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Originally published In Press as doi:10.1074/jbc.M302211200 on April 30, 2003

J. Biol. Chem., Vol. 278, Issue 28, 25591-25599, July 11, 2003
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Ganglioside GM3 Inhibits Matrix Metalloproteinase-9 Activation and Disrupts Its Association with Integrin*

Xiao-Qi Wang, Ping Sun and Amy S. Paller {ddagger}

From the Departments of Pediatrics and Dermatology, Children's Memorial Institute for Education and Research, Northwestern University Medical School, Chicago, Illinois 60614

Received for publication, March 3, 2003 , and in revised form, April 29, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Gangliosides GM3 and GT1b both inhibit epithelial cell adhesion and migration on fibronectin. GT1b binds to integrin {alpha}5{beta}1 and blocks the integrin-fibronectin interaction; GM3 does not interact with integrin, and its effect is poorly understood. We evaluated the effects of endogenous modulation of GM3 expression on epithelial cell motility on several matrices and the mechanism of these effects. Endogenous accumulation of GM3 decreased cell migration on fibronectin, types I, IV, and VII collagen matrices; depletion of GM3 dramatically increased cell migration, regardless of matrix. GM3 overexpression and depletion in vitro correlated inversely with the expression and activity of matrix metalloproteinase-9; consistently, the cell migration stimulated by GM3 depletion is reversed by inhibition of matrix metalloproteinase-9 activity. Accumulation and depletion of GM3 in epithelial cells grown on fibronectin also correlated inversely with epidermal growth factor receptor and mitogen activated protein kinase phosphorylation and with Jun expression. Ganglioside depletion facilitated the co-immunoprecipitation of matrix metal-loproteinase-9 and integrin {alpha}5{beta}1, while endogenous accumulation of GM3, but not GT1b, blocked the co-immunoprecipitation. These data suggest modulation of epidermal growth factor receptor signaling and dissociation of integrin/matrix metalloproteinase-9 as mechanisms for the GM3-induced effects on matrix metalloproteinase-9 function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Epithelial cell motility plays a fundamental role in both physiologic and pathologic conditions, such as tissue resorption and remodeling during embryogenesis, wound healing, and metastasis. However, the mechanisms involved in cell motility are not well understood. Gangliosides are sialylated membrane glycosphingolipids that modulate several biologic processes of keratinocytes and keratinocyte-derived cell lines in vitro, including cell proliferation, adhesion, migration, differentiation, and apoptosis (1–8). The highly sialylated ganglioside of keratinocytes, GT1b,1 strongly inhibits adhesion, spreading, and migration of cultured epithelial cells, specifically when cells are plated on a fibronectin (FN) matrix (2, 8, 9). GT1b has been shown to bind specifically to the {alpha}5 subunit of {alpha}5{beta}1 integrin, leading to competitive inhibition of integrin interaction with FN and explaining the specificity of the GT1b inhibitory effect (5). GM3, the predominant ganglioside of epithelial cells (5) is also able to regulate the adhesion and migration of several carcinoma and other tumor cell lines grown on FN (10–16). However, GM3 is unable to interact with {alpha}5{beta}1 integrin directly (5), suggesting an alternate means of inhibition of cell motility (15, 17, 18).

Recent studies have shown that GM3, in addition to its suppression of cell motility, also inhibits tumor cell invasion (16, 18). Both cell migration and invasion are facilitated by the proteolytic degradation of extracellular matrices (ECM) by matrix metalloproteinases (MMPs). Cell-matrix contact signals the cells to express and activate proteinases specific for the encountered matrix protein (19). The human MMP family is composed of at least 25 zinc-dependent endopeptidases, which require proteolytic N-terminal processing for functional activity (19, 20). MMP-9, a metalloproteinase expressed by human epithelial cells, is capable of degrading several matrices, including FN, laminin, gelatin, and types I, IV, and VII collagen (21–24). MMP-9 expression and activation is stimulated by many effectors, including epidermal growth factor receptor (EGFR) ligands and the EGFR signaling pathway-related molecules (25–31). In human epithelial malignancies, expression of MMP-9 correlates strongly with tumor infiltration (32, 33).

We have evaluated the effects of GM3 on squamous carcinoma cell migration and invasion and have addressed the possibility that GM3 expression modulates MMP activity. These studies show that endogenous alterations in GM3 expression in this carcinoma cell line affect migration and invasion potential by a mechanism that is not FN-specific. Instead, GM3 content dictates MMP-9 expression and activation through the effect of GM3 on EGFR signaling. The content of ganglioside GM3, but not of GT1b, also affects the ability of MMP-9 and {alpha}5{beta}1 integrin to associate in the membrane, suggesting a regulatory role for GM3 in membrane complex formation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells—The human keratinocyte-derived SCC12F2 cell line (SCC12), a generous gift from Dr. James Rheinwald (Harvard, Boston, MA) was maintained in DMEM/F12 (1:1, v/v) (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) without antibiotics in 5% CO2 at 37 °C.

Overexpression of GM3 by Treatment with Antisense Oligodeoxynucleotides—Membrane content of GM3 on SCC12 cells was endogenously increased as described previously (34) by treatment concurrently of SCC12 cells with antisense oligodeoxynucleotides of both GM2/GD2 synthase and GD3 synthase, leading to blockade of synthetic pathways downstream of GM3.

Total Depletion of Membrane Gangliosides—Gangliosides were depleted both by stable gene transfection and biochemically as previously described (34). SCC12 cells were stably transfected with human plasma membrane ganglioside-specific sialidase cDNA (GenBankTM accession number AB008185 [GenBank] , courtesy of Dr. T. Miyagi, Tokyo, Japan) (35) in a pcDNA3 vector using LipofectAMINE reagent (4, 7). Gene and protein expression in the resultant "SSIA cells" were demonstrated by Northern blot, sialidase activity measurements, and ganglioside depletion shown by thin layer chromatography (TLC) immunostaining (4, 7, 36). Four SSIA cell lines (SSIA3, SSIA6, SSIA12, and SSIA25), and 2 mock-transfected pcDNA cell lines were studied. Gangliosides were also eliminated by inhibition of glycosphingolipid synthesis. Cells were treated with 2 µM of racemic threo-1-phenyl-2-hexadecanoyl-amino-3-pyrrolidinopropan-1-ol, HCl (PPPP, Calbiochem, La Jolla, CA) for 5 days. PPPP inhibits the activity of glucosylceramide synthase, and thus prevents the formation of glucosylceramide, a precursor for GM3. In contrast to 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, PPPP does not significantly increase ceramide content (37). Overexpression and depletion of GM3 was confirmed using ganglioside ELISA (34), TLC immunostaining (4, 7, 36), and immunofluorescence microscopy.

