Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M512566200 on April 24, 2006

J. Biol. Chem., Vol. 281, Issue 26, 18145-18155, June 30, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/26/18145    most recent
M512566200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, Q.
Right arrow Articles by Furukawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, Q.
Right arrow Articles by Furukawa, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Metastatic Potential of Mouse Lewis Lung Cancer Cells Is Regulated via Ganglioside GM1 by Modulating the Matrix Metalloprotease-9 Localization in Lipid Rafts*

Qing Zhang{ddagger}, Keiko Furukawa{ddagger}, Ho-Hsiang Chen{ddagger}§, Takumi Sakakibara{ddagger}§, Takeshi Urano{ddagger}, and Koichi Furukawa{ddagger}1

From the {ddagger}Department of Biochemistry II and the §Department of Surgery II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan

Received for publication, November 23, 2005 , and in revised form, April 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
To analyze mechanisms for cancer metastasis, we established high metastatic sublines from mouse Lewis lung cancer (P29) by repeated injection. Sublines established from the two subclones H7 and C4 commonly exhibited increased proliferation and invasion activity and reduced expression of ganglioside GM1, although they showed different preferences in their target organs of metastasis. The high metastatic sublines secreted higher levels of activated matrix metalloprotease (MMP)-9 as well as pro-MMP-9 in the culture medium than the parent lines. Furthermore, they contained MMP-9 at the glycolipid-enriched microdomain (GEM)/rafts fractionated by the sucrose density gradient ultracentrifugation of Triton X-100 extracts, whereas the parent cells showed faint bands at the fraction. When high metastatic sublines were treated with methyl-beta-cyclodextrin, their invasion activities were dramatically suppressed, and the MMP-9 secretion was also suppressed. All these results indicated that GEM/rafts play crucial roles in the increased invasion and high metastatic potential. To clarify the implication of reduced GM1 expression, low GM1-expressing cell lines were established using an RNA interference-expression vector of the GM1 synthase. Low GM1-expressing cell lines showed increased proliferation and invasion, enrichment in the GEM/rafts, and increased secretion of MMP-9. Among adhesion molecules, only integrin beta1 was detected in GEM/rafts with stronger intensity in high metastatic lines and low GM1-expressing cells. Taken together, integrins seemed to be enriched in the GEM/rafts by reduced GM1 levels, and subsequently MMP-9 was recruited to the GEM/rafts, resulting in its efficient secretion and activation, and eventually in the increased invasion and metastatic potentials.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of metastasis is a major cause of death in many human cancers. Mechanisms for the acquisition of metastatic potential are, however, not well understood. Cancer metastasis includes many processes, such as invasion into surrounding tissues, release from the primary tumor, intravasation, adhesion to vascular walls, extravasation, and formation of new foci. Consequently, multiple factors are involved in individual steps of metastasis. In the past decade, a number of systems and molecules involved in the metastasis have been identified, and their mutual correlations have also been widely investigated (1). Among them, cell adhesion (2), angiogenesis (1, 3), lymphangiogenesis (4, 5), and protease-mediated migration and invasion (6) are the most advanced fields, and their findings might provide new clues for the novel approaches to overcome cancer metastasis.

Roles of proteolysis and proteases in cancer metastasis have been dramatically elucidated (6). Above all, the matrix metalloproteases (MMP)2-2 and MMP-9 are known as gelatinases and have been considered to be major proteolytic enzymes in the degradation of extracellular matrix during cancer cell progression and invasion (7). Recently, a number of nonmatrix proteins have also been identified as substrates of these gelatinases, leading to the knowledge on the roles of theses enzymes in many other biological processes (6).

Microdomains in the cell surface membrane are recognized as particularly important sites in many physiological and pathological cellular events (8, 9). They include cholesterol metabolism, endocytosis, infection of microorganisms, apoptosis, and signal transduction. Historically, these microdomains have been called caveolae (10) based on their morphological features, lipid rafts (12), or glycolipid-enriched microdomain (GEM), detergent-insoluble (or -resistant) microdomain based on their biochemical natures (11). In this study we call them GEM/rafts. Little is known about the roles of GEM/rafts in cancer metastasis to date, although their implications in the cell transformation and oncogenesis have been extensively studied (13).

We established high metastatic sublines from mouse Lewis lung cancer P29 (14) by repeated intravenous or subcutaneous injection, and we analyzed cellular changes between the parent cells and the high metastatic sublines with focus on the cell surface molecules. In this study, we found that ganglioside GM1 was specifically down-regulated in high metastatic sublines. Furthermore, we demonstrated that GM1 expression is one of crucial factors to regulate cancer metastatic potential via the modulation of MMP-9 localization and secretion, and to eventually suppress the tumor invasion activity. This study should be the first report on the roles of GM1 in the regulation of metalloproteases in GEM/rafts.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Culture—P29, a low metastatic subline (with subcutaneous inoculation) of the Lewis lung cancer cell line, was subcloned to establish monoclonal sublines H7 and C4. They were repeatedly injected into C57BL/6 mice (intravenous or subcutaneous), and high metastatic sublines were established. These sublines were then maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 7.5% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. GM1 synthase RNAi transfectant (GM1-siRNA) cells and vector control cells were cultured in DMEM supplemented with 7.5% FBS and puromycin (6 µg/ml) (Calbiochem).

Antibodies—Rabbit anti-mouse MMP-9 (AB19047) was purchased from Chemicon International Inc. (Temecula, CA). Rabbit anti-integrin beta1 (sc-8978) and mouse anti-HCAM (CD44) mAb (sc-9960) were purchased from Santa Cruz Biotechnology. Rabbit anti-DMP-1 (M176) was purchased from Takara Bio Inc. (Otsu, Shiga, Japan). Horseradish peroxidase-conjugated anti-rabbit IgG was purchased from Cell Signaling Technology (Beverly, MA). CTB-Alexa555 was from Molecular Probes (Eugene, OR). The following anti-ganglioside mAbs were used as described previously (15, 16): mAbM2590 (anti-GM3), mAb10-11 (anti-GM2), mAb229 (anti-GA1), mAb92-22 (anti-GD1a), mAbR24 (anti-GD3), mAb220-51 (anti-GD2), and mAb370 (anti-GD1b). GM1 expression was analyzed as described in flow cytometric analysis. Cell surface adhesion molecules were analyzed using rat mAbKMI6 (antimouse integrin beta1 chain), hamster mAbHa31/8 (anti-mouse integrin {alpha}1 chain), hamster mAbHM2 (anti-mouse integrin {alpha}2 chain), rat mAbR1-2 (anti-mouse integrin {alpha}4 chain), rat mAb5H10-27 (anti-mouse integrin {alpha}5 chain), mAb2H5 (anti-sialyl Lewis x), rat mAbIM7 (anti-mouse CD44), rat mAb281-2 (anti-mouse syndecan-1), and rat mAb KY/8.2 (anti-mouse syndecan-4) from Pharmingen and rat mAb ECCD-2 (anti-mouse E-cadherin) from Takara (Otsu, Japan).

Assay of Metastasis—Cells were detached from culture dishes by 5 min of treatment with 0.02% EDTA in PBS, centrifuged, and resuspended in 200 µl of PBS. For the spontaneous metastasis assay, cells (2 x 106/mouse) were inoculated subcutaneously into the thigh of age-matched female C57BL/6 mice (Nippon SLC, Hamamatsu, Japan). Mice were sacrificed at 4 weeks after injection, and metastatic nodules on the surface of lungs were counted by naked eye. The criteria used for the identification of metastatic nodules were as follows: round shape, whitish and uniformly colored, more than 1 mm in diameter, and a different quality of appearance from surrounding normal tissues. Preliminary microscopic examination of tissues had been performed to confirm the identity of metastatic foci compared with the macroscopic observation. For the experimental metastasis assay, cells (1.0 or 0.5 x 106/mouse) were injected into the tail vein of C57BL/6 mice. Mice were sacrificed 3 weeks after injection, and metastatic nodules on the surface of lungs were counted. When mice died before this term, metastatic nodules were counted at death. Metastatic nodules from lungs or other tissues such as ovary and adrenal gland were isolated and cultured to be used for the next injection and/or other functional assays. To examine the effects of MbetaCD treatment, H7-O cells were incubated for 30 min at 37 °C with or without methyl-beta-cyclodextrin (MbetaCD) (10 mg/ml) before injection into the tail vein of mice.

