Activation of Membrane-Type Matrix Metalloproteinase 3 Zymogen by the Proprotein Convertase Furin in the Trans-Golgi Network1

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[CANCER RESEARCH 62, 675–681, February 1, 2002] Activation of Membrane-type Matrix Metalloproteinase 3 Zymogen by the Proprotein Convertase Furin in the trans-Golgi Network1

Tiebang Kang, Hideaki Nagase, and Duanqing Pei2 Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455 [T. K., D. P.], and Kennedy Institute of Rheumatology, London W6 8LH, United Kingdom [H. N.]

ABSTRACT recognized that centers on the membrane-bound MMPs that can process some soluble MMPs, such as proMMP-2 and proMMP-13, Matrix metalloproteinases (MMPs), a family of zinc-dependent endo- into active forms (16, 19–22). The first member of this group was peptidases implicated in tumor invasion and metastasis, must undergo MT1-MMP, which can specifically cleave at Asn-Leu within the zymogen activation prior to expressing any proteolytic activity. Although prodomain of MMP-2, triggering an autoactivation process to gener- the cysteine-switch model predicts the well-established autoactivation 112 process, ϳ40% of the known MMPs possess a conserved RXKR motif ate the active form of MMP-2 at Y (20, 21, 23). MT3-MMP and between their pro- and catalytic domains and, thus, could be activated MT5-MMP were subsequently cloned and shown to be able to acti- directly by members of the proprotein convertase family. To further vate proMMP-2 as well (24, 25). Yet, the predominant pathway for understand this process, we analyzed the activation of proMT3-MMP as MMP activation appears to be mediated by furin or its related PCs (19, a model system. We demonstrated that the conversion of MT3-MMP 26). The PCs are a family of serine proteinases that recognize dibasic zymogen into active form is dependent on both the furin-type convertase or RXK/RR motifs and cleave the peptide bond on the COOH side 116 activity and the R RKR motif. Consistently, MT3-MMP was colocalized (27–29). Responsible for the processing of various proproteins in the with furin in the trans-Golgi network by confocal microscopy. However, secretory pathway, furin or its related PCs have been demonstrated to neither furin activity nor its recognition site in MT3-MMP is required for process proMMP-11 and proMT1-MMP, or non-MMP enzymes like the observed colocalization. In fact, the colocalization pattern remains ␣ ␤ intact, even in the presence of brefeldin A, an agent known to block tumor necrosis factor- convertase, -amyloid converting enzyme, endoplasmic reticulum to Golgi trafficking. Yet, brefeldin A completely and aggrecanases (5–7, 19, 26, 30, 31). Within the MMP superfamily, blocked the activation of MT3-MMP. A23187, a calcium ionophore known at least 9 MMPs (of a total of ϳ24) contain RXK/RR motifs and, thus, to block furin maturation, also blocked proMT3-MMP activation but had could be processed into active forms by furin or its related PCs (19, minimal effect on the colocalization between MT3-MMP and furin. Thus, 24, 25, 32–37). Despite the potential importance of this activation furin processes MT3-MMP zymogen in the trans-Golgi network, where mechanism, the cellular events regulating the interactions between they colocalize independently of their apparent enzyme-substrate relation- this subset of MMPs and the PCs remain poorly defined. ship. MT3-MMP was initially cloned from malignant oral melanoma and subsequently identified in malignant tumors, smooth muscle cells, and INTRODUCTION inflamed cornea tissues (25, 38–44), suggesting that MT3-MMP may play a significant role in mediating ECM degradation during inflam- A key regulatory mechanism in controlling proteolysis is zymogen mation and tumor cell invasion and metastasis. Mechanistically, MT3- activation, as demonstrated in several biological processes including MMP was demonstrated to interact with proteoglycans on the mela- apoptosis (1, 2), Alzheimer’s disease (3–6), and tumor necrosis fac- noma cell surface to confer an invasive phenotype (39). On the other tor-␣-mediated inflammatory responses (7). During tumor invasion 3 hand, the