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Biochemistry and Biophysics |
5ß1 Integrin Clustering and Stimulates Cell Migration1
Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 [H.-B. G., I. L., M. K., M. P.], and Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709 [S. K. A.]
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ABSTRACT |
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5 and ß1 integrin subunits of the fibronectin receptor. Cell migration toward fibronectin and invasion through Matrigel were both substantially stimulated in cells with induced expression of GnT-V. Induction of GnT-V had no effect on the level of cell surface expression of 5 and ß1 integrin subunits but did result in a more diffuse staining of the 5 and ß1 integrin subunits on the cell surface, suggesting that inhibition of integrin clustering may be causing these cells to be less adhesive and more motile. Surprisingly, there was no detectable expression of N-linked ß1,6 branching on the 5 subunit purified from HT1080 cells before and after induction of GnT-V; by contrast, however, the ß1 subunit showed a basal level of ß1,6 branching that was greatly increased after induction of GnT-V. These results suggest that changes in N-linked ß1,6 branching that occur during oncogenesis alter cell-matrix adhesion and migration by modulating integrin clustering and subsequent signal transduction pathways. These effects most likely result from altered N-linked carbohydrate expression on the ß1 integrin subunit. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1,6-mannose of the trimannosyl core (1)
. This GlcNAcß1,6Man structure is synthesized by GnT-V (or Mgat5, EC 2.1.4.155), a key enzyme in the processing of multiantennary asparagine-linked glycans during the synthesis of glycoproteins (2
, 3)
. Results from previous studies showed the expression of ß1,6-GlcNAc branched N-linked oligosaccharides in human mammary, colon, hepatic, and glial tumors (4, 5, 6, 7, 8)
and suggest that a relationship exists between N-linked ß1,6 branching glycans on one hand and tumorigenesis and tumor invasion on the other. Recent experiments have revealed the mechanisms by which oncogenesis causes changes of GnT-V activity. GnT-V transcription was stimulated by several oncogenes including src, her-2/neu, H-ras, and v-sis (9, 10, 11) , and this overexpression was regulated through the ras-raf-ets pathways. Stimulation of GnT-V activity has also been observed via the protein kinase C and phosphatidylinositol 3-kinase-protein kinase B signaling pathways (9, 10, 11, 12, 13) . GnT-V-deficient mice (GnT-V-/- or Mgat5-/-) have been used to study the effects of eliminating GnT-V activity on tumor progression (14) . When crossed with mice that express the polyoma middle T antigen under control of the mouse mammary tumor virus promoter, the progression of mammary tumors in the GnT-V-/- mice was significantly reduced compared with that in the GnT-V+/- mice. This study provides evidence that changes in N-linked ß1,6 branching can affect carcinoma progression in vivo.
Since GnT-V cDNA was cloned by our and Taniguchi’s group (15 , 16) , several studies have transfected GnT-V sense or antisense cDNA into cultured cells, selected for clones with altered GnT-V expression, and investigated cell-adhesive properties (8 , 17, 18, 19) . Although these studies show that alterations in N-linked ß1,6 branching may affect cell-adhesive behaviors, including cell adhesion and migration, the exact relationship were often inconsistent among the clones under study, and mechanisms involved in these events remain unclear. In one study, for example, two clones overexpressing GnT-V were selected and shown to have altered cell migration on plastic, high levels of apoptosis, and decreased adhesion to fibronectin and laminin, but the clone with intermediate GnT-V expression and ß1,6 branching levels showed a greater alteration of adhesive properties than the clone with higher levels of GnT-V and ß1,6 branches (17) .
Altered cell-cell and cell-ECM interactions are clearly critical during the acquisition of the invasive phenotype. Cell surface integrins, especially those binding to fibronectin and laminin, play essential roles in tumor cell invasion and metastasis. The fibronectin receptor 5ß1 contains a total of 26 putative N-linked glycosylation sites (14 on 5 and 12 on ß1), although it is not known which of these sites are used. Inhibitors of N-linked oligosaccharide processing have been shown to inhibit the ability of 5ß1 to bind fibronectin but not to affect its cell surface levels or ability to dimerize (20
, 21) . The mechanisms by which changes in distal glycosylation, such as ß1,6 branching, could affect cell adhesion and migration are, however, only now being understood.
To investigate in detail the relationship between increased N-linked ß1,6 branching and 5ß1-mediated adhesion to fibronectin and explore its possible mechanisms, we have used an inducible retroviral expression system. Our results show that increased expression of GnT-V activity and resulting increased levels of ß1,6 branching inhibits attachment and spreading of cells on fibronectin but stimulates cell migration toward fibronectin and invasion through a reconstituted Matrigel basement membrane. Increased GnT-V expression cause increased levels of N-linked ß1,6 branching on the ß1 subunit of the fibronectin receptor, but there is no evidence of these oligosaccharide structures on the 5 subunit, even after increased GnT-V activity. Increased ß1,6 oligosaccharide branching inhibits clustering of the 5ß1 integrins and organization of F-actin into extended microfilaments when cells are plated on fibronectin-coated plates.