Specific Endogenous Depletion of GM3 Expression—Sialidase overexpression or treatment with PPPP deplete all SCC12 cell gangliosides, although GM3 is the predominant ganglioside. To compare the results of total ganglioside depletion and specific depletion of GM3, a model of specific GM3 depletion was generated. Up-regulation of GD3 synthase expression by gene transfection results in a specific decrease in expression of GM3 and an increase in expression of GD3. Because in vitro studies had suggested that overexpression of GD3 would result in cell apoptosis, an inducible system was employed (a generous gift from Dr. S. Tsai and X. Wang, Houston, TX) (38, 39). The GD3 synthase cDNA-containing construct (GenBankTM accession number X77922 [GenBank] , courtesy of Dr. Lloyd, New York) (40) was introduced following the technique previously described for GM2/GD2 synthase (8), except that the linearized gene was inserted into purified p17 x 4-tkA vector opened at XhoI and EcoRV after treatment with XhoI and StuI restriction enzymes. The purified p17 x 4-tkA/GD3 synthase construct was cotransfected into SCC12 cells with the purified pCEP4·GL-VP (hygromycin-resistant) transactivator construct using LipofectAMINE reagent. Putative cotransfectants were selected by antibiotic resistance to both 500 µg/ml G418 and 60 µg/ml hygromycin B, followed by limiting dilution to ensure clonality.

The time course of gene expression after stimulation with mifepristone (RU-486) and the leakiness of the in vitro inducible system was tested initially by the concurrent generation and testing of {beta}-galactoseexpressing cells as described previously (8). Peak expression was demonstrated at 24–48 h after addition of RU-486, and no leakiness was detected in vitro. GD3 synthase expression in the p17 x 4-tkA/GD3-pGL-VP cells after 24–48 h stimulation with RU-486 (100 nM) was examined by Northern blotting assay using a digoxigenin (DIG)-labeled 636-bp GD3 synthase cDNA probe and by RT-PCR. For Northern assays, a DIG-labeled 316-bp fragment of the constitutively expressed human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as a control for RNA loading. Expression was detected with a DIG-high Prime DNA Labeling and Detection Starter kit II as indicated by the manufacturer's instructions (Roche Applied Science). For RTPCR, total RNA was isolated from cells using an RNeasy Isolation kit (Qiagen) following the manufacturer's instructions. The RNA was quantified by measuring the absorption at 260 nm. An aliquot of purified total RNA was electrophoresed in a 1.2% agarose, 2.2 M formaldehyde gel to verify its integrity, and the remaining purified RNA was stored at –80 °C. GD3 synthase primers for RT-PCR were: 5'-AGAGGGGCCATGGCTGTACTG-3' (GD3 synthase sense primer) and 5'-CAGTACAGCCATGGCCCCTCT-3' (GD3 synthase antisense primer). Equal loading of total RNA was confirmed by determining the expression of GAPDH with both sense primer 5'-GCCAAGGTCATCCATGACAAC-3' and antisense primer 5'-CTTTGGTTCTCCAGCTTCAGG. The PCR reaction was activated with HotStar TaqDNA polymerase and performed in a Perkin-Elmer DNA Thermal Cycler 480 as follows: 2 min denaturation at 95 °C followed by 30 cycles of denaturation for 1 min at 95 °C, annealing for 1 min at 60 °C for GADPH and 68 °C for GD3 synthase, and extension for 1 min at 72 °C; cycles were followed by a 10-min extension phase at 72 °C. The RT-PCR products of GADPH (498-bp) and GD3 synthase (285-bp) were visualized on 1.5% agarose gels. Gene expression after RU-486 stimulation was compared with expression in transfected cells without RU-486 induction, mock-transfected, and parental SCC12 cells. Successful transfection of cells with human GD3 synthase cDNA was also further confirmed by ganglioside ELISA assays as previously reported (8), and by TLC immunostaining with anti-GM1, -GM2, -GM3, -GD2, -GT1b (Seikagaku Corp., Tokyo) and anti-GD3 (Sigma) antibodies as described (36).

Cell Migration Assays—Cell migration assays and other studies were largely performed in the absence of EGF ligands, but in the presence of FN, to evaluate strictly the effect of matrix activation on signaling, in the absence of additional stimulation by EGFR ligands. Cell migration assays were performed using both chemotaxis migration assay (Transwell cell culture system from BD Biosciences) and scratch analysis. The Transwell insert with a polycarbonate filter of an 8-µm pore size was coated with or without 20 µg/cm2 of FN or 30 µg/cm2 poly-L-lysine (Sigma) overnight at room temperature. After air-drying, the insert was placed into a 6-well cell culture plate, and the lower portion of the plate was filled with 500 µl of serum-free DMEM/F12 containing 1 mg/ml bovine serum albumin (BSA). SCC12 cells, pretreated with or without antisense oligodeoxynucleotides or PPPP, the SSIA cells, the pcDNA3 vector mock-transfected SCC12 cells, and the GD3 synthase overexpressors with or without 100 nM RU-468 induction for 30 h were plated onto the upper surface of the filter. Cells were allowed to grow for 18 h at 37 °C in same medium as in the lower level with the continued addition of oligodeoxynucleotides, PPPP or RU-486. Cells that migrated into the lower level were collected and counted. Results were presented as mean ± S.D. from three different experiments with triplicate wells per experiment.

To perform scratch assays, cells were plated into 8-well cell culture plate, precoated with or without FN (5 µg/cm2), type I collagen (20 µg/cm2), type IV collagen (20 µg/cm2), type VII collagen (5 µg/cm2), or poly-L-lysine (10 µg/cm2) as described above. Cells were allowed to grow in 10% FBS containing DMEM/F12 medium for 4 h, then were washed with serum-free medium and starved of both serum and growth factors for overnight. A 1-mm wide scratch was made across the cell layer using a pipette tip. After washing with serum-free medium twice, DMEM/F12 medium containing 10 µg/ml FN, 40 µg/ml type I or IV collagen, 10 µg/ml type VII collagen or 20 µg/ml poly-L-lysine was added to replace matrix depleted with the SCC12 cells. Plates were photographed after 6, 12, and 24 h. All experiments were performed at least 6 times.

Quantitative scratch assays were performed as described above except that a deep permanent gouge was made with a razor blade into the culture plate at the border of the scratch to allow definitive separation of migrating cells. After 6, 12, and 24 h in culture, cells that had migrated into the scratched area were identified and counted. Results were presented as mean ± S.D. from 3 high power fields per experiment in at least four different experiments.

