Chlorotoxin Inhibits Glioma Cell Invasion via Matrix Metalloproteinase-2*

Jessy Deshane, Craig C. GarnerDagger, and Harald Sontheimer§

From the Department of Neurobiology and Civitan International Research Center, University of Alabama, Birmingham, Alabama 35294

Received for publication, June 7, 2002, and in revised form, November 15, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Primary brain tumors (gliomas) have the unusual ability to diffusely infiltrate the normal brain thereby evading surgical treatment. Chlorotoxin is a scorpion toxin that specifically binds to the surface of glioma cells and impairs their ability to invade. Using a recombinant His-Cltx we isolated and identified the principal Cltx receptor on the surface of glioma cells as matrix metalloproteinase-2 (MMP-2). MMP-2 is specifically up-regulated in gliomas and related cancers, but is not normally expressed in brain. We demonstrate that Cltx specifically and selectively interacts with MMP-2 isoforms, but not with MMP-1, -3, and -9, which are also expressed in malignant glioma cells. Importantly, we show that the anti-invasive effect of Cltx on glioma cells can be explained by its interactions with MMP-2. Cltx exerts a dual effect on MMP-2: it inhibits the enzymatic activity of MMP-2 and causes a reduction in the surface expression of MMP-2. These findings suggest that Cltx is a specific MMP-2 inhibitor with significant therapeutic potential for gliomas and other diseases that invoke the activity of MMP-2.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Malignant gliomas are the most common primary intracranial tumors in man. They constitute a heterogeneous group of tumors associated with significant morbidity and mortality (1). Gliomas are characterized by a high invasive potential and display a wide diversity of histological features. They are derived from glial support cells in the brain and the vast majority are thought to be of astrocytic origin (2). Even low-grade gliomas infiltrate the entire brain, a feature that precludes their successful therapy (3). Although systemic spread is a rarity in malignant gliomas, invasion of tumor cells has been tracked predominantly in the basement membrane of blood vessels, the subependymal space, the glia limitans externa, and parallel and intersecting nerve fiber tracts in the white matter (4). Significant progress has been made in understanding pathways of glioma migration and invasion. Molecular mechanisms of brain tumor invasion are complex. They involve modification of receptor-mediated adhesive properties of tumor cells, degradation and remodeling of extracellular matrix (ECM)1 by tumor-secreted metalloproteinases, and creation of an intercellular space for invasion of tumor cells (5). Spatial and temporal regulation of ECM proteolytic degradation is maintained to ensure successful invasion of tumor cells through the ECM. It has been established through in vitro and in vivo glioma models that the deposition of ECM components occurs at the confrontation zone between glioma cells and normal brain tissue (6). In addition, paths of invading tumor cells are also enriched in ECM molecules that promote cell motility (7, 8). Cells interact with the ECM via integrins that play a major role in cell invasion. Indeed, functional blocking anti-integrin antibodies (9) and disintegrins, a group of snake venom toxins, can block migration and invasion via an RGD-sequence dependent interaction (10-12).

In addition to these interactions with the extracellular brain environment, invading glioma cells undergo dramatic shape and cell volume changes allowing them to make their way through the narrow extracellular spaces (13). This process appears to require the secretion of fluid along with Cl- ions, as several Cl- channel inhibitors including DIDS and tamoxifen have been shown to inhibit glioma cell invasion in vitro (13). Chlorotoxin (Cltx) is a 36-amino acid peptide that was originally isolated from Leiurus quinquestriatus venom (14) and has been shown to inhibit small conductance Cl- channels in colonic epithelial cells (14, 15). Cltx also inhibits Cl- fluxes across glioma membranes (13, 16). Immunohistochemical studies show that Cltx specifically and selectively binds to glioma cells (17) and radiolabeled Cltx targets tumor cells in mice bearing xenografted glioma tumors. Glioma cell migration and invasion into fetal brain aggregates is significantly reduced by Cltx (13). A recent survey of over 200 tissue biopsies from patients with various malignancies suggests that Cltx binds to the surface of gliomas and other embryologically related tumors of neuroectodermal origin (18) but not to normal brain.

The unusual specificity of Cltx for glioma tissues and its remarkable anti-invasive effect prompted us in the current study to isolate and identify the membrane receptor for Cltx. Affinity purification with a recombinant His6-Cltx followed by mass spectroscopy and sequencing identified the putative Cltx receptor as the matrix metalloproteinase-2 (MMP-2). MMP-2 co-purified as a major component of a stable macromolecular complex comprised of MMP-2, alpha vbeta 3 integrin, MT1-MMP, and TIMP-2, which are proteins involved in cell migration and invasion. Cltx inhibited the enzymatic activity of MMP-2 in vitro and reduced the surface expression of MMP-2 in glioma cells. Cltx was found to specifically bind to MMP-2 but not to MMP-1, MMP-3, and MMP-9, which are also expressed by glioma cells. These findings explain the specificity of Cltx for glioma in the intracranial animal model studies and also the previously documented anti-invasive potential of Cltx. Cltx may thus be a highly effective drug of therapeutic potential for gliomas and possibly other diseases that invoke the activity of MMP-2.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Cell Culture

Human glioma cell lines D54-MG (obtained from Dr. D. D. Bigner, Duke University, Durham, NC) and CCF-STTG-1 (ATCC, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium/F-12 supplemented with 2 mM L-glutamine (Invitrogen) and 7% heat inactivated fetal bovine serum (Hyclone). Cortical astrocytes were isolated from Sprague-Dawley rat pups at postnatal day 0 and cultured as described earlier (19).

Antibodies and Reagents

Polyclonal antibodies directed against MMP-2, MT1-MMP, TIMP-2, actin, as well as alpha -rabbit and alpha -mouse horseradish peroxidase-conjugated secondary antibodies, pure MMP-1, MMP-3, MMP-9, and filipin were purchased from Sigma. Monoclonal alpha -MMP-2 antibody was obtained from RDI Inc. (Flanders, NJ). Monoclonal antibodies for alpha vbeta 3 integrin and recombinant human MMP-2 were purchased from Chemicon (Temecula, CA). Alexa Fluor (488) goat anti-mouse IgG2a and Alexa red (546) goat anti-rabbit conjugated secondary antibodies (Molecular Probes) were used for immunofluorescence at the manufacturers recommended dilutions.