MTT Assay—Cells (2 x 103) were seeded into 96-well plates and cultured in DMEM containing 7.5% FBS. Twenty µl of 5 mg/ml MTT (Sigma) in PBS was added to each well. After incubation for 3.5 h at 37 °C, 150 µl of n-propyl alcohol containing 0.1% Nonidet P-40 and 4 mM HCl was added. The color reaction was quantitated using the automatic plate reader Immuno-Mini NJ-2300TM (Nihon InterMed, Tokyo, Japan) at 590 nm with a reference filter of 620 nm.

Matrigel Invasion Assay—Cells (0.5 x 106) were suspended in serum-free DMEM and seeded in the upper chamber of Matrigel-coated transwell filters (8 µm pore) (BD Biosciences). The plain medium with or without serum was added to the lower chamber and incubated at 37 °C for 20 h. Noninvading cells remaining on the upper surface of the filter were removed, and the cells that appeared on the lower surface of the filter were fixed with 75% ethanol for 30 min and then stained and counted under a microscope. To examine the effects of MbetaCD treatment, cells (0.5 x 106) were seeded in upper chambers after being incubated for 30 min at 37 °C with MbetaCD.

Flow Cytometric Analysis—Cell surface expression of gangliosides and adhesion molecules was analyzed by flow cytometry (BD Biosciences). Cells were incubated with first mAbs for 40 min on ice and then stained with FITC-conjugated goat anti-mouse IgM or anti-rat IgG or anti-hamster IgG (Cappel, Durham, NC) after being washed. To analyze GM1 expression, cells were incubated with the cholera toxin B (CTB) subunit-biotin conjugates (List Biological Laboratories, Inc., Campbell, CA) for 40 min on ice and then stained with FITC-conjugated avidin (EY Laboratories, San Mateo, CA). Control samples were prepared using the second antibody alone. Intensity of fluorescence was measured and presented in arbitrary units.

Gelatin Zymography—Cells (2 x 106) were seeded in 10-cm culture dishes and cultured in DMEM containing 7.5% FBS up to 80% confluency. Cells were washed twice with PBS and incubated in serum-free DMEM for another 24 h and then conditioned medium was harvested and concentrated 80-fold with centrifugal filter Amicon Ultra-15TM (Millipore, Bedford, MA). Samples were electrophoresed under nonreducing conditions using 10% SDS-polyacrylamide gels containing 0.1% gelatin (Wako, Tokyo, Japan). After electrophoresis, the gel was washed twice in 2.5% Triton X-100 in 10 mM Tris-HCl (pH 7.4) for 30 min to remove SDS. After overnight incubation in 50 mM Tris-HCl (pH 7.4) containing 10 mM CaCl2 and 0.02% NaN3 at 37 °C, gels were stained with Coomassie Brilliant Blue in 50% methanol, 10% acetic acid and destained in 20% methanol, 10% acetic acid. Gelatinolytic activity was identified as a clear band on a blue background. The density of the MMP-9 band was measured with a scanner using the NIH image program 1.62 (Bethesda, MD). For MbetaCD treatment, cells were incubated for 30 min at 37 °C with MbetaCD. Cells were further incubated for 5 or 20 h in plain DMEM, and the supernatants were concentrated.

Isolation of Raft Fraction—GEM/rafts were isolated using a detergent extraction method essentially as described by Mitsuda et al. (15). Cells were plated at a density of 1 x 107/15-cm dish and cultured up to 90% confluency, and five dishes of cells were used for each preparation. After washing twice with ice-cold PBS, the cells were collected, resuspended in 1 ml of MNE/Triton X-100 buffer (1% Triton X-100, 25 mM MES-NaOH, pH 6.5, 150 mM NaCl, 5 mM EGTA, 1 mM Na3VO4, 1 mM PMSF, 1 µg/ml aprotinin), and then Dounce-homogenized 15 times. Samples were placed on the bottom of Ultra-ClearTM centrifuge tubes (Beckman Instruments) and mixed with an equal volume of 80% (w/v) sucrose in MNE buffer without Triton X-100. Then 2 ml of 30% sucrose (w/v) in MNE buffer without Triton X-100 was overlaid, and 1 ml of 5% (w/v) sucrose in MNE buffer without Triton X-100 was layered on the top. The samples were centrifuged at 36,000 rpm in an SW50.1 rotor for 16 h at 4 °C. The entire procedure was performed at 4 °C. From the top of the gradient, 0.5 ml of each fraction was collected to yield 10 fractions. For MbetaCD treatment assay, cells were incubated for 30 min at 37 °C with MbetaCD before the fractionation.

Cell Lysis and Immunoblotting—Cells (0.5 x 106) were seeded in 6-cm culture dishes and cultured in DMEM containing 7.5% FBS. Cells were lysed separately in 0.5 ml of ice-cold lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF) (Cell Signaling), and insoluble material was removed by centrifugation at 4 °C at 15,000 x g for 10 min. Lysates were separated with SDS-PAGE using 10-15% gels. The separated proteins were transferred onto a polyvinylidene difluoride membrane (Millipore). Blots were blocked with in 0.05% Tween 20 in PBS (PBST) containing 8% bovine serum albumin for 1 h. The membrane was first probed for 1 h with primary antibodies at the dilution suggested by the suppliers. After washing three times with PBST, the blots were then incubated for 1 h with goat anti-rabbit IgGs or goat anti-mouse IgGs conjugated with horseradish peroxidase (Cell Signaling) (1:4000). After the membranes were washed three times with PBST, bound conjugates were visualized with an ECLTM detection system (PerkinElmer Life Sciences). For the detection of GM1, the membranes were probed with CTB/biotin (1:200) and detected with an ABCTM kit (Vector Laboratories, Burlingame, CA).

Coprecipitation Experiments—Cells (1 x 107) were solubilized in 1 ml of the lysis buffer (Cell Signaling), and the lysates were incubated with 10 µl of anti-integrin beta1 (2 µg of IgG) or normal rabbit IgG overnight, and then 30 µl of protein A-Sepharose (1:1) (Amersham Biosciences) was added and incubated for 6 h at 4°C. After being washed with immunoprecipitation washing buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.5% Nonidet P-40, 1 mM Na3VO4) four times, the precipitates were solubilized in 40 µl of a Laemmli sample buffer and heated for 3 min at 100 °C. The immunoprecipitates were subjected to SDS-PAGE and then blotted onto a polyvinylidene difluoride membrane, as described above.

Coimmunoprecipitation experiments were performed under different conditions, changing temperature and detergent. First, cells were solubilized in the Triton X-100-containing lysis buffer (Cell Signaling) at room temperature, and the lysates were incubated with 10 µl of anti-integrin beta1 (2 µg of IgG) or normal rabbit IgG for 2 h at room temperature. Then 30 µl of protein A-Sepharose (1:1) (Amersham Biosciences) was added and also incubated for 1 h at room temperature. Finally, washing and electrophoresis were performed as described above. Second, cells were solubilized in another lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Nonidet P-40, 1 mM Na3VO4,1 µg/ml leupeptin, and 1 mM PMSF), and then immunoprecipitation was performed as described under "Cell Lysis and Immunoblotting."

Extraction and Analysis of Gangliosides—Ganglioside extraction was performed as described previously (17). Briefly, lipids were extracted with chloroform/methanol (2:1, 1:1, and 1:2, sequentially) and desalted using a Sep-Pak C18 cartridge (Waters Associates). Acidic glycolipids were separated using DEAE-dextran (A-50) ion exchange chromatography. Ganglioside fractions were analyzed by TLC using a solvent system of chloroform, methanol, 0.2% CaCl2 (55:45:10). For TLC immunostaining, glycolipids were separated by high performance TLC with aluminum sheet silica gel plates (Merck) using a solvent system of chloroform, methanol, 0.2% CaCl2 (55:45: 10, v/v/v). The plates were dipped in isopropyl alcohol, 0.2% CaCl2, methanol (4:20:7, v/v/v), and glycolipids were transferred onto a clear blot membrane-PTM (Atto, Tokyo) using TLC thermal blotter AC-5970TM (Atto, Tokyo). After incubation in PBS with 5% bovine serum albumin, the membranes were probed with CTB/biotin (1:200), and bands were detected with an ABC kit (Vector Laboratories) and immunostained with horseradish peroxidase-1000 kit from Konica (Tokyo, Japan).