expression of MT3-MMP appears to enhance the growth of and metastasis, the ECM barriers are breached in part by members of MDCK cells in a three-dimensional gel lattice made of purified type the MMP family that all require zymogen activation (8–12). The I collagen (45). The observed enhancement is dependent on MT3- latency and activation of MMPs is known to be governed by a MMP activity because its catalytically inactive mutant failed to pro- cysteine-switch; a cysteine within the conserved PRCGVPD motif duce any growth advantage under similar conditions (45). Consis- keeps MMP latent by binding to the catalytic zinc (13). The disen- tently, we have observed the processing of MT3-MMP zymogen into gagement of this Cys-Zinc bond may lead to autoactivation (13). The an active form by MDCK cells (45). In this report, we continue our direct visualization of this Cys-Zinc interaction was achieved in study on the structure and function of MT3-MMP in this model proMMP-2 by X-ray crystallography (14), validating the latency system by analyzing the cellular mechanisms leading to its zymogen mechanism (13). In in vitro studies, MMP activations have been activation. We demonstrate that MT3-MMP is activated by the arche- shown with conditions such as oxidation, proteolysis, freeze/thaw, and typal PC, furin, which accomplishes the activation process by colo- mild denaturation that can disrupt the Cys-Zinc bond and trigger the calizing with its substrate in the trans-Golgi network. autoactivation process (reviewed in Refs. 11, 15, and 16). In vivo, the urokinase-type plasminogen activator/tissue-type plasminogen activa- MATERIALS AND METHODS tor and plasminogen axis has been implicated in the activation of some proMMPs, especially proMMP-1 and proMMP-9 zymogens Cell Culture and Reagents. MDCK and its derivatives are generated and (17, 18). Recently, an emerging paradigm of MMP activation has been maintained as described (46). COS cells are maintained in 5% FBS DMEM. Cell culture media and supplements were purchased from Life Technologies, Received 5/29/01; accepted 12/3/01. Inc. (Rockville, MD). Rabbit anti-MT3-MMP antisera were raised against a The costs of publication of this article were defrayed in part by the payment of page GST-MT3-MMP fusion protein as described (47). Rabbit polyclonal anti-furin charges. This article must therefore be hereby marked advertisement in accordance with convertase antibody was purchased from Affinity Bioreagents, Inc. (Golden, 18 U.S.C. Section 1734 solely to indicate this fact. CO). Brefeldin A, M2 antibody, and other immunological reagents were from 1 This work was supported in part by NIH Grant CA76308. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, Sigma Chemical Co. (St. Louis, MO). CMK-based furin inhibitor was pur- University of Minnesota, 321 Church Street, S.E., 6–120 Jackson Hall, Minneapolis, MN chased from Bachem (Philadelphia, PA). 55455. Phone: (612) 626-1468; Fax: (612) 625-8408; E-mail: [email protected]. Expression Constructs. Wild-type and EA mutant constructs are detailed 3 The abbreviations used are: ECM, extracellular matrix; MT-MMP, membrane-type elsewhere (45). The MT3-MMP RA mutation was created by sequential PCR matrix metalloproteinase; BFA, brefeldin A; PC, proprotein convertase; FBS, fetal bovine serum; CMK, chloromethylketone; ADAM, a disintegrin and metalloproteinase; MDCK, using two primers covering the furin motifs C7307 (CCT GTC AAT GCA Madin-Darby canine kidney; WT, wild type. TAT GCC GCT GCA GCA ATA TGA AAT) and C7308 (TAT GCA TTG 675

ACA GGA CAG) and cloned into pCR3.1 expression vector as described Immunostaining and Confocal Microscopy. Cells seeded on coverslips previously (24, 26). in six-well plates were treated for 24 h with or without CMK, BFA, or A23187. DNA Transfection and Generation of Stable MT3-MMP Transfectants. After fixations for 30 min at room temperature, the cells were permeabilized