Using the quantitative scratch assays, cell migration was also observed in the presence of MMP inhibitors including 200 nM anti-MMP-9 blocking antibody, 10 nM recombinant TIMP-1, and 5 µM cyclic peptide inhibitor CTTHWGFTLC (CTT) [H-Cys1-Thr-Thr-His-Trp-Gly-Phe-Thr-Leu-Cys10-OH (cyclic: 1->10)] (CalBioChem, Ref. 41).

Invasion Assays—Cell invasion assays were performed using BD BioCoatTM MatrigelTM Invasion Chambers (BD Biosciences) following the manufacturer's instruction. The lower portion of both invasion and control chambers was filled with 500 µl of serum-free DMEM/F12 containing 1 mg/ml BSA with or without either 10 nM EGF or, as a stimulant of MMP-9 expression, 100 ng/ml PMA. SCC12 cells pretreated with or without sense or antisense oligodeoxynucleotides, PPPP, or Me2SO (<0.1% v/v, vehicle control), SCC12 cells stably transfected with sialidase cDNA or mock-transfected with the pcDNA vector, SCC12 cells transfected with GD3 synthase cDNA and treated with or without RU-486 were plated onto the upper surface of the filter. Cells were allowed to grow for 48 h at 37 °C. Cells that invaded the lower level were collected and counted. Results are presented as mean ± S.D. from three different experiments, performed in duplicate.

Immunoblotting—Immunoblotting was carried out as described (6) using an enhanced chemiluminescence (ECL) detection system (PerkinElmer Life Sciences) with total protein from either whole cell lysates or conditioned cell culture medium. In brief, cells were treated with or without antisense oligodeoxynucleotides or PPPP, or were stably transfected with sialidase cDNA or pcDNA3 vector. Cells were incubated for 48 h in serum-free medium with or without either 20 µg/ml plasma FN or 10 ng/ml EGF after starvation overnight of serum, FN, and growth factors. Cells were also allowed to grow in FN or EGF-containing, serum-free medium with or without either 250 nM AG1478, a specific inhibitor of EGFR-related kinase, 100 µM PD98059, a specific mitogen-activated protein kinase (MAPK) inhibitor, or 100 nM PMA, a broad spectrum stimulant of MMP expression, for 48 h at 37 °C. Incubation of cells with oligodeoxynucleotides or PPPP was continued in cell culture throughout the treatment with inhibitors. Total protein from the cell culture conditioned medium was concentrated using Protein-Concentrate Kit (Chemicon) and mixed with the non-denatured whole cells lysate. Total protein was quantified by colorimetric assay (Bio-Rad, Hercules, CA) to ensure equal loading. 8–50 µg of total protein from whole cell lysates, conditioned cell culture medium, or the protein mixture was boiled in Laemmli buffer (42), and loaded onto SDS-PAGE mini-gels. After transfer to polyvinylidene difluoride or nitrocellulose membranes, the separated proteins were detected by immunoblotting with anti-phosphotyrosine, anti-MAPK, anti-EGFR, anti-Jun, or anti-MMP-9 antibodies. Blots were reprobed as previously described (6) with anti-actin antibody to confirm equal loading. All blots were repeated in at least three different experiments.

Immunoprecipitation—After starvation of serum, FN, and EGF overnight, cells were stimulated with or without either 20 µg/ml plasma FN, or both FN and 100 nM PMA for 30 min. Cells were harvested and lysed in cold immunoprecipitation buffer (6). Total protein (1 mg) from the cell lysates was mixed with 5 µg of anti-{alpha}5{beta}1 integrin polyclonal antibody and the total reaction volume was adjusted to 1 ml in the immunoprecipitation buffer. After incubation with the antibodies for 2 h at 4 °C, protein A-agarose was added, and incubated for an additional 30 min at 4 °C (6).

Gelatin Zymography—The effect of ganglioside modulation of MMP-9 activity was analyzed by gelatin zymography (43). In brief, 50 µg of protein from the mixture prepared as described (see "Immunoblotting") was applied onto 10% SDS-PAGE min-gels containing 0.1% (w/v) gelatin after treating the protein with non-denatured sample buffer (17.4% SDS, 7% sucrose, and 0.01% phenol red) for 30 min at 25 °C. Following electrophoresis, gels were washed with 2.5% Triton X-100 for 30 min at 37 °C to remove the SDS. Gels were incubated at 37 °C overnight in developing buffer containing 50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 5 mM CaCl2, and 5 mM ZnCl2. Gels were then stained with 0.5% Coomassie Brilliant Blue R250 in 30% methanol, 7.5% glacial acetic acid for 30 min and destained. Gelatin-degrading enzymes were identified as clear bands against the blue background of the stained gel. Images of stained gels were captured by the AlphaImagerTM 2200 (San Leandro, CA). The intensity of the bands was measured by densitometric analysis by Storm 800 fluorescence PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA), and comparisons were made within each gel to determine relative changes in MMP activity. Data for each zymograph were expressed as relative changes in MMP activity within the gel and in comparison with other zymographs. Each experiment was repeated at least four times.

Detection of MMP-9 Expression by RT-PCR—Total RNA was isolated from cells using an RNeasy Isolation kit (Qiagen) following manufacturer's instructions. The RNA was quantified by measuring the absorption at 260 nm. An aliquot of purified total RNA was electrophoresed in a 1.2% agarose, 2.2 M formaldehyde gel to verify its integrity, and the remaining RNA was stored at –80 °C.

MMP-9 expression after stimulation with or without FN was analyzed by RT-PCR using the Qiagen OneStep RT-PCR Kit following the manufacturer's instruction (Qiagen). Primers were designed and prepared as described before (44): 5'-ATCCAGTTTGGTGTCGCGGAGC-3' (MMP-9 sense primer) and 5'-GAAGGGGAAGACGCACAGCT-3' (MMP-9 antisense primer). GAPDH expression was measured to ensure equal loading. GAPDH primers included 5'-GCCAAGGTCATCCATGACAAC-3' (sense) and 5'-CTTTGGTTCTCCAGCTTCAGG (antisense). The PCR reaction was activated with HotStar TaqDNA polymerase and performed in a Perkin-Elmer DNA Thermal Cycler 480 as follows: 2 min denaturation at 95 °C followed by 30 cycles of denaturation for 1 min at 95 °C, annealing for 1 min at 60 °C for GADPH and 65 °C for MMP-9, and extension for 1 min at 72 °C; cycles were followed by a 10-min extension phase at 72 °C. The RT-PCR products of GADPH (498-bp) and MMP-9 (498-bp) were visualized on 1.5% agarose gels.

Effects of Ganglioside GT1b on the Association of Integrin {alpha}5{beta}1 and MMP-9 —To determine the specificity of the effects of GM3, the effects of modulation of GT1b, another epidermal ganglioside that affects cell motility, were evaluated. To increase the content of GT1b exogenously, SCC12 cells were incubated with 0.1 µM GT1b for 48 h in 2% FBS containing DMEM/F12 medium (6). To upregulate GT1b endogenously, SCC12 cells were stably transfected with GM2/GD2 synthase cDNA using an inducible system as previously described (8). The increase in GT1b after exogenous addition and endogenous modification was documented by ganglioside ELISA. The effects of increased GT1b expression on MMP-9 expression were assessed by immunoblotting as described above. The effect of increased GT1b on the association of {alpha}5{beta}1 and MMP-9 was also examined as described, but these studies with increased GT1b were only performed in the absence of FN, because GT1b is a potent inhibitor of FN-stimulated cell signaling, leading to SCC12 cell apoptosis and loss of adhesion in the presence of FN (2, 5, 8, 9).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Endogenous Modulation of GM3 Affects Cell Migration on Several Matrices—Ganglioside GM3 was increased 1.9-fold after SCC12 cells were treated with antisense oligodeoxynucleotides of both GM2/GD2 synthase and GD3 synthase as detected by TLC immunostaining and ganglioside enzyme-linked immunosorbent assays (34). No detectable ganglioside was found on the cell membrane after cells were treated with either PPPP or stable transfection with human plasma ganglioside-specific sialidase (7, 34). GM3 expression was decreased 1.8-fold in cultured SCC12 cells stably transfected with human GD3 synthase cDNAs as detected by TLC immunostaining (not shown) and ganglioside ELISA (Fig. 1B) after induction with or without 100 nM RU-486 for 48 h. GD3 synthase expression was also confirmed by Northern blotting (not shown) and RT-PCR (Fig. 1A). Cells transfected with GD3 synthase cDNA but not stimulated with RU-486 showed only baseline expression of GD3 synthase and no ganglioside alteration.



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  		FIG. 1.
Induction of stably transfected GD3 synthase cDNA increases the expression of GD3 synthase and endogenously decreases GM3 expression. GD3 synthase cDNA was cotransfected with the transactivator pCEP4· GL-VP into SCC12 cells, as described under "Experimental Procedures." Studies with the {beta}-galactosidase reporter gene showed maximal gene expression after exposure to 100 nM RU-486 for 48 h. Parental SCC12 cells, mock transfectant p17 x 4-tkA/pCEP4·GL-VP cells, and each of the 4 selected clones transfected with GD3 synthase were treated with or without 100 nM RU-486 for 48 h. Cells were then either collected for RNA extraction (A) or plated on 96-well microtiter plates (B) and grown for an additional 6–8 h in the presence or absence of 100 nM RU-486. Increased expression of GD3 synthase mRNA was detected by RT-PCR in the presence of 100 nM RU-486 for 48 h, as shown for 4 selected positive transfectants (Clones 2, 5, 10, and 17) (A). Ganglioside expression was detected by ganglioside ELISA using specific anti-ganglioside antibodies and was expressed as the mean ± S.D. of the four GD3 synthase-expressing clones and the two mock-transfected clones, each run 6 times in quadruplicate (B). *, p < 0.05; ***, p < 0.001.

 

Significant inhibition of migration induced by endogenous GM3 overexpression was first noted within 12 h of culture in scratch assays in the face of FN (Fig. 2A-b) compared with control untreated SCC12 cells (Fig. 2A-a) and sense-treated SCC12 cells (not shown). By 18 h in culture, endogenous accumulation of GM3 led to a 1.7–3.1-fold decrease in cell migration by both chemotaxis migration assay (not shown) and quantitative scratch assay in comparison with sense-treated or untreated SCC12 cells, regardless of plating on FN, or on type I, type IV, or type VII collagen (Fig. 2B, p < 0.01). Ganglioside depletion by PPPP and sialidase gene transfection markedly increased migration within 12 h in scratch assays in the face of FN (Fig. 2A, c and d) in comparison with parental SCC12 cells (Fig. 2A-a) and mock or vehicle controls (not shown). After 18 h in culture on FN, both ganglioside depletion by PPPP treatment or sialidase gene transfection and GM3 reduction by GD3 synthase overexpression increased cell migration; quantitative scratch assay showed 3.6-fold, 4.4-fold, and 3.9-fold increases, respectively, when compared with controls (Fig. 2B, p < 0.001 for GD3 synthase and sialidase transfectants, p < 0.01 for PPPP). Depletion of GM3 expression also at least tripled cell migration in the face of type I, IV, and VII collagen matrices in comparison with their respective controls as detected by quantitative scratch assay (Fig. 2B). Ganglioside modulation did not affect cell migration when cells were plated onto poly-L-lysine or directly on plastic (not shown).



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  		FIG. 2.
Modulation of GM3 content affects cell migration on several matrices. SCC12 cells (8 x 106/well) were treated with or without sense or antisense oligodeoxynucleotides of both GM2/GD2 synthase and GD3 synthase for 5 days, or were treated with 2 µM PPPP or its Me2SO vehicle for 4 days. Studies were also performed with established cell lines that overexpressed sialidase cDNA, GD3 synthase, or their mock vectors. GD3 synthase transfected cells and their control cells were treated with or without RU-486. Cells were then plated onto 8-well cell culture plates and grown in DMEM/F12 medium with 10% FBS until almost confluent (about 4 h after plating). Cells were starved of serum, growth factor, and FN overnight and a 1-mm wide scratch was made with a pipette tip (A). After washing with serum-free medium, cells were incubated in DMEM/F12 medium containing 20 µg/ml plasma FN for 12 h at 37 °C. a, SCC12 cells; b, antisensetreated GM3 overexpressors; c, PPPP; d, SSIA cells. Bar = 70 µm. B, cells (1 x 106/well) treated as described in A were plated onto 8-well plates precoated with FN (5 µg/cm2), type I collagen (20 µg/cm2), type IV collagen (20 µg/cm2), type VII collagen (5 µg/cm2), or poly-L-lysine (10 µg/cm2). Deep scratches were created as described under "Experimental Procedures," and cells that migrated beyond the scratch were counted with at least 5 high power fields counted per well. Results are expressed as the mean ± S.D. from 2 wells in each of 6 different experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 

Depletion of GM3 Increases Tumor Cell Invasion—The SCC12 cells, although derived from a facial squamous cell carcinoma, show little tendency to invade through a Matrigelcoated transwell (Fig. 3A). Similarly, other control cells grown in DMEM/F12 medium with BSA (SCC12 cells treated with sense oligodeoxynucleotides, mock transfectants, GD3 synthase-transfected cells without RU-486, and SCC12 cells treated with Me2SO) showed minimal invasiveness. In contrast, cells depleted of ganglioside by PPPP treatment, overexpression of sialidase, or induced overexpression of GD3 synthase were 3.3–4.2-fold more invasive than control cells (Fig. 3A). Both EGF (Fig. 3B) and PMA (Fig. 3C) increased the ability of control cells to invade slightly, but dramatically triggered cell invasion when GM3 was decreased. The subtle decrease in SCC12 cell invasion with overexpression of GM3 ganglioside was accentuated by cell exposure to EGF (Fig. 3B, p < 0.05) or PMA (Fig. 3C, p < 0.01).



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  		FIG. 3.
GM3 overexpression or depletion affects cell invasion. Cell invasion was performed using BD BioCoatTM MatrigelTM Invasion Chambers (BD Biosciences). Cells were prepared as described under "Experimental Procedures," and were plated onto the upper surface of the filter. Cells were allowed to grow for 48 h at 37 °C in DMEM/F12 medium with 1 mg/ml BSA and without (A) or with either 10 nM EGF (B) or 100 nM PMA (C). Cells that had invaded the lower level were collected and counted. Invasion was quantified as the total number of cells in the lower level of chamber per well and expressed as the mean ± S.D. Studies were performed at least three different times with duplicate wells per experiment. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 

Expression of GM3 Modulates Expression and Activation of MMP-9 —Endogenous accumulation of GM3 decreased the expression of MMP-9, as detected by both immunoblotting (Fig. 4A-a, top row, lane 3) and RT-PCR (Fig. 4A-b, top row, lane 3) and inhibited the activation of MMP-9 (Fig. 4A-c, lane 3) induced by both FN (Fig. 4A) and EGF (not shown) in comparison with untreated cells (Fig. 4A, a and b, top rows, and c, lane 1), and cells treated with sense oligodeoxynucleotides (Fig. 4A, a and b, top rows, c, lane 2). In contrast, ganglioside depletion increased the expression of MMP-9 as determined by both immunoblotting (Fig. 4A, a, top row, lanes 5, 6, and 8) and RT-PCR (Fig. 4A, b, top row, lanes 5, 6, and 8). Ganglioside depletion also promoted the activation of MMP-9, leading to increased expression of the 84 kDa activated form of MMP-9 (Fig. 4A, a, lower band of top panel and lower band of c, lanes 5, 6, and 8) in comparison with parental SCC12 cells (Fig. 4A, top rows of a and b, lanes 1 and c, lane 1), mock-transfected pcDNA cells (Fig. 4A, a and b, top rows, and c, lane 4) and Me2SO vehicle-treated control cells (Fig. 4A, a and b, top rows, and c, lane 7). Neither GM3 overexpression nor ganglioside depletion affected the expression or activity of MMP-9 when cells were starved of serum, EGF, and FN (Fig. 4B).



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  		FIG. 4.
Endogenous modulation of GM3 affects MMP-9 expression and activation in the presence of FN. Cells were prepared as described in Fig. 2. Cells were starved of serum, FN, and growth factor overnight, and then incubated in serum-free medium with (A) or without (B) 20 µg/ml plasma FN for 48 h at 37 °C. Protein from whole cell lysates was obtained by incubating cells with 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 3 mM phenylmethylsulfonyl fluoride, and 0.1 M octyl glucoside for1hat4 °C. After brief centrifugation, the supernatant was mixed with concentrated conditioned medium. Concentration of the total protein was determined by colorimetric assay (Bio-Rad). 30 µg of total protein was boiled in Laemmli buffer, and applied onto an 8% SDS-PAGE mini-gel. Immunoblotting with anti-MMP-9 monoclonal antibody was performed (A-a, top row and B-a). Equal loading of protein was confirmed by probing with anti-actin antibody using a gel run in parallel (A-a, bottom row). MMP-9 expression was also detected by RT-PCR (A-b and B-b, top rows), using GAPDH as a control (A-b and B-b, bottom rows). To perform zymography, 50 µg of total protein was incubated with non-denaturing sample buffer (17.4% SDS, 7% sucrose, and 0.01% phenol red) for 30 min at room temperature. Samples were then applied onto a 10% SDS-PAGE mini-gel containing 1 mg/ml gelatin (A-c). Lane 1, SCC12 cells; lane 2, sense-treated cells; lane 3, antisense-treated cells; lane 4, pcDNA mock control cells; lane 5, SSIA 3 clone; lane 6, SSIA 6 clone; lane 7, Me2SO-treated vehicle control cells; lane 8, PPPP-treated cells.

 

Stimulation of Cell Migration by Ganglioside Depletion Requires MMP-9 Activation—Anti-MMP-9 blocking antibody, recombinant TIMP-1, and CTT, all inhibitors of MMP-9 activity, significantly decreased the accelerated cell migration of ganglioside-deficient cells in comparison with untreated cells (Fig. 5) and cells treated with normal IgG control (not shown). This inhibition was observed regardless of whether cells were grown on a FN matrix (Fig. 5A), or on matrices of type VII collagen (Fig. 5B), type I collagen or type IV collagen (not shown). Blockade of MMP-9 activation did not further diminish the reduction in cell migration modulated by GM3 overexpression (Fig. 5, A and B). Treatment with anti-MMP-9 blocking antibody, recombinant TIMP-1, or CTT did not affect MMP-9 expression (not shown).



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  		FIG. 5.
Ganglioside-modulated cell migration requires MMP-9 activation. Quantitative scratch assay was performed on FN-(A) or collagen VII-coated (B) 8-well cell culture plate as described under "Experimental Procedures" and in the legend to Fig. 2. After making the deep scratch, cells were incubated in DMEM/F12 medium with either 10 µg/ml FN or 10 µg/ml collagen VII, with or without 200 nM anti-MMP-9 blocking antibody, 10 nM recombinant TIMP-1, 5 µM CTT or, as a control, 200 nM normal IgG. Migrating cells were counted after incubation for 18 h at 37 °C. The extent of migration was quantified as the total number of cells per high power field with 5 fields counted, and expressed as the mean number of cells ± S.D. Studies were performed at least six times. **, p < 0.01; ***, p < 0.001.

 

The Activation of MMP-9 by Ganglioside Depletion Requires EGFR-related Signaling—We have previously shown that accumulation of GM3 inhibits EGFR and MAPK phosphorylation in the presence of EGF or transforming growth factor-{alpha} (4, 34). GM3 overexpression also inhibits EGFR (Fig. 6A, lane 3) and MAPK (Fig. 6B, lane 3) phosphorylation in the absence of EGF but in the presence of FN. In contrast, ganglioside depletion stimulates FN-induced phosphorylation of both the EGFR and MAPK (Fig. 6, A and B, lanes 5, 6, and 8). Endogenous accumulation of GM3 also decreased expression of Jun (Fig. 6C, lane 3), while ganglioside depletion increased Jun expression (Fig. 6C, lanes 5, 6, and 8) in comparison with control cells (Fig. 6C, lanes 1, 2, 4, and 7). GM3 accumulation or depletion did not affect the expression of EGFR or MAPK in the presence of either FN (not shown) or EGF (4, 34). Inhibition of EGFR activation by AG1478 (Figs. 7, A and B, lane 2) or of MAPK activation by PD98059 (Figs. 7, A and B, lane 3) decreased the expression of MMP-9 and activated MMP-9 induced by ganglioside depletion via either sialidase overexpression (Fig. 7A) or PPPP treatment (Fig. 7B) in comparison with untreated cells (Fig. 7, A and B, lane 1). PMA significantly stimulated expression of MMP-9 and the activated form of MMP-9 in ganglioside-deficient cells (Fig. 7, A and B, lane 4) and slightly enhanced the expression and activation of MMP-9 in parental SCC12 cells (not shown).



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  		FIG. 6.
GM3 expression in the presence of FN affects the phosphorylation of EGFR and MAP kinase and the expression of Jun. Cells were prepared as indicated in Fig. 2. After starvation of serum, growth factors, and FN overnight, cells were stimulated with 20 µg/ml plasma FN for 30 min and lysed. 8–20 µg total protein from whole cell lysates were applied onto an 8% (A) or a 12% (B and C) SDS-PAGE mini-gel to perform immunoblotting with anti-EGFR-phosphotyrosine (A), rabbit anti-human phospho-p44/42 MAP kinase polyclonal antibody (B), or anti-Jun antibody (C). Lane 1, SCC12 cells; lane 2, sense-treated cells; lane 3, antisense-treated cells; lane 4, pcDNA mock control cells; lane 5, SSIA 3 clone; lane 6, SSIA 6 clone; lane 7, Me2SO-treated vehicle control cells; lane 8, PPPP-treated cells.

 


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  		FIG. 7.
MMP-9 activation by ganglioside depletion is blocked by inhibitors of EGFR and MAPK signaling. Ganglioside was depleted by either stably transfecting SCC12 cells with sialidase (A) or treatment with PPPP (B) as described under "Experimental Procedures." Ganglioside-deficient cells were starved of serum, growth factors and FN overnight, and grown in DMEM/F12 medium containing 20 µg/ml plasma FN with or without 250 nM EGFR inhibitor AG1478, 100 µM MAPK inhibitor PD98059, or 100 nM MMP inducer PMA for 48 h at 37 °C. Cell lysates and concentrated conditioned cell culture medium were mixed, and 30 µg of total protein from the mixture was applied onto 8% SDS-PAGE to perform immunoblotting with anti-MMP-9 antibody.

 

Ganglioside Depletion Dramatically Increases Membrane Expression of Activated MMP-9 —Ganglioside depletion significantly increased activated MMP-9 in SCC12 cell membrane (Fig. 8B, bottom band, lanes 5, 6, and 8) when cells were grown on FN in the absence of EGF. Ganglioside depletion also increased cell-associated expression of the inactive latent form of MMP-9 and secretion of the activated form (Fig. 8A, bottom bands, lanes 5, 6, and 8) in comparison with parental SCC12 cells (Fig. 8, A and B, lane 1), mock pcDNA control cells (Fig. 8,A and B, lane 4), and Me2SO vehicle-treated control cells (Fig. 8, A and B, lane 7) (p < 0.001). Overexpression of GM3 markedly decreased the content of secreted MMP-9 (Fig. 8A, lane 3) and eliminated detectable cell-associated MMP-9 (Fig. 8B, lane 3) in comparison with untreated (Fig. 8, A and B, lane 1) and sense-treated cells (Fig. 8, A and B, lane 2) (p < 0 .001). Activated MMP-9 was never detectable in the presence of GM3.



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  		FIG. 8.
Ganglioside depletion increases membrane expression of activated MMP-9. Samples were prepared as described in Fig. 4. 50 µg of total protein from the concentrated conditioned cell culture medium (A) or 40 µg total protein from the cell lysate (B) was applied onto an 8% SDS-PAGE mini-gel. MMP-9 expression was detected by immunoblotting with anti-MMP-9 monoclonal antibody. Lane 1, SCC12 cells; lane 2, sense-treated cells; lane 3, antisense-treated cells; lane 4, pcDNA mock control cells; lane 5, SSIA 3 clone; lane 6, SSIA 6 clone; lane 7, Me2SO-treated vehicle control cells; lane 8, PPPP-treated cells.

 

GM3 Overexpression Prevents the Co-immunoprecipitation of MMP-9 with Integrin {alpha}5{beta}1—MMP-9 was co-immunoprecipitated with integrin {alpha}5{beta}1 from cells grown in the presence (Fig. 9A) or absence (Fig. 9B) of FN. In parental SCC12 cells, the latent 92-kDa form of MMP-9 was co-immunoprecipitated with the {alpha}5{beta}1, regardless of the presence of FN (Fig. 9, A-c and B-b, lane 1). Overexpression of GM3 in the presence or absence of FN blocked the association of {alpha}5{beta}1 integrin (Fig. 9A-b, lane 3) and MMP-9 (Fig. 9, A-c and B-b, lane 3) that was seen in untreated cells (Fig. 9, A-c and B-b, lane 1) and sense-treated control cells (Fig. 9, A-c and B-b, lane 2). Ganglioside depletion by either stable overexpression of ganglioside-specific sialidase or treatment with PPPP activated (i.e. lead to cleavage of latent to the activated 84-kDa form) and increased the expression of MMP-9 in the presence, but not absence, of FN (Fig. 9, A-a and B-a, lanes 5, 6, and 8). Regardless of the presence or absence of FN, ganglioside depletion also increased the association of both cleaved and precursor MMP-9 with integrin {alpha}5{beta}1 (Fig. 9, A-c and B-b, lanes 5, 6, and 8) in comparison with parental SCC12 (lane 1) and mock (lane 4) or Me2SO vehicle-treated (lane 7) control cells.