Expression Vector of RNAi against GM1 Synthase and Transfection—The RNAi expression vectors were constructed using an expression vector pH1-RNApuro (18), which contained a DNA template for the synthesis of (siRNA). The 21-mer candidate target sequences were selected from the open reading frame of mouse beta1,3-galactosyltransferase (B3galt4, GM1 synthase, GenBankTM accession number NM_019420 [GenBank] ), corresponding to nucleotides 463-483 (relative to the start codon) of B3galt4. The siRNA sequence contained a 5' BamHI cloning site followed by 21-mer target sequences, a 9-mer spacer, another 21-mer reverse complementary target sequence, the transcription terminator (TTTTT), and the HindIII (3') cloning site. The full-length of the sequence was 68-nucleotide oligonucleotides, which were synthesized in the forward and reverse directions and annealed to form double-stranded DNA. This double-stranded DNA was cloned into pH1-RNA-puro to form pH1-RNApuro/siB3galt4.

For stable transfection of the RNAi expression vector, H7 cells (50-60% confluent) were grown in 6-cm culture dishes, and either 8 µg of pH1-RNApuro/siB3galt4 or pH1-RNApuro alone was introduced using Lipofectamine 2000TM (Invitrogen) according to the manufacturer's instructions. After 24 h, 6 µg/ml of puromycin (Calbiochem-Novabiochem) was added to the culture medium to select puromycin-resistant clones. Two weeks later, independent colonies were picked up, subcultured, and tested for expression of GM1 by flow cytometric analysis with biotin-conjugated cholera toxin B subunit as described above. The selected stable clones with decreased levels of GM1 were maintained in a complete culture medium containing puromycin (6 µg/ml).

Real Time RT-PCR—Total RNA was isolated in TrizolTM (Invitrogen), and template cDNA was synthesized from total RNA using Moloney murine leukemia virus reverse transcriptase kit (Invitrogen). Real time RT-PCR was done using DNA Engine Opticon2TM System (Bio-Rad). cDNA product (8 ng) was amplified in a 20-µl reaction containing 10 µl of DyNAmoTM SYBR Green qPCR kit (Finnzymes, Espoo, Finland) and 1 µl of each primer (5 mM). The primers for beta1,3-Gal-T were 5'-GTCAGCAGCACACAGGGATA-3' and 5'-CTGGGACGTTGACATACACG-3', to amplify a 158-bp fragment corresponding to nucleotides 751-594 in the open reading frame of the beta1, 3-Gal-T gene (GenBankTM accession number NM_019420 [GenBank] ). The PCR program consisted of an initial denaturation at 95 °C for 10 min followed by amplification with 40 cycles (95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s). Data were analyzed using Opticon Monitor2TM software (Bio-Rad).

Immunofluorescence—Cells grown on coverslips were fixed in 3.7% paraformaldehyde for 30 min at room temperature. After blocking with 10% normal goat serum (Chemicon International Inc.) for 30 min, cells were incubated with a mixture of anti-MMP-9 antibody (Ab19047) (10 µg/ml) and CTB-Alexa555 in 10% normal goat serum at 4 °C overnight. After washing, antibody binding was detected with FITC-conjugated goat anti-rabbit IgG (MP Biomedicals, Inc. Eschwege, Germany) for 1 h at room temperature. The stained cells were analyzed with a confocal laser microscope Fluoview FV500TM (Olympus, Tokyo, Japan).

Statistical Analysis—Statistical significance of data were determined using Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of High Metastatic Sublines—To establish high metastatic sublines using a low metastatic line of Lewis lung carcinoma (P29), we adopted a consecutive in vivo selection strategy. Before in vivo selection, we established two sublines H7 and C4 from P29 by a limiting dilution method. Then we repeatedly injected H7 or C4 cells into C57BL/6 mice with intravenous or subcutaneous injection. After five rounds of injection (Table 1), metastatic nodules from lung, ovary, adrenal gland, or lymph node were harvested to establish several high metastatic sublines growing in vitro, and they were designated as H7-Lu (lung metastasis of H7 by intravenous), H7-O (ovary metastasis of H7 by intravenous), H7-A (adrenal metastasis of H7 by intravenous), C4-sc (lung metastasis of C4 by subcutaneous), and C4-Ly (lymph node metastasis of C4 by subcutaneous) (Fig. 1A). Malignant properties of high metastatic sublines were analyzed by MTT assay and invasion assay. High metastatic sublines showed higher proliferation and invasion activity than the parent lines (Fig. 1, B and C). High metastatic sublines from C4 showed very high invasion activity when serum was present in the lower chamber.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Lung metastatic potentials of P29 cells * indicates p < 0.05 (n = 4); ** indicates p < 0.001 (n = 5), as compared with the values at the first metastatic experiment.

 


Figure 1
View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 1.
Establishment of high metastatic sublines. Two sublines H7 and C4 isolated by a limiting dilution from P29 cells were repeatedly injected into C57BL/6 mice with intravenous or subcutaneous injection as described under "Experimental Procedures." After five rounds of selection, the metastatic nodules from lung, ovary, adrenal gland, or lymph node were harvested to establish several high metastatic sublines growing in vitro. A, examples of metastasis in lung (a) and ovary and adrenal gland (b) with intravenous injection of H7 cells, and lung (c) and lymph node (d) with subcutaneous injection of C4 cells. B, in vitro proliferation was examined by MTT assay. Error bars represent S.D. (n = 3). C, 5 x 105 cells were seeded in the upper chamber in the presence or absence of serum and were incubated for 20 h. The noninvading cells remaining on the upper surface of the filter were removed, and the cells that appeared on the lower surface of the filter were fixed with 75% ethanol for 30 min, then stained, and counted under a microscope. Invading cell numbers were counted, and results were presented with serum-free H-7 or C4 samples as 100%. Columns represent the means ± S.D. A representative of three independent experiments was shown. P, Student's t test. Lu, lung; O, ovary, A, adrenal gland.

 
Expression of Gangliosides and Adhesion Molecules on the Cell Surface Membrane—We examined expression profiles of gangliosides on the cell surface membrane of high metastatic lines and parent lines by flow cytometry (Fig. 2A). Among the gangliosides examined, definite expression of GM1 and GD1a and minimal or low percentage expression of GM2, GA1, GD3, GD2, and GT1b were observed. Expression levels of GM1 markedly decreased in the high metastatic lines (H7-Lu, H7-O, H7-A, C4-sc, and C4-Ly). Expression levels of other gangliosides showed no apparent change compared with those in the individual parent lines.


Figure 2
View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 2.
Expression of gangliosides and adhesion molecules on the cells. Expression of gangliosides (A) and adhesion molecules (B) on the cells was analyzed by flow cytometry using anti-ganglioside mAbs and anti-adhesion molecule antibodies as described under "Experimental Procedures." Ordinate and abscissa represent cell numbers and relative fluorescence intensity, respectively. Thin lines represent samples with first antibodies, and dark shadings represent controls with the second antibody alone.

 
Expression of 10 adhesion molecules on the cell surface membrane was also analyzed by flow cytometry (Fig. 2B). In the high metastatic lines, slightly lower expression of integrin subunit {alpha}1 was observed, but there were no obvious changes in the expression levels of other antigens between high metastatic lines and the individual parent lines, suggesting that changes in GM1 expression levels might be relevant to the increased metastatic potential.

Increased Secretion and Activation Levels of MMP-9 in High Metastatic Lines—The secretion and activation levels of MMP-2 and MMP-9, main proteolytic enzymes for extracellular matrix components, in the culture supernatants were analyzed by gelatin zymography (Fig. 3). Both pro-form and active form of MMP-9 levels were significantly increased in the supernatants of high metastatic lines compared with those of the parent lines, although there were no persistent changes in the expression levels of MMP-2 between the high metastatic and the parent lines. These results suggested that the increased metastatic activities in the high metastatic lines depend mainly on the increased secretion and activity of MMP-9.

