Galectin-3 mediates nuclear beta-catenin accumulation and Wnt signaling in human colon cancer cells by regulation of glycogen synthase kinase-3beta activity

Abstract

Wnt/beta-catenin signaling plays an essential role in colon carcinogenesis. Galectin-3, a beta-galactoside-binding protein, has been implicated in Wnt signaling, but the precise mechanisms by which galectin-3 modulates the Wnt pathway are unknown. In the present study, we determined the effects of galectin-3 on the Wnt/beta-catenin pathway in colon cancer cells, as well as the mechanisms involved. Galectin-3 levels were manipulated in human colon cancer cells by stable transfection of galectin-3 antisense, short hairpin RNA, or full-length galectin-3 cDNA, and effects on beta-catenin levels, subcellular distribution, and Wnt signaling were determined. Galectin-3 levels correlated with beta-catenin levels in a variety of colon cancer cell lines. Down-regulation of galectin-3 resulted in decreased beta-catenin protein levels but no change in beta-catenin mRNA levels, suggesting that galectin-3 modulates beta-catenin by another mechanism. Reduction of galectin-3 led to reduced nuclear beta-catenin with a concomitant decrease in TCF4 transcriptional activity and expression of its target genes. Conversely, transfection of galectin-3 cDNA into colon cancer cells increased beta-catenin expression and TCF4 transcriptional activity. Down-regulation of galectin-3 resulted in AKT and glycogen synthase kinase-3beta (GSK-3beta) dephosphorylation and increased GSK activity, increasing beta-catenin phosphorylation and degradation. Ly294002, an inhibitor of phosphatidylinositol 3-kinase, and dominant-negative AKT, suppressed TCF4 transcriptional activity induced by galectin-3 whereas LiCl, a GSK-3beta inhibitor, increased TCF4 activity, mimicking the effects of galectin-3. These results suggest that galectin-3 mediates Wnt signaling, at least in part, by regulating GSK-3beta phosphorylation and activity via the phosphatidylinositol 3-kinase/AKT pathway, and, thus, the degradation of beta-catenin in colon cancer cells.

Figure 1. Down-regulation of galectin-3 reduces β-catenin levels in human colon cancer cells

A, The expression of galectin-3 and β-catenin in a variety of colon cancer cell lines was determined by immunoblotting. B, Protein levels of galectin-3, β-catenin, and cyclin D1 were assessed by Western blotting in paired MC1 (vector control) and M22 (galectin-3 antisense) cells and paired GV3 (control) and E2 (galectin-3 shRNA) cells. C&D, MC1/M22 and GV3/E2 cells were prepared in cytospin slides and stained for galectin-3 and β-catenin as described in Materials and Methods.

Figure 2. Subcellular localization of β-catenin in galectin-3 antisense M22 cells and vector control MC1 cells

A, Cytoplasmic and nuclear fractions of colon cancer cells were subjected to SDS-PAGE and then immunoblotted with anti-β-catenin, β-actin and histone-1. β-actin was used as a cytoplasmic protein loading control, and histone1 was used for a nuclear protein loading control. B, Indirect immunofluorescence were performed on MC1 and M22 cells using anti-galectin-3 antibody (TIB166 1:100, red) and anti-β-catenin antibody (1:100, green), followed by DAPI nuclear counterstaining (blue). The merge of galectin-3 (red) and β-catenin (green) with DAPI (blue) is also shown.

Figure 3. Alterations in galectin-3 levels lead to corresponding changes in TCF4 transcriptional activity

A left panel, MC1 and M22 cells were cotransfected with 1 µg of SuperTop TCF4 reporter and 0.2 µg of pCH110. Luciferase reporter activity was measured after 48 hours (left panel). A right panel, AG1 cells containing galectin-3 antisense under control of a tetracycline-inducible promoter were cotransfected with 1 µg of SuperTop TCF4 reporter and 0.2 µg of pCH110 and treated with or without doxycycline 1 µg/ml for 48 hours (right panel). B, Cell lines GB2 and E2 (containing galectin-3 shRNA) and vector transfected GV3 control cells were cotransfected with 1 µg of SuperTop TCF4 reporter and 0.2 µg of pCH110. Luciferase reporter activity was measured after 48 hours. C, LS174T and LiM6 cells were cotransfected with 1 µg of pCNC10gal-3 vector or control vector pCNC10 and 1 µg of SuperTop TCF4 reporter and 0.2 µg of pCH110. Luciferase reporter activity was measured after 48 hours. For all transfection experiments, values shown represent the mean and SD of triplicate assays, and experiments were repeated at least twice (* P<0.001, **P<0.05).

Figure 4. Up-regulation of galectin-3 increases β-catenin level and TCF4 activity in human colon cancer cells

A. Protein levels of galectin-3, β-catenin, and cyclin D1 were assessed by Western blotting in RKO cells stably transfected with galectin-3 sense (RKO-gl3) or a vector control plasmid (RKO-v). B. RKO-gl3 and RKO-v cells were transfected with 1 µg of SuperTop TCF4 reporter and 0.2 µg of pCH110. Luciferase reporter activity was measured after 48 hours. For all experiments, values shown represent the mean and SD of at least triplicate assays (* P<0.005).

Figure 5. Galectin-3 regulates GSK-3β activity/phosphorylation via the PI3K/AKT pathway

A, Total cell lysates of paired MC1/ M22 cells and RKO-v /RKO-gl3 cells were prepared and immunoblots were performed for galectin-3, phospho-AKT and total AKT, phospho-GSK-3β and total GSK-3β and total β-catenin, and β-actin expression levels. B, In vitro kinase assay. Endogenous GSK-3β was immunoprecipitated by 2µg rabbit anti-GSK-3β antibody from 1000µg MC1 and M22 cell lysates, then specific GSK-3β substrate peptide (Calbiochem, San Diego, CA) was used to perform an in vitro kinase assay. Kinase activity was determined by scintillation counting. 0.2 µg purified GSK-3β (Upstate Biotechnology, Charlottesville, VA) was used as positive kinase control, and negative GSK-3β substrate peptide (Calbiochem, San Diego, CA) was used to detect background phosphorylation (GSK-3β autophosphorylation). The data expressed represents activity after subtraction of background phosphorylation (* p<0.005) C, Human colon cancer cells (LiM6 or LS174T) were transfected with 1 µg of SuperTop TCF4 reporter alone (bars 1, 3), or cotransfected with 1 µg of pCNC10 gal-3 vector (bars 2, 4, 6), cotransfected with 1 µg of dominant-negative AKT construct (AKT AAA) (bars 5, 6) for 48 hours. Cells were then treated with Ly294002 5µM for an additional 24 hours (bars 3, 4) and luciferase reporter activity was measured (*p< 0.05, **p<0.01). D, Cells were cotransfected with 1 µg of SuperTop TCF4 reporter and 1 µg of pCNC10gal-3 vector and then treated with 20 mM LiCl for 24 hours. Luciferase reporter activity was measured after 48 hours. For all experiments, values shown represent the mean and SD of at least triplicate assays (*p<.005).

Figure 6. Proposed model by which galectin-3 mediates Wnts signaling in colon cancer cells

Galectin-3 mediates AKT phosphorylation, thereby increasing phosphorylation of GSK-3β and decreases its activity. Inactivation of GSK-3β leads to a reduction in β-catenin degradation, and increased cellular β-catenin levels. β-catenin can then translocate to the nucleus, bind to TCF4 and activate the transcription of its specific target genes.