Sialylation of β1 Integrins Blocks Cell Adhesion to Galectin-3 and Protects Cells against Galectin-3-induced Apoptosis

In previous studies, we determined that β1 integrins from human colon tumors have elevated levels of α2-6 sialylation, a modification added by β-galactosamide α-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the β1 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 α2-6 sialylation of the β1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking α2-6 sialylation exhibit β1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of α2-6 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 α2-6-sialylated, β1 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 β1 subunit, suggesting that β1 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. In previous studies, we determined that β1 integrins from human colon tumors have elevated levels of α2-6 sialylation, a modification added by β-galactosamide α-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the β1 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 α2-6 sialylation of the β1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking α2-6 sialylation exhibit β1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of α2-6 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 α2-6-sialylated, β1 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 β1 subunit, suggesting that β1 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. Aberrant cell surface carbohydrates are highly associated with tumor invasion and metastasis. In particular, N-linked glycans on tumor cells tend to be more highly branched, with greater levels of terminal sialylation (1Hakomori S. Cancer Res. 1996; 56: 5309-5318PubMed Google Scholar, 2Gorelik E. Galili U. Raz A. Cancer Metastasis Rev. 2001; 20: 245-277Crossref PubMed Scopus (255) Google Scholar, 3Dennis J.W. Semin. Cancer Biol. 1991; 2: 411-420PubMed Google Scholar, 4Dall'Olio F. Glycoconj. J. 2000; 17: 669-676Crossref PubMed Scopus (94) Google Scholar, 5Bellis S.L. Biochim. Biophys. Acta. 2004; 1663: 52-60Crossref PubMed Scopus (132) Google Scholar). The ST6Gal-I 2The abbreviations used are: ST6Gal-Iβ-galactosamide α-2,6-sialyltranferase IAPalkaline phosphatasegalgalectinParparentalEVempty vectorBSAbovine serum albuminPBSphosphate-buffered salineFITCfluorescein isothiocyanatePVDFpolyvinylidene difluoride. sialyltransferase, which adds α2-6-linked sialic acids to glycoproteins (4Dall'Olio F. Glycoconj. J. 2000; 17: 669-676Crossref PubMed Scopus (94) Google Scholar, 6Chammas R. McCaffery J.M. Klein A. Ito Y. Saucan L. Palade G. Farquhar M.G. Varki A. Mol. Biol. Cell. 1996; 7: 1691-1707Crossref PubMed Scopus (21) Google Scholar), is up-regulated in a number of tumors including colon adenocarcinoma, and its expression positively correlates with tumor metastasis and poor patient survival (4Dall'Olio F. Glycoconj. J. 2000; 17: 669-676Crossref PubMed Scopus (94) Google Scholar, 5Bellis S.L. Biochim. Biophys. Acta. 2004; 1663: 52-60Crossref PubMed Scopus (132) Google Scholar, 7Bresalier R.S. Rockwell R.W. Dahiya R. Duh Q.Y. Kim Y.S. Cancer Res. 1990; 50: 1299-1307PubMed Google Scholar). Moreover, both in vitro and animal studies have implicated ST6Gal-I in regulating tumor cell invasiveness and metastasis (7Bresalier R.S. Rockwell R.W. Dahiya R. Duh Q.Y. Kim Y.S. Cancer Res. 1990; 50: 1299-1307PubMed Google Scholar, 8Le Marer N. Stehelin D. Glycobiology. 1995; 5: 219-226Crossref PubMed Scopus (50) Google Scholar, 9Lin S. Kemmner W. Grigull S. Schlag P.M. Exp. Cell Res. 2002; 276: 101-110Crossref PubMed Scopus (165) Google Scholar, 10Zhu Y. Srivatana U. Ullah A. Gagneja H. Berenson C.S. Lance P. Biochim. Biophys. Acta. 2001; 1536: 148-160Crossref PubMed Scopus (88) Google Scholar). However, the mechanisms linking elevated α2-6 sialylation to tumor progression are still poorly understood. Previously, our group identified the β1 integrin subunit as a target for oncogenic Ras-induced ST6Gal-I activity (11Seales E.C. Jurado G.A. Singhal A. Bellis S.L. Oncogene. 