Changes in the Floatation Pattern of MMP-9—To analyze the mechanisms for higher secretion and activation of MMP-9 in the high metastatic lines, we examined the intracellular localization of MMP-9 by isolating GEM/rafts fraction with Triton X-100 extracts of cells. Ten fractions from the discontinuous sucrose density gradient were prepared and analyzed for distribution of MMP-9 as well as raft markers, such as caveolin-1 and GM1 (Fig. 4). Immunoblot analysis showed that most of caveolin-1 and GM1 were found in fractions 3 and 4, indicating that they contained the GEM/rafts fraction (data not shown). Interestingly, MMP-9 bands (105 kDa) appeared mainly in the raft fraction of high metastatic lines, but only faint or no bands were present in that of the parent lines. These results suggested that localization of MMP-9 to the GEM/rafts fraction resulted in the increased MMP-9 secretion and activation, leading to the increased invasion activity.


Figure 3
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 3.
Increased MMP-9 secretion and activation levels in high metastatic lines. A, MMP-2 and MMP-9 secretion and activation levels of the cells were determined by gelatin zymography as described under "Experimental Procedures." B, bands of pro-MMP-9 and act-MMP-9 in A were quantitated by a scanner, and the relative intensities of the bands were presented. Note that thick bands at 105 kDa were of a proform activated during the gel electrophoresis.

 


Figure 4
View larger version (74K):
[in this window]
[in a new window]
  		FIGURE 4.
Localization of MMP-9 to the GEM/rafts fraction in high metastatic lines. Cells were lysed using Triton X-100, and the extracts were fractionated with discontinuous sucrose density gradient centrifugation. Fractions were subjected to immunoblotting using the antibodies against the proteins indicated. Bands of mouse MMP-9 (105 kDa) were found in fractions 3 and 4 in the high metastatic lines derived from both sublines.

 
MbetaCD Suppressed MMP-9 Secretion, Invasion Activity, and Metastatic Potential—Depletion of cholesterol with MbetaCD for 30 min suppressed MMP-9 activity in a dose-dependent manner in the culture medium of a high metastatic line H7-O (Fig. 5A). Activity of MMP-9 was decreased in the supernatant of cells treated with 5 mg/ml MbetaCD, and both MMP-9 and MMP-2 activities disappeared with 10 or 15 mg/ml MbetaCD. Then we examined the effect of MbetaCD treatment on the cell invasion in vitro. As expected, the MbetaCD treatment resulted in marked suppression of the invasion activity in H7-O cells in a dose-dependent manner (Fig. 5B, right). Similar suppression of invasion activity was also observed in the other high metastatic lines (H7-Lu and H7-A) (Fig. 5B, left). Then we examined intracellular localization of MMP-9 in MbetaCD-treated H7-O cells by isolating GEM/rafts (Fig. 5C). To our surprise, the MMP-9 band could not be detected in GEM/rafts fraction in the MbetaCD-treated cells. Furthermore, we analyzed the effect of MbetaCD on the metastatic potential in vivo. The numbers of lung metastases in the MbetaCD-treated H7-O cells were definitely reduced (Fig. 5D). These results strongly suggested that MMP-9 secretion and cell invasion were tightly associated with intracellular localization of MMP-9 in GEM/rafts.


Figure 5
View larger version (62K):
[in this window]
[in a new window]
  		FIGURE 5.
MbetaCD treatment suppressed MMP-9 activity and invasion in a dose-dependent manner in high metastatic lines. A, H7-O cells were incubated for 30 min at 37 °C with MbetaCD (10 mg/ml). Cells were further incubated for 5 or 20 h in plain DMEM, and culture supernatants were analyzed by gelatin zymography after concentration (left). H7-O cells were incubated for 30 min at 37 °C with various concentrations of MbetaCD. Culture supernatants after 5 h of incubation were analyzed by gelatin zymography (right). B, H7, H7-Lu, H7-O, and H7-A cells were treated by DMEM with or without MbetaCD (10 mg/ml) (left), or H7-O cells were treated by DMEM with MbetaCD (0, 1, 5, 10, and 15 mg/ml) (right) for 30 min at 37 °C, and then 5 x 105 were seeded to the upper chamber in the absence of serum and incubated for 20 h as described in Fig. 1C. Invaded cell numbers were counted, and results were presented with nontreated H7 (left) or nontreated H7-O (right) samples as 100%. Columns represent the means ± S.D. A representative of two independent experiments was shown. P, Student's t test. (right). C, H7-O cells were treated with MbetaCD (10 mg/ml) and then were lysed, and the extracts were fractionated as described in the legend for Fig. 4. Immunoblotting with these fractions using the antibodies indicated was performed. Bands of MMP-9 (105 kDa) were found in the fractions 3 and 4 in untreated cells. D, H7-O cells were treated with MbetaCD (10 mg/ml), and cells (0.5 x 106/mouse) were injected into the tail vein of C57BL/6 mice. Mice were sacrificed at 3 weeks after injection, and metastatic nodules on the lung surface were counted. Columns represent the mean ± S.D. A representative of three independent experiments is shown.

 
Reduced Expression of GM1 in the High Metastatic Lines—Expression levels of gangliosides on cell surface membranes of the high metastatic lines and the parent lines are shown in Fig. 2. Among the gangliosides examined, only GM1 expression was reduced in the high metastatic lines. In TLC of ganglioside fractions, similar changes in ganglioside composition were shown, i.e. marked decrease of GM1 bands in high metastatic lines. Band intensities of GM3, GM2, and GD1a showed no clear change in the individual groups, i.e. H7 and C4 (Fig. 6A). These results were also confirmed in TLC immunostaining using biotin-conjugated CTB (Fig. 6, B and C). GM1 levels in the cell lysates were also analyzed by immunoblotting. As shown in Fig. 6, D and E, GM1 levels were apparently suppressed in the high metastatic lines compared with those in the parent lines. Because ganglioside GM1 has been considered to be one of the constituents of lipid rafts or microdomains, where cellular signals are exchanged on the cell membrane (12), these results strongly suggest that GM1 was involved in the regulation of MMP-9 secretion and activity.

Down-regulation of GM1 with RNAi Resulted in the Enhanced Invasion—To further analyze the role of GM1 in tumor metastasis, we took advantage of the siRNA approach. After the transfection of the parent line H7 cells with a GM1 synthase RNAi expression vector (pH1-RNApuro/siB3galt4) or pH1-RNA-puro, two stable GM1-siRNA lines (Si-1 and Si-2) and two vector control lines (V-1 and V-2) were established. To confirm the knockdown of GM1 expression in H7 cells, we analyzed the expression of the GM1 synthase gene and of GM1 in siRNA lines and vector control cells by real time RT-PCR (Fig. 7A) and flow cytometry (Fig. 7B), respectively. GM1 synthase mRNAs were markedly suppressed in GM1-siRNA cells (Si-1 and Si-2). Cell surface expression of GM1 in GM1-siRNA cells completely disappeared. Cell invasion and growth were analyzed (Fig. 7, C and D), showing significantly increased cell invasion (p < 0.05, n = 2) and mildly enhanced proliferation in GM1-siRNA cells. These results supported the idea for the role of GM1 in suppressing signals for cell growth and invasion.

Effects of GM1 Knockdown on the Intracellular Localization of MMP-9 and Its Secretion—To clarify the regulatory mechanisms for the MMP-9 secretion with GM1 expression, we examined the intracellular localization of MMP-9 by isolating GEM/rafts. The MMP-9 bands (105 kDa) appeared more strongly in the GEM/rafts fraction (fractions 3 and 4) in GM1-siRNA cells compared with those of the vector control cells (Fig. 8A). We then analyzed the secreted MMP-9 in the culture medium of GM1-siRNA cells, showing higher MMP-9 activity in GM1-siRNA cells compared with that in the vector control cells (Fig. 8, B and C). These results strongly suggested that down-regulation of GM1 enhanced the MMP-9 secretion and its activation based on the increased MMP-9 localization in the GEM/rafts, probably leading to the enhanced metastatic potential.