Synthesis and Purification of Recombinant Chorotoxin

As a first step toward biochemical isolation of the receptor for Cltx, a recombinant fusion protein, His-Cltx, was synthesized in Escherichia coli. To produce His-Cltx, we cloned Cltx into HindIII and EcoRI sites of pGHBE (a modified pGEX2T vector in which the EcoRI, SmaI, and BamHI sites were replace with HindIII, BamHI, and EcoRI). The cloned Cltx was then subcloned into the BamHI and EcoRI sites in pRSETB vector (Invitrogen). pRSETB has an N-terminal polyhistidine tag (His6), which allowed the purification of His-Cltx by immobilized metal affinity chromatography (Talon Resin, Clontech Labs). His-tagged fusion protein was purified as a 8-kDa protein using buffer conditions as described by the manufacturer.

Biological Activity of His-Cltx Was Assessed by Two Assays

Matrigel Invasion Assay-- Matrigel invasion chamber (BD Biosciences) inserts of 8 µm pore size were rehydrated following the manufacturer's recommendations. These chambers were then coated on the lower surface with vitronectin and used for invasion assay as described earlier for transwell migration assays (13). Briefly, glioma cells were plated at a density of 5 × 104 cells/filter. Cells were treated with 30 nM to 3 µM of either commercial peptide Cltx (Alomone, Jerusalem, Israel) or His-Cltx and further incubated at 37 °C for 24 h. Cells which remained in the upper chamber were scrubbed off the inserts and the invaded cells were fixed and stained with Diff-Quik stain kit (Dade Behring, VWR Scientific, Switzerland). The relative inhibition of invasion was calculated as decrease in the number of migrated cells in the presence of Cltx compared with untreated vitronectin-coated control. Dose-response curves were obtained from six samples each and data were averaged. Half-maximal inhibition of matrigel cell invasion was achieved with ~200 nM Cltx and did not differ between His-Cltx and commercial Cltx (Alomone).

Chloride Flux Measurements Using MEQ-- Glioma cells plated in a 96-well plate at 5000 cells/well and loaded with MEQ and Cl- fluxes across glioma membranes were assayed in the presence of 30 nM to 3 µM His-Cltx or native peptide Cltx (Alomone) using previously described methods (13) with minor modifications in preparation of MEQ dye. Briefly, MEQ was reduced to a yellow oil by 12% sodium borohydride under constant flow of nitrogen. When yellow oil separated out, the oil was subjected to two consecutive extractions in a 50% ether:water (v/v) mixture and then further dried under nitrogen. Experiments were performed at room temperature with gluconate-based buffers as described earlier. Inhibition of Cl- fluxes was directly compared for His6-Cltx and commercially available Cltx and found to be indistinguishable.

Overlay Assays (Far Westerns)

Briefly, proteins processed from membrane fractions, cytosolic fractions, or total cell lysates were analyzed by Far Western blots using protocols described before (20). Membranes were probed with 300-500 nM His-Cltx followed by detection of the bound proteins using an anti-His monoclonal antibody (Clontech, 1:5000).

Cell Lysis and Fractionation

Cultured cells were washed twice with cold PBS, scraped with cell scrapers, and pelleted at 2000 × g for 5 min at 4 °C. The cell homogenates were prepared by resuspending the cell pellet in 1.0 ml of homogenization buffer (10 mM Tris-Cl, pH 7.5, 0.32 M sucrose, 1 mM MgCl2, 5 mM CaCl2 supplemented with 10 µl/ml protease inhibitor mixtures I and II (mixture I: 1 mg/ml leupeptin, 1 mg/ml antipain, 5 mg/ml aprotinin, 10 mg/ml benzamidine hydrochloride, 10 mg/ml soybean trypsin inhibitor; and mixture II: 1 mg/ml pepstatin, 30 mM phenylmethanesulfonyl fluoride in dimethyl sulfoxide)) and homogenizing in tissue grinders for 1 min with incubations on ice at 1-min intervals. Cell debris was spun down at 2000 × g for 5 min at 4 °C and the supernatant was collected and fractionated and membrane fractions were processed for analysis as described earlier (21). Western blot analysis was performed as described (22). For immunoprecipitation analysis, samples were prepared and precleared as described earlier (22). The precleared lysate was incubated with Protein A beads already pre-reacted with rabbit serum (60 min at room temperature) for 2 h at room temperature. Beads were then centrifuged gently and supernatant was collected. Protein A beads were pre-reacted with primary antibody (1:1, v/v) and followed procedures for immunoprecipitation as described (22).

Biotinylation of Cell Surface Proteins

Cell surface proteins were biotinylated as previously described (21). Cells at 80% confluence in a 100-mm dish were incubated with 3.8 ml of a 1.5 mg/ml sulfo-NHS biotin (Pierce) in PBS/calcium/magnesium solution for 30 min at 4 °C with gentle shaking. Following quenching of biotinylation in PBS/calcium/magnesium plus 100 mM glycine, and further incubation for 30 min at 4 °C with gentle shaking, glycine solution was aspirated and rinsed twice with PBS/calcium/magnesium. Cells were then lysed and biotinylated cell surface fractions and intracellular fractions were separated and analyzed by immunoblotting as described (21).

Coupling Recombinant Cltx to Actigel-ALD

Recombinant chlorotoxin was coupled to Actigel-ALD (Sterogene, Carlsbad, CA) and stored following the manufacturer's recommendations. His-Cltx was added to Actigel-ALD (0.5 mg/ml of resin) followed by ALD-coupling solution (1 M NaCNBH3) to a final concentration of 0.1 M (0.2 ml/ml resin).