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MATERIALS AND METHODS |
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Construction of Retrovirus Plasmids Containing GnT-V cDNA.
A 2.5-kb fragment containing the full-length mouse GnT-V cDNA was cut from the pMMVGnT-V plasmid with SmaI. This fragment was then ligated with SfiI adapters (formed using 3'-GATCCGGA and 5'-CTAGGCCTACA) and cloned into the noncohesive universal cloning SfiI site of retroviral plasmid pTJ66 to produce pTJ66GnT-V. The orientation of the insert was confirmed by BstXI restriction digestion mapping. This vector uses a 5'-long terminal repeat promoter, includes an internal ribosome entry signal, and produces a chimera mRNA that simultaneously expresses the protein of interest and the Zeo:eGFP reporter/selection marker. pTJ68-IZG denotes the retroviral plasmid with inducible cytomegalovirus/tet-operon (tetO-CMV) promoter, which expresses the tetracycline trans-activator (tTA) protein (tet-off) and uses Zeo:eGFP as the reporter marker. For the inducible expression of GnT-V, pTJ68-IZG was linearized by digestion with BstXI, followed by dephosphorylation, and then ligated, after its 3'-overhang was removed using T4 polymerase, with the 2.5-kb fragment of GnT-V to produce pTJ68-IZGGnT-V. BstXI and SapI digestions were used to identify the orientation of the insert.
Cell Culture, Retroviral Production, and Infection.
HT1080 and Phoenix producer cells were maintained and passaged routinely at 37°C in 5% CO2 in DMEM growth medium containing 10% heat-inactivated fetal bovine serum (Atlanta Biologicals) supplemented with 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg of streptomycin (Sigma-Aldrich).
Retroviral production and infection were performed as described previously (22) . Briefly, infectious retroviral supernatants were produced by a transient, helper virus-free protocol with pTJ66GnT-V and pTJ68-IZGGnT-V plasmids. Phoenix producer cells were grown to 50–80% confluence in DMEM in 100-mm culture dishes and transfected with retroviral plasmids (pTJ66GnT-V and pTJ68-IZGGnT-V, respectively) using 2 M CaPO4 and 25 mM chloroquine in 50 mM 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid buffer, pH 7.0, for 6–8 h, and then the dishes were refed with 25 ml of growth medium. Twenty-four hours after transfection, the growth medium was aspirated, replaced with 9 ml of growth medium, and incubated in 5% CO2 at 32°C to increase the retroviral titer. Supernatants containing infectious retrovirus were collected every 12 h up to 106 h after transfection and then filtered using a 0.45-µm pore size filter, aliquoted, snap frozen in liquid nitrogen, and stored at -80°C. At the same time, pTJ66 and pTJ68-IZG plasmids without GnT-V cDNA insert were used to generate control retroviral supernatants.
For the infection, HT1080 cells were grown in six-well plates and infected by adding retroviral supernatant and Polybrene (8 µg/ml). The plates were then incubated at 32°C for 15 min, followed by centrifuging at 2500 rpm for 30 min at 32°C. The infectious supernatant was aspirated; the cells were refed with growth medium and then placed back to the incubator with 37°C. The infection was repeated 8–12 h later. Cells were then cultured for 48 h after the second infection before selection in medium containing 800 µg/ml Zeocin for 3–5 days. The surviving cells (expressing GFP) were transferred into a culture flask and used for experiments. After two rounds of retroviral infections, >95% of the cells showed GFP fluorescence in each infection experiment when viewed with an inverted fluorescence microscope.
Transfected cells were maintained in DMEM plus 10% FBS. The cells with tet-inducible promoter were maintained in growth medium with 0.1 µg/ml tetracycline to suppress exogenous GnT-V expression (noninduced). Before each experiment, the cells containing the inducible promoter were induced to express GnT-V by the withdrawal of tetracycline from the culture medium (induced), and levels of expression were determined by altering the duration of time that cells were maintained in the medium without tetracycline. Unless specified, induction time was at least 36 h. For noninduced cells, parallel cultures were maintained with growth medium containing 0.1 µg/ml tetracycline.
GnT-V Activity Assay.
Cells were trypsinized, pelleted, and lysed with 50 mM 4-morpholinepropanesulfonic acid (pH 6.5), 150 mM NaCl, and 1% Triton X-100. Insoluble debris was pelleted by microcentrifugation (10 min at 4°C), and the supernatant was used for the GnT-V activity assay with UDP-[3
H]GlcNAc as donor substrate. Sep-Pak columns (Waters) were used to separate the substrates and products as described (23)
. The column methanol eluant was added to scintillation fluid and radioactivity measured by scintillation counting. Assays were performed in duplicate, the data were averaged, and results were expressed as specific activity (nmol/h/mg). Protein concentration in the cell lysates was determined using the bicinchoninic acid assay (Pierce).