Enrichment of Integrin beta1 in the GEM/Rafts Fraction in the High Metastatic Lines and GM1-siRNA Transfectants—To further analyze the mechanisms for the increased secretion and activation of MMP-9 in the high metastatic lines, we examined the intracellular localization of adhesion molecules such as CD44, DMP-1, and integrins by immunoblotting of the sucrose density gradient fractions of Triton X-100 extracts (Fig. 9). Both CD44 (Fig. 9A) and DMP-1 (Fig. 9B) were found only in non-GEM/rafts fractions in the all cell lines examined. On the other hand, definite bands of integrin beta1 were detected in the GEM/rafts fractions as well as in the non-GEM/rafts fractions, and band intensities of integrin beta1 in GEM/rafts fractions were apparently stronger in high metastatic lines and in the GM1-siRNA transfectants than those of individual control cells (Fig. 9C). These results suggested that enrichment of integrin beta1 in the GEM/rafts was implicated in the increased MMP-9 secretion and activation.


Figure 6
View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 6.
Lower expression levels of GM1 in high metastatic lines. Biochemical analysis of ganglioside compositions of cell lines. A, TLC patterns of gangliosides extracted from P29 lines (H7 and C4) were compared as described under "Experimental Procedures." Bands were visualized with resorcinol spray. M, ganglioside standards from bovine brain. B, to clearly visualize GM1 bands, TLC immunostaining with biotin/CTB was performed. C, bands of GM1 in B were quantitated by a scanner, and the relative intensities of the bands were plotted. D, Western immunoblotting (IB) was performed to detect GM1 at the dye front. Fifty µg of total protein were loaded in each lane, and beta-actin expression levels were used as a control for equal protein loading. E, bands of GM1 in D were quantitated by a scanner, and the relative intensities of the bands were plotted after correction with beta-actin bands.

 


Figure 7
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 7.
Knockdown of GM1 with RNAi resulted in increased invasion and proliferation. Transfection of an expression vector for RNAi against GM1 synthase based on the pH1-RNApuro vector resulted in the establishment of two stable RNAi-expressing lines (Si-1 and Si-2) as well as two vector control lines (V-1 and V-2). A, expression levels of GM1 synthase as analyzed by real time RT-PCR. Error bars represent S.D. (n = 3). B, expression levels of cell surface GM1 were analyzed by flow cytometry. C, invasion of Si-1, Si-2, V-1, and V-2 cells were analyzed by Matrigel invasion assay as described under "Experimental Procedures." Columns represent the mean ± S.D. A representative of two independent experiments is shown. D, in vitro proliferation of Si-1, Si-2, V-1, and V-2 cells was examined by MTT assay. Error bars represent S.D. (n = 3). P, Student's t test.

 
Complex Formation of GM1, Integrins, and MMP-9 on the Cell Surface—To clarify the implication of integrins in the regulatory mechanisms for the MMP-9 secretion and activation, associations of integrin beta1 with MMP-9, DMP-1, CD44, and GM1 were examined by immunoprecipitation and subsequent immunoblotting of the immunoprecipitates using antibodies for individual molecules (Fig. 10). First of all, MMP-9 was coprecipitated with integrin beta1 in all cell lines examined, whereas DMP-1 and CD44 were not found in the immunoprecipitates with anti-integrin beta1. In the H7 lines, coprecipitated MMP-9 bands with integrin beta1 were stronger in high metastatic lines than that in the parent line (Fig. 10B). As for GM1, it was more efficiently coprecipitated with anti-integrin beta1 in the two patent lines (H-7 and C4) and in the GM1-siRNA vector controls than the individual counterparts (Fig. 10A). However, this might be due to the differences in the expression levels of GM1. Furthermore, similar results were obtained when coimmunoprecipitation experiments were performed under different conditions using Nonidet P-40 to prepare the lysates (Fig. 10, C and D), or incubating the lysates with antibodies at room temperature instead of at 4 °C (Fig. 10, E and F). Taken together, integrin molecules seemed to form a complex with MMP-9 and/or GM1 on the cell surface of P29 cells, probably at the GEM/rafts.


Figure 8
View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 8.
The effects of the knockdown of GM1 on the localization and secretion of MMP-9. A, localization of MMP-9 in V-1 and Si-1 cell was analyzed as described in Fig. 4. B, MMP-2 and MMP-9 secretion and activation levels in Si-1, Si-2, V-1, and V-2 cells were determined by gelatin zymography as described under "Experimental Procedures." C, bands of pro-MMP-9 and act-MMP-9 in B were quantitated by a scanner, and the relative intensities of the bands are presented.

 


Figure 9
View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 9.
Enrichment of integrin beta1 in the GEM/rafts fractions in high metastatic lines and GM1-siRNA transfectants. Cells were lysed using Triton X-100, and the extracts were fractionated with discontinuous sucrose density gradient centrifugation. Fractions were subjected to immunoblotting (IB) using the antibodies against CD44 (A), DMP-1 (B), and integrin beta1(C). Note that high metastatic lines showed stronger bands of integrin beta1 in the GEM/rafts than individual parent cells or control cells.

 


Figure 10
View larger version (63K):
[in this window]
[in a new window]
  		FIGURE 10.
Integrins and MMP-9 form a complex as analyzed by immunoprecipitation. Cells of GM1-siRNA transfectants (A, C, and E) and P29 lines (B, D, and F) were solubilized in the lysis buffers containing either Triton X-100 (A-D) or Nonidet P-40 (E and F), and the lysates were immunoprecipitated (IP) with 2 µg of anti-integrin beta1 or normal rabbit IgG at either 4 °C (A, B, E, and F)or at room temperature (C and D) as described under "Experimental Procedures." After SDS-PAGE of the immunoprecipitates with an anti-integrin beta1 antibody, immunoblotting (IB) was performed with antibodies against the proteins indicated. GM1 was detected with biotin/CTB.

 
Distribution Patterns of MMP-9 and GM1 as Analyzed by Immunocytostaining—To analyze the distribution patterns of MMP-9 and GM1 in the cells, double immunofluorescence staining was performed using H7, H7-O, and GM1-siRNA cells. As shown in Fig. 11, MMP-9 was stained with higher intensity and GM1 was stained with lower intensity in H7-O and GM1-siRNA cells compared with the parent H7 cells. When these two stainings merged, high metastatic sublines H7-O, Si-1, and Si-2 showed MMP-9-dominant patterns, whereas the parent H7 and a vector control V-1 showed GM1-dominant patterns with partial colocalization of GM1 and MMP-9. These results confirmed the results obtained by biochemical studies, i.e. expression levels of GM1 regulate intracellular localization of MMP-9 and integrins by modulating GEM/rafts and forming a molecular complex consisting of GM1-integrin-MMP-9 or integrin-MMP-9 in the GEM/rafts. The latter complex might facilitate the release and activation of MMP-9, resulting in the promotion of tumor metastasis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Membrane microdomains such as GEM/rafts have been considered to be involved in the regulation of cellular signals critical for the cell proliferation, differentiation, and apoptosis (19). Well known constituting components in GEM/rafts are caveolins and flotillin (20). Ganglioside GM1 has also been used as a GEM/rafts marker in various experimental systems (21). Among the caveolin families, caveolin-1 is most frequently down-regulated in many oncogenically transformed cells (22) and tumors, with some exception (13), and is considered to be a sort of tumor suppressor molecule. In fact, suppression of caveolin-1 expression enhanced tumor development in mice (23), and targeted disruption of caveolin-1 gene also resulted in the enhanced proliferation (24) or tumorigenesis (25). Furthermore, expression of oncoproteins such as c-Myc, Bcr-Abl, activated Ras families, Src, and ErbB2 down-regulated the expression of caveolin-1 (13).

As for GM1, we have analyzed the effects of overexpression of GM1 synthase cDNAs (26) on cell proliferation and differentiation (15, 27). Generally, GM1 expression caused reduced cell proliferation in Swiss3T3 cells (15) and NIH3T3 cells (28) or reduced response of PC12 cells to nerve growth factor (27). In these situations, GM1 expression reduced cell signals from platelet-derived growth factor or nerve growth factor, and also induced changes in the intracellular localization of platelet-derived growth factor receptor (15) or nerve growth factor receptors (27). These data indicated that GM1 is not only a mere marker of GEM/rafts but a significant regulatory molecule of important signals probably based on the modulation of the physicochemical properties of GEM/rafts (15, 27).