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  		FIG. 9.
Overexpression of GM3, but not GT1b, prevents the association of MMP-9 with integrin {alpha}5{beta}1, while ganglioside depletion facilitates the association. Cells were prepared as indicated in Fig. 4, and were lysed with 20 mM Hepes, pH 7.2, 1% Nonidet P-40, 10% (v/v) glycerol, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 10 µg/ml leupeptin. Cells were exposed to FN (A), treated in the absence of FN (B), or were exposed to PMA (C). In other studies, SCC12 cells were pretreated with or without 0.1 µM GT1b for 48 h, or were stably transfected with GM2/GD2 synthase cDNA and induced with or without 100 nM RU-486 as described previously (8) (D). The overall expression of MMP-9 was determined by application of the mixture of cell lysate and concentrated conditioned cell culture medium to an 8% SDS-PAGE minigel and immunoblotting with anti-MMP-9 antibody (A-a, B-a, C-a, D-a). 5 µg of anti-integrin {alpha}5{beta}1 polyclonal antibody was incubated with 1 mg of cell-free lysates at 4 °C for 2 h to immunoprecipitate integrin {alpha}5{beta}1 as previously described (5). After boiling the immunoprecipitates in Laemmli buffer for 10 min, aliquots of immunoprecipitate were applied to 8% SDS-PAGE mini-gels to assess the purity of integrin {alpha}5{beta}1 by immunoblotting with anti-integrin {alpha}5{beta}1 antibody (A-b, D-b; not shown for cells without FN exposure or for cells exposed to PMA) and the co-immunoprecipitated MMP-9 by immunoblotting with anti-MMP-9 antibody (A-c, B-b, C-b, and D-c). A–C, lane 1, SCC12 cells; lane 2, sense-treated cells; lane 3, antisense-treated cells; lane 4, pcDNA mock control cells; lane 5, SSIA 3 clone; lane 6, SSIA 6 clone; lane 7, Me2SO-treated vehicle control cells; lane 8, PPPP-treated cells. Denatured IgG bands (55 kDa and 25 kDa) are not shown.

 

Although expression of both the latent and activated forms of MMP-9 was increased by ganglioside depletion in the presence of FN, the ratio of the total co-immunoprecipitated MMP-9 to total MMP-9 expression was 3.28-fold and 2.69-fold that of vector control cells for the sialidase-overexpressing clones 3 (Fig. 9, lane 5) and 6 (Fig. 9, lane 6), respectively. In addition, densitometric evaluation of immunoblots showed that the ratio of latent and activated forms of MMP-9 in PPPP-treated SCC12 cells was 2.85-fold that of the Me2SO vehicle-treated cells (Fig. 9, lane 8). The activated form of the MMP-9 was more selectively co-immunoprecipitated with the {alpha}5{beta}1; although the activated form of MMP-9 represented a mean of 17% of the overall MMP-9 expression in ganglioside-depleted cells, the activated form represented 78% of the total MMP-9 co-immunoprecipitated with {alpha}5{beta}1. (Fig. 9A-c, lanes 5, 6, and 8). In ganglioside-deficient cells without FN stimulation, the amount of MMP-9 co-immunoprecipitating with integrin {alpha}5{beta}1 was increased by 2.2–2.9-fold (Fig. 9B-b, lanes 5, 6, and 8), despite the lack of change in MMP-9 expression (Fig. 9B-a).

To address the possibility that the diminution in the MMP-9 concentration itself accounted for the failure of MMP-9 to coimmunoprecipitate with {alpha}5{beta}1 integrin in the presence of GM3, the expression and activation of MMP-9 was stimulated by treatment with PMA (Fig. 9C). Despite increases in both expression and activation of the MMP-9 in the presence of the PMA (Fig. 9C-a), endogenous increases in GM3 prevented the association of either the latent form of MMP-9 or the activated form of the MMP-9 with {alpha}5{beta}1 (Fig. 9C-b, lane 3).

To assess the specificity of the effect of ganglioside GM3, cells were treated by exogenous addition of 0.1 µM GT1b (6) or stable transfection of GM2/GD2 synthase using an inducible system (8) to upregulate endogenous expression of of ganglioside GT1b. Overexpression of GT1b did not affect either the expression of MMP-9 (Fig. 9D-a) or the association of MMP-9 with integrin {alpha}5{beta}1 (Fig. 9D, bottom row of b).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we have shown that endogenous increases in the expression of ganglioside GM3 suppress MMP-9 expression and activation, which likely plays a role in the demonstrated ganglioside-induced inhibition of SCC12 carcinoma cell motility and invasion. This mechanism is distinct from the means by which GT1b, another epithelial ganglioside, inhibits SCC12 carcinoma cell motility. GT1b has no effect on cell-associated MMP-9 activity; instead, the inhibitory effect of GT1b on motility is specific for cells grown on a FN matrix (2, 9), because GT1b directly binds the {alpha}5 subunit of integrin (5), thereby affecting downstream signaling (4, 6, 8). GM3 is unable to bind to {alpha}5{beta}1 directly (5) and, in contrast to the specific interaction of GT1b with FN, inhibits cell motility on a variety of matrices, consistent with the ability of MMP-9 to modulate cell motility on several matrices.

The inhibitory effect of the endogenous increase in GM3 is mirrored by the marked stimulation of MMP-9 expression and activation induced by GM3 depletion, whether global ganglio-side depletion from overexpression of sialidase or biochemical treatment with PPPP, or specific depletion of GM3 by overexpression of GD3 synthase, which converts GM3 to GD3 and 9-O-acetyl-GD3.2 By inhibiting MMP-9 activity, we have shown that the effect on cell migration of ganglioside depletion requires the activation of MMP-9, regardless of the underlying matrix.

Studies from our laboratory and others have shown that GM3 inhibits cell proliferation by suppressing EGFR activation (4, 45) and downstream signaling (4). The mechanism for this ganglioside-specific inhibition appears to involve both binding of GM3 directly to the EGFR, an interaction that requires receptor glycosylation (45), and direct post-binding effects on signal transduction. Consistently, endogenous depletion of gangliosides, including GM3, by stable transfection of SCC12 cells with sialidase cDNA leads to cell hyperproliferation and stimulation of EGFR signaling, especially in response to EGFR ligands (4). Given the known stimulation by EGFR ligands of MMP-9 expression, we hypothesized that EGFR/MAPK signaling contributed to the increased expression and activation of MMP-9 with GM3 depletion. Studies were performed in the absence of EGF ligands, but in the presence of FN, to evaluate strictly the effect of matrix activation on signaling, rather than the additive effect of direct EGFR ligand stimulation. Our studies provide evidence that activation or suppression of EGFR/MAPK signaling and of the expression of Jun, a component of AP-1, is required for the effect of modulation of GM3 content on MMP-9 expression and activation. EGFR activation also increases the expression of stromelysin (MMP-3) (46) and matrilysin (MMP-7) (47), two metalloproteinases that induce activation of MMP-9 in keratinocytes (48, 49). The marked increase in MMP-9 activation seen with GM3 depletion suggests that the activation of EGFR signaling also triggers expression of these additional metalloproteinases, which in turn amplifies the effect on MMP-9 activation.

Recent studies emphasize that cell-matrix and cell-cell interactions are key in the modulation of MMP gene expression (50–57), suggesting that matrix stimulation occurs either directly or indirectly through integrin signaling (58). We have now demonstrated that increased expression of GM3, in addition to its effect on MMP-9 activation itself, prevents the association of {alpha}5{beta}1 with MMP-9. The blockade of co-immunoprecipitation of MMP-9 with {alpha}5{beta}1 is not merely a function of MMP-9 concentration, since treatment with PMA is able to overcome the inhibitory effect of endogenous accumulation of GM3 on MMP-9 expression and activation, but does not significantly overcome the prevention of MMP-9 association with {alpha}5{beta}1 by GM3. This GM3-induced disruption of the association between MMP-9 and {alpha}5{beta}1 appears to be specific to GM3 and is not seen with GT1b. Although a putative direct interaction between {alpha}5{beta}1 integrin and MMP-9 has not been explored, direct interaction of matrix metalloproteinases with other integrins are well known. For example, MMP-2 directly binds to integrin {alpha}v{beta}3 (59) and disruption of this binding inhibits angiogenesis and tumor growth in vivo (60). In addition, MMP-1 direct binds to {alpha}2{beta}1 integrin in keratinocytes plated on type I collagen (61) via the I domain of the {alpha}2 integrin subunit (62).

One means by which GM3 may inhibit cell motility in SCC12 carcinoma cells is through the association of GM3 with CD9, a tetraspan membrane protein. CD9 forms complexes with integrins to facilitate cell adhesion and motility but, in the presence of increased GM3, CD9 inhibits cell motility (13). Endogenous increases in GM3 expression in CD9-expressing IdlD cells lead to complexing of CD9 with {alpha}3 integrin, leading to the inhibition of cell motility on a laminin-5 matrix by GM3 (15); the increased GM3 expression also promotes the association of CD9 with {alpha}5 integrin (15). The mechanism by which GM3 content affects the association of MMP-9 and {alpha}5{beta}1 integrin is unclear. We have recently shown that GM3 is able to shift caveolin-1 into membrane domains with the EGFR, promoting the formation of caveolin-1/EGFR complexes and inhibition of ligand-induced EGFR autophosphorylation (34). The possibility that depletion or accumulation of GM3 induces shifts in membrane domains that facilitate or prevent, respectively, the complex formation between {alpha}5{beta}1 and MMP-9, and perhaps with CD9 and the EGFR (63) as well, deserves further investigation.

Aberrant expression of gangliosides has been reported in a variety of malignant tumors, and has been associated with invasive growth and metastatic potential of the tumors (46, 64). The changes in ganglioside content and distribution that occur in epithelial malignancies and dictate invasiveness and ability to spread have been relatively unexplored. GM3 remains the predominant ganglioside of neoplastic epithelial cells. However, the de novo synthesis of 9-O-acetyl-GD3 has clearly been described in neoplastic epithelial cells of squamous and basal cell carcinomas of skin (5, 65, 66), breast carcinomas (67), and several other carcinoma cells (68). Although altered expression of specific enzymes has not yet been explored, synthesis of 9-O-acetyl-GD3 suggests the increased expression of GD3 synthase in these neoplastic cells and concomitant depletion of GM3. In fact, bladder carcinomas have recently been shown to have high levels of GM3 when non-invasive and low levels of GM3 when invasive (18). Renal cell carcinomas and colonic carcinomas also show an inverse relationship between expression of GM3 and degree of malignancy (69, 70). The mechanism of the suppressive effect of GM3 on invasion of bladder carcinoma cells has been hypothesized to involve the recently demonstrated inhibitory effect on CD9. Our studies suggest an alternative or supplemental mechanism for the effect of modulation of GM3 on carcinoma cell invasiveness, by direct suppression by GM3 of MMP-9 expression and activation. Furthermore, these investigations provide a rationale for therapy of epithelial malignancies with agents that increase expression of GM3, such as antisense oligodeoxynucleotides or brefeldin A (71).


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant R01 AR44619. 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. Back

{ddagger} To whom correspondence should be addressed: Division of Dermatology 107, Children's Memorial Hospital, 2300 Children's Plaza, Chicago, IL 60614. Tel.: 773-880-3681; Fax: 773-880-3025; E-mail: apaller{at}northwestern.edu.

1 The abbreviations used are: GT1b, NeuAc{alpha}2->3Gal{beta}1->3GalNAc-{beta}1->4(NeuAc{alpha}2->8NeuAc{alpha}2->3)Gal{beta}1->4Glc{beta}1-Cer; AP-1, activator protein transcription factor-1; BSA, bovine serum albumin; CTT, H-Cys1-Thr-Thr-His-Trp-Gly-Phe-Thr-Leu-Cys10-OH (cyclic: 1->10); DIG, digoxigenin; DMEM, Dulbecco's modified Eagle's medium; Me2SO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GD2, GalNAc-{beta}1->4(NeuAc{alpha}2->8NeuAc{alpha}2->3)Gal{beta}1->4Glc{beta}1-Cer; GD3, NeuAc{alpha}2-> 8NeuAc{alpha}2->3Gal{beta}1->4Glc{beta}1-Cer; GM1, Gal{beta}1->3GalNAc{beta}1->4-(NeuAc{alpha}2->3Gal{beta}1->4Glc{beta}1-Cer; GM2, GalNAc{beta}1 ->4(NeuAc{alpha}2->3)-Gal{beta}1->4Glc{beta}1-Cer; GM3, NeuAc{alpha}2->3Gal{beta}1->4Glc{beta}1-Cer; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; pcDNA, SCC12 cells stably transfected with pcDNA3 vector; PMA, phorbol 12-myristate 13-acetate; PPPP, threo-1-phenyl-2-hexadecanoyl-amino-3-pyrrolidinopropan-1-ol HCl; RU-486, mifepristone; SSIA, SCC12 cells stably transfected with ganglioside-specific human sialidase cDNA; TIMP, tissue inhibitor of matrix metalloproteinase; MAPK, mitogenactivated protein kinase. Back

2 X.-Q. Wang, P. Sun, and A. S. Paller, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Sharon Stack for review of the manuscript.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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