Lectin Blotting.
Cells were harvested, rinsed with PBS, and lysed with 1% Triton X-100 in PBS. Cell lysates containing 30 µg of protein were boiled in SDS sample buffer with or without ß-mercaptoethanol, loaded on 4–20% SDS-PAGE gels, and then transferred onto a PVDF membrane. After being blocked with 1% BSA, the membrane was incubated with 1 µg/ml biotinylated L-PHA in blocking solution for 1 h followed by the incubation with a 1:5000 dilution of streptavidin-HRP for another 30 min at room temperature. The blots were washed and developed with the ECL detection system using X-ray film. Quantification was obtained using a Fluor-S scanner (Bio-Rad).
Assays of Cell-Fibronectin Adhesion and Spreading.
Cell attachment was assayed as described (24)
. Briefly, 96-well microtiter plates were coated with 0.1 ml of human plasma fibronectin in PBS, incubated at 37°C for 1 h, and blocked by 1% BSA at 37°C for 30 min after washing. Cells (3 x 104) suspended in 100 µl of serum-free DMEM were added to each coated well and incubated at 37°C for 30 min. Wells were gently washed three times with 100 µl of ice cold PBS to remove unbound cells, followed by fixation of adherent cells using 3.5% formaldehyde for 15 min. Cells were then stained with a 0.5% crystal violet solution. After the wells were washed twice with PBS, the absorbance of each well at 595 nm was measured using an automated microtiter plate spectrophotometer. For the antibody inhibition experiments (25)
, function-blocking mAb13 (50 µg/ml) against ß1 integrin or mAb16 (2 mg/ml) against 5 integrin was added to the cell suspension before addition of the cell suspension to the fibronectin-coated wells. The data were expressed as the mean of triplicate wells.
For assessment of cell spreading on fibronectin, the same procedure as above was used. After the adherent cells were fixed and stained with crystal violet, the number of spread cells was quantitated by counting the percentage of spread cells versus the total number of cells. Cells were considered as spread if they became flattened and lost nuclear refractivity. Each experimental point was the result of counting cells in 3–5 fields/well in at least 2 wells viewed at x100 magnification. The values from 6–10 fields were used to calculate the mean and SE. Typically, 50–150 cells were present per field.
Wound Healing Assay.
Cells (3 x 105) were seeded into fibronectin (10 µg/ml)-coated six-well plates in 2 ml of serum-free DMEM overnight. A clear area was then scraped in the monolayer with a 200-µl yellow plastic tip. After being washed with serum-free DMEM, the plate was incubated for 6 h at 37°C in serum-free DMEM. In some experiments, 1 µg/ml swainsonine was added to the cells for 24 h as GnT-V was induced to express. For inhibitory experiments, mAb13 (50 µg/ml), or mAb16 (2 mg/ml) was added into the well after wounding. Migration of cells into wounded areas was evaluated with an inverted microscope and photographed.
Transwell Cell Migration and Invasion Assays.
Migration assays were performed using 12-well Transwell units with 8-µm pore size polycarbonate inserts. Briefly, the undersides of insert membranes were coated with fibronectin (10 µg/ml) overnight at 4°C and blocked with 1% BSA for 1 h at 37°C. Cells (3 x 104) suspended in 400 µl DMEM plus 0.1% BSA were added to the upper compartment of the Transwell unit, and 1.5 ml DMEM plus 0.1% BSA were added to the lower chamber. Cells were allowed to migrate for 6 h at 37°C in a humidified atmosphere containing 5% CO2. The cells on the upper side of the membrane were removed using a cotton swab, whereas the cells that migrated to the underside were fixed and stained with crystal violet. The number of cells on the underside of the membrane was counted in five different fields with a light microscope at x100, and the mean and SD were calculated. Typically, each field contained 
300 cells. For some experiments, mAb13 (50 µg/ml) or mAb16 (2 mg/ml) was added into the cells before they were added to the Transwell apparatus. For swainsonine-treated cells, 1 µg/ml swainsonine was added to the culture medium for 24 h before cells were removed for addition to the upper chamber of the Transwell apparatus. Using Matrigel-coated 24-well Boyden chambers, invasion assays were performed using the same procedure as the migration assays, except that the incubation time of the experiment was prolonged to 21 h. Cells that migrated through the membrane were stained and counted as described above.
Gelatin Gel Zymography.
Cells were cultured to subconfluence in six-well plates coated with fibronectin (10 µg/ml). Then, cells were washed and incubated with 2 ml of serum-free medium for 24 h. The media were collected, centrifuged, and frozen until experiment. To detect gelatinase, 10 µl of conditioned medium were loaded to 10% SDS-PAGE gel containing 1% gelatin at 4°C under nonreducing conditions. After electrophoresis, the gels were rinsed in renaturing buffer (2.5% Triton X-100) for 1 h and incubated at 37°C overnight in buffer containing 50 mM Tris-HCl (pH 7.7), 5 mM CaCl2, and 0.02% NaN3. MMP-9 and MMP-2 were indicated by clear bands at 92 and 72 kDa that appeared after staining with Coomassie Brilliant Blue. Gels were then treated with resin and dried.
Cell Surface Labeling and Immunoprecipitation.
Cell labeling with biotin and 5ß1 immunoprecipitation were performed as described by Rigot et al. (26)
with minor modifications. Subconfluent cells were washed and detached using 2 mM EDTA. Cells were then washed twice with ice-cold PBS and incubated with 1 mg/ml NHS-LC-biotin in PBS for 20 min at 4°C on a rocking platform. After a washing with PBS, cells were lysed by incubation with buffer I (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; Complete mini protein inhibitor cocktail; 0.7 µg/ml pepstatin; and 1% Triton X-100). Lysates were cleared by centrifugation and incubated with protein-L-agarose (50 µl of beads/ml of lysate) at 4°C under agitation for 3–5 h to remove nonspecific adsorption to the agarose beads. After the determination of total protein using a bicinchoninic acid assay, cell lysates were incubated with 5 µg/ml mAb13 or mAb16 overnight at 4°C under agitation, followed by incubation with 50 µl/ml protein-L-agarose at 4°C for 3–5 h under agitation. The pellets were washed twice in buffer I, followed by two washes in buffer II (50 mM Tris-HCl, pH 7.5; 500 mM NaCl; 0.1% NP40; 0.05% sodium deoxycholate), and once in buffer III (50 mM Tris-HCl, pH 7.5; 0.1% NP40; 0.05% sodium deoxycholate) and subjected to SDS-PAGE under reducing and nonreducing conditions. The gels were then transferred onto a polyvinylidine difluoride membrane and probed with streptavidin-HRP (1:5000).
Fluorescence Staining.
Cells were plated onto fibronectin-coated chamber slides (10 µg/ml) at 37°C in serum-free medium for either 30 or 120 min. Cells were then fixed with 3.5% formaldehyde in PBS for 15 min followed by three washes with PBS, permeabilized with 0.5% Triton X-100 (for actin staining only) for 15 min, and blocked with 1% BSA. For the localization of 5ß1 integrins, chamber slides were incubated with 10 µg/ml mAb13 for ß1 or mAb11 for 5 integrin at 37°C for 30 min, followed by incubation with Rhodamine-conjugated antirat IgG (1:250) at 37°C for another 30 min. For actin filament staining, chamber slides were incubated with Rhodamine-phalloidin (1:40). After a washing with PBS, the chamber slides were mounted and imaged using an immunofluorescence confocal microscope (Bio-Rad).
5ß1 Integrin Purification.
Purification of 5ß1 integrin was performed according to the procedure of Pytela et al. (27)
with minor modifications. Briefly, 2 x 108 cells were harvested and lysed in PBS buffer containing mini protease inhibitor cocktail (1 tablet/10 ml), 0.7 µg/ml pepstatin, and 100 mM N-octylglucoside plus 0.5% SB3-10 (Sigma-Aldrich). Lysed cells were loaded onto an immunoaffinity column consisting of mAb16 (against 5 integrin) coupled to agarose beads using the Ultralink immobilization kit. Briefly, 2 mg of mAb16 in 200 µl of PBS were mixed with 0.25 g of dry beads in 4 ml of 4-morpholinepropanesulfonic acid coupling buffer and allowed to swell and couple for 1 h. The reaction was terminated by incubating beads in 5 ml of 3 M ethanolamine for 3 h. Noncoupled protein was removed by washing the beads with 8 M urea in PBS, followed by rinsing with at least 10 ml of equilibration buffer (PBS containing 0.5% SB3-10). Beads were incubated on ice for 1 h before use. After cell lysate was applied to the 1-ml column of beads at 4°C, the column was washed with 10 column volumes of equilibration buffer and eluted with low pH elution buffer (0.1 M glycine adjusted to pH 2.9 with acetic acid) at 4°C. Fractions of 1 ml each were collected and subjected to SDS-PAGE under nonreducing conditions. The identity of the two distinct bands corresponding to 140 and 120 kDa under nonreducing conditions was confirmed to be the 5 and ß1 subunits, respectively, by Western blotting with a combination of 20 µg/ml mAb11 and 20 µg/ml mAb13.
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RESULTS |
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After infection of HT1080 cells with a plasmid with no GnT-V sequence and a noninducible promoter (mock transfected cells), cells showed a GnT-V specific activity of 0.18 ± 0.02 nmol/h/mg, whereas cells infected with a similar plasmid that encoded GnT-V (GnT-V-transfected cells) showed a specific activity of 10.7 ± 1.3 nmol/h/mg. As shown in Fig. 1A
,cells infected with the plasmid containing GnT-V insert and the tet-off inducible promoter (GnT-V-transfected cells) showed an increase in GnT-V activity as early as 3 h after the removal of tetracycline and reached maximum induction (about 10–12-fold) 24 h after withdrawal of tetracycline from culture medium. These cells also showed a dose-dependent increase in GnT-V activity as the concentration of tetracycline in the culture medium was reduced (Fig. 1B)
. When maintained in tetracycline concentrations of >0.1 µg/ml, these cells showed 1.5-fold higher GnT-V activity than did mock transfected cells, showing that the promoter activity of the GnT-V plasmid could not be completely suppressed. In these cells, mRNA that encoded both GnT-V and GFP showed a tetracycline dose-dependent induction as the concentration of tetracycline in the culture medium decreased (data not shown).
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1,6. Cell lysates were subjected to SDS-PAGE, followed by lectin blotting and ECL detection of L-PHA binding. The results (Fig. 1C)
confirmed that there were substantial increases in the levels of ß1,6 branching of glycoproteins after induction of GnT-V activity.
We first studied the growth properties of HT1080 cells overexpressing GnT-V. The growth rate of GnT-V-transfected cells cultured on plastic in serum-containing medium was similar to that of mock transfected cells, and no significant difference in the number of colonies was observed when both types of transfected cells were grown in soft agar (data not shown), showing that high levels of GnT-V activity did not influence cell proliferation. Then, we measured the effects of GnT-V-increased expression on cell spreading and adhesion to fibronectin. A population of HT1080 cells overexpressing GnT-V activity showed significantly reduced (>50%) spreading on fibronectin-coated plates after 30 min (Fig. 2A)
. Spreading on fibronectin was inhibited by the addition of either of two function-blocking mAbs, mAb13, specific for the human ß1 integrin subunit, and mAb16, specific for the 5 subunit. These results demonstrate that the spreading of the cells on fibronectin resulted from adhesion through the 5ß1 integrin receptors and that increased expression of GnT-V caused significantly reduced spreading. When HT1080 cells transfected with GnT-V under control of the tet-off promoter were induced to overexpress GnT-V (Fig. 2B)
, inhibition was also observed on spreading on fibronectin. In fact, some inhibition of spreading could be observed when noninduced cells with GnT-V insert were compared with mock transfected cells, suggesting that even relatively small increases in GnT-V activity, caused by the leaky tet-off promoter, could affect spreading on fibronectin.
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. The attachment of noninduced cells was slightly but consistently inhibited compared with that of control vector-infected cells. Adhesion of the HT1080 cells was inhibited by mAb13 and mAb16, showing that the adhesion was 5ß1 dependent (data not shown). These results show that the adhesion of HT1080 cells to fibronectin was significantly inhibited by increased GnT-V expression.
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show that induction of GnT-V activity caused a striking increase of cell migration and that this effect was strongly inhibited by antibodies against the integrin ß1 subunit and to a lesser extent by antibodies against the integrin 5 subunit. Swainsonine inhibits the Golgi -mannosidase II, ultimately causing the inhibition of N-linked ß1,6 oligosaccharide expression upstream of the action of GnT-V. Cells preincubated in swainsonine before wounding but after induction of GnT-V showed little or no increase in migration.
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2-fold increase in rate of migration compared with mock transfected cells (Fig. 5A)
. These increased rates were inhibited by antibodies to 5 and ß1 and by preincubation with swainsonine. Taken together, these results are consistent with the conclusion that increased ß1,6 oligosaccharide branching caused altered adhesion to fibronectin mediated by the 5ß1 integrin that results in increased rates of migration.
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shows that HT1080 cells with induced GnT-V activity showed almost a 3-fold increase in in vitro invasiveness after 21 h of incubation and that this effect was inhibited by antibodies to 5 and ß1 or by pretreatment of cells with swainsonine. Noninduced cells, which showed a 1.5-fold increase in GnT-V activity, migrated only slightly faster than control cells, suggesting that the ability of cells to penetrate and to migrate through Matrigel was very sensitive to relatively small increases in GnT-V activity.
We also detected the secretion of MMP-9 and MMP-2 in the culture medium of HT1080 cells grown on the fibronectin-coated plates, but no significant difference in the MMP-9 or MMP-2 levels was found after induction of GnT-V overexpression (Fig. 5C)
. Taken together, these results show that the increased ability of HT1080 cells overexpressing GnT-V to migrate through Matrigel resulted from increased expression of ß1,6 branching.