pCR3.1 MT3-MMPRA were transfected into MDCK cells by Lipofectamine for 1 h with buffer A (0.3% Triton, 1% BSA, and 0.01% NaN3 in PBS) and (Life Technologies, Inc., Rockville, MD), and stable clones were selected in incubated with primary antibody (1:50 or 1:100 dilution in buffer A) for 3 h the presence of G418. The stable clones were screened by Western analysis of and then secondary antibody conjugated with FITC or Rhodamine Red (Jack- the cell lysates with M2 antibody and zymographic analysis of proMMP-2 son ImmunoResearch Laboratory, Inc., West Grove, PA) for 1 h. The cells activation. Positive cells were further analyzed by Northern blotting using were washed three times in PBS and mounted. Confocal microscopy was MT3-MMP cDNA as a probe (see below). Seven representative clones named carried out in the Biomedical Image Processing Laboratories at the University WT2, WT7, RA16, RA17, EA6, and EA15 were included in the present study. of Minnesota as described (45). Western Blotting, Immunoprecipitation, and Gelatin Zymography. The basic protocols for these procedures are essentially the same as described RESULTS previously (20, 24). Briefly, for zymography, confluent cultures were washed three times with PBS and allowed to incubate in the presence of DMEM General Consideration of MMP Latency and Zymogen Activa- supplemented with the proMMP-2 from 5% fetal bovine serum in the medium. tion. If the cysteine-switch can be considered as the “off” signal for dec-Arg-Val-Lys-Arg-CMK, a specific inhibitor of furin convertase (48, 49), MMP latency with the exception of MMP-23 (13, 34), the RXKR calcium ionophore A23187, or BFA (a blocker of ER to Golgi trafficking; Ref. motif initially recognized for its role in MMP-11 zymogen activation 50), were subsequently included in the medium as needed. After 24 h of could be viewed as a switch turning on MMP activity (Fig. 1; Refs. incubation, medium was collected and cleared of cell debris by centrifugation and analyzed by SDS-PAGE impregnated with gelatin (1 mg/ml) as described 26, 51). To date, a list of MMPs possessing a similar RXKR motif has (20, 24). For immunoprecipitation and Western Blot, cells grown in six-well been discovered and presented in Fig. 1 in the context of the plates were lysed in 250 ␮l of RIPA buffer [50 mM Tris (pH 7.5), 150 mM PRCXXPD latency motif. We propose that this subfamily of MMPs NaCl, 0.25% sodium deoxycholate, 0.1% NP40, 10 ␮M leupeptin, 0.1 ␮M has clearly defined “off/on” signals for latency and activation (Fig. 1). 5-p-amidinophylmethanesulfonyl fluoride, and 1 ␮M aprotinin] supplemented Because the activation process mediated by furin or its related PCs is with 10 mM of EDTA to protect the active forms of MT3-MMP from degra- accomplished in a single step without evoking any autocatalytic dation. The lysates were centrifuged at 14,000 ϫ g for 20 min to remove the mechanism, this mode of activation should be distinguished from the ␮ cell debris. Rabbit polyclonal anti-MT3-MMP antiserum (1.5 l/reaction) was autocatalytic activation process predicted by the cysteine-switch added to the resulting supernatants and allowed to incubate at 4°Cfor1h.The model (13). immune-complex was collected with protein-A/G PLUS agarose (10 ␮l; Santa Activation of proMT3-MMP by a Furin-like Activity. Previ- Cruz Biotechnology), washed with RIPA buffer four times, and then eluted with 2ϫ SDS-PAGE sample buffer under reducing conditions. After electro- ously, we have shown that MT3-MMP stable transfectants are able to phoresis, the proteins were transferred to polyvinylidene difluoride membranes activate proMMP-2 (45). We infer that transfected MT3-MMP be and probed with M2 anti-FLAG mouse monoclonal antibody and developed as activated by the cellular machinery (45). On the basis of the presence 116 described (20, 24). of the R RKR motif between its pro- and catalytic domain (Fig. 1), Northern Blot. Total RNAs were isolated from cells with TRI-Reagent as we hypothesize that MT3-MMP is activated via a furin-mediated described by the manufacturer (Molecular Research Center, Columbus, OH). pathway as described for MMP-11 and MMP-14 (26, 31, 51). To test Equal amounts of total RNAs (5 ␮g) were denatured with glyoxal and DMSO, fractionated on a 1% agarose gel in 10 mM phosphate buffer at a constant 55 V for 5 h, and then transferred to nylon membrane overnight. The