2003; 22: 7137-7145Crossref PubMed Scopus (107) Google Scholar) and further determined that α2-6 sialylation of β1 integrins stimulated cell attachment and migration on collagen I (12Seales E.C. Jurado G.A. Brunson B.A. Wakefield J.K. Frost A.R. Bellis S.L. Cancer Res. 2005; 65: 4645-4652Crossref PubMed Scopus (265) Google Scholar). We also reported that β1 integrins in colon tumor tissues carry elevated levels of α2-6-linked sialic acid (12Seales E.C. Jurado G.A. Brunson B.A. Wakefield J.K. Frost A.R. Bellis S.L. Cancer Res. 2005; 65: 4645-4652Crossref PubMed Scopus (265) Google Scholar). Collectively, these results suggest that hypersialylation of the β1 integrin may contribute to the invasive tumor cell phenotype by modulating cell-matrix interactions. β-galactosamide α-2,6-sialyltranferase I alkaline phosphatase galectin parental empty vector bovine serum albumin phosphate-buffered saline fluorescein isothiocyanate polyvinylidene difluoride. There is a vast literature directed at understanding integrin association with traditional extracellular matrix ligands such as collagen, fibronectin, etc. However, accumulating data suggest that integrins also bind to galectins, a family of lectins that can associate with the matrix through interactions with laminin and fibronectin (13Elola M.T. Wolfenstein-Todel C. Troncoso M.F. Vasta G.R. Rabinovich G.A. CMLS Cell Mol. Life Sci. 2007; 64: 1679-1700Crossref PubMed Scopus (288) Google Scholar, 14Ochieng J. Furtak V. Lukyanov P. Glycoconj. J. 2004; 19: 527-535Crossref PubMed Scopus (287) Google Scholar). Galectins are β-galactoside-binding proteins that bind to target molecules through conserved carbohydrate recognition domains. So far, at least 15 mammalian galectins have been identified, and these are known to regulate numerous biological processes including cell adhesion/migration, apoptosis, angiogenesis, and immune responses (13Elola M.T. Wolfenstein-Todel C. Troncoso M.F. Vasta G.R. Rabinovich G.A. CMLS Cell Mol. Life Sci. 2007; 64: 1679-1700Crossref PubMed Scopus (288) Google Scholar, 15Liu F.T. Rabinovich G.A. Nat. Rev. Cancer. 2005; 5: 29-41Crossref PubMed Scopus (1196) Google Scholar). One unique member of the galectin family is galectin-3 (gal-3), which has only one carbohydrate recognition domain, in combination with an extended N-terminal domain that is thought to promote oligomerization (16Dumic J. Dabelic S. Flogel M. Biochim. Biophys. Acta. 2006; 1760: 616-635Crossref PubMed Scopus (853) Google Scholar). Galectin-3 is expressed intracellularly but can also be secreted through a nonclassical pathway (17Hughes R.C. Biochim. Biophys. Acta. 1999; 1473: 172-185Crossref PubMed Scopus (547) Google Scholar). Intracellular gal-3 generally mediates anti-apoptotic effects through carbohydrate-independent processes, whereas extracellular gal-3 binds to cell surface oligosaccharides and thereby induces apoptosis and also modulates cell-matrix interactions (15Liu F.T. Rabinovich G.A. Nat. Rev. Cancer. 2005; 5: 29-41Crossref PubMed Scopus (1196) Google Scholar, 16Dumic J. Dabelic S. Flogel M. Biochim. Biophys. Acta. 2006; 1760: 616-635Crossref PubMed Scopus (853) Google Scholar, 18Nakahara S. Oka N. Raz A. Apoptosis. 2005; 10: 267-275Crossref PubMed Scopus (254) Google Scholar). Given the diverse locales and functions of gal-3, the ultimate effect of this lectin on tumor cell behavior, and particularly survival, likely depends upon the balance of signals arising from intracellular versus extracellular gal-3. Like ST6Gal-I, gal-3 appears to play important roles in neoplastic cell metastasis. Transfection of gal-3 into low metastatic colon cancer cells enabled the cells to become more metastatic after inoculation into spleen or cecum of nude mice. In contrast, transfection of antisense gal-3 into a highly metastatic colon cancer cell line reduced metastatic capability (19Bresalier R.S. Mazurek N. Sternberg L.R. Byrd J.C. Yunker C.K. Nangia-Makker P. Raz A. Gastroenterology. 1998; 115: 287-296Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Galectin-3 is reported to be highly expressed in several tumor types including colon carcinoma, and up-regulated gal-3 expression is associated with tumor aggressiveness (15Liu F.T. Rabinovich G.A. Nat. Rev. Cancer. 2005; 5: 29-41Crossref PubMed Scopus (1196) Google Scholar, 16Dumic J. Dabelic S. Flogel M. Biochim. Biophys. Acta. 2006; 1760: 616-635Crossref PubMed Scopus (853) Google Scholar). Several studies have suggested that galectin binding to β-galactosides may be sensitive to terminal sialylation. For example, Hirabayashi's group (20Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W.E. Yagi F. Kasai K. Biochim. Biophys. Acta. 2002; 1572: 232-254Crossref PubMed Scopus (829) Google Scholar) used frontal affinity chromatography to show that the binding of gal-3 to synthetic oligosaccharides (as opposed to cellular glycoproteins) could occur in the presence of α2-3, but not α2-6, sialylation of β-galactose. Studies of immune cells support this general concept; cell surface α2-6 sialylation was reported to block apoptosis induced by galectin-1 (gal-1) (21Amano M. Galvan M. He J. Baum L.G. J. Biol. Chem. 2003; 278: 7469-7475Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 22Suzuki O. Nozawa Y. Abe M. Int. J. Oncol. 2006; 28: 155-160PubMed Google Scholar). However, the potential roles of gal-3 and ST6Gal-I in regulating epithelial cell survival have received little attention despite the fact that both gal-3 and ST6Gal-I are implicated in tumor progression. Accordingly, we compared the effects of gal-3 on SW48 cells that either lack endogenous ST6Gal-I (parental cells) or have forced expression of ST6Gal-I. These studies showed that gal-3 preferentially binds to cells with unsialylated β1 integrins. Moreover, α2-6 sialylation of the β1 integrin was found to protect cells against gal-3-mediated cell apoptosis, implicating a role for ST6Gal-I in regulating tumor cell survival. Cell Lines—The human SW48 colon epithelial cell line was purchased from the ATCC (Manassas, VA). These cells are derived from a stage IV colon adenocarcinoma, and subcutaneous inoculation of SW48 cells into nude mice reportedly results in tumor formation 100% of the time (23Hay R. Caputo J. Chen T.R. Macy M. McClintock P. Reid Y. ATCC Cell Lines and Hybridomas. 8th Ed. ATCC, Rockville, MD1994Google Scholar). SW48 cells have no detectable α2-6 or α2-3 sialyltransferase activity (24Dall'Olio F. Chiricolo M. Lollini P. Lau J.T. Biochem. Biophys. Res. Commun. 1995; 211: 554-561Crossref PubMed Scopus (30) Google Scholar). SW48 cells stably expressing either empty vector (EV) or ST6Gal-I (ST6) were established as described previously using a lentiviral vector (12Seales E.C. Jurado G.A. Brunson B.A. Wakefield J.K. Frost A.R. Bellis S.L. Cancer Res. 2005; 65: 4645-4652Crossref PubMed Scopus (265) Google Scholar), and pooled populations of lentiviral-infected clones were utilized for all studies. The cells were grown in Leibovitz's L-15 medium with 2 mmol/liter l-glutamine (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Hyglone, Logan, UT). EV and ST6 cells were maintained with 0.5 μg/ml puromycin (Sigma) in complete culture medium (the puromycin was removed from the medium at least 1 day in advance of all experiments). All cells were cultured at 37 °C in a CO2-free incubator. Galectin-1/3 Cell Adhesion Assay—InnoCyte ECM adhesion assay gal-1/gal-3 kits were purchased from Calbiochem. According to the vendor, the wells were coated with 100 μl/well of either 5 μg/ml gal-1 or 5 μg/ml gal-3 in PBS followed by blocking with 2% BSA in PBS. BSA- and poly-l-lysine-coated wells were used as negative and positive controls, respectively. The wells were never exposed to serum-containing medium during commercial processing. Cells were harvested by cell stripper (Mediatech) and washed extensively in PBS. Then the cells (at subconfluent density) were seeded onto each well in serum-free medium and incubated for 2 h at 37°C. The 2-h attachment interval was selected because epithelial cells typically bind maximally to most substrates within 2 h. As well, allowing cells to adhere for longer time points increases the possibility that cells will secrete matrix molecules such as fibronectin or collagen, which could compromise the interpretation of results. After a 2-h incubation, cells were washed with PBS twice and labeled with calcein AM for 1 h at 37°C. The number of calcein AM-labeled cells was measured by VersaFluor fluorometer (Bio-Rad) at 520 nm. For some experiments, cells were incubated in the presence or absence of either lactose (Sigma) or sucrose (Bio-Rad). For the β1 integrin blocking study, 35 μg/ml function blocking antibodies against the β1 integrin (catalog number MAB1965, Chemicon International, Temecula, CA) or a mouse IgG1 isotype control (R&D Systems) were preincubated with SW48 cells for 45 min at 37 °C prior to seeding cells onto the galectin-coated plates. All data were analyzed by a Student's paired t test. Flow Cytometry—Cells were harvested by cell stripper (Mediatech) and were adjusted to 1 × 106 cells/tube with 100 μl of 0.2% BSA in PBS. 10 μl of FITC-conjugated anti-β1 integrin antibody (Chemicon International), FITC-conjugated mouse IgG1 (Invitrogen), or PBS only were added to each tube and incubated on ice for 60 min. The cells were washed three times with 0.2% BSA in PBS and resuspended in 500 μl of 0.2% BSA in PBS for flow cytometric analysis on FACScan (BD Biosciences). Western Blotting—Cells were lysed in 50 mm Tris-HCl (pH 7.4) with 1% Triton X-100 and protease inhibitors (Roche Applied Science). Cell lysates were resolved by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20. The membranes were incubated with a primary antibody against gal-1 (R&D Systems), gal-3 (R&D Systems), β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA), cleaved caspase-3 (Cell Signaling Technology, Danvers, MA), or β1 integrins (BD Biosciences). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) and developed with Immobilon chemiluminescent horseradish peroxidase substrate (Millipore, Billerica, MA). Neuraminidase Treatment of Cell Lysates—90 μg of cell lysates were treated in 50 mm sodium citrate buffer (pH 6.0) in the presence or absence of different concentrations of neura-minidase from Clostridium perfringens (New England Biolabs, Ipswich, MA) for 1 h at 37°C. The cell lysates were subsequently boiled in 2× SDS-PAGE sample buffer, resolved by SDS-PAGE, and immunoblotted for the β1 integrin (BD Biosciences). Neuraminidase Treatment of Cells—4 × 105 cells were treated in the presence or absence of 50 units of neuraminidase from C. perfringens (New England Biolabs) in PBS (pH 7.0) for 1 h at 37 °C. Then the cells were washed three times with PBS and resuspended in serum-free culture medium for gal-3 cell adhesion assay. Immunofluorescent Staining—Round German glass coverslips (Electron Microscopy Science, Hatfield, PA) were coated with 20 μg/ml collagen I (Inamed Biomaterials, Fremont, CA) overnight. The cells were plated on the collagen-coated coverslips for 3 h at 37°C and then fixed with 3.7% formaldehyde at room temperature for 15 min. The cells were treated with 0.2% Triton X-100 in PBS for 2 min and blocked with 5% donkey serum. The cells were subsequently incubated with anti-gal-3 antibody (R&D Systems), no primary antibody, or mouse IgG2b isotype control (R&D Systems) for 1 h at room temperature followed by Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen) for 45 min at room temperature. The coverslips were mounted with Aqua-Poly/Mount medium (Polysciences Inc., Warrington, PA), and cells were viewed under ×60 oil objective of a fluorescent microscope (Nikon, Tokyo, Japan). Images were taken by a Nikon CoolSNAP camera. Galectin-3 Overlay—Cell lysates (500 μg of total protein/sample) were incubated overnight at 4 °C with a glycosylation-insensitive anti-β1 integrin antibody, MAB 2000 (Chemicon International). Protein A/G agarose beads (Santa Cruz Biotechnology Inc.) were then added, and samples were incubated for 2 additional hours at 4 °C with rotation. The agarose beads were washed with lysis buffer and then boiled in 2× SDS-PAGE sample loading buffer. Precipitated proteins were resolved by on 7% SDS-PAGE and transferred to PVDF membranes. Membranes were exposed to alkaline phosphatase (AP)-conjugated gal-3 and developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (25de Melo F.H. Butera D. Medeiros R.S. Andrade L.N. Nonogaki S. Soares F.A. Alvarez R.A. Moura da Silva A.M. Chammas R. J. Histochem. Cytochem. 2007; 55: 1015-1026Crossref PubMed Scopus (18) Google Scholar). Galectin-3-induced Apoptosis—Cells were seeded in 4-well chamber slides (BD Biosciences) or 6-well plates in complete medium for 24 h. The adherent cells (40-60% confluency) were then washed with PBS twice and incubated in serum-free medium in the presence or absence of gal-3 (R&D Systems) or gal-3 plus β-lactose (Sigma) for 24 h. Direct visualization of the cells following the gal-3 incubation did not reveal any significant increases in the number of detached cells, suggesting that the addition of soluble gal-3 had no effect on cell adhesion to tissue culture plastic. After gal-3 treatment, the cells were either fixed for Hoechst 33258 staining or solubilized in protein lysis buffer for cleaved caspase-3 Western blots. The 24-h time point for analyzing apoptotic responses to gal-3 was selected because this is a common time interval for evaluating apoptosis of epithelial cells and has been used previously to study gal-1- and gal-3-induced apoptosis of lymphocytes. We recognize that soluble gal-3 was used in this assay, rather than the immobilized form of gal-3 used for binding assays. We considered examining apoptosis in response to immobilized gal-3 but concluded that such experiments might be difficult to interpret. Specifically, ST6 cells do not adhere well on immobilized gal-3 (as shown in Fig. 1), and extended incubations would likely result in anoikis. Accordingly, it might be difficult to discriminate between anoikis-related apoptosis versus gal-3-induced apoptosis. To evaluate integrin involvement in gal-3-induced apoptosis, cells were allowed to adhere to tissue culture plates for 24 h as before, and then anti-β1 integrin function-blocking antibodies were added to the cells for 45 min followed by the addition of the gal-3-containing medium. Four different antibodies were tested (at 35 μg/ml): two distinct anti-β1 function-blocking antibodies (catalog numbers MAB1965 and MAB2253, Chemicon International) and two distinct IgG1 isotype controls (Chemicon International, catalog number PP100; and R&D Systems, catalog number MAB002). Neither the anti-β1 nor the control antibodies had any apparent effect on cell attachment or morphology (data not shown). Hoechst 33258 Staining—Cells were fixed with 3.7% formaldehyde in PBS and treated with 0.2% Triton X-100 in PBS for 2 min. The cells were then washed with PBS and stained with 1 μg/ml Hoechst 33258 (Invitrogen) for 30 min at room temperature. The slides were mounted with Aqua-Poly/Mount medium (Polysciences Inc.), and the percentage of apoptotic cells was calculated from 10 different randomly selected fields at ×40 objective. Apoptotic nuclei were visualized by nuclear condensation and fragmentation. All data were analyzed by a Student's paired t test. Staurosporine-induced Apoptosis—Cells were seeded in 6-well plates in complete medium for 24 h before treatment. The cells were washed with PBS twice and incubated in 1 μm staurosporine (Cell Signaling Technology) for 4 h. The cells were lysed in protein lysis buffer for cleaved caspase-3 Western blots. α2-6 Sialylation Inhibits the Binding of SW48 Cells to Galectin-3—Parental (Par), EV-transduced, or ST6Gal-I-expressing (ST6) SW48 cells were evaluated for cell adhesion to either gal-1 or gal-3. Briefly, cells were allowed to adhere for 2 h to tissue culture wells precoated with either gal-1 or gal-3. BSA- and poly-l-lysine-coated wells were used as negative and positive controls, respectively. As shown in Fig. 1A, none of the cell lines exhibited any specific binding to gal-1; therefore this lectin was not studied further. In contrast, Par and EV cells demonstrated substantial binding to gal-3, whereas the binding of ST6Gal-I-expressing cells was limited. Cell attachment to gal-3 was significantly inhibited by β-lactose but not by an equivalent concentration of sucrose, indicating that gal-3 binds to specific carbohydrate structures (Fig. 1B). Taken together, these data suggest that α2-6 sialic acids, added by the ST6Gal-I sialyltransferase, block the binding of gal-3 to cell surface glycan targets. Cell Adhesion to Galectin-3 Is Dependent upon β1 Integrins—The dual findings that β1 integrins can be differentially sialylated (5Bellis S.L. Biochim. Biophys. Acta. 2004; 1663: 52-60Crossref PubMed Scopus (132) Google Scholar, 11Seales E.C. Jurado G.A. Singhal A. Bellis S.L. Oncogene. 2003; 22: 7137-7145Crossref PubMed Scopus (107) Google Scholar, 12Seales E.C. Jurado G.A. Brunson B.A. Wakefield J.K. Frost A.R. Bellis S.L. Cancer Res. 2005; 65: 4645-4652Crossref PubMed Scopus (265) Google Scholar) and are binding partners for galectins (26Fukumori T. Takenaka Y. Yoshii T. Kim H.R. Hogan V. Inohara H. Kagawa S. Raz A. Cancer Res. 2003; 63: 8302-8311PubMed Google Scholar) prompted us to test whether gal-3 binding was mediated by the β1 subunit. Of note, Par and ST6 cells express equivalent amounts of cell surface β1 integrins (Fig. 1C); therefore diminished attachment of ST6 cells is not due to a reduction in integrin expression. To assess the role of the β1 integrin in gal-3 binding, cell attachment was evaluated in the presence of anti-β1 integrin function blocking antibodies. As shown in Fig. 1D, anti-β1 blocking antibodies markedly inhibited the binding of parental cells to gal-3, and interestingly, also attenuated the binding of ST6Gal I expressors. These results suggest that β1 integrins play a major role in regulating cell adhesion to this lectin. Desialylation of ST6 Cells Increases Cell Adhesion to Galectin-3—To verify that α2-6 sialylation inhibits gal-3 binding, cells were treated with neuraminidase to remove sialic acids and then evaluated for binding to gal-3-coated plates. We first tested neuraminidase efficacy by monitoring the electrophoretic mobility of the β1 integrin following neuraminidase treatment. As shown in Fig. 2A, the mature form of the β1 integrin from ST6 cells has a reduced electrophoretic mobility as compared with integrins from Par cells; this altered mobility was previously confirmed to result from increased α2-6 sialylation (12Seales E.C. Jurado G.A. Brunson B.A. Wakefield J.K. Frost A.R. Bellis S.L. Cancer Res. 2005; 65: 4645-4652Crossref PubMed Scopus (265) Google Scholar). Neuraminidase treatment of integrins from ST6 cells caused a dose-dependent increase in the electrophoretic mobility of the mature β1 isoform, consistent with the enzymatic removal of sialic acids. In contrast, the electrophoretic mobility of mature β1 integrins from Par cells was not altered by neuraminidase treatment, consistent with the fact that parental SW48 cells do not express either α2-3 or α2-6 sialyltransferases (24Dall'Olio F. Chiricolo M. Lollini P. Lau J.T. Biochem. Biophys. Res. Commun. 1995; 211: 554-561Crossref PubMed Scopus (30) Google Scholar). Neuraminidase treatment also had no effect on the mobility of the precursor β1 isoform from either Par or ST6 cells, which was expected given that the precursor is thought to be localized to the endoplasmic reticulum (and is therefore not a substrate for ST6Gal-I). As shown in Fig. 2B, ST6 cells treated with neuraminidase demonstrated a 3-fold increase in gal-3 binding as compared with untreated ST6 cells. In contrast, neuraminidase treatment had no effect on the binding of parental cells to gal-3. Collectively, these results strongly suggest that α2-6 sialylation inhibits the binding of SW48 cells to gal-3. α2-6 Sialylation of β1 Integrins Abolishes Galectin-3 Binding—To determine whether gal-3 binds directly to β1 integrins, we used a gal-3 overlay technique. Briefly, β1 integrins were immunoprecipitated from either parental or ST6Gal-I-expressing cells, resolved by SDS-PAGE, and transferred to PVDF membrane. The membrane was then overlaid with an AP-conjugated gal-3. As shown in Fig. 3, AP-conjugated gal-3 can directly bind to the mature form of β1 integrins immunoprecipitated from parental, but not ST6Gal-I-expressing, cells. These data suggest that α2-6 sialylation of β1 integrins blocks gal-3 binding. Interestingly, a higher molecular weight band was also detected in the gal-3 overlay of β1 immunoprecipitates, suggesting that another gal-3 ligand associates with, and is co-precipitated by, β1. The identity of this glycoprotein is currently unknown. To verify that equal amounts of β1 were precipitated, duplicate immunoprecipitated samples were immunoblotted for β1 (Fig. 3). Notably, gal-3 does not appear to bind to the precursor β1 isoform, which is consistent with the hypothesis that this endoplasmic reticulum-resident β1 isoform is modified with high mannose-type glycans that are not substrates for galectins. α2-6 Sialylation of Cell Surface Proteins Does Not Alter the Expression Level or Subcellular Localization of Endogenous Galectin-3—A number of studies have reported that gal-3 is highly expressed in tumor cells, including colon cancer cells (27Lahm H. Andre S. Hoeflich A. Kaltner H. Siebert H.C. Sordat B. von der Lieth C.W. Wolf E. Gabius H.J. Glycoconj. J. 2004; 20: 227-238Crossref PubMed Scopus (144) Google Scholar); therefore we tested whether SW48 cells produce endogenous gal-3. To evaluate the levels of endogenous gal-3, lysates from parental, EV, and ST6Gal-I-expressing SW48 cells were immunoblotted. Results from these experiments showed that Par, EV, and ST6 cells express similar levels of endogenous gal-3 but no detectable gal-1. Promonocytic U937 cells were included as a positive control for gal-1 (Fig. 4A). In addition to assessing gal-3 protein levels, gal-3 subcellular distribution was monitored by immunofluorescent microscopy. As shown in Fig. 4, B and C, the staining of endogenous gal-3 was mainly cytoplasmic in all three SW48 populations, which is noteworthy in light of studies suggesting that a cytoplasmic localization of gal-3 is pro-tumorigenic (15Liu F.T. Rabinovich G.A. Nat. Rev. Cancer. 2005; 5: 29-41Crossref PubMed Scopus (1196) Google Scholar, 16Dumic J. Dabelic S. Flogel M. Biochim. Biophys. Acta. 2006; 1760: 616-635Crossref PubMed Scopus (853) Google Scholar). Thus, our data suggest that Par, EV, and ST6 cells all produce robust amounts of endogenous, cytoplasmic gal-3, and forced expression of ST6Gal-I does not alter either gal-3 levels or subcellular distribution. ST6Gal-I Protects Cells from Galectin-3-induced Apoptosis—Given our results suggesting that α2-6 sialylation blocks gal-3 binding, we hypothesized that ST6Gal-I expression would attenuate gal-3-induced apoptosis in SW48 cells. To test this hypothesis, Par and ST6 cells were seeded onto chamber slides, treated with different concentrations of human recombinant gal-3 for 24 h, and then evaluated for nuclear morphology. Hoechst 33258 staining was used to