To investigate the mechanisms underlying the effects of GnT-V expression on cell-adhesive behavior, F-actin filaments were first visualized in GnT-V-overexpressing cells. At early times (30 min) after plating cells on fibronectin-coated wells, phalloidin staining of permeabilized cells revealed increased F-actin staining in membrane protrusions in cells overexpressing GnT-V, suggesting increased motility of these cells (Fig. 6)
. After longer times on fibronectin (120 min), the network of F-actin stress fibers appeared to be less organized in the GnT-V-induced cells, as compared with mock transfected cells, suggesting that increased ß1,6 branching had a destabilizing effect on F-actin filaments.
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5ß1 integrin expression on the cell surface. To test this possibility, cell surface 5ß1 integrin receptors were quantitated by biotinylation, immunoprecipitation, and blotting using streptavidin-peroxidase. As shown in Fig. 7A
,levels of 5 or ß1 on HT1080 cells were similar after induction of GnT-V expression. In addition, the total mRNA levels of ß1 integrin determined by RT-PCR were also unchanged in GnT-V-expressing cells compared with control cells (data not shown). These results suggest that the effects observed on adhesion/migration after induction were not the result of altered levels of 5ß1 on the cell surface.
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5 and ß1 using mAbs, however, showed clear differences after induction of GnT-V activity (Fig. 7B)
. Cells with induced GnT-V grown on fibronectin plates for 2 h demonstrated a more diffuse surface staining of both 5 and ß1 subunits compared with mock transfected cells, suggesting decreased 5ß1 integrin clustering. This observation is consistent with a less adhesive and more motile cellular phenotype (28)
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To determine whether levels of ß1,6 branching on the 5ß1 subunits were specifically altered after induction of GnT-V, the 5ß1 receptor was purified from HT1080 cells before and after induction of GnT-V using an antibody affinity column, followed by chromatography on immobilized wheat germ agglutinin. Equal amounts of the purified receptor were subjected to SDS-PAGE under nonreducing conditions, blotted, and probed with various antibodies and lectins. The results of these experiments (Fig. 7C)
showed that the subunits could be distinguished and that each bound the lectin ConA. Surprisingly, however, only the ß1 subunit and not the 5 subunit was bound by L-PHA, demonstrating that ß1,6 branching could be detected only on the ß1 subunit. When GnT-V activity was induced, significant increases in L-PHA staining (>3-fold) were observed on the ß1 subunit. Even after induction, however, no L-PHA binding to the 5 subunit was observed, suggesting a selective glycosylation of the ß1 subunit by GnT-V. These results demonstrate that induction of GnT-V activity caused increased ß1,6 branching on the ß1 subunit and that the effects of increased GnT-V expression on fibronectin-mediated adhesive phenotypes likely result from this altered glycosylation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We first investigated the effect of GnT-V expression on cell proliferation and found that GnT-V levels had minimal effects on the rate of cell growth on plastic or anchorage-independent growth in soft agar. In addition, no effect of GnT-V overexpression was observed on the apoptotic behavior of cell populations (data not shown), in contrast to the results of an earlier report (17)
. Altering GnT-V expression did, however, cause significant changes in cell-matrix interaction. We found that both cell adhesion to and cell spreading on fibronectin was markedly decreased after the induction of GnT-V expression in HT1080 cells, and these events were most likely mediated by 5ß1 integrin confirmed by antibodies that blocked 5ß1 adhesion. After exogenous expression of GnT-V, reduced cell-matrix adhesion was observed in mink lung epithelial cells (17)
and hepatocarcinoma cells (31)
, but not in mouse mammary cancer cells (19)
. This difference in cell adhesion might result from the variation between individual clones used in the experiment. Our results together with previous studies suggest strongly, therefore, that changes in N-linked glycosylation caused by increased ß1,6 branching had direct effects on 5ß1-mediated cell spreading and adhesion.
We used different types of assays to test whether GnT-V overexpression could affect rates of migration. We observed with both the wound healing assay and the haptotaxis assay that cell motility was significantly increased after induced expression of GnT-V. The increased migration of HT1080 cells induced by GnT-V expression could be suppressed by adding function-blocking antibodies against 5 or ß1, indicating that this receptor was involved in regulating this migratory behavior. The increased migration of HT1080 cells overexpressing GnT-V was inhibited by swainsonine treatment, consistent with the conclusion that ß1,6 oligosaccharide branching may play a direct role in 5ß1 integrin-mediated cell motility. Cell migration is a complex behavior regulated by multiple mechanisms (32)
. A critical factor that regulates the rate of cell migration is the degree of adhesion of cells to their ECM substrata. Migration is maximal under conditions of intermediate levels of cell adhesion (33)
. Thus, the increased rate of migration on fibronectin is consistent with decreased cell adhesion and spreading observed for GnT-V overexpressing cells.