membrane was then stained with methylene blue for the 28S and 18Sϩ rRNA to establish equivalence in sample loading. The membrane was prehybridized at room temperature for at least 30 min, hybridized at 62°C for overnight with 32P- labeled MT3-MMP cDNA as a probe, washed, exposed to an ABI screen, and scanned on a phosphorimager (ABI, Foster City, CA). Purification of MT3-MMP RA Proteins. A stable line MT3-MMP RA-17 was expanded to 12 dishes (150 ϫ 20 mm). After reaching confluence, these cells were washed with PBS three times and then lysed with 36 ml of 1% Triton X-100 in TBS containing 10 ␮M BB94 for 15 min in 4°C. The cell extracts were centrifuged at 14,000 rpm for 20 min in 4°C, subsequently loaded onto a M2 column, washed, and eluted as described (46). The eluted materials were analyzed by SDS-PAGE, Western blotting using rabbit polyclonal anti-MT3-MMP antisera, and gelatin zymography, respectively, as described (20, 24, 26). Deglycosylation. Proteins from MT3-MMP WT, RA, and EA mutants were immunoprecipitated as described above, and the immune complexes were eluted in 1% SDS and 5% 2-mercaptoethanol by boiling at 100°C for 10 min. The eluted materials were treated with or without N-glycosidase F for 15–20 hat37°Cin20mM sodium phosphate (pH 8.0), 30 mM EDTA, 0.5% NP40, 0.1% SDS, and 0.5% ␤-mercaptoethanol as suggested by the supplier (Roche Diagnostics, Indianapolis, IN). The fractions were subsequently analyzed by Western blot using M2 antibody as described in previous sections (45). Growth of MDCK and Its Derivatives in Three-Dimensional Collagen Gel. Cells (1.2 ϫ 103) were mixed with 250 ␮l of collagen (2 mg/ml; Fig. 1. A summary model for MMP latency and activation. Upper panel, the sequences surrounding the junctions between pro- and catalytic domains for selected MMPs are Collaborative Research, Bedford, MA) and allowed to gel at 37°C in 24-well aligned to show the conservation of the cysteine-switch (underlined) as the signal for plates to give rise to a three-dimensional collagen matrix. Fresh medium latency (13) and the RXKR motifs (boxed) as the signal for zymogen activation (26). containing 95% DMEM and 5% fetal bovine serum were added to the wells Downward arrow, the site for maturation cleavage. Lower panel, representatives from and changed every 2 days. After 12 days, MDCK and its derivatives were each MMP class are diagrammed to show the presence of various domains and motifs. S, signal peptide; Pro, prodomain; C, cysteine-switch; R, RXKR motif; Cat, catalytic photographed by a video camera attached to a Nikon microscope at the domain; H, hinge region; Pexin, hemopexin-like domain; TM, transmembrane domain; C, University of Minnesota Bioimaging Processing facility as described (45). cytosolic domain. 676

of proMT3-MMP zymogen (Fig. 3B, b, Lanes 4 and 5 versus Lanes 2 and 3). Inhibitions of proMT3-MMP Activation Alter Its Pattern of Posttranslational Modification. The RA species in Fig. 3B exhib- ited higher molecular weights than its WT counterparts (Fig. 3B, b, Lanes 4 and 5). A similar shift in molecular mass to higher ones was also evident for WT MT3-MMP when treated with CMK inhibitor (Fig. 2, Lanes 14 and 15), thus suggesting that CMK inhibition of MT3-MMP processing also results in altered posttranslational modifications such as glycosylations. To resolve this possibility, the immunoprecipitates from WT, EA, and RA mutants were treated with glycanase F prior to Western blotting. As described previously (45), Fig. 2. CMK furin inhibitor blocks the processing of proMT3-MMP. MDCK cells deglycosylation converted the four bands in WT and EA mutants to stably transfected with vector (Lanes 1, 2, and 9) or MT3-MMP (WT2, Lanes 3–8 and two species, representing pro and active forms, respectively (Fig. 3C, 10–15) were grown in six-well plates to 50% confluence, treated with serum-free medium Lanes 1 and 2 and Lanes 4 and 5). In contrast, deglycosylation of RA overnight, and then assayed in a 24-h period for the activation of proMMP-2 supplied from 5% FBS in DMEM medium containing 1:1000 (v/v) methanol (Lanes 1, 3, 9, and converted only some of the proforms to the one detected