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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (2124). MMP-9 expression and activation is stimulated by many effectors, including epidermal growth factor receptor (EGFR) ligands and the EGFR signaling pathway-related molecules (2531). 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 5
1 integrin to associate in the
membrane, suggesting a regulatory role for GM3 in membrane complex
formation.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Overexpression of GM3 by Treatment with Antisense OligodeoxynucleotidesMembrane 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 GangliosidesGangliosides 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 ExpressionSialidase 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
-galactoseexpressing cells as described previously
(8). Peak expression was
demonstrated at 2448 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 2448 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 AssaysCell 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: 110)] (CalBioChem, Ref.
41).
Invasion AssaysCell 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.
ImmunoblottingImmunoblotting 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. 850 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.
ImmunoprecipitationAfter 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-5
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 ZymographyThe 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-PCRTotal 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
5
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
5
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 1.73.1-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).
|
Depletion of GM3 Increases Tumor Cell InvasionThe 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 3.34.2-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).
|
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).
|
Stimulation of Cell Migration by Ganglioside Depletion Requires MMP-9 ActivationAnti-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).
|
The Activation of MMP-9 by Ganglioside Depletion Requires EGFR-related
SignalingWe have previously shown that accumulation of GM3
inhibits EGFR and MAPK phosphorylation in the presence of EGF or transforming
growth factor- (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).
|
|
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.
|
GM3 Overexpression Prevents the Co-immunoprecipitation of MMP-9 with
Integrin
5
1MMP-9
was co-immunoprecipitated with integrin
5
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
5
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
5
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
5
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.
|
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 5
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
5
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
5
1 was increased by 2.22.9-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
5
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
5
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
5
1 (Fig.
9D, bottom row of b).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
(5057),
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
5
1 with MMP-9. The blockade of
co-immunoprecipitation of MMP-9 with
5
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
5
1 by GM3. This
GM3-induced disruption of the association between MMP-9 and
5
1 appears to be specific to GM3 and is not
seen with GT1b. Although a putative direct interaction between
5
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
v
3
(59) and disruption of this
binding inhibits angiogenesis and tumor growth in vivo
(60). In addition, MMP-1
direct binds to
2
1 integrin in
keratinocytes plated on type I collagen
(61) via the I domain of the
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
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
5 integrin
(15). The mechanism by which
GM3 content affects the association of MMP-9 and
5
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
5
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 |
---|
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,
NeuAc2
3Gal
1
3GalNAc-
1
4(NeuAc
2
8NeuAc
2
3)Gal
1
4Glc
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-
1
4(NeuAc
2
8NeuAc
2
3)Gal
1
4Glc
1-Cer;
GD3, NeuAc
2
8NeuAc
2
3Gal
1
4Glc
1-Cer;
GM1,
Gal
1
3GalNAc
1
4-(NeuAc
2
3Gal
1
4Glc
1-Cer;
GM2, GalNAc
1
4(NeuAc
2
3)-Gal
1
4Glc
1-Cer; GM3,
NeuAc
2
3Gal
1
4Glc
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.
2 X.-Q. Wang, P. Sun, and A. S. Paller, unpublished results.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|