Increased ß1,6 branching also promoted the ability of cells to invade through Matrigel. Antibodies against both the ß1 and 5 integrin subunits and swainsonine treatment blocked increased HT1080 invasion caused by the induced expression of GnT-V, indicating the involvement of 5ß1 integrin and ß1,6 branching in the invasion process. HT1080 cells produce MMPs, including both MMP-2 and MMP-9, which can degrade the components of basement membranes and ECM and increase cell invasion (34)
. To determine whether GnT-V overexpression enhanced the secretion of MMP-2 and MMP-9 in HT1080 cells, levels of MMP-2 and MMP-9 in culture medium were analyzed by zymography using gelatin as substrate. No significant differences were observed in levels of either MMP-2 or MMP-9 after induction expression of GnT-V in HT1080 cells. This result supports the conclusion that increased cell invasion because of increased ß1,6 branching is the result of increased cell migration rather than an increased secretion of MMPs.
Focal adhesions are formed when cells contact the ECM, linking the cytoplasmic tails of integrins to the actin cytoskeleton, and play a pivotal role in cell spreading and migration (35 , 36) . In our study, we found increased F-actin staining in membrane protrusion at early times and decreased stress fiber formation in the cells with overexpressed GnT-V. These observations are consistent with a less adhesive, more motile phenotype, as suggested by Duband et al. (28) . It was shown that after cell spreading on fibronectin, activation of FAK, and transient inhibition of a small GTPase, RhoA, proved to be essential for promoting membrane protrusion and polarity (37 , 38) ; Moreover, FAK-dependent transient inhibition of RhoA is required to promote focal adhesion turnover and actin filament reorganization (39 , 40) . It is reasonable, therefore, to postulate that the signaling pathway mediated by FAK and/or RhoA activity might be affected in GnT-V overexpressing cells.
Integrin receptors regulate cell adhesion, spreading, migration, and proliferation, as well as gene transcription (25
, 32
, 41 , 42)
. The 5ß1 integrin, a primary receptor of fibronectin, contains N-linked glycans. Previous studies demonstrated that 5ß1 function can be modulated by N-linked oligosaccharide expression (20
, 43)
. To investigate further glycosylation differences after GnT-V induction, the 5ß1 integrin was purified from induced and noninduced cells and subjected to lectin blotting with L-PHA. Increased ß1,6 branching was observed on the ß1 subunit after induction of GnT-V, but no ß1,6 branching could be detected on the subunit either before or after induction. ConA bound to both 5 and ß1 subunits, by contrast, and little change in binding could be observed before and after the induction of GnT-V expression. More information on the N-linked oligosaccharide expression on the integrin subunits will await further detailed structural characterization. The effect of increased ß1,6 oligosaccharide branching on 5ß1 function, however, appears to result solely from changes in ß1 glycosylation. Kobata’s laboratory suggested that changes in ß1,6 branching of the ß1 subunit of transformed 3T3 cell lines correlated well with increased tumorigenicity and metastatic potential (44)
. More recently, Skinner et al. (45)
reported that overexpression of the 16-kDa membrane subunit of vacuolar ATPase could inhibit ß1,6 branching of ß1 integrin subunit, resulting in decreased tumor cell invasiveness. These studies are consistent with our direct demonstration of increased ß1,6 branching on the ß1 subunit after induction of GnT-V expression.
In our study, the levels of both 5 and ß1 subunits on HT1080 cell surfaces remained relatively unchanged when GnT-V was induced (Fig. 7)
. We did observe changes in cell surface 5ß1 integrin clustering on fibronectin, however, after induction of GnT-V overexpression, using antibodies to visualize separately the 5 and ß1 subunits. Formation of focal adhesions is a critical event as cells attach to matrix components, initiated by the binding of integrins to specific ECM ligands and subsequent clustering of these receptors (46)
. We found more diffuse staining of 5ß1 integrins on cells spreading on fibronectin after induced GnT-V expression, indicating that the clustering of 5ß1 integrins was reduced in GnT-V overexpressing cells. This conclusion was further supported by our recent experiments with 12G10, an activating anti-ß1integrin mAb which was reported to induce cell-cell adhesion and ß1 integrin clustering in HT1080 cells (47)
. We found that overexpression of GnT-V in HT1080 cells could significantly inhibit ß1 integrin clustering induced by 12G10 (unpublished data), suggesting that N-linked glycosylation of the ß1 integrin may be a critical factor for regulating integrin clustering. In another study (8)
, human glioma cells transfected with GnT-V resulted in different localization pattern of 3ß1 integrin, where the immunostaining of 3ß1 integrin showed a strong localization of the integrin heterodimer to leading lamellipodia of GnT-V-transfected cells, but not of parental or mock transfected cells. It is therefore clear that glycosylation of integrins by GnT-V causes functional alteration in ß1-containing integrin heterodimers.