in the WT 10), 6 ␮M (Lanes 4 and 11), 12 ␮M (Lanes 5 and 12 ), 25 ␮M (Lanes 6 and 13), 50 ␮M clones (Fig. 3C, Lanes 3 and 6), revealing a novel higher species of (Lanes 2, 7, and 14), and 100 ␮M (Lanes 8 and 15) CMK. Aliquots (5 ␮l; Lanes 1–8)of the conditioned medium (600 ␮l total for each well) were analyzed by gelatin zymography (1 mg/ml gelatin in 7.5% PAGE, incubated at 37°C for 12 h). As described previously, the proMMP-2 migrated at 72 kDa and the activation intermediate at 62 kDa, whereas little activation of MMP-2 at 58 kDa was detected (Lanes 1–8; Ref. 45). The cells were washed with PBS (three times) and lysed in RIPA buffer. The lysates (Lanes 9–15) were immunoprecipitated with rabbit anti-MT3-MMP antisera and analyzed by Western blot with anti-FLAG M2 antibody as described in “Materials and Methods.” The pro- and active MT3-MMP species are marked on the right (Lanes 9 and 10). The IgGs from the immunoprecipitations are shown as indicated. Arrowhead, the extra species of proMT3- MMP accumulated during the inhibition of PCs. IP, immunoprecipitation; Blot, Western blotting. the involvement of furin in proMT3-MMP activation, we incubated the cells with dec-Arg-Val-Lys-Arg-CMK, a synthetic inhibitor known to inhibit furin (48–50). We first analyzed the effect of CMK on the processing of proMMP-2 that is a direct indicator of MT3- MMP activity in vivo. As shown in Fig. 2, CMK blocked MT3-MMP mediated processing of proMMP-2 in a dose-dependent manner (upper panel). To assess the role of MT3-MMP activity in the observed proMMP-2 activation, we monitored the processing of proMT3-MMP in cell lysates by immunoprecipitation (anti-MT3-MMP antisera) coupled with Western blotting (anti-FLAG M2 monoclonal antibody). In agreement with the zymography data (Fig. 2, upper panel), a dose- dependent inhibition of MT3-MMP activation by CMK was observed (Fig. 2, lower panel). These results suggest that furin activity is required for the activation of proMT3-MMP. The R116RKR Motif Is Required for proMT3-MMP Activation. In both MMP-11 and MMP-14, the RXKR motifs have been shown to be required for furin-mediated processing (26, 31, 51). Thus, a similar motif, R116RKR in MT3-MMP, should also be required for the Fig. 3. Mutational analysis of the PC recognition motif R116RKR in MDCK stable observed processing. To prove this hypothesis, we generated a mutant transfectants. A, a schematic illustration of WT and RA mutants of MT3-MMP. Full- designated MT3-MMP-RA that converts the R116RKR motif into length MT3-MMP cDNA was cloned downstream of the cytomegalovirus promoter/ A116AAA (Fig. 3A). A panel of stable MT3-MMP-RA transfectants enhancer in the pCR3.1 (Invitrogen) vector. A FLAG tag (F) was fused to its COOH terminus for detection (45). Substitution of the R116RKR motif with four Ala residues were generated in MDCK cells, expressing low to moderate levels of resulted in the RA mutant, as described in “Materials and Methods.” Abbreviations are as MT3-MMP-RA (data not shown). Two clones were analyzed further in Fig. 1B. Characterizations of MT3-MMP transfectants. MDCK cells transfected with and presented along with two wild-type clones in Fig. 3. We measured vector (Lane 1), WT (WT 2 and 7; Lanes 2 and 3), or RA mutants (RA 16 and 17; Lanes 4 and 5) of MT3-MMP were analyzed by: a, Northern blotting for MT3-MMP mRNA as the levels of expression in these clones by Northern blot analysis and described in “Materials and Methods.” Note that 28S rRNAs were stained to show demonstrated in Fig. 3B that WT and RA clones all express a com- equivalency in loading/lane as indicated, and the size for recombinant MT3-MMP transcript is around 2.5 kb; b, Western blotting for MT3-MMP protein products by parable amount of mRNA (a, Lanes 2–5). When their ability to immunoprecipitation and immunoblotting as described in Fig. 2; and c, zymography to process proMMP-2 was assessed, it was apparent that the RA trans- detect proMMP-2 activation by WT and RA MT3-MMP transfectants. Note that the fectants could no longer cleave proMMP-2, whereas the WT clones proMMP-2 (Pro) is from the 5% fetal bovine serum supplemented in the culture medium. The processed intermediate at 68 kDa and a faint 58 kDa active form of MMP-2 are could (Fig. 3B, c, Lanes 2 and 3 versus Lanes 4 and 5). To confirm indicated by horizontal bars on the right. C, posttranslational modifications of WT, EA, the activation status of MT3-MMP in these stable clones, cell-asso- and RA mutant proteins of MT3-MMP. Immunoprecipitates from WT (WT2, Lanes 1 and ciated MT3-MMPs were analyzed by immunoprecipitation coupled 4), EA (EA6, Lanes 2 and 5), or RA (RA17, Lanes 3 and 6) stable clones were obtained as described in Fig. 3, subjected to treatment with N-glycanase F (Lanes 4–6) overnight, with Western blotting as described in Fig. 2. Shown in b of Fig. 3B are and analyzed by Western blot with anti-FLAG M2 antibody as described in “Materials and the protein species for WT and RA clones. In contrast to the WT Methods.” The relationships among protein species before and after deglycosylation were estimated as described (45) and indicated by the bracket lines. The pro-, active, as well as clones, the RA type mutants failed to express any active species, thus the hyperglycosylated MT3-MMP species are indicated by arrows, as indicated on the demonstrating that the R116RKR motif is required for the processing right. 677

be activated in the ER compartment without being transported to the Golgi apparatus (53). Furthermore, this result suggests that the trans- activation of MT3-MMP by furin requires the proper microenviron- ment of the trans-Golgi network. A similar blockade of MT3-MMP activation was observed with A23187, a calcium ionophore known to inhibit the autoactivation of furin (53). Shown in Fig. 5B, a gradual increase of A23187 shifted the MT3-MMP species from active to proforms on Western blots (Lanes 7–13), accompanied by the inhibitions of proMMP-2 activation (Lanes 1–6). Together, these results suggest that furin-mediated activation of proMT3-MMP occurs in the Golgi complex and requires proper calcium signaling. Colocalization between Furin and MT3-MMP. The requirement Fig. 4. Proteolytic activity of RA mutant transfectants. A, RA mutant of MT3-MMP of ER to Golgi trafficking for MT3-MMP activation, as evident with retains an intact catalytic mechanism. Cells from MT3-MMP RA mutant stable line 17 BFA treatment in Fig. 5, suggests strongly that MT3-MMP and furin (RA17) were expanded. Confluent cultures were washed with PBS three times and lysed may be colocalized in the trans-Golgi network, where furin is highly in 36 ml of 1% Triton X-100 in TBS containing 10 ␮M BB94 for 15 min at 4°C. The cell extracts were centrifuged at 14,000 rpm for 20 min at 4°C, and the supernatants were concentrated (27). To gain further support for the idea that furin loaded onto an M2 column. After extensive wash with the extraction buffer, the column activates proMT3-MMP in the trans-Golgi network, we investigated was eluted with FLAG peptide (75 ␮g in 5 ml of TBS). The purified RA MT3-MMP proteins were analyzed by Western blotting (5 ␮l) using rabbit polyclonal anti-MT3-MMP the cellular localization of furin and MT3-MMP by confocal micros- antisera (Lane 1) or gelatin zymography (20 ␮l; Lane 2) as described in “Materials and copy. MT3-MMP-transfected MDCK cells were fixed and incubated Methods.” Note that the MT3-MMP RA protein degrades gelatin in the zymogram (Lane with rabbit anti-furin antibody (Affinity Laboratories) and anti-FLAG 2). B, the RA mutation renders the MT3-MMP mutant inactive in enhancing MDCK growth in type I collagen lattice. MDCK stable clones transfected with control vector monoclonal antibody (Sigma Chemical Co.) and then Rhodamine Red (panel 1), WT (WT2, Lane 2), RA mutants (RA17, Lane 3), or EA mutants (EA6, Lane 4) conjugated goat antirabbit antibody and FITC-conjugated rabbit anti- were mixed with 250 ␮l of collagen (2 mg/ml; Collaborative Research) and allowed to gel at 37°C in 24-well plates as described in “Materials and Methods.” After 12 days of mouse antibody, respectively. The double-labeled cells were subse- incubation, the cells in three-dimensional collagen gel were photographed and presented quently sectioned optically with z-series on a Bio-Rad confocal as indicated (45). These four panels were representatives of cells from three independent system and presented in Fig. 6, A–C. Extensive overlaps between experiments taken under the same microscope with similar settings. MT3-MMP and furin were observed in the perinuclear ER/Golgi complex. In contrast, some of the MT3-MMP signals were observed MT3-MMP (Fig. 3C, Lane 6). These data suggest that furin-mediated around the edges of plasma membrane, where very little furin was processing of MT3-MMP zymogen may regulate its trafficking present (Fig. 6, A–C). To see whether the colocalization pattern is through the Golgi network where glycosylation takes place. dependent on furin activity or the RXKR motif on MT3-MMP, we Zymogen Activation Is Required for MT3-MMP to Exert Its analyzed the distribution patterns of furin and the RA mutants. Sur- Biological Function. In our previous studies, we have established an prisingly, the colocalization pattern between furin and MT3-MMP is in vitro biological assay for MT3-MMP activity, i.e., the enhanced also observed for RA mutants and furin, suggesting that the observed cell growth in three-dimensional collagen lattice (45). To assess the role of zymogen activation in MT3-MMP-mediated function in a biologically relevant system, we seek to determine the ability of RA mutants to grow as cysts in type I collagen gels (45). Although RA mutants are no longer activated by furin, these mutants have intact metalloproteinase domains and may be activated by mechanisms independent of furin cleavage of the propeptide, such as perturbation of zymogen structure through detergents used in zymography. Indeed, as shown in Fig. 4A, the RA mutants can be activated by the zymographic procedure to express gelatinolytic activity in gel, indicating that these mutants retain an intact catalytic mechanism (Lane 2). We then assayed for the ability of the RA mutants to form cysts from dispersed singular cells in three-dimensional collagen gels as described (45). Shown as representatives in Fig. 4B, the WT MT3- MMP-transfected MDCK cell formed a bigger cyst than cells transfected with the expression vector alone or the RA and EA mutants (panels 2, 1, 3, and 4, respectively). In fact, the RA mutant behaved Fig. 5. BFA and A23187 blocks the processing of WT MT3-MMP. A, control- similar to the catalytically inactive mutant EA (Fig. 4B, panels 3 transfected MDCK cells (Lane 7) and a MT3-MMP stable clone (WT2, Lanes 1–6 and 8–13) were seeded in six-well plates to 50% confluence, grown in serum-free medium versus 4), suggesting that the RA mutant expresses little or no pro- overnight, before being assayed for proMMP-2 activation in a 24-h period in DMEM teolytic activity in three-dimensional collagen gel. Thus, we conclude supplemented with 5% FBS containing 1:1000 methanol (Lanes 1, 7, and 8), 0.5 (Lanes 2 and 9), 1.0 (Lanes 3 and 10), 2.5 (Lanes 4 and 11), 5.0 (Lanes 5 and 12), and 10.0 (Lanes that zymogen activation of MT3-MMP is required for the enhanced 6 and 13) ␮g/ml of BFA. Aliquots (5 ␮l; Lanes 1–8) of the conditioned medium (600 ␮l growth of MDCK cells in three-dimensional type I collagen lattice. total/well) were analyzed by gelatin zymography as described in Fig. 2. PBS washed cells Inhibition of MT3-MMP Activation by BFA and A23187. To were lysed and analyzed by immunoprecipitation and Western blot as described in Fig. 2 (Lanes 9–15). Note the pro- (top) and activation intermediate (lower) for MMP-2 bands further define the intracellular compartment for MT3-MMP activa- in the upper panel for zymography. The pro- and active species of MT3-MMP are marked tion, we treated the cells with BFA, which blocks the trafficking on the right. B, similar cells from A were treated with vehicle alone (Lanes 1, 7, and 8), between the ER to Golgi apparatus (52) but permits furin autoactiva- 50 (Lanes 2 and 9), 125 (Lanes 3 and 10), 250 (Lanes 4 and 11), 500 (Lanes 5 and 12), and 1000 (Lanes 6 and 13) ␮g/ml A23187. The supernatants were analyzed for proMMP-2 tion (53). As shown in Fig. 5A, BFA inhibited the MT3-MMP- activation, and lysates were analyzed by Western blot for MT3-MMP processing as mediated activation of proMMP-2 in a dose-dependent fashion (Lanes described in A. Note that MMP-9 (arrowheads) is expressed by MDCK cells constitu- tively, and the proMMP-2 (arrows) is from the 5% fetal bovine serum supplemented into 1–6), apparently by preventing the activation of proMT3-MMP the culture medium as described (45). The activation intermediates at 62 kDa were marked (Lanes 7–13). This is quite surprising, given the fact that furin could by horizontal bars beneath the arrow in A, Lanes 1–6, and B, Lanes 1–6. 678

