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Volume 129, Issue 5, Pages (November 2005)

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1 Volume 129, Issue 5, Pages 1581-1591 (November 2005)
Galectin-3 Modulates MUC2 Mucin Expression in Human Colon Cancer Cells at the Level of Transcription via AP-1 Activation  Shumei Song, James C. Byrd, Nachman Mazurek, Kaifeng Liu, Ja Seok Koo, Robert S. Bresalier  Gastroenterology  Volume 129, Issue 5, Pages (November 2005) DOI: /j.gastro Copyright © 2005 American Gastroenterological Association Terms and Conditions

2 Figure 1 Galectin-3 up-regulation of MUC2 protein expression. (A) MUC2 and galectin-3 protein levels were assessed by Western blotting in galectin-3 sense clone (LS5), the vector control clone (LSC2), in the galectin-3 antisense clone (M22), the vector control clone (MC1), and in the cells with either lower metastatic capacity (LM12) or higher metastatic capacity (LiM6). (B) Immunocytochemical staining of MUC2 and galectin-3 proteins in LSC2 and LS5 cell by anti-MUC2 monoclonal antibody (CCP58) and anti–galectin-3 monoclonal antibody (TIB166). Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions

3 Figure 2 Subcellular location of galectin-3 in galectin-3 sense and antisense clones. (A) The cytoplasmic and nuclear fractions of the cells were subjected to SDS–polyacrylamide gel electrophoresis and then immunoblotted with anti–galectin-3 (TIB166), β-actin for cytoplasm loading control, and histone 1 for nucleus loading control. (B) Indirect immunofluorescence was performed in LS5 and MC1 cells using anti–galectin-3 antibody (TIB166 1:100, green), followed by DAPI nuclear counterstaining (blue). The merge of galectin-3 (green) with DAPI (blue) also is shown. Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions

4 Figure 3 Galectin-3 up-regulation of MUC2 transcriptional activity. MUC2 luciferase activity (assayed by transient transfection) in LS5 cells compared with LSC2 cells, in M22 cells compared with MC1 cells, and in LM12 cells compared with LiM6 cells. The assays were performed in triplicate and repeated 3 times. Differences between pairs all were significant (P < .001 for each comparison). Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions

5 Figure 4 Down-regulation of MUC2 protein and transcriptional activity by tetracycline-inducible galectin-3 antisense. (A) MUC2 and galectin-3 protein levels were measured by Western blotting in AG4 cells, a clonal derivative of human colon cancer cell line HM7 that contains galectin-3 antisense under the control of a tetracycline-inducible promoter grown in the absence (−) or presence (+) of 1 μg/mL doxycycline for 48 hours. (B) MUC2 transcription was measured in AG4 cells incubated in the presence or absence of doxycycline (1 μg/mL). Experiments were performed in triplicate (P < .01). Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions

6 Figure 5 Deletion analysis of MUC2 promoter activity. (A) Schematic representation of luciferase reporter constructs containing various lengths of the MUC2 promoter. ○, Locations of potential AP-1 binding sites. (B) Deletion constructs of the MUC2 promoter were transfected transiently into galectin-3 sense clone LS5 cells and control LSC2 cells. □, LSC2; ■, LS5. (C) Deletion constructs of MUC2 promoter were transfected transiently into the galectin-3 antisense clone M22 and MC1 vector control. Cells were harvested and analyzed for luciferase activity 48 hours after transfection. □, MC1; ■, M22. Assays were performed in triplicate and repeated 3 times. Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions

7 Figure 6 Correspondence of AP-1 activity and MUC2 transcription in cell lines differing in galectin-3 levels. Luciferase activities of MUC2 and AP-1 in cell lines with lower or higher intrinsic levels of galectin-3 (LM12 vs LiM6), in cells stably transfected with galectin-3 sense cDNA (LSC2 vs LS5), and in cells stably transfected with galectin-3 antisense cDNA (MC1 vs M22). Cells were harvested and analyzed for luciferase activity 48 hours after transient transfection with either MUC2 or AP-1 luciferase reporter constructs as detailed in the Materials and Methods section. The assays were performed in triplicate and repeated 3 times. Differences between pairs all were significant for both MUC2 and AP-1 activity (P < .001 for each comparison). □, MUC2; ■, AP-1. Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions

8 Figure 7 Reduction of MUC2 promoter activity by mutation of the AP-1 site in the MUC2 promoter or by cotransfection with an AP-1 dominant-negative (AP-1dn) expression vector. (A) A MUC2 promoter reporter mutated at the AP-1 site (−1871) and its WT promoter (−2186 MUC2 promoter) reporter were transfected into cells stably transfected with galectin-3 sense cDNA (LSC2 vs LS5), and in cells stably transfected with galectin-3 antisense cDNA (MC1 vs M22). MUC2 promoter activity was significantly lower in LS5 and MC1 cells transfected with mutated AP-1 compared with wild-type AP-1 (P < .001). □, Wild-type; ■, mutated. (B) Cells were cotransfected with the −2205 MUC2 promoter construct and the AP-1 dominant-negative expression vector TAM67 or the control pCMV vector. After 2 days, cells were harvested and luciferase activities were measured. □, Control; ■, AP-1dn. P < .001 for differences in each cell line. Assays were performed in triplicate and repeated 3 times. Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions

9 Figure 8 Effect of galectin-3 levels on binding of nuclear proteins to the AP-1 consensus site of the MUC2 promoter. (A) A 3′-end biotin-labeled double-stranded oligonucleotide probe corresponding to the AP-1 site located −1871 bp upstream from the MUC2 translation start site was incubated with 5-ug aliquots of nuclear protein from cell lines with lower or higher intrinsic levels of galectin-3 (LM12 vs LiM6), in cells stably transfected with galectin-3 sense cDNA (LSC2 vs LS5), and in cells stably transfected with galectin-3 antisense cDNA (MC1 vs M22), and subjected to a mobility shift assay as described in Materials and Methods. As controls, nuclear protein was omitted, or a specific competitor, a 200-fold molar excess of cold probe (200× cold AP-1), was added to the reaction. (B) Specificity of AP-1 binding