In this study, reduced expression levels of ganglioside GM1 were one of the conspicuous changes in the expression levels of cell surface molecules commonly detected among the high metastatic P29 sublines compared with the individual parent cells (Fig. 2A). Although expression levels of GD1a were generally very high in these cell lines, no definite changes in its expression levels could be detected in the H7 group, and a mild decrease was found in the C4 group in TLC. However, there were no definite differences in the flow cytometry patterns, suggesting GD1a might not be relevant to their metastatic potential. In fact, reduced GM1 expression had profound implications in the tumor properties such as cell proliferation and invasion activity (Fig. 7). Here, it was demonstrated that GM1 is capable of modulating molecules directly involved in tumor metastasis like metalloproteases and integrins as well as the regulation of receptors for growth/differentiation factors.

Gelatinases such as MMP-2 and MMP-9 have been rigorously studied on their structures, substrates (6), regulation of the gene expression (29, 30), and proenzyme activation (31). In particular, MMP-9 is inducible and is associated on the cell surface in multiple ways (6, 32). It was reported that adherent cell types do not contain storage granules of gelatinases and are dependent on direct secretion of the newly synthesized enzymes (33), whereas leukocytes have large amounts of protease-containing granules named as gelatinase granules (34). In our study, MMP-9 could be definitely detected in the GEM/raft fraction only in the high metastatic sublines (Fig. 4). Disruption of lipid rafts with MbetaCD resulted in the disappearance of MMP-9 from the lipid raft fraction (Fig. 5C). Furthermore, reduction of GM1 levels with RNAi dramatically enhanced invasion activity (Fig. 7C) and clearly enriched MMP-9 to the GEM/rafts fraction in the transfectant cells (Fig. 8A). All these results suggested that MMP-9 could be enriched in GEM/rafts leading to smooth secretion and subsequent activation in low GM1 cells. Although MMP-9 bands were generally stronger in the high metastatic lines than in the parent lines, suggesting that transcriptional regulation might be more important, real time RT-PCR experiments revealed that mRNA levels of MMP-9 were not altered among those cell lines (data not shown). Thus, we have elucidated that the GM1 level is one of potent factors to regulate intracellular localization of MMP-9.


Figure 11
View larger version (82K):
[in this window]
[in a new window]
  		FIGURE 11.
Distinct localization of MMP-9 and GM1 as analyzed by immunocytostaining. Cells were fixed with paraformaldehyde and double stained with anti-MMP-9 antibody (AB19047) and CTB-Alexa555 combined with FITC-conjugated goat anti-rabbit IgG. Images were obtained by a confocal microscopy as described under "Experimental Procedures." Bars indicate 10 or 20 µm as indicated.

 
Although the enrichment of gelatinases in GEM/rafts seems critical for the expression of their enzyme activity (35), complex regulatory mechanisms in the subsequent processes, until the actual proteolytic degradation of the substrates, have been elucidated (33). Caveolin-1 was essential in the regulation of the secretion of MMP-2/9, as shown by the enhanced tumorigenesis and metastasis in caveolin-1 gene disrupted mice (36). However, the mere arrest of gelatinases at the GEM/rafts was not necessarily efficient in the angiogenesis (37) or tumorigenicity (38). Forced expression of MMP-9 on the MCF-7 cell surface in a chimeric form with a glycosylphosphatidylinositol anchor resulted in less angiogenesis than the wild type MMP-9 due to reduced release (37). A mutant MT1-MMP lacking the cytoplasmic tail showed reduced tumorigenesis, indicating that the cytoplasmic portion was crucial in the release of the enzyme and in the appropriate turnover (37). Our results might indicate that MMP-9 could localize in GEM/rafts and also could be released efficiently for the higher invasion and metastasis in low GM1 cells.

There have been a number of reports on molecules that bind to MMP-9 on the cell surface (32). In particular, adhesion molecules such as CD44 (39, 40) and small integrin binding ligand N-linked glycoproteins (SIBLINGs) (41, 42) have been demonstrated to be physically and functionally associated with MMP-9 and/or other metalloproteases, leading to the generation of invasion and metastatic activity in cancer cells. Dentin matrix protein I (DMP1), one of SIBLINGs, also interacts with CD44 and/or integrin {alpha}vbeta3 to enhance the cofactor activity of factor H (41). Integrins have been also reported to contribute in the regulation of MMP-9 expression (44-46) and secretion (47-50). Therefore, we examined the intracellular localization of CD44, DMP-1, and integrins as shown in Fig. 9. Only bands with anti-integrin beta1 were detected in the GEM/rafts fractions. Moreover, the band intensities of integrin beta1 were stronger in the cell lines with high metastatic and/or high invasive features than in the individual control lines. These results strongly suggested that changes in the floating pattern of integrin molecules as well as those of MMP-9, as shown in Fig. 4, were essentially important and mutually related.

Knockdown of GM1 using RNAi expression vectors against the GM1 synthase gene was very successful (Fig. 7, A and B) and clearly revealed that GM1 is needed to regulate the metabolism, intracellular localization, and probably secretion of MMP-9 (Fig. 8). On the other hand, overexpression of GM1 with GM1 synthase cDNA in the high metastatic sublines was not achieved for unknown reasons, although repeated transfection was tried with different expression vectors. Although the mechanisms for the modulation of MMP-9 and integrin localization with GM1 are not clear at the present time, expression levels of GM1 at GEM/rafts might change the physicochemical nature of the microdomain, resulting in the dispersion/enrichment of integrins and MMP-9 as shown for many other surface molecules (15, 27). Changes in glycolipid composition might induce drastic alterations in the distribution of not only receptors but of caveolin-1 as shown by overexpression of GM3 (51).

Gelatinases have been reported to be intimately regulated by integrins (6). The fact that MMP-9 mRNA stability was regulated by {alpha}3beta1 integrin (52) and that the integrin cytoplasmic tail motif was sufficient to promote tumor cell invasion mediated by MMP-2/9 (53) indicate that integrins regulate MMP-9 in an outside-in manner. However, integrins also directly interacted with MMP-9 on the cell surface (54-55). Karadag et al. (56) reported an interesting finding that DMP-1 bridged MMP-9 to integrins and/or CD44 on colon cancer cells to enhance cell invasion.

In our study, only MMP-9 could be coprecipitated with integrin beta1, and CD44 and DMP-1 could not, corresponding with the results of GEM/rafts fractionation experiments. Therefore, it is assumed that the residence of integrin molecules in the GEM/rafts was primarily determined by expression levels of GM1, and MMP-9 was subsequently recruited by integrins into the GEM/rafts, leading to its efficient secretion and activation. The fact that integrin molecules were coprecipitated with MMP-9 even by incubation at room temperature or from lysates prepared with different detergents suggested that these molecules were significantly associated directly or indirectly, not simply because of the cofractionation in the GEM/rafts. Whether integrins and MMP-9 directly bind or unknown molecules bridge these two should be clarified in the future. Thus, the remaining issues are the mechanisms by which GM1 expression levels regulate intracellular distribution of integrins, and by which MMP-9 binds to integrins on the cell surface of P29 cells.


Figure 12
View larger version (62K):
[in this window]
[in a new window]
  		FIGURE 12.
A schematic to show that MMP-9 secretion is regulated by the modulation of GEM/rafts based on the expression levels of GM1 and the complex formation of GM1, integrin beta1, and MMP-9. In the parent cells, high expression levels of GM1 result in the localization of integrins and MMP-9 mainly outside of GEM/rafts. On the other hand, low expression levels of GM1 in high metastatic lines induce enrichment of GEM/rafts-localizing MMP-9 and integrins, forming a complex of integrin and MMP-9 (with/without GM1), leading to the enhancement of MMP-9 secretion and activation, and eventually to the promotion of tumor metastasis.