Affinity Purification

Four gorilla flasks of D54-MG cells were grown to confluence, yielding about 20 mg of total protein. Cell debris was removed by centrifugation at 2000 × g for 5 min at 4 °C and the supernatant was collected and re-centrifuged at 100,000 × g in a Beckman T70.1 rotor for 60 min at 4 °C. Pellets, representing the total cell membrane fraction, were resuspended in homogenization buffer (supplemented with protease inhibitors) containing 1% SDS followed by addition of a 7-fold excess volume of 1% Triton X-100. The samples were then heated to 48 °C for 5 min. This lysate was then precleared with unconjugated Actigel-ALD beads with end-over-end rotation (100-µl bead slurry per 1-ml lysate) for 4 h at 4 °C. Following a brief centrifugation at 100 × g, the supernatant was removed and incubated with the His-Cltx-conjugated Actigel-ALD beads (250 µl/ml of precleared lysate) for 4 h at 4 °C or overnight. The beads were then centrifuged for 2 min at 100 × g and unbound material was removed. The beads were extensively washed with homogenization buffer (supplemented additionally with 0.1% Nonidet P-40 and 0.01% Tween). The bound proteins were then eluted by boiling with Laemmli SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, and 600 mM 2-mercaptoethanol) for 5 min. Samples were separated on denaturing 8, 10, or 4-15% gradient gels by SDS-PAGE and further analyzed by Western blots and overlay assays. The identity of the receptor was determined following electrophoresis of the affinity purified fractions on a 4-15% gradient polyacrylamide gel, staining with Bio-Safe Coomassie (Bio-Rad), and excising the band of interest. The proteins were then destained and trypsinized and the protein digest extract was analyzed by a MALDI-TOF mass spectrometer (PE Biosystems, Framingham, MA). The peptide masses were entered into MASCOT to identify the protein by searching the NCBI data base. Sequence information was obtained with a Micromass Q-TOF-2 mass spectrometer.

Gelatin Zymography

Gelatin zymography was performed using 10% polyacrylamide gels containing 0.1% gelatin (Bio-Rad). Briefly, eluates from the affinity purification column were separated by SDS-PAGE on the gel, and following electrophoresis the gel was washed with 2.5% Triton X-100 for 1 h to remove SDS and incubated at 37 °C for 24 h in a buffer containing 50 mM Tris-Cl (pH 8.0), 5.0 mM CaCl2, and 1 µM ZnCl2, and 0.005% Brij buffer. The gel was then stained with Coomassie for 30 min and destained quickly for 5 min to reveal gelatinolytic activity as opaque unstained bands.

Gelatinase Activity Assay for Analysis of Modulation of Enzymatic Activity by Cltx

Gelatinase activity was analyzed utilizing a MMP gelatinase activity assay kit (Chemicon) provided with recombinant human MMP-2 following the manufacturer's recommendations. Briefly, purified human MMP-2 (Chemicon) was diluted in reaction buffer (50 mM Tris-Cl, pH 7.5, 0.15 M NaCl, 5 mM CaCl2) at concentrations ranging from 2.8 to 280 nM. 50-µl samples of these different concentrations of MMP-2 were transferred to a rehydrated biotin-binding plate and incubated for 30 min with 160 µl of 30-1000 nM Cltx in a 96-well plate in the presence of biotinylated gelatin. After incubation at 37 °C for 15 or 30 min, 100 µl of each sample was analyzed for MMP-2 activity (n = 8) as described by the manufacturer (Chemicon).

In Situ Zymographic Analysis of Cell Surface Gelatinolytic Activity in Vitro

To localize net gelatinolytic activity of MMPs by in situ zymography, fluorescein isothiocyanate-labeled DQ gelatin that is intramolecularly quenched (Molecular Probes) was used as a substrate for degradation by gelatinases as reported earlier (23). Proteolysis by gelatinases yields cleaved fluorescein isothiocyanate-gelatin peptides and the localization of this fluorescence indicates the sites of net gelatinolytic activity. Briefly, glioma cells were plated on 12-mm coverslips. After 24 h incubation, cells were treated with 30 nM Cltx, 300 nM Cltx, or 50 µM 1,10-phenanthroline for 30 min at 37 °C. Untreated cells served as negative control for this experiment. Cells were washed with PBS and then incubated with zymography reaction buffer (0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl2, and 0.2 mM NaN3, pH 7.6, the high concentration of azide prevented the gelatin from being phagocytosed and thus allowing cell surface gelatinolytic activity to occur) containing 100 µg/ml DQ gelatin at 37 °C overnight. At the end of the incubation period, without fixation or further washes, gelatinolytic activity of the MMPs was localized and photographed by fluorescence microscopy and images were acquired by Spot digital camera.

Immunofluorescence Studies of Glioma Cells

Glioma cells were plated at 40% confluency onto 12-mm round coverslips in appropriate tissue culture medium. After overnight incubation at 37 °C, medium was aspirated and replaced by serum-free medium containing irrelevant His-protein (pRsetB-His) for untreated controls and serum-free media containing His-Cltx (300 nM) and further incubated at 37 °C for 30 min. Cells were then washed with PBS and fixed for 10 min at room temperature in PBS containing 4% paraformaldehyde. Cells were rinsed, after fixation, with PBS (3 × 10 min). After a 30-min incubation at room temperature with blocking buffer (PBS containing 3% goat serum, 0.1% azide, and 0.3% Triton X-100 (for permeabilized samples), alpha -MMP-2 primary antibodies were added to the cells (1:500) and incubated overnight at room temperature in blocking buffer with or without Triton X-100. Cells were then rinsed (3 × 10 min) with PBS containing blocker and then incubated with Alexa red (1:750) secondary antibodies in blocker for 1 h at room temperature. Coverslips were mounted using gel mount following another 10-min wash in PBS. Immunofluorescence study was carried out using a Leica epifluorescence microscope (×40 oil and ×100 oil objectives), and images were captured using a digital spot camera.

Internalization Assay

Glioma cells were plated at a density of 5 × 105 cells in 100-mm tissue culture dishes in serum containing media. After overnight incubation at 37 °C, cells were washed and then incubated with serum-free media containing 300 nM His-Cltx, irrelevant His-protein (pRsetB-His), or 50 µM 1,10-phenanthroline for 30 min at 37 °C to allow sufficient internalization of His-Cltx. Cells were then washed and cell surface biotinylation was carried out as described above. Biotinylated cell surface fractions and intracellular fractions were analyzed using Western blots for quantitative levels of Cltx. Alternatively, cells were treated with filipin at 5 µg/ml for 15 min at 4 °C to flatten the caveolae prior to treatments with Cltx. Cells were then processed for cell surface biotinylation as described above to analyze the level and route of internalization of His-Cltx and associated MMP-2.

Statistical Analysis

Data were analyzed with Origin (version 6.0, MicroCal, Northampton, MA). All curve fitting was performed using curve fitting routine provided by this software. Analysis of variance was used for multiple comparisons of data. All values are reported as mean ± S.E. and percentages calculated from mean values.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Cltx Receptor on Glioma Membranes Is MMP-2-- To isolate and characterize the putative Cltx receptor, we synthesized recombinant Cltx with a His6 tag (His-Cltx) in E. coli. The biological activity of the recombinant His-Cltx was indistinguishable from that of commercially available peptide (see "Experimental Procedures"). Total cell lysates as well as crude membrane preparations of glioma cells were probed with His-Cltx and showed binding to a prominent protein with a corresponding molecular weight of ~70,000. This protein was not present in cultured rat astrocytes (Fig. 1a).


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Fig. 1.   The putative Cltx receptor is MMP-2, which is expressed in glioma cells but not in astrocytes. a, far Western blot of cell lysates from glioma cells and cortical astrocytes were probed with His-Cltx (500 nM) showing specific binding to a 72-kDa protein in D54-MG cells but not in astrocytes. b, expression of a Cltx receptor in plasma membrane and intracellular fractions of glioma cells. Immunoblot of cell surface-biotinylated D54-MG (WHO grade IV) and CCF-STTG1 (WHO grade III) glioma cell lines was initially probed with an anti-actin monoclonal antibody, stripped, and then overlaid with His-Cltx. The actin immunoreactive band is a control to positively identify the intracellular fractions. The 72-kDa band, bound by His-Cltx, is detected in both the plasma membrane and intracellular fractions. c, far Western blot of glioma cell membranes with His-Cltx. Cell membranes were affinity purified using His-Cltx-conjugated Actigel-ALD beads under stringent conditions (see "Experimental Procedures") to break interactions between proteins. Far Western blot with His-Cltx (500 nM) shows specific binding to a 72-kDa protein in D54-MG cell membranes but not to astrocyte membranes. d, mass spectrometric analysis and mass profile of the affinity purified receptor, in which the mass profile of the Cltx receptor was compared with internal bovine serum albumin standards.

To demonstrate that the expression of the Cltx-binding protein is on the cell surface, we used a cell surface biotinylation approach to distinguish between cell surface proteins and proteins contained in intracellular membranes. In Far Western blots with His-Cltx, we found that the Cltx-binding protein was present both in the cell surface membrane fractions in two glioma cell lines as well as intracellullar fractions (D54-MG and CCF-STTG1; Fig. 1b). Actin immunoreactivity, which served as a control, was only found associated with intracellular fractions (Fig. 1b).

For isolation and further purification of the Cltx receptor, His-Cltx was immobilized to Actigel ALD beads by chemical conjugation. Affinity purification of glioma membrane fractions (20 mg) using this column revealed that the putative Cltx receptor could be purified together with a number of other proteins that were mostly insoluble in 1% Triton X-100 (Fig. 3a). To disrupt this complex and selectively purify only Cltx-binding proteins, membrane fractions were sequentially treated with buffers containing 1% SDS and then a 7-fold excess volume of 1% Triton X-100. Samples were subsequently heated to 48 °C for 5 min before binding to the affinity column. These conditions were able to disrupt the complex and allow the isolation of a ~70-kDa protein, which retained binding to His-Cltx as assessed by Far Western blots (Fig. 1c). MALDI-TOF mass spectrometric analysis of this affinity purified 70-kDa protein following tryptic digestion of the excised protein band from SDS-PAGE revealed its identity to be the matrix metalloproteinase, MMP-2 (see mass profile Fig. 1d). Sequence information obtained with a Micromass Q TOF-2 mass spectrometer further confirmed the identity of the Cltx receptor as MMP-2 (Fig. 3b).

The overlay assays of affinity purified membrane fractions in Fig. 1c suggest that the Cltx receptor, most likely MMP-2, is only expressed in glioma cell membranes but not in astrocytes. To demonstrate that this lack of Cltx binding in astrocytes is because of the absence of MMP-2, we performed Western blot analysis of astrocyte and glioma cell lysates with rabbit alpha -MMP-2 antibodies (Fig. 2a). MMP-2 immunoreactivity was only seen in glioma cells but not in astrocytes explaining the lack of His-Cltx binding to astrocytes.


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Fig. 2.   Cltx specifically binds to MMP-2. a, gliomas express the various species of MMP-2 whereas astrocytes do not. Whole cell lysates from D54 glioma cells and primary astrocyte cultures were probed with MMP-2 antibodies showing binding to several molecular weight bands consistent with the 62,000, 68,000, and 72,000 species of MMP-2 in glioma cells but not in astrocytes. A ~20-kDa band that binds MMP-2 antibodies in astrocytes was not characterized. b, immunoblot with MMP-2 antibodies demonstrates binding to a 72-kDa band in His-Cltx affinity purified membrane fractions. This is the same molecular weight as that identified by His-Cltx in Western blots of glioma extracts. c, His-Cltx interacts with recombinant MMP-2, and shows the same immunoreactivity observed in membrane fractions from two glioma cells lines. His-Cltx Far Western blot of recombinant purified MMP-2 shown side-by-side with D54-MG and CCF-STTG1 cell membranes show protein bands at ~72,000 (arrow), the same molecular weight as recombinant MMP-2. Specific binding with astrocytic membranes was not observed. d, gelatin zymography of the His-Cltx affinity purified fractions show 3 opaque gelatinolytic protein bands. The most prominent two bands, between ~60,000 and ~75,000, and ~45,000 have similar molecular weights to that of various species of MMP-2 isoforms (45,000, 62,000, 65,000, 68,000, and 72,000) that have gelatinase activity. Left lane contains protein markers only. e, Cltx interacts specifically with MMP-2. Far Western blot with His-Cltx of purified recombinant MMP-2, MMP-9, MMP-1, and MMP-3 resulted in binding only to MMP-2. Coomassie-stained gel (bottom) indicates that the comparable amount of each protein was loaded on the SDS gel.

MMP-2 can exist in several molecular weight forms including a latent proenzyme of 72,000, a ~64,000 activation intermediate, a ~62,000 mature protease (24), and a 45,000 process active form (25, 26). In Western blots, MMP-2 antibodies recognized three bands at these molecular weights in total cell lysates from glioma cells (Fig. 2a). Following affinity purification of glioma membranes with His-Cltx, MMP-2 antibodies recognize primarily a 72-kDa band (Fig. 2b). This is consistent with previous reports showing this species to be the major form of MMP-2 on glioma membranes (27). The ability of Cltx to interact with MMP-2 in glioma cells and not astrocytes is supported by overlays demonstrating that His-Cltx binds to recombinant MMP-2, as well as bands of the same mobility in D54-MG and CCF-STTG1 glioma cell lines but not astrocytes (Fig. 2c). Proteins affinity purified from D54 membranes with His-Cltx were found to exhibit gelatinase activity in gelatin zyomograms (Fig. 2d). The mobility of the zymographically active bands appeared between ~60,000 and 75,000 and ~45,000, which are molecular weights consistent with the presence of the various MMP-2 species. Whether Cltx inhibits MMP-2 catalytic activity will be examined below.

Given that glioma cells express several MMPs including MMP-1, MMP-2, MMP-3, and MMP-9 (28) we sought to evaluate whether the zymographically active bands in Fig. 2d were because of an association of Cltx with MMP-2 or one of these other MMPs. To do this, purified MMP-1, -3, and -9 were run on gels and probed with His-Cltx. Our data show that Cltx only interacts with MMP-2 (Fig. 2e). Taken together, these findings suggest that MMP-2 is the principal receptor for Cltx on the surface of glioma cells.

Characterization of the Cltx Receptor Protein Complex-- As mentioned above, His-Cltx affinity purification of its receptor also led to the isolation of additional protein bands (Fig. 3a) that could be separated from MMP-2 by heating to 48 °C in SDS and Triton X-100. These data indicate that in the plasma membrane of glioma cells, the Cltx receptor is present in a macromoelcular complex with other proteins. To better understand the nature of this protein complex, membrane samples solubilized in Triton X-100 and affinity purified with His-Cltx were separate by SDS-PAGE, stained with Coomassie Blue, excised, and processed for mass spectrometry. Of the additional six major proteins bands seen by Coomassie Blue staining (Fig. 3a), mass spectrometric analysis was able to determine the identity of three as MT1-MMP, alpha v integrin, and TIMP-2 (Fig. 3b). Details pertaining to the identification by mass spectrometry are given in Fig. 3b. The presence of these proteins in His-Cltx-isolated material was confirmed by Western blot analysis with antibodies against alpha vbeta 3 integrin, MT1-MMP, and TIMP-2 (Fig. 3c). The co-existence of MMP-2 (Fig. 2) in a complex with MT1-MMP, alpha vbeta 3 integrin, and TIMP-2 is intriguing as it has previously been shown that MMP-2 is docked on the surface of cells via a protein complex that includes MT1-MMP, alpha vbeta 3 integrin, and TIMP-2 (29). MT1-MMP is presumed to play an important role in mediating the docking of MMP-2 proenzyme on the plasma membrane for efficient activation of the protease (24, 26, 30). To assess whether in glioma cells this protein complex occurs naturally or whether it is induced to form by His-Cltx, we immunoprecipitated glioma membranes with MMP-2 antibodies and probed the purified fractions with antibodies to alpha vbeta 3 integrin, MMP-2, MT1-MMP, or with His-Cltx (Fig. 3d). These immunoblots demonstrate that MMP-2, alpha vbeta 3 integrin, and MT1-MMP are indeed present in a complex on the surface of glioma cells in the absence of His-Cltx.


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Fig. 3.   Cltx binds to a protein complex containing several proteins implicated in glioma cell invasion. a, Coomassie Blue-stained SDS gel of the His-Cltx affinity purified glioma membrane fraction obtained under stringent conditions that break apart the interactions between the purified proteins (see text) shows several prominent protein bands. The molecular weight of these bands is consistent with their identity being integrin alpha v, MT1-MMP, TIMP-2, and MMP-2. b, mass spectrometric analysis of His-Cltx affinity purified macromolecular complexs. The table lists a mascot match score for protein bands affinity purified with His-Cltx from glioma membrane fractions and characterized by mass spectrometric analysis. Accession numbers and Swiss protein data bank identity numbers are also shown in the table. c, His-Cltx affinity purified complex contains integrin, MT1-MMP, and TIMP-2. Immunoblots of His-Cltx affinity purified D54-MG cell membrane fractions incubated with antibodies directed against alpha vbeta 3 integrin, MT1-MMP, and TIMP-2 confirming the presence of these proteins in the affinity purified Cltx-complex. D, the macromolecular complex of MMP-2, MT1-MMP, and integrin exist in the absence of His-Cltx. Immunoblots of D54-MG membranes after immunoprecipitation with MMP-2 antibodies were probed with antibodies to alpha vbeta 3 integrin, then stripped and re-probed with MMP-2, MT1-MMP, or His-Cltx (consecutively), to show existence of this protein complex in the absence of His-Cltx.

Cltx Binds to MMP-2 and Inhibits Its Catalytic Activity-- Tumor invasion, metastasis, and angiogenesis require controlled degradation of ECM. Increased expression of MMPs has been associated with these processes in malignant tumors of different histogenetic origin (31). In gliomas, up-regulation of MMP-2, MMP-9, and MT1-MMP characterizes high grade gliomas (glioblastoma multiformae) as opposed to low grade gliomas or nontransformed control brain tissues (27, 32, 33). Moreover, MMP-2 activity also modulates glioma cell migration and contributes significantly to their invasive potential (34). Consequently (35) several MMP inhibitors including 1,10-phenanthroline, cyclic peptides, and hydroxamate derivatives have been found to effectively block migration and invasion of tumor cells (36).

To gain a better understanding of how Cltx inhibits glioma cell invasion, we investigated whether Cltx could bind to and modulate the metalloprotease activity of MMP-2. To assess binding of His-Cltx to MMP-2, we used an enzyme-linked immunosorbent based binding assay with purified MMP-2 (Chemicon) immobilized on the plate. When we compare the efficiency of binding of His-Cltx versus an irrelevant control His-protein (Fig. 4a, pRsetB-His) to MMP-2, we found that His-Cltx binds MMP-2 in a dose-dependent manner with 50% binding achieved at ~115 nM (Fig. 4a).


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Fig. 4.   Cltx binds to MMP-2 and inhibits its catalytic activity. a, His-Cltx binds to pure recombinant MMP-2. Concentration dependent increase in binding of His-Cltx (3 nM to 3 µM) to MMP-2 was detected in a enzyme-linked immunosorbent based assay. y axis represents percent binding compared with irrelevant His-protein (pRsetB-His). 50% binding was achieved at 115 nM Cltx. b, Cltx inhibits gelatinase activity of purified MMP-2. Quantitative analysis of gelatinase activity of recombinant MMP-2 as a function of varying MMP-2 concentration (0.7 to 280 nM) in the presence or absence of 500 nM Cltx. c, dose-dependent inhibition of MMP-2 activity following treatment with Cltx. Dose-response curve of gelatinase activity of recombinant MMP-2 (0.7 nM) treated for 30 min at 37 °C with increasing concentrations of Cltx. Cltx inhibits MMP-2 in a dose-dependent manner.

We next examined the effect of Cltx on the catalytic activity of MMP-2. In the presence of 500 nM Cltx, a concentration reported to inhibit glioma cell invasion (13), gelatinase activity of MMP-2 was markedly reduced, as shown for MMP-2 concentrations of up to 70 nM in Fig. 4b. To examine the dose-dependent inhibition of MMP-2 by Cltx, we used a fixed concentration of MMP-2 (0.7 nM) that was within the linear range of the relative MMP-2 activity (Fig. 4b). Under these conditions, Cltx showed dose-dependent inhibition of MMP-2, which saturated at 1 µM Cltx (Fig. 4c). Both the enzymatic activity of recombinant MMP-2 and the inhibitory effect of Cltx on its activity were potentiated by in vitro activation of MMP-2 with 1 mM aminophenylmercuric acetate but without a change in relative affinity (data not shown).

Cltx Inhibits Cell Surface Gelatinase Activity-- We next investigated the effect of Cltx on the cell surface gelatinolytic activity by in situ zymography using fluorescein isothiocyanate-labeled DQ gelatin. Untreated glioma cells exhibited significant cell surface gelatinolytic activity. A significant decrease in surface gelatinolytic activity was observed following treatment with 30 nM Cltx, with complete inhibition in the presence of 300 nM Cltx (Fig. 5). The inhibition by 300 nM Cltx was comparable with that achieved with 50 µM 1,10-phenanthroline, a well established MMP-2 inhibitor (37), for which we determined an IC50 of ~8 µM for MMP-2 inhibition (data not shown). These data suggest that Cltx is much more potent in inhibiting cell surface gelatinolytic activity than suggested by our in vitro enzyme assay with purified MMP-2 (Fig. 5). This suggests that perhaps, secondarily, Cltx may also affect the surface expression of MMP-2.


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Fig. 5.   Cltx inhibits cell surface gelatinase activity. Representative fluorescent micrographs of D54-MG cells plated on coverslips coated with DQ-gelatin in the presence or absence of 30 nM Cltx, 300 nM Cltx, or 50 µM 1,10-phenanthroline. Cell surface gelatinolytic activity was assayed as a measure of fluorescence emitted by degrading gelatin. Untreated cells exhibited significant cell surface gelatinase activity, which was completely inhibited in the presence of 300 nM Cltx or 50 µM 1,10-phenanthroline. An intermediate effect was observed with 30 nM Cltx.

Cltx Alters Surface Expression of MMP-2-- MMP-2 is a major matrix metalloproteinase in glioma cells (27) and the vast majority of MMP-2 is thought to stay in association with the cell surface (34). It has previously been demonstrated that certain toxins, MT1-MMP, and integrins can be internalized via caveolae (38). We therefore examine the possibility that Cltx may cause an internalization of cell surface MMP-2. To this end, we treated D54-MG glioma cells for 30 min with 500 µM Cltx at 37 °C. Cells were then fixed and stained under either unpermeabilized or permeabilized conditions (Fig. 6a). The former only detects cell surface MMP-2, whereas the latter reveals the distribution of both surface and intracellular MMP-2. Prominent expression of MMP-2 was observed in untreated glioma cells both at the cell surface and intracellularly (Fig. 6a). However, following a 30-min treatment with Cltx, surface staining for MMP-2 was essentially absent whereas intracellular staining remained. These data suggest that His-Cltx does indeed reduce the surface expression of MMP-2, perhaps by inducing an increased internalization of MMP-2. This was also examined biochemically using a cell surface biotinylation approach. Therefore, cell surface membrane proteins were labeled with biotin and isolated using avidin beads, from either untreated control cells or cells treated with Cltx for 30 min at 37 °C. The majority of membrane-associated MMP-2 disappeared from the cell surface upon Cltx exposure (Fig. 6b).


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Fig. 6.   Cltx reduces the surface association of MMP-2 and Cltx/MMP-2. a, surface expression of MMP-2 is regulated by Cltx. Representative fluorescent micrographs of D54-MG cells that have been treated (bottom panels) with His-Cltx for 30 min at 37 °C. Untreated cells are shown in the top panels. Nonpermeabilized cells (left panels top and bottom) reveal the surface expression of MMP-2, whereas cells permeabilized (right panels top and bottom) reveal the total amount of MMP-2 expressed by D54-MG cells. Magnification ×40. Cltx treatment reduced the surface expression of MMP-2. b, filipin prevents internalization of MMP-2 induced by Cltx. Immunoblots from cell surface-biotinylated D54-MG glioma cells either treated with Cltx (30 min at 37 °C) or untreated in the presence or absence of filipin (+) (4 °C for 10 min). Blots were probed with antibodies to MMP-2 or anti-His antibodies to detect the presence of MMP-2 and His-Cltx, respectively. Treatment with Cltx enhances the internalization of MMP-2 in the absence of filipin resulting in less membrane-associated MMP-2. In the presence of filipin, the membrane-associated MMP-2 is much enhanced and Cltx fails to reduce surface localization of MMP-2.

Receptor-mediated endocytosis has been shown to occur either by a clathrin-mediated pathway (39) or by caveoli (40). To assess whether this observed MMP-2 internalization by Cltx is via caveolae, we examined cell surface expression of MMP-2 in the presence and absence of filipin, a sterol binding drug known to disrupt caveolae (41). In the presence of filipin, a significantly larger fraction of His-Cltx as well as MMP-2 remained on the cell surface, whereas in the absence of filipin, a significant reduction in membrane-associated His-Cltx and MMP-2 was observed (Fig. 6b). These data suggest that a filipin-sensitive mechanism, possibly caveolae, is involved in the internalization of MMP-2 following binding of Cltx.

Cltx Inhibits Matrigel Invasion of Glioma Cells by Its Interactions with MMP-2-- It has previously been demonstrated that MMP-2 activity is essential for matrigel invasion of glioma cells (42). Hence, one would expect, based on the above data, that Cltx binding to MMP-2 should reduce glioma cell invasion either by the inhibition of its enzymatic activity or by decreasing the surface expression and/or release of MMP-2. We examined the inhibitory properties of Cltx on glioma matrigel invasion by directly comparing the effect of His-Cltx to the commercially available peptide. Both peptides inhibited invasion in a concentration-dependent manner with an IC50 of about 200 nM (Fig. 7a), a value consistent with the inhibition of MMP-2 activity by Cltx (see Fig. 4c). The maximal inhibition obtained with Cltx was between 70 and 80% as compared with untreated control cells. Interestingly, addition of 1,10-phenanthroline yielded essentially identical inhibition of invasion as did Cltx (Fig. 7b). However, when we examined the effect of either Cltx or 1,10-phenanthroline in the presence of filipin, the effect of Cltx was reduced by over 50%, suggesting that a significant component of the Cltx effect on matrigel invasion presumably involves the endocytosis of MMP-2 via caveolae. Consistent with this concept, we find that the inhibition of glioma invasion by 1,10-phenanthroline, which specifically inhibits the enzymatic activity of MMP-2, was only minimally affected by treatment with filipin. Taken together, these findings suggest a novel mechanism of action for this scorpion toxin, wherein Cltx regulates invasion of glioma cells by modulating the surface expression of enzymatically active MMP-2.


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Fig. 7.   Cltx inhibits matrigel invasion of glioma cells. a, His-Cltx inhibits glioma cell migration. Dose-response curve of D54-MG cells treated with His-Cltx or commercial Cltx peptide (Alomone) and analyzed by matrigel invasion assay at 24 h post-treatment. Half-maximal inhibition (IC50) for Cltx was 184 nM. Percent inhibition was calculated as the decrease in the number of migrated cells normalized to control. b, His-Cltx inhibition of matrigel invasion is filipin-sensitive. Normalized percent inhibition of glioma cell invasion following filipin (5 µg/ml) treatment, with/without His-Cltx (500 nM) as well as filipin (5 µg/ml) treatment with/without 1,10-phenanthroline (30 µM). The data suggest that the reduction in matrigel invasion by Cltx was at least in parts mediated by a filipin-sensitive mechanism, possibly involving internalization of MMP-2 via caveoli.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cltx is a 36-amino acid peptide that belongs to a large family of insect toxins. Several recent studies have suggested that Cltx is a highly specific ligand for malignant human gliomas, which shows no significant binding to normal brain (17). Cltx selectively targets xenografted gliomas in scid mice and remains associated with the tumor for at least 24 h (17). Moreover, Cltx also inhibits glioma cell invasion in vitro (13). However, the receptor for Cltx and the mechanism underlying its anti-invasive effect have been unknown.

In this report, we show that MMP-2 is the primary receptor for Cltx on the surface of glioma cells. MMP-2 belongs to a superfamily of zinc-dependent endopeptidases. It is secreted as a latent zymogen requiring activation. Once activated, the latent 72-kDa MMP-2 is converted via a ~64-kDa activation intermediate to a ~62-kDa mature protease (24, 26). Efficient activation of the pro-enzyme to the mature enzyme has been shown to contribute to the invasive potential of tumor cells (43). MMP-2 has been demonstrated to be expressed in numerous tumor types (31) and also in diseases that involve tissue remodeling (44). However, MMP-2 appears to be a major matrix metalloproteinase in gliomas (27) and is absent from normal brain tissue (45, 46). Hence, the finding that MMP-2 is the principal receptor for Cltx is consistent with the recent finding that the Cltx receptor is primarily expressed in glioma and tumors of neuroectodermal origin (18). Our data suggests that Cltx does not interact with MMP-1, MMP-3, or MMP-9, which are also expressed by glioma cells (28).

Importantly, we show that the anti-invasive effect of Cltx on glioma cells is mediated predominantly by its interactions with MMP-2. Our data, discussed sequentially below, suggest that Cltx exerts a dual effect on MMP-2: it inhibits the enzymatic activity of MMP-2 and it causes a reduction in surface expression.

Our in vitro affinity purification and binding assays indicate that Cltx can interact with both the latent 72 kDa and the activated forms of MMP-2. The 72-kDa form of MMP-2 is generally referred to as a latent form of the enzyme, but it constitutes the dominant MMP-2 species in glioma cells, which, when activated exhibits significant gelatinolytic activity in these cells. Interestingly this activation is thought to occur on the cell surface of glioma cells in an MT1-MMP-specific manner (34).

Our biochemical and cell biological assays indicate that Cltx binding to MMP-2 inhibits its catalytic activity. For example, Cltx inhibits MMP-2 activity in vitro in a dose-dependent manner, and both the catalytic activity and the inhibition by Cltx are enhanced upon activation of recombinant MMP-2 with aminophenylmercuric acetate. In addition, we found that Cltx can completely inhibit the cell surface gelatinolytic activity. Taken together, this data suggests that the binding of Cltx to the surface of glioma cells occurs through its direct association with MMP-2 and that this binding inhibits the catalytic activity of MMP-2.

MMP-2 plays a crucial role in degradation and remodeling of the ECM (34) thereby allowing the penetration of normal and tumor cells through tissue barriers. The mechanism of activation and regulation of MMP-2 is tightly regulated by several other proteins that form a macromolecular complex (47). Specifically, this involves interactions with membrane-associated MT1-MMP and alpha vbeta 3 integrin, matrix proteins, and its endogenous inhibitor TIMP-2. These proteins are stoichiometrically balanced in equimolar ratios to facilitate tumor cell migration, invasion, and control and maintenance of ECM proteolysis (34, 48, 49). MT1-MMP, a membrane-type MMP activates MMP-2, and alpha vbeta 3 integrin promotes the maturation and release of MMP-2 (49). It has also been demonstrated that MT1-MMP forms a homophilic complex through the hemopexin domain keeping the MT1-MMP molecules together facilitating pro-MMP-2 activation (50). Integrins have been suggested to localize proteases to invadopodia (51), cell extensions in the active process of invasion. As mentioned above, activation of MMP-2 and other MMPs and expression of MT1-MMP and alpha vbeta 3 have been shown to correlate with tumor invasion, neovascularization, and metastasis of glioma (43) melanoma cells both in vivo and in vitro (29) and breast cancer (52).

Based on these macromolecular interactions, it is not surprising that Cltx, by binding to MMP-2, would indirectly interact with this entire protein complex. Our data suggests that the anti-invasive effects of Cltx occur at least at two levels. In the first, Cltx inhibits the enzymatic activity of the enzyme in vitro. Second, our studies suggest that treatment of cells with Cltx also causes a reduction of surface expression of MMP-2 from glioma cells. Although not investigated in detail, our studies using filipin, a sterol binding drug known to disrupt caveolae (41), suggests that endocytosis of the MMP-2·Cltx complex via caveolae may be one of the underlying pathways that regulates the surface expression of MMP-2. The endocytosis of receptor-ligand complexes is generally because of dynamin-mediated endocytosis via clathrin coated pits (39). However, caveolin-1 expression has been demonstrated in several rat and human astroglioma cell lines (40) and the entry of certain pathogens and toxins into host cells has also been shown to occur via caveolae (53-55). Indeed, the internalization of cholera toxin into CACO-2 cells can be inhibited by filipin (39) a finding that is consistent with our findings on Cltx.

Our results from matrigel invasion assays suggest that the interaction of Cltx with MMP-2 and the resulting decrease of surface expression are sufficient to inhibit glioma cell invasion. Although the enzymatic activity of MMP-2 was directly inhibited by Cltx, internalization of MMP-2 appears to be an important step in the functional inhibition, because Cltx looses much of its effectiveness in the presence of filipin.

We currently do not know which site on the MMP-2 molecule interacts with Cltx. We performed preliminary competition experiments to examine two well characterized sites, namely that for TIMP-2 and the integrin binding site. Neither recombinant TIMP-2 nor functional blocking alpha vbeta 3 integrin antibodies were able to significantly compete for binding of Cltx to recombinant MMP-2. Clearly future studies need to examine these molecular interactions in greater detail.

The findings presented in this study have significant therapeutic implications. The anti-invasive effects of Cltx on glioma cells suggest that this drug may be highly useful in the treatment of malignant gliomas. Indeed, Cltx has passed preclinical safety studies and has recently won FDA approval for use in a phase I/II clinical trial. Several embryologically related tumors, including melanomas, have also been shown to express MMP-2 and to bind Cltx (18). Clinical use of Cltx may thus be expanded to include these tumors as well. Importantly, however, potentially being a specific MMP-2 inhibitor, Cltx may have even broader utility. MMP-2 is involved in a range of diseases that involve tissue remodeling in disease progression. Several chemical inhibitors of MMP-2 are in various stages of clinical testing but most have failed because of toxicity or lack of specificity. Cltx may be a safer and more specific drug, worthy of further exploration in this context.

    ACKNOWLEDGEMENTS

We acknowledge the valuable contributions made by Xiaojin Liu and Joon Wook Chung toward fusion protein constructs. We also acknowledge Lori Coward (Mass spectrometry Facility, University of Alabama at Birmingham) for expertise in mass spectrometric analysis of proteins.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant 2RO1-NS-36692 and American Cancer Society Grant RPG-97-083-04-CDD. Operation of the UAB Comprehensive Cancer Center Mass Spectrometry Shared Facility is supported in part by National Institutes of Health NCI Core Research Support Grant P30 CA-13148 to the UAB Comprehensive Cancer Center. The mass spectrometer was purchased by funds from National Institutes of Health shared instrumentation Grant S10RR11329.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.

Dagger Current address: Dept. of Psychiatry and Behavioral Science, Stanford University, Palo Alto, CA 94304-5485.

§ To whom correspondence should be addressed: 1719 6th Ave., South CIRC 589, Birmingham, AL 35294-0021. Tel.: 205-975-5805; Fax: 205-975-5518; E-mail: hws@nrc.uab.edu.

Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M205662200

    ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; Cltx, chlorotoxin; MMP-2, matrix metalloproteinase-2; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TIMP-2, tissue inhibitor of matrix metalloproteinase-2.

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