysis, protein disulfide isomerase cleaves the disulfide bond that connects the noncatalytic domains with the serine protease domain of activated matriptase. For single-chain matriptase, if cleavage of this disulfide linkage by protein disulfide isomerase occurs, the serine protease domain will not separate from the noncatalytic domain, because the protease will still be held as single-chain matriptase by peptide bonds. Figure 2C summarizes our current theory that the 85-kDa matriptase complex is derived from the 120-kDa complex and shows the positions of epitopes recognized by the three MAb that we used.
S1P and suramin induce accumulation and activation of matriptase at activation foci. While both S1P and suramin induce matriptase activation in 184 A1N4 cells and share several common characteristics, such as dose-response relationship, rapid kinetics, and rapid inhibition of active matriptase by HAI-1, we have observed some differences between them. Specifically, suramin requires micromolar concentration, compared with nanomolar concentration for S1P, to induce matriptase activation. Also, compared with S1P-treated cells, much higher proportions of latent matriptase were converted to activated matriptase in suramin-treated cells. In addition, S1P simultaneously induced changes in cell shape under the light microscope, but such changes were not observed with suramin. The S1P-induced changes in cell shape were associated mainly with the S1P-induced actin cytoskeletal rearrangement, that is, the formation of actin subcortical belts. Furthermore, destruction of actin cytoskeletal rearrangement inhibits S1P-induced matriptase activation (7). Therefore, it is of interest to investigate the role of the cellular events, particularly actin cytoskeletal rearrangement, in suramin-induced matriptase activation. In Fig. 3, we compared suramin with S1P for their abilities to induce the redistribution of matriptase and cause actin cytoskeletal rearrangement. After growing cells in IMEM supplemented with 0.5% FBS for 2 days, matriptase was observed mainly in the cytoplasm with diffuse staining patterns (Fig. 3A). Little F-actin was observed in these cells (Fig. 3B). After 30 min of suramin treatment, while matriptase was still located in cytoplasm, the protease apparently concentrated at vesicle-like structures with various sizes (Fig. 3D), in striking contrast to the cell-cell contact localization of matriptase induced by S1P (Fig. 3G). While filamentous actin structures were increased by S1P treatment (Fig. 3H), they were much less organized in suramin-treated cells (Fig. 3E). Interestingly, pretreatment with suramin apparently did not affect the S1P-induced formation of subcortical actin belts and translocation of matriptase to cell-cell contacts (Fig. 3, J and K).
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DISCUSSION |
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HAI-1 was initially identified as a cognate matriptase inhibitor (18) and was subsequently shown also to be required for matriptase activation (25). Its inhibitory role results from the binding of its first Kunitz domain with active matriptase (14). However, its participation in matriptase activation is mainly via its LDL receptor class A domain, which could provide the structural basis for protein-protein interactions in the formation of matriptase activation complexes. The translocation and concentration of both matriptase and HAI-1 in activation foci in response to cellular stimulation by suramin or S1P further support the close temporospatial relationship between these two molecules during matriptase activation. Therefore, HAI-1 may directly or indirectly contact latent matriptase in the activation complex. This close spatial proximity of HAI-1 to active matriptase must result in a very efficient and rapid inhibition of active matriptase immediately after matriptase activation. This inhibitor-dependent activation could be the explanation for all activated matriptase in HAI-1 complexes (Figs. 1 and 2). The coupling of matriptase activation with its inhibition apparently makes the half-life of active matriptase very short. Therefore, it is plausible that the substrates of matriptase may also be present in the activation foci or that the matriptase substrates could even be required for full matriptase activation. This mechanism could allow matriptase activation, action on substrates, and inhibition to occur simultaneously.
The dual role of HAI-1 in matriptase activation and inhibition appears to be reminiscent of the role of tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in the regulation of matrix metalloproteinase-2 (MMP-2). It has been established that MMP-2 activation occurs via a mechanism to form a trimolecular complex of TIMP-2, MMP-2 zymogen, and MT1-MMP in 1:1:1 stoichiometry (11). When the concentration of TIMP-2 is low, TIMP-2 cannot bind to active MMP-2, leading to collagen degradation by these active MMP-2. At the high concentration of TIMP-2, TIMP-2 functions as a great inhibitor to block MMP-2 activity (13). Similarly, HAI-1 is necessary to form oligomeric complexes with matriptase zymogens and/or other yet identified proteins to facilitate matriptase activation. The coupling of protease activation with inhibition ensures a tight control in the protease function, avoiding harmful effects of proteolysis.
Because matriptase is likely to be activated via autoproteolytic activation and because only the first protease in a protease cascade needs this activation mechanism, we have hypothesized that matriptase may be at the pinnacle of an as yet poorly characterized protease cascade. The complicated regulatory mechanisms that govern matriptase activation, action on its substrates, inhibition, and subcellular localization seem parallel to its potential leading position in a protease cascade. By these regulatory mechanisms, activation of matriptase and the putative protease cascade would occur only at the right time, when the exogenous inducers were present, and only at the right place, such as at cell-cell junctions where its substrates might be colocalized or cotranslocated. Depletion of the exogenous inducers, as well as HAI-1 inhibition, provides the mechanisms for fast and timely switch-off of the protease cascade. Ectodomain shedding of matriptase-HAI-1 complexes could be the mechanism for their cellular clearance.
Although S1P and suramin induce matriptase activation at different subcellular locations, both activation inducers cause translocation and accumulation of matriptase, HAI-1, and probably other required components at activation foci. These common processes for matriptase activation indicate some basic mechanisms for matriptase activation: the involvement of the cell membrane and intracellular signaling. Both cell-cell junctions and vesicle-like structures contain lipid biolayers for the anchoring and trafficking of these proteins, consistent with the transmembrane protein characteristics of matriptase and HAI-1. The involvement of PKC-like protein kinases in both S1P- and suramin-induced matriptase activation suggests that intracellular signaling may contribute to the trafficking and oligomerization of these proteins at activation foci, consistent with the role of PKC in vesicle trafficking (4). Interestingly, this protein kinase-based intracellular signaling appears to depend on the assembly of F-actin and adherens junctions in response to S1P stimulation; suramin, however, could induce this intracellular signaling in the absence of F-actin.
In conclusion, in response to exogenous activation inducers, matriptase, its cognate inhibitor HAI-1, and possibly other unidentified proteins translocate and accumulate at activation foci, either at cell-cell junctions or at vesicle-like structures inside cells. Activation foci provide a good milieu in which matriptase can interact with those essential components and initiate matriptase activation. After its activation, active matriptase is rapidly bound to HAI-1. Subsequently, the matriptase-HAI-1 complex is shed into the extracellular milieu. Thus our data indicate that activation and HAI-1-mediated inhibition of matriptase are well organized and controlled at activation foci in human mammary epithelial cells.
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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.
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