The function of integrin clustering in the regulation of cell adhesion strength has been studied by several groups. Yauch et al. (48)
reported that the deletion of the 4 integrin tail region significantly impaired static cell adhesion by severely restricting ß1 integrin clustering. Without affecting ligand-binding affinity, integrin-mediated adhesion appeared to be regulated by the rate of receptor diffusion/clustering. Another study showed that leukocyte function-associated antigen-1 or integrin Lß2 ligand binding strength to intercellular adhesion molecule-1 was regulated by lateral diffusion of the integrins and their interaction with the cytoskeleton (49)
. Kassner et al. (50)
showed that an 4 integrin chimera expressing 2 or 5 tails greatly enhanced localization of the chimeric very late antigen-4 into focal adhesions and concluded that relatively more mobile 4 integrin, less engaged with cytoskeletal interaction, is likely to augment cell migration and diminish cell spreading and adhesion strengthening. Consistent with these reports, our data suggest that altered integrin clustering is one of the major consequences of increased ß1,6 branching of ß1 integrin and results in a less adhesive and more motile phenotype through regulating cell spreading, adhesion, as well as actin-filament reorganization.
There are now clear examples of the inhibition of signal transduction pathways by mutations in specific N-acetylglucosaminyltransferases. Altered glycosylation of the Notch cell surface receptor during Drosophila development can clearly affect the signaling function of this receptor (51)
and is caused by mutations in the Fringe N-acetylglucosaminyltransferase in the O-linked pathway. In addition, a recent study has documented that inactivating mutations in a human N-acetylglucosaminyltransferase, which also functions in the biosynthesis of specific O-linked glycans, causes an inhibition of a particular cell surface signaling pathway, which, in turn, results in a form of muscular dystrophy known as muscle-eye-brain disease (52)
. Glycosylation of the CD8ß coreceptor stalks on native thymocytes has recently been shown to regulate interactions between the CD8 "head" domain and major histocompatibility complex class I (MHCI) tetramers, thereby modulating thymic selections (53)
. In light of these results, it is reasonable to suggest that increased ß1,6 branching of the ß1 integrin subunit by GnT-V that is up-regulated after oncogenesis results in increased cell migration and invasion by modulating integrin clustering and subsequent signal transduction pathways.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by National Cancer Institute NCI Grant 5RO1CA64462 to M. P. 
2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, Life Science Building, B-314, University of Georgia, Athens, GA 30602. Phone: (706) 542-1701; Fax: (706) 542-1759; E-mail: hawkeye@arches.uga.edu.
3 The abbreviations used are: GnT-V, N-acetylglucosaminyltransferase V (Mgat5); GlcNAc, N-acetylglucosamine; ECM, extracellular matrix; L-PHA, leukoagglutinating phytohemagglutinin; ConA, concanavalin A; mAb, monoclonal antibody; ECL, enhanced chemiluminescence; tet-off, tetracycline inducible; HRP, horseradish peroxidase; GFP, green-fluorescent protein; MMP, matrix metalloproteinase; FAK, focal adhesion kinase; RhoA, a small GTPase.
Received 7/ 2/02. Accepted 10/ 4/02.
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REFERENCES |
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-mannoside ß1,6 N-acetylglucosaminyltransferase is an early event in hepatocarcinogenesis. Int. J. Cancer, 91: 631-637, 2001.[Medline]
5ß1 integrin receptor: de-N-glycosylation induces dissociation or altered association of 5 and ß1 subunits and concomitant loss of fibronectin binding activity. J. Biol. Chem., 269: 12325-12331, 1994.-6-D-mannoside ß-1,6-N-acetylglucosaminyltransferase. J. Biol. Chem., 267: 2920-2927, 1992.vß6 as a fibronectin-binding protein. J. Biol. Chem., 267: 5790-5796, 1992.5ß1 analyzed by a three-dimensional mapping technique. Eur. J. Biochem., 237: 76-85, 1996.[Medline]
4 cytoplasmic domain. Mol. Biol. Cell, 6: 661-674, 1995.[Abstract]
, mock transfected HT1080 cells; 
, GnT-V-transfected HT1080 cells. C, lectin blotting was performed with biotinylated L-PHA using cell lysates from GnT-V-transfected cells cultured in tetracycline-free (Lane 1) and tetracycline-containing (Lane 2, 0.001 µg/ml; Lane 3, 1 µg/ml) medium and mock transfected HT1080 cells (Lane 4). MW, molecular mass.
, mock transfected cells; 
, noninduced GnT-V-transfected cells; 
0.05 and **, P 