Fig. 6. Colocalization between MT3- MMP and furin. MDCK cells transfected with WT MT3-MMP were either treated alone (A–C) or with CMK (D–F), BFA (G– I), or A23187 (J–L) for 24 h. The fixed slides were stained for MT3-MMP with M2 monoclonal antibody (A, D, G, and J) and anti-furin antibody (C, F, I, and L). Z-series of confocal images were acquired on a Bio- Rad MRC confocal system, and one representative section selected from the middle of the cells is shown here. Merged pictures for both MT3 and furin were presented in the middle column as indicated. Note that MT3- MMP is colocalized with furin, a pattern not disturbed by CMK, BFA, and A23187.

colocalization does not depend on the furin motif (data not shown). To DISCUSSION see whether the catalytic activity of furin is required, we treated cells with CMK, which blocked MT3-MMP activation, as shown in Fig. 3. Zymogen activation constitutes a critical step in proteolysis- As shown in D–F of Fig. 6, CMK did not alter the colocalization regulated biological functions. For apoptosis, the caspase-based apo- pattern between furin and MT3-MMP either, suggesting that furin ptotic mediators are prepackaged inside every cell and regulated activity is not required for the observed colocalization. BFA, a com- through a cascade of zymogen activation steps (2). For members of pound known for blocking trafficking between ER to Golgi, was the MMP and ADAM family of proteinases, zymogens seem to be shown to block proMT3-MMP activation (Fig. 5A, M) but also failed maintained by a cysteine-switch, and activation involves the removal to prevent the colocalization between furin and MT3-MMP (Fig. 6, of the prodomain located NH2-terminal to the catalytic domains (13, G–I). Similarly, A23187, a calcium ionophore known to block furin 55). Although various mechanisms have been proposed for the acti- activation (53) and processing of proMT1-MMP (54) and proMT3- vation of proMMPs, the furin pathway has been recognized increas- MMP (Fig. 5B), did not interfere with the colocalization pattern ingly as a physiologically significant mechanism (26, 56, 57). In between furin and MT3-MMP (Fig. 6, J–L). Together, the interactions MMP-11, furin mediates zymogen activation by recognizing a con- between furin and MT3-MMP as evaluated by colocalizations are served RXK/RR motif sandwiched between its pro- and catalytic relatively strong and resistant to a wide variety of interventions. domains and cleaving off the prodomain in the trans-Golgi network 679

(26, 51). The same mechanism of activation may be used by nine the trans-Golgi network may regulate ECM remodeling by activating MMPs, all harboring a similar RXK/RR motif (Fig. 1). In this report, those proMMPs with RXKR motifs in transit to cell surface. we analyzed the activation of MT3-MMP by furin and demonstrated 116 that both the R RKR motif and furin are required for the activation REFERENCES process. However, neither the furin recognition motif nor its enzymic 1. Green, D. R. Apoptotic pathways: paper wraps stone blunts scissors. Cell., 102: 1–4, activity appears to be required for the observed colocalization. 2000. Implication for MMP Latency and Activation. The cysteine- 2. Stennicke, H. R., and Salvesen, G. S. Caspases—controlling intracellular signals by switch model predicts that MMPs are kept latent by the interaction of protease zymogen activation. Biochim. Biophys. Acta, 1477: 299–306, 2000. 3. 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Tiebang Kang, Hideaki Nagase and Duanqing Pei

Cancer Res 2002;62:675-681.

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