Sialylation of beta1 integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis

Abstract

In previous studies, we determined that beta1 integrins from human colon tumors have elevated levels of alpha2-6 sialylation, a modification added by beta-galactosamide alpha-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the beta1 integrin is thought to be a ligand for galectin-3 (gal-3), a tumor-associated lectin. The effects of gal-3 are complex; intracellular forms typically protect cells against apoptosis through carbohydrate-independent mechanisms, whereas secreted forms bind to cell surface oligosaccharides and induce apoptosis. In the current study, we tested whether alpha2-6 sialylation of the beta1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking alpha2-6 sialylation exhibit beta1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of alpha2-6 sialic acids from ST6Gal-I expressors by neuraminidase treatment restores gal-3 binding. Additionally, using a blot overlay approach, we determined that gal-3 binds directly and preferentially to unsialylated, as compared with alpha2-6-sialylated, beta1 integrins. To understand the physiologic consequences of gal-3 binding, cells were treated with gal-3 and monitored for apoptosis. Galectin-3 was found to induce apoptosis in parental SW48 colonocytes (unsialylated), whereas ST6Gal-I expressors were protected. Importantly, gal-3-induced apoptosis was inhibited by function blocking antibodies against the beta1 subunit, suggesting that beta1 integrins are critical transducers of gal-3-mediated effects on cell survival. Collectively, our results suggest that the coordinate up-regulation of gal-3 and ST6Gal-I, a feature that is characteristic of colon carcinoma, may confer tumor cells with a selective advantage by providing a mechanism for blockade of the pro-apoptotic effects of secreted gal-3.

FIGURE 1.

α2-6 sialylation inhibits the attachment of SW48 cells to gal-3 and cell adhesion to gal-3 is dependent upon β1 integrins. Par, EV, or ST6Gal-I-expressing (ST6) SW48 cells were seeded onto cell culture plates coated with either gal-3 or gal-1, and binding was quantified by measuring the fluorescence of calcein AM-labeled cells. BSA- and poly-l-lysine (PL)-coated wells were used as negative and positive controls respectively. A, expression of ST6Gal-I inhibits cell adhesion to gal-3 (^* denotes significant difference relative to parental cells on gal-3, p < 0.001). However, none of the cell lines tested adhered to gal-1. B, cell binding to gal-3 is inhibited by 20 mm β-lactose but not 20 mm sucrose, indicating that gal-3 binding is carbohydrate-dependent (^* denotes significant difference from parental cells on gal-3; # denotes significant inhibition by β-lactose, p < 0.001). C, Par and ST6 cells were stained with FITC-conjugated anti-β1 integrin antibody (dotted line) or FITC-conjugated mouse IgG1 (solid line) and analyzed by flow cytometry for cell surface staining of β1 integrins. Par and ST6 cells have similar β1 integrin expression on the cell surface. Mean fluorescent intensity (MFI) ± S.D. from three independent experiments are given in each plot. D, parental and ST6 cells were preincubated with either an anti-β1 integrin function blocking antibody or a mouse IgG1 isotype control for 45 min at 37 °C. The cells were subsequently seeded onto gal-3-coated wells, and adhesion was quantified as described previously. As shown, gal-3 binding was significantly inhibited by the anti-β1 integrin function blocking antibody (^* denotes significant difference from parental cells; # denotes significant inhibition from ST6 cells, p < 0.001). All values from adhesion experiments represent the means and S.E. for three independent experiments performed with at least three samples per cell line.

FIGURE 2.

Treatment with neuraminidase removes α2-6-linked sialic acid of β1 integrins and increases ST6 cell adhesion to gal-3. A, lysates from Par and ST6 cells were treated with varying concentrations of neuraminidase and immunoblotted for the β1 integrin. The blot was then stripped and reprobed for β-actin. Cont, control. B, Par and ST6 cells were treated with 50 units of neuraminidase and washed with PBS. The cells were resuspended in serum-free medium and subsequently seeded onto gal-3-coated wells, and adhesion was quantified as described previously. As shown, the adhesion of ST6 cells to gal-3 was significantly increased by neuraminidase treatment (^* denotes significant difference from parental cells; # denotes significant difference between ST6 cells with and without neuraminidase treatment, p < 0.001). PL, poly-l-lysine.

FIGURE 3.

α2-6 sialylation of β1 integrins abolishes gal-3 binding. β1 integrins were immunoprecipitated (IP) from parental or ST6Gal-I-expressing cells, resolved by 7% SDS-PAGE, and transferred to PVDF membrane. The membrane was overlaid with an AP-conjugated gal-3. As shown, the AP-conjugated gal-3 bound the mature β1 integrin from parental cells but not ST6Gal-I expressors. Duplicate immunoprecipitated samples were immunoblotted with an anti-β1 integrin antibody to verify equivalent levels of β1 in the immunoprecipitates.

FIGURE 4.

α2-6 sialylation did not change the expression or subcellular localization of gal-3. A, lysates from Par, EV, ST6, and the promonocytic cell line, U937, were Western blotted with antibody against gal-1 and then stripped and reblotted for gal-3 and β-actin. B, immunofluorescent staining of gal-3 in Par, EV, and ST6 cells seeded onto coverslips and allowed to adhere for 3 h at 37°C. C, immunofluorescent staining of Par cells with anti-gal-3 antibody, or alternately, with a control IgG2b or no primary antibody (no Ab) as negative controls. Hoechst 33258 was used for nuclear staining.

FIGURE 5.

ST6Gal-I protects cells from gal-3-induced apoptosis. A, Par and ST6 cells were seeded onto chamber slides and treated with different concentrations of human recombinant gal-3 (0, 1, 10, and 20 μg/ml) in serum-free medium for 24 h. The cells were fixed and stained with Hoechst 33258 to detect apoptotic nuclei. The percentage of apoptotic cells was counted from 10 different randomly selected fields at ×40 objective. Values represent means and S.E. for two independent experiments (^* denotes significant difference from parental cells, p < 0.001). B, Par, EV, and ST6 cells were incubated in serum-free medium in the presence or absence of 20 μg/ml gal-3 or 20 μg/ml gal-3 + 20 mm β-lactose for 24 h. The percentage of apoptotic cells stained by Hoechst 33258 was counted as above. Values represent means and S.E. for two independent experiments (^* denotes significant difference from parental cells, p < 0.001; # denotes significant inhibition by β-lactose, p < 0.001). C, cells were treated as described in panel B and then lysed and Western blotted for cleaved (activated) caspase-3 and β-actin.

FIGURE 6.

Galectin-3-induced apoptosis is dependent upon β1 integrins. A, parental cells were preincubated with two distinct function-blocking antibodies against the β1 integrin or with two mouse IgG isotype controls for 45 min at 37 °C. The cells were incubated in serum-free medium in the presence or absence of 20 μg/ml gal-3 for 24 h and then lysed and Western blotted for cleaved caspase-3 and β-actin. B, staurosporine induces apoptosis in both parental and ST6Gal-I-expressing cells. Par and ST6 cells were treated with 1 μm staurosporine for 4 h. The cell lysates were Western blotted for cleaved caspase-3 and β-actin.