 
As shown in Fig. 12, GM1 probably forms a complex with MMP-9 and integrins at a low level in the GEM/rafts of the parent cells. In turn, a complex of MMP-9 and integrins lacking GM1 is mainly formed in the GEM/rafts in high metastatic lines and GM1-siRNA cells, facilitating the efficient secretion and activation of MMP-9. As for roles of gangliosides, MMP-9 activation was inhibited by ganglioside GM3, and its association with integrin was also disrupted by GM3 (57). Association of integrins with gangliosides has also been reported by Cheresh et al. (58), Chen et al. (59), and Aixinjueluo et al. (60). In particular, gelatinase secretion was reported to be dependent on integrin {alpha}3beta1 (61) or regulated by focal adhesion kinase (62), probably based on the interaction between integrins and extracellular matrix. Therefore, it seems possible that MMP-9 is regulated by gangliosides through interactions between gangliosides and integrins. Furthermore, integrins ({alpha}vbeta3) and MMP-9 sometimes cooperate in regulating cancer cell migration (50) or angiogenesis (43). Therefore, GM1 ganglioside should play its role in not only modulating GEM/rafts features but in direct modification of integrins. Whether and how a molecular complex is formed among these molecules should be key issues for further understanding of the mechanisms by which gangliosides regulate gelatinases, and tumor metastasis remains to be investigated.

In this study, we cloned two different sublines of P29 with limiting dilution methods based on the expression levels of GM1. Repeated injection of cells from the metastatic foci resulted in several high metastatic lines for both sublines. Although they showed similar high frequency of lung metastasis by intravenous injection, H7-derived lines also metastasized to distant organs such as adrenal gland and ovary. On the other hand, C4 showed higher metastatic frequency to lung, when injected subcutaneously. Thus, they showed different tropism in preferential tissues and sites of tumor metastasis. These results suggested that these two sublines should have distinct features. Nevertheless, high metastatic lines derived from these two sublines showed similar changes in the GM1 expression (Fig. 2A), cell proliferation (Fig. 1B), invasion activity (Fig. 1C), MMP-9 activity in the culture supernatants (Fig. 3C), and intracellular localization of MMP-9 (Fig. 4) and integrins (Fig. 9C). Moreover, the involvement of MMP-9 and integrins and the reduced GM1 expression level in the enhanced metastatic potential of these sublines should be common mechanisms, indicating that GM1 expression levels are generally crucial in the determination of metabolic behaviors of proteolytic enzymes, and eventually of malignant properties of the tumors.


   FOOTNOTES
 
* This work was supported by Grant-in-aid for Scientific Research on Priority Areas 14082102, by Grant-in-aid for Scientific Research 16390075 from the Ministry of Education, Culture, Science, Sports and Technology of Japan, and by CREST, Japan Science and Technology Agency. 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

1 To whom correspondence should be addressed: Dept. of Biochemistry II, Nagoya University Graduate School of Medicine, Tsurumai, Showa-ku, Nagoya 466-0065 Japan. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi{at}med.nagoya-u.ac.jp

