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[Cancer Research 62, 675-681, February 1, 2002]
© 2002 American Association for Cancer Research

Biochemistry and Biophysics

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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases implicated in tumor invasion and metastasis, must undergo zymogen activation prior to expressing any proteolytic activity. Although the cysteine-switch model predicts the well-established autoactivation process, ~40% of the known MMPs possess a conserved RXKR motif between their pro- and catalytic domains and, thus, could be activated directly by members of the proprotein convertase family. To further understand this process, we analyzed the activation of proMT3-MMP as a model system. We demonstrated that the conversion of MT3-MMP zymogen into active form is dependent on both the furin-type convertase activity and the R116RKR motif. Consistently, MT3-MMP was colocalized with furin in the trans-Golgi network by confocal microscopy. However, neither furin activity nor its recognition site in MT3-MMP is required for the observed colocalization. In fact, the colocalization pattern remains intact, even in the presence of brefeldin A, an agent known to block endoplasmic reticulum to Golgi trafficking. Yet, brefeldin A completely blocked the activation of MT3-MMP. A23187, a calcium ionophore known to block furin maturation, also blocked proMT3-MMP activation but had minimal effect on the colocalization between MT3-MMP and furin. Thus, furin processes MT3-MMP zymogen in the trans-Golgi network, where they colocalize independently of their apparent enzyme-substrate relationship.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A key regulatory mechanism in controlling proteolysis is zymogen activation, as demonstrated in several biological processes including apoptosis (1 , 2) , Alzheimer’s disease (3, 4, 5, 6) , and tumor necrosis factor-{alpha}-mediated inflammatory responses (7) . During tumor invasion and metastasis, the ECM3 barriers are breached in part by members of the MMP family that all require zymogen activation (8, 9, 10, 11, 12) . The latency and activation of MMPs is known to be governed by a cysteine-switch; a cysteine within the conserved PRCGVPD motif keeps MMP latent by binding to the catalytic zinc (13) . The disengagement of this Cys-Zinc bond may lead to autoactivation (13) . The direct visualization of this Cys-Zinc interaction was achieved in proMMP-2 by X-ray crystallography (14) , validating the latency mechanism (13) . In in vitro studies, MMP activations have been shown with conditions such as oxidation, proteolysis, freeze/thaw, and mild denaturation that can disrupt the Cys-Zinc bond and trigger the autoactivation process (reviewed in Refs. 11 , 15 , and 16 ). In vivo, the urokinase-type plasminogen activator/tissue-type plasminogen activator and plasminogen axis has been implicated in the activation of some proMMPs, especially proMMP-1 and proMMP-9 zymogens (17 , 18) . Recently, an emerging paradigm of MMP activation has been recognized that centers on the membrane-bound MMPs that can process some soluble MMPs, such as proMMP-2 and proMMP-13, into active forms (16 , 19, 20, 21, 22) . The first member of this group was MT1-MMP, which can specifically cleave at Asn-Leu within the prodomain of MMP-2, triggering an autoactivation process to generate the active form of MMP-2 at Y112 (20 , 21 , 23) . MT3-MMP and MT5-MMP were subsequently cloned and shown to be able to activate proMMP-2 as well (24 , 25) . Yet, the predominant pathway for MMP activation appears to be mediated by furin or its related PCs (19 , 26) . The PCs are a family of serine proteinases that recognize dibasic or RXK/RR motifs and cleave the peptide bond on the COOH side (27, 28, 29) . Responsible for the processing of various proproteins in the secretory pathway, furin or its related PCs have been demonstrated to process proMMP-11 and proMT1-MMP, or non-MMP enzymes like tumor necrosis factor-{alpha} convertase, ß-amyloid converting enzyme, and aggrecanases (5, 6, 7 , 19 , 26 , 30 , 31) . Within the MMP superfamily, at least 9 MMPs (of a total of ~24) contain RXK/RR motifs and, thus, could be processed into active forms by furin or its related PCs (19 , 24 , 25 , 32, 33, 34, 35, 36, 37) . Despite the potential importance of this activation mechanism, the cellular events regulating the interactions between this subset of MMPs and the PCs remain poorly defined.

MT3-MMP was initially cloned from malignant oral melanoma and subsequently identified in malignant tumors, smooth muscle cells, and inflamed cornea tissues (25 , 38, 39, 40, 41, 42, 43, 44) , suggesting that MT3-MMP may play a significant role in mediating ECM degradation during inflammation and tumor cell invasion and metastasis. Mechanistically, MT3-MMP was demonstrated to interact with proteoglycans on the melanoma cell surface to confer an invasive phenotype (39) . On the other hand, the expression of MT3-MMP appears to enhance the growth of MDCK cells in a three-dimensional gel lattice made of purified type I collagen (45) . The observed enhancement is dependent on MT3-MMP activity because its catalytically inactive mutant failed to produce any growth advantage under similar conditions (45) . Consistently, we have observed the processing of MT3-MMP zymogen into an active form by MDCK cells (45) . In this report, we continue our study on the structure and function of MT3-MMP in this model system by analyzing the cellular mechanisms leading to its zymogen activation. We demonstrate that MT3-MMP is activated by the archetypal PC, furin, which accomplishes the activation process by colocalizing with its substrate in the trans-Golgi network.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents.
MDCK and its derivatives are generated and maintained as described (46) . COS cells are maintained in 5% FBS DMEM. Cell culture media and supplements were purchased from Life Technologies, Inc. (Rockville, MD). Rabbit anti-MT3-MMP antisera were raised against a GST-MT3-MMP fusion protein as described (47) . Rabbit polyclonal anti-furin convertase antibody was purchased from Affinity Bioreagents, Inc. (Golden, CO). Brefeldin A, M2 antibody, and other immunological reagents were from Sigma Chemical Co. (St. Louis, MO). CMK-based furin inhibitor was purchased from Bachem (Philadelphia, PA).

Expression Constructs.
Wild-type and EA mutant constructs are detailed elsewhere (45) . The MT3-MMP RA mutation was created by sequential PCR using two primers covering the furin motifs C7307 (CCT GTC AAT GCA TAT GCC GCT GCA GCA ATA TGA AAT) and C7308 (TAT GCA TTG ACA GGA CAG) and cloned into pCR3.1 expression vector as described previously (24 , 26) .

DNA Transfection and Generation of Stable MT3-MMP Transfectants.
pCR3.1 MT3-MMPRA were transfected into MDCK cells by Lipofectamine (Life Technologies, Inc., Rockville, MD), and stable clones were selected in the presence of G418. The stable clones were screened by Western analysis of the cell lysates with M2 antibody and zymographic analysis of proMMP-2 activation. Positive cells were further analyzed by Northern blotting using MT3-MMP cDNA as a probe (see below). Seven representative clones named WT2, WT7, RA16, RA17, EA6, and EA15 were included in the present study.

Western Blotting, Immunoprecipitation, and Gelatin Zymography.
The basic protocols for these procedures are essentially the same as described previously (20 , 24) . Briefly, for zymography, confluent cultures were washed three times with PBS and allowed to incubate in the presence of DMEM supplemented with the proMMP-2 from 5% fetal bovine serum in the medium. dec-Arg-Val-Lys-Arg-CMK, a specific inhibitor of furin convertase (48 , 49) , calcium ionophore A23187, or BFA (a blocker of ER to Golgi trafficking; Ref. 50 ), were subsequently included in the medium as needed. After 24 h of incubation, medium was collected and cleared of cell debris by centrifugation and analyzed by SDS-PAGE impregnated with gelatin (1 mg/ml) as described (20 , 24) . For immunoprecipitation and Western Blot, cells grown in six-well plates were lysed in 250 µl of RIPA buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 0.25% sodium deoxycholate, 0.1% NP40, 10 µM leupeptin, 0.1 µM 5-p-amidinophylmethanesulfonyl fluoride, and 1 µM aprotinin] supplemented with 10 mM of EDTA to protect the active forms of MT3-MMP from degradation. The lysates were centrifuged at 14,000 x g for 20 min to remove the cell debris. Rabbit polyclonal anti-MT3-MMP antiserum (1.5 µl/reaction) was added to the resulting supernatants and allowed to incubate at 4°C for 1 h. The immune-complex was collected with protein-A/G PLUS agarose (10 µl; Santa Cruz Biotechnology), washed with RIPA buffer four times, and then eluted with 2x SDS-PAGE sample buffer under reducing conditions. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes and probed with M2 anti-FLAG mouse monoclonal antibody and developed as described (20 , 24) .

Northern Blot.
Total RNAs were isolated from cells with TRI-Reagent as described by the manufacturer (Molecular Research Center, Columbus, OH). Equal amounts of total RNAs (5 µg) 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 18S+ 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 x 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 µM 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 h at 37°C in 20 mM sodium phosphate (pH 8.0), 30 mM EDTA, 0.5% NP40, 0.1% SDS, and 0.5% ß-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 x 103) were mixed with 250 µl of collagen (2 mg/ml; Collaborative Research, Bedford, MA) and allowed to gel at 37°C in 24-well plates to give rise to a three-dimensional collagen matrix. Fresh medium containing 95% DMEM and 5% fetal bovine serum were added to the wells and changed every 2 days. After 12 days, MDCK and its derivatives were photographed by a video camera attached to a Nikon microscope at the University of Minnesota Bioimaging Processing facility as described (45) .

Immunostaining and Confocal Microscopy.
Cells seeded on coverslips in six-well plates were treated for 24 h with or without CMK, BFA, or A23187. After fixations for 30 min at room temperature, the cells were permeabilized for 1 h with buffer A (0.3% Triton, 1% BSA, and 0.01% NaN3 in PBS) and incubated with primary antibody (1:50 or 1:100 dilution in buffer A) for 3 h and then secondary antibody conjugated with FITC or Rhodamine Red (Jackson ImmunoResearch Laboratory, Inc., West Grove, PA) for 1 h. The cells were washed three times in PBS and mounted. Confocal microscopy was carried out in the Biomedical Image Processing Laboratories at the University of Minnesota as described (45) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
General Consideration of MMP Latency and Zymogen Activation.
If the cysteine-switch can be considered as the "off" signal for MMP latency with the exception of MMP-23 (13 , 34) , the RXKR motif initially recognized for its role in MMP-11 zymogen activation could be viewed as a switch turning on MMP activity (Fig. 1Citation ; Refs. 26 , 51 ). To date, a list of MMPs possessing a similar RXKR motif has been discovered and presented in Fig. 1Citation in the context of the PRCXXPD latency motif. We propose that this subfamily of MMPs has clearly defined "off/on" signals for latency and activation (Fig. 1)Citation . Because the activation process mediated by furin or its related PCs is accomplished in a single step without evoking any autocatalytic mechanism, this mode of activation should be distinguished from the autocatalytic activation process predicted by the cysteine-switch model (13) .



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  		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 aligned to show the conservation of the cysteine-switch (underlined) as the signal for latency (13) and the RXKR motifs (boxed) as the signal for zymogen activation (26) . Downward arrow, the site for maturation cleavage. Lower panel, representatives from 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 domain; H, hinge region; Pexin, hemopexin-like domain; TM, transmembrane domain; C, cytosolic domain.

 
Activation of proMT3-MMP by a Furin-like Activity.
Previously, we have shown that MT3-MMP stable transfectants are able to activate proMMP-2 (45) . We infer that transfected MT3-MMP be activated by the cellular machinery (45) . On the basis of the presence of the R116RKR motif between its pro- and catalytic domain (Fig. 1)Citation , we hypothesize that MT3-MMP is activated via a furin-mediated pathway as described for MMP-11 and MMP-14 (26 , 31 , 51) . To test 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, 49, 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. 2Citation , 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. 2Citation , upper panel), a dose-dependent inhibition of MT3-MMP activation by CMK was observed (Fig. 2Citation , lower panel). These results suggest that furin activity is required for the activation of proMT3-MMP.



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  		Fig. 2. CMK furin inhibitor blocks the processing of proMT3-MMP. MDCK cells stably transfected with vector (Lanes 1, 2, and 9) or MT3-MMP (WT2, Lanes 3–8 and 10–15) were grown in six-well plates to 50% confluence, treated with serum-free medium 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 10), 6 µM (Lanes 4 and 11), 12 µM (Lanes 5 and 12 ), 25 µM (Lanes 6 and 13), 50 µM (Lanes 2, 7, and 14), and 100 µM (Lanes 8 and 15) CMK. Aliquots (5 µl; Lanes 1–8) of the conditioned medium (600 µl 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 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 observed processing. To prove this hypothesis, we generated a mutant designated MT3-MMP-RA that converts the R116RKR motif into A116AAA (Fig. 3A)Citation . A panel of stable MT3-MMP-RA transfectants were generated in MDCK cells, expressing low to moderate levels of MT3-MMP-RA (data not shown). Two clones were analyzed further and presented along with two wild-type clones in Fig. 3Citation . We measured the levels of expression in these clones by Northern blot analysis and demonstrated in Fig. 3BCitation that WT and RA clones all express a comparable amount of mRNA (a, Lanes 2–5). When their ability to process proMMP-2 was assessed, it was apparent that the RA transfectants could no longer cleave proMMP-2, whereas the WT clones could (Fig. 3BCitation , c, Lanes 2 and 3 versus Lanes 4 and 5). To confirm the activation status of MT3-MMP in these stable clones, cell-associated MT3-MMPs were analyzed by immunoprecipitation coupled with Western blotting as described in Fig. 2Citation . Shown in b of Fig. 3BCitation are the protein species for WT and RA clones. In contrast to the WT clones, the RA type mutants failed to express any active species, thus demonstrating that the R116RKR motif is required for the processing of proMT3-MMP zymogen (Fig. 3BCitation , b, Lanes 4 and 5 versus Lanes 2 and 3).



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  		Fig. 3. Mutational analysis of the PC recognition motif R116RKR in MDCK stable transfectants. A, a schematic illustration of WT and RA mutants of MT3-MMP. Full-length MT3-MMP cDNA was cloned downstream of the cytomegalovirus promoter/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 resulted in the RA mutant, as described in "Materials and Methods." Abbreviations are as in Fig. 1BCitation . Characterizations of MT3-MMP transfectants. MDCK cells transfected with 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 described in "Materials and Methods." Note that 28S rRNAs were stained to show 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 immunoprecipitation and immunoblotting as described in Fig. 2Citation ; and c, zymography to detect proMMP-2 activation by WT and RA MT3-MMP transfectants. Note that the 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 indicated by horizontal bars on the right. C, posttranslational modifications of WT, EA, and RA mutant proteins of MT3-MMP. Immunoprecipitates from WT (WT2, Lanes 1 and 4), EA (EA6, Lanes 2 and 5), or RA (RA17, Lanes 3 and 6) stable clones were obtained as described in Fig. 3Citation , subjected to treatment with N-glycanase F (Lanes 4–6) overnight, and analyzed by Western blot with anti-FLAG M2 antibody as described in "Materials and 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 the hyperglycosylated MT3-MMP species are indicated by arrows, as indicated on the right.

 
Inhibitions of proMT3-MMP Activation Alter Its Pattern of Posttranslational Modification.
The RA species in Fig. 3BCitation exhibited higher molecular weights than its WT counterparts (Fig. 3BCitation , 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. 2Citation , 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) , deglycosylation converted the four bands in WT and EA mutants to two species, representing pro and active forms, respectively (Fig. 3CCitation , Lanes 1 and 2 and Lanes 4 and 5). In contrast, deglycosylation of RA converted only some of the proforms to the one detected in the WT clones (Fig. 3CCitation , Lanes 3 and 6), revealing a novel higher species of MT3-MMP (Fig. 3CCitation , Lane 6). These data suggest that furin-mediated processing of MT3-MMP zymogen may regulate its trafficking through the Golgi network where glycosylation takes place.

Zymogen Activation Is Required for MT3-MMP to Exert Its Biological Function.
In our previous studies, we have established an in vitro biological assay for MT3-MMP activity, i.e., the enhanced 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. 4ACitation , 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. 4BCitation , 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 similar to the catalytically inactive mutant EA (Fig. 4BCitation , panels 3 versus 4), suggesting that the RA mutant expresses little or no proteolytic activity in three-dimensional collagen gel. Thus, we conclude that zymogen activation of MT3-MMP is required for the enhanced growth of MDCK cells in three-dimensional type I collagen lattice.



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  		Fig. 4. Proteolytic activity of RA mutant transfectants. A, RA mutant of MT3-MMP retains an intact catalytic mechanism. Cells from MT3-MMP RA mutant stable line 17 (RA17) were expanded. Confluent cultures were washed with PBS three times and lysed in 36 ml of 1% Triton X-100 in TBS containing 10 µM 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 loaded onto an M2 column. After extensive wash with the extraction buffer, the column was eluted with FLAG peptide (75 µg in 5 ml of TBS). The purified RA MT3-MMP proteins were analyzed by Western blotting (5 µl) using rabbit polyclonal anti-MT3-MMP antisera (Lane 1) or gelatin zymography (20 µl; Lane 2) as described in "Materials and Methods." Note that the MT3-MMP RA protein degrades gelatin in the zymogram (Lane 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 (panel 1), WT (WT2, Lane 2), RA mutants (RA17, Lane 3), or EA mutants (EA6, Lane 4) were mixed with 250 µl 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 incubation, the cells in three-dimensional collagen gel were photographed and presented as indicated (45) . These four panels were representatives of cells from three independent experiments taken under the same microscope with similar settings.

 
Inhibition of MT3-MMP Activation by BFA and A23187.
To further define the intracellular compartment for MT3-MMP activation, we treated the cells with BFA, which blocks the trafficking between the ER to Golgi apparatus (52) but permits furin autoactivation (53) . As shown in Fig. 5ACitation , BFA inhibited the MT3-MMP-mediated activation of proMMP-2 in a dose-dependent fashion (Lanes 1–6), apparently by preventing the activation of proMT3-MMP (Lanes 7–13). This is quite surprising, given the fact that furin could be activated in the ER compartment without being transported to the Golgi apparatus (53) . Furthermore, this result suggests that the transactivation of MT3-MMP by furin requires the proper microenvironment 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. 5BCitation , 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.



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  		Fig. 5. BFA and A23187 blocks the processing of WT MT3-MMP. A, control-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 overnight, before being assayed for proMMP-2 activation in a 24-h period in DMEM 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 6 and 13) µg/ml of BFA. Aliquots (5 µl; Lanes 1–8) of the conditioned medium (600 µl total/well) were analyzed by gelatin zymography as described in Fig. 2Citation . PBS washed cells were lysed and analyzed by immunoprecipitation and Western blot as described in Fig. 2Citation (Lanes 9–15). Note the pro- (top) and activation intermediate (lower) for MMP-2 bands in the upper panel for zymography. The pro- and active species of MT3-MMP are marked on the right. B, similar cells from A were treated with vehicle alone (Lanes 1, 7, and 8), 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) µg/ml A23187. The supernatants were analyzed for proMMP-2 activation, and lysates were analyzed by Western blot for MT3-MMP processing as described in A. Note that MMP-9 (arrowheads) is expressed by MDCK cells constitutively, and the proMMP-2 (arrows) is from the 5% fetal bovine serum supplemented into the culture medium as described (45) . The activation intermediates at 62 kDa were marked by horizontal bars beneath the arrow in A, Lanes 1–6, and B, Lanes 1–6.

 
Colocalization between Furin and MT3-MMP.
The requirement of ER to Golgi trafficking for MT3-MMP activation, as evident with BFA treatment in Fig. 5Citation , suggests strongly that MT3-MMP and furin may be colocalized in the trans-Golgi network, where furin is highly concentrated (27) . To gain further support for the idea that furin activates proMT3-MMP in the trans-Golgi network, we investigated the cellular localization of furin and MT3-MMP by confocal microscopy. MT3-MMP-transfected MDCK cells were fixed and incubated with rabbit anti-furin antibody (Affinity Laboratories) and anti-FLAG monoclonal antibody (Sigma Chemical Co.) and then Rhodamine Red conjugated goat antirabbit antibody and FITC-conjugated rabbit antimouse antibody, respectively. The double-labeled cells were subsequently sectioned optically with z-series on a Bio-Rad confocal system and presented in Fig. 6, A–CCitation . Extensive overlaps between MT3-MMP and furin were observed in the perinuclear ER/Golgi complex. In contrast, some of the MT3-MMP signals were observed around the edges of plasma membrane, where very little furin was present (Fig. 6, A–C)Citation . To see whether the colocalization pattern is dependent on furin activity or the RXKR motif on MT3-MMP, we analyzed the distribution patterns of furin and the RA mutants. Surprisingly, the colocalization pattern between furin and MT3-MMP is also observed for RA mutants and furin, suggesting that the observed colocalization does not depend on the furin motif (data not shown). To see whether the catalytic activity of furin is required, we treated cells with CMK, which blocked MT3-MMP activation, as shown in Fig. 3Citation . As shown in D–F of Fig. 6Citation , CMK did not alter the colocalization pattern between furin and MT3-MMP either, suggesting that furin activity is not required for the observed colocalization. BFA, a compound known for blocking trafficking between ER to Golgi, was shown to block proMT3-MMP activation (Fig. 5A, M)Citation but also failed to prevent the colocalization between furin and MT3-MMP (Fig. 6, G–I)Citation . Similarly, A23187, a calcium ionophore known to block furin activation (53) and processing of proMT1-MMP (54) and proMT3-MMP (Fig. 5B)Citation , did not interfere with the colocalization pattern between furin and MT3-MMP (Fig. 6, J–L)Citation . Together, the interactions between furin and MT3-MMP as evaluated by colocalizations are relatively strong and resistant to a wide variety of interventions.



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  		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.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Zymogen activation constitutes a critical step in proteolysis-regulated biological functions. For apoptosis, the caspase-based apoptotic mediators are prepackaged inside every cell and regulated through a cascade of zymogen activation steps (2) . For members of the MMP and ADAM family of proteinases, zymogens seem to be maintained by a cysteine-switch, and activation involves the removal of the prodomain located NH2-terminal to the catalytic domains (13 , 55) . Although various mechanisms have been proposed for the activation of proMMPs, the furin pathway has been recognized increasingly as a physiologically significant mechanism (26 , 56 , 57) . In MMP-11, furin mediates zymogen activation by recognizing a conserved RXK/RR motif sandwiched between its pro- and catalytic domains and cleaving off the prodomain in the trans-Golgi network (26 , 51) . The same mechanism of activation may be used by nine MMPs, all harboring a similar RXK/RR motif (Fig. 1)Citation . In this report, we analyzed the activation of MT3-MMP by furin and demonstrated that both the R116RKR motif and furin are required for the activation process. However, neither the furin recognition motif nor its enzymic activity appears to be required for the observed colocalization.

Implication for MMP Latency and Activation.
The cysteine-switch model predicts that MMPs are kept latent by the interaction of the conserved Cys residue in the prodomain and the catalytic zinc, and the disruption of this interaction leads to autocatalytic removal of the prodomain, i.e., autoactivation (13) . This model applies universally to both MMPs and the newly discovered ADAM family, with MMP-23 as the only exception (11 , 32 , 34) . Autoactivation plays a significant part in the generation of mature and active enzymes for a subset of MMPs such as proMMP-1 and proMMP-2 (21 , 58, 59, 60, 61) . MMPs with a RXKR furin recognition motif, on the other hand, may be activated by furin or its related PCs with a single specific cleavage at the COOH side of the basic motif, as defined for MMP-11 and MT1-MMP (20 , 26 , 31 , 51 , 57) and MT3-MMP (this report). The apparent activation of the catalytically inactive MT3-MMP EA mutant by furin further distinguishes this activation mechanism from autoactivation. Thus, for MMPs including MMP-11, MMPs 14–18, MMP-24, MMP-25, and MMP-27, both the latency mechanism as encoded by the cysteine-switch and the activation process signaled by the RXKR motif are present as clearly identifiable sequence signals for latency and activation (Fig. 1)Citation . For the rest of the MMPs except MMP-23, they contain only the latency switch, i.e., the cysteine switch, but lack a clearly identifiable signal for activation. The only exception may be the N37-L bond within the prodomain of proMMP-2, which is apparently recognized and cleaved by MT1-MMP (20 , 21 , 23) . Because the cleavage at N37-L does not result in the activation of proMMP-2, this N37-L only qualifies as a quasi activation switch. MMP-23, on the other hand, possesses an RXKR activation signal but lacks a cysteine-switch for latency (34) . Because all proteinases are encoded as zymogens, MMP-23 must contain an entirely novel mechanism for latency (34) . Evolved separately, the convergence of the cysteine-switch for latency and the RXKR motif for furin-mediated activation in this subset of MMPs offers a unique combination of efficiency, economy, and precision in controlling their proteolytic activities through latency and activation.

Significance of the Furin/PC Pathway in Zymogen Activations.
The CMK inhibitor of furin was effective in inhibiting the invasiveness of HT1080 cells, presumably through the inhibition of MMP zymogen activation including proMMP-2 and any endogenous MT-MMPs (56) . To date, nine MMPs including MMPs-11, MMPs 14–17, MMP-23, MMP-24, MMP-25, and MMP-27 all contain a cognate furin-cleavage site, RXK/RR and, thus, could be regulated by both the cysteine-switch latency mechanism and the furin-mediated activation process (19 , 24 , 25 , 34, 35, 36, 37 , 62 , 63) . In fact, all members of the ADAM family also contain a similar RXK/RR motif and thus could potentially be regulated by a similar pathway in the trans-Golgi network (30 , 64, 65, 66) . Because these non-MMPs seem to use a similar cysteine-switch as a latency mechanism (64) , we expect the proposed latency and activation model in Fig. 1Citation to be directly applicable in the ADAM enzymes. In addition to these MMPs and ADAMs, the ß-amyloid converting enzyme also contains a similar furin-recognition site for zymogen processing (5 , 6 , 67) . Given the fact that furin knockout mice are embryonic lethal, these proteinases are all potential downstream substrates and may participate in contributing to the lethal phenotype (68) . Future comparative investigations between mice lacking furin expression and its proteinase substrates such as MT3-MMP may yield a more comprehensive understanding of furin function in vivo. Nevertheless, the resident furin-type convertases of the trans-Golgi network may regulate ECM remodeling by activating those proMMPs with RXKR motifs in transit to cell surface.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by NIH Grant CA76308. Back

2 To whom requests for reprints should be addressed, at Department of Pharmacology, University of Minnesota, 321 Church Street, S.E., 6–120 Jackson Hall, Minneapolis, MN 55455. Phone: (612) 626-1468; Fax: (612) 625-8408; E-mail: peixx003{at}tc.umn.edu Back

3 The abbreviations used are: ECM, extracellular matrix; MT-MMP, membrane-type matrix metalloproteinase; BFA, brefeldin A; PC, proprotein convertase; FBS, fetal bovine serum; CMK, chloromethylketone; ADAM, a disintegrin and metalloproteinase; MDCK, Madin-Darby canine kidney; WT, wild type. Back

Received 5/29/01. Accepted 12/ 3/01.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Differential Inhibition of Membrane Type 3 (MT3)-Matrix Metalloproteinase (MMP) and MT1-MMP by Tissue Inhibitor of Metalloproteinase (TIMP)-2 and TIMP-3 Regulates Pro-MMP-2 Activation
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Co-recycling of MT1-MMP and MT3-MMP through the Trans-Golgi Network: IDENTIFICATION OF DKV582 AS A RECYCLING SIGNAL
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E. M. Majerus, X. Zheng, E. A. Tuley, and J. E. Sadler
Cleavage of the ADAMTS13 Propeptide Is Not Required for Protease Activity
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J. Nie and D. Pei
Direct Activation of Pro-Matrix Metalloproteinase-2 by Leukolysin/Membrane-type 6 Matrix Metalloproteinase/Matrix Metalloproteinase 25 at the Asn109-Tyr Bond
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M. Leighton and K. E. Kadler
Paired Basic/Furin-like Proprotein Convertase Cleavage of Pro-BMP-1 in the trans-Golgi Network
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G. Mayer, G. Boileau, and M. Bendayan
Furin interacts with proMT1-MMP and integrin {alpha}V at specialized domains of renal cell plasma membrane
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T. Kang, Y.-G. Zhao, D. Pei, J. F. Sucic, and Q.-X. A. Sang
Intracellular Activation of Human Adamalysin 19/Disintegrin and Metalloproteinase 19 by Furin Occurs via One of the Two Consecutive Recognition Sites
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