2 The abbreviations used are: MMP, matrix metalloprotease; GM1-siRNA, GM1 synthase RNAi transfectant; GEM, glycolipid-enriched microdomain; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MbetaCD, methyl-beta-cyclodextrin; CTB, cholera toxin B; RT, reverse transcription; siRNA, small interfering RNA; RNAi, RNA interference; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; MES, 4-morpholineethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank T. Mizuno and Y. Nakayasu for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Harlozinska, A. (2005) Anticancer Res. 25, 3327-3333[Abstract/Free Full Text]
  2. Kannagi, R. (2004) Glycoconj. J. 20, 353-364[CrossRef][Medline] [Order article via Infotrieve]
  3. Ellis, L. M. (2004) Semin. Oncol. 31, 3-9[Medline] [Order article via Infotrieve]
  4. Nathanson, S. D. (2003) Cancer 98, 413-423[CrossRef][Medline] [Order article via Infotrieve]
  5. Cao, Y. (2005) Nat. Rev. Cancer 5, 735-743[CrossRef][Medline] [Order article via Infotrieve]
  6. Bjorklund, M., and Koivunen, E. (2005) Biochim. Biophys. Acta 1755, 37-69[Medline] [Order article via Infotrieve]
  7. Egeblad, M., and Werb, Z. (2002) Nat. Rev. Cancer 2, 161-174[Medline] [Order article via Infotrieve]
  8. Brown, D. A., and London, E. (1998) Annu. Rev. Cell Dev. Biol. 14, 111-136[CrossRef][Medline] [Order article via Infotrieve]
  9. Laude, A. J., and Prior, I. A. (2004) Mol. Membr. Biol. 21, 193-205[CrossRef][Medline] [Order article via Infotrieve]
  10. Stan, R. V. (2005) Biochim. Biophys. Acta, 1746, 334-348[Medline] [Order article via Infotrieve]
  11. Hakomori, S. I. (2000) Glycoconj. J. 17, 143-151[CrossRef][Medline] [Order article via Infotrieve]
  12. Simons, K., and Toomre, D. (2000) Nat. Rev. Mol. Cell Biol. 1, 31-39[CrossRef][Medline] [Order article via Infotrieve]
  13. Williams, T. M., and Lisanti, M. P. (2005) Am. J. Physiol. 288, C494-C506[CrossRef]
  14. Nakanishi, H., Oguri, K., Yoshida, K., Itano, N., Takenaga, K., Kazama, T., Yoshida, A., and Okayama, M. (1992) Biochem. J. 288, 215-224[Medline] [Order article via Infotrieve]
  15. Mitsuda, T., Furukawa, K., Fukumoto, S., Miyazaki, H., Urano, T., and Furukawa, K. (2002) J. Biol. Chem. 277, 11239-11246[Abstract/Free Full Text]
  16. Zhao, J., Furukawa, K., Fukumoto, S., Okada, M., Miyazaki, H., Shiku, H., Aizawa, S., Matsuyama, M., and Furukawa, K. (1999) J. Biol. Chem. 274, 13744-13747[Abstract/Free Full Text]
  17. Yamashiro, S., Ruan, S., Furukawa, K., Tai, T., Lloyd, K. O., Shiku, H., and Furukawa, K. (1993) Cancer Res. 53, 5395-5400[Abstract/Free Full Text]
  18. Takamizawa, J., Konishi, H., Yanagisawa, K., Tomida, S., Osada, T., Harano, T., Yatabe, Y., Nagino, M., Nimura, Y., Mitsudomi, T., and Takahashi, T. (2004) Cancer Res. 64, 3753-3760[Abstract/Free Full Text]
  19. Hakomori, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10231-10233[Free Full Text]
  20. Parkin, E. T., Turner, A. J., and Hooper, N. M. (1999) Biochem. J. 344, 23-30[CrossRef][Medline] [Order article via Infotrieve]
  21. Harder, T., Scheiffele, P., Verkade, P., and Simons, K. (1998) J. Cell Biol. 141, 929-942[Abstract/Free Full Text]
  22. Koleske, A. J., Baltimore, D., and Lisanti, M. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1381-1385[Abstract/Free Full Text]
  23. Galbiati, F., Volonte, D., Engelman, J. A., Watanabe, G., Burk, R., Pestell, R. G., and Lisanti, M. P. (1998) EMBO J. 17, 6633-6648[CrossRef][Medline] [Order article via Infotrieve]
  24. Razani, B., Engelman, J. A., Wang, X. B., Schubert, W., Zhang, X. L., Marks, C. B., Macaluso, F., Russell, R. G., Li, M., Pestell, R. G., Di Vizio, D., Hou, H., Jr., Kneitz, B., Lagaud, G., Christ, G. J., Edelmann, W., and Lisanti, M. P. (2001) J. Biol. Chem. 276, 38121-38138[Abstract/Free Full Text]
  25. Williams, T. M., Lee, H., Cheung, M. W., Cohen, A. W., Razani, B., Iyengar, P., Scherer, P. E., Pestell, R. G., and Lisanti, M. P. (2004) J. Biol. Chem. 279, 24745-24756[Abstract/Free Full Text]
  26. Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., Furukawa, K., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799[Abstract/Free Full Text]
  27. Nishio, M., Fukumoto, S., Furukawa, K., Ichimura, A., Miyazaki, H., Kusunoki, S., Urano, T., and Furukawa, K. (2004) J. Biol. Chem. 279, 33368-33378[Abstract/Free Full Text]
  28. Hamamura, K., Tanahashi, K., Furukawa, K., Hattori, T., Hattori, H., Mizutani, H., Ueda, M., Urano, T., and Furukawa, K. (2005) Int. J. Oncol. 26, 897-904[Medline] [Order article via Infotrieve]
  29. Huhtala, P., Tuuttila, A., Chow, L. T., Lohi, J., Keski-Oja, J., and Tryggvason, K. (1991) J. Biol. Chem. 266, 16485-16490[Abstract/Free Full Text]
  30. Mook, O. R. F., Frederiks, W. M., and Van Noorden, C. J. F. (2004) Biochim. Biophys. Acta 1705, 69-89[Medline] [Order article via Infotrieve]
  31. Van Wart, H. E., and Birkedal-Hansen, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5578-5582[Abstract/Free Full Text]
  32. Fridman, R., Toth, M., Chvyrkova, I., Meroueh, S. O., and Mobashery, S. (2003) Cancer Metastasis Rev. 22, 153-166[CrossRef][Medline] [Order article via Infotrieve]
  33. Taraboletti, G., D'Ascenzo, S., Borsotti, P., Giavazzi, R., Pavan, A., and Dolo, V. (2002) Am. J. Pathol. 160, 673-680[Abstract/Free Full Text]
  34. Stefanidakis, M., Ruohtula, T., Borregaard, N., Gahmberg, C. G., and Koivunen, E. (2004) J. Immunol. 172, 7060-7068[Abstract/Free Full Text]
  35. Puyraimond, A., Fridman, R., Lemesle, M., Arbeille, B., and Menashi, S. (2001) Exp. Cell Res. 262, 28-36[CrossRef][Medline] [Order article via Infotrieve]
  36. Williams, T. M., Medina, F., Badano, I., Hazan, R. B., Hutchinson, J., Muller, W. J., Chopra, N. G., Scherer, P. E., Pestell, R. G., and Lisanti, M. P. (2004) J. Biol. Chem. 279, 51630-51646[Abstract/Free Full Text]
  37. Mira, E., Lacalle, R. A., Buesa, J. M., de Buitrago, G. G., Jimenez-Baranda, S., Gomez-Mouton, C., Martinez-A, C., and Manes, S. (2004) J. Cell Sci. 117, 1847-1857[Abstract/Free Full Text]
  38. Rozanov, D. V., Deryugina, E. I., Monosov, E. Z., Marchenko, N. D., and Strongin, A. Y. (2004) Exp. Cell Res. 293, 81-95[CrossRef][Medline] [Order article via Infotrieve]
  39. Bourguignon, L. Y., Gunja-Smith, Z., Iida, N., Zhu, H. B., Young, L. J., Muller, W. J., and Cardiff, R. D. (1998) J. Cell. Physiol. 176, 206-215[CrossRef][Medline] [Order article via Infotrieve]
  40. Yu, Q., and Stamenkovic, I. (1999) Genes Dev. 13, 35-48[Abstract/Free Full Text]
  41. Fedarko, N. S., Jain, A., Karadag, A., and Fisher, L. W. (2004) FASEB J. 18, 734-736[Abstract/Free Full Text]
  42. Fisher, L. W., Jain, A., Tayback, M., and Fedarko, N. S. (2004) Clin. Cancer Res. 10, 8501-8511[Abstract/Free Full Text]
  43. Hamano, Y., Zeisberg, M., Sugimoto, H., Lively, J. C., Maeshima, Y., Yang, C., Hynes, R. O., Werb, Z., Sudhakar, A., and Kalluri, R. (2003) Cancer Cell 3, 589-601[CrossRef][Medline] [Order article via Infotrieve]
  44. Larjava, H., Lyons, J. G., Salo, T., Makela, M., Koivisto, L., Birkedal-Hansen, H., Akiyama, S. K., Yamada, K. M., and Heino, J. (1993) J. Cell. Physiol. 157, 190-200[CrossRef][Medline] [Order article via Infotrieve]
  45. Xie, B., Laouar, A., and Huberman, E. (1998) J. Biol. Chem. 273, 11576-11582[Abstract/Free Full Text]
  46. Thomas, G. J., Lewis, M. P., Hart, I. R., Marshall, J. F., and Speight, P. M. (2001) Int. J. Cancer 92, 641-650[CrossRef][Medline] [Order article via Infotrieve]
  47. Wize, J., Sopata, I., Smerdel, A., and Maslinski, S. (1998) Inflamm. Res. 47, 325-327[CrossRef][Medline] [Order article via Infotrieve]
  48. Agrez, M., Gu, X., Turton, J., Meldrum, C., Niu, J., Antalis, T., and Howard, E. W. (1999) Int. J. Cancer 81, 90-97[CrossRef][Medline] [Order article via Infotrieve]
  49. Morini, M., Mottolese, M., Ferrari, N., Ghiorzo, F., Buglioni, S., Mortarini, R., Noonan, D. M., Natali, P. G., and Albini, A. (2000) Int. J. Cancer 87, 336-342[CrossRef][Medline] [Order article via Infotrieve]
  50. Rolli, M., Fransvea, E., Pilch, J., Saven, A., and Felding-Habermann, B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 9482-9487[Abstract/Free Full Text]
  51. Wang, X. Q., Sun, P., and Paller, A. S. (2002) J. Biol. Chem. 277, 47028-47034[Abstract/Free Full Text]
  52. Iyer, V., Pumiglia, K., and DiPersio, C. M. (2005) J. Cell Sci. 118, 1185-1195[Abstract/Free Full Text]
  53. Morgan, M. R., Thomas, G. J., Russell, A., Hart, I. R., and Marshall, J. F. (2004) J. Biol. Chem. 279, 26533-26539[Abstract/Free Full Text]
  54. Karamessinis, P. M., Tzinia, A. K., Kitsiou, P. V., Stetler-Stevenson, W. G., Michael, A. F., Fan, W. W., Zhou, B., Margaritis, L. H., and Tsilibary, E. C. (2002) Lab. Investig. 82, 1081-1093[Medline] [Order article via Infotrieve]
  55. Hassanieh, L., Rodriguez, D., Xu, J., Brooks, P. C., and Broek, D. (2003) Hybrid. Hybridomics 22, 285-292[CrossRef][Medline] [Order article via Infotrieve]
  56. Karadag, A., Fedarko, N. S., and Fisher, L. W. (2005) Cancer Res. 65, 11545-11552[Abstract/Free Full Text]
  57. Wang. X., Q, Sun, P., and Paller, A. S. (2003) J. Biol. Chem. 278, 25591-25599[Abstract/Free Full Text]
  58. Cheresh, D. A., Pytela, R., Pierschbacher, M. D., Klier, F. G., Ruoslahti, E., and Reisfeld, R. A. (1987) J. Cell Biol. 105, 1163-1173[Abstract/Free Full Text]
  59. Chen, H.-H., Fukumoto, S., Furukawa, K., Nakao, A., Akiyama, S., Urano, T., and Furukawa, K. (2003) Int. J. Cancer 103, 169-176[CrossRef][Medline] [Order article via Infotrieve]
  60. Aixinjueluo, W., Furukawa, K., Zhang, Q., Hamamura, K., Tokuda, N., Yoshida, S., Ueda, R., and Furukawa, K. (2005) J. Biol. Chem. 280, 29828-29836[Abstract/Free Full Text]
  61. DiPersio, C. M., Shao, M., Di Costanzo, L., Kreidberg, J. A., and Hynes, R. O. (2000) J. Cell Sci. 113, 2909-2921[Abstract]
  62. Sein, T. T., Thant, A. A., Hiraiwa, Y., Amin, A. R., Sohara, Y., Liu, Y., Matsuda, S., Yamamoto, T., and Hamaguchi, M. (2000) Oncogene 19, 5539-5542[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
W. J. Lubbe, D. S. Zuzga, Z. Zhou, W. Fu, J. Pelta-Heller, R. J. Muschel, S. A. Waldman, and G. M. Pitari
Guanylyl Cyclase C Prevents Colon Cancer Metastasis by Regulating Tumor Epithelial Cell Matrix Metalloproteinase-9
Cancer Res., April 15, 2009; 69(8): 3529 - 3536.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
C. Suzuki and N. Kojima
A Cholesterol-independent Membrane Microdomain Serves as a Functional Counter-receptor for E-selectin at the Colo201 Cell Surface and Initiates Signalling on E-selectin Binding
J. Biochem., July 1, 2007; 142(1): 55 - 64.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/26/18145    most recent
M512566200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, Q.
Right arrow Articles by Furukawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, Q.
Right arrow Articles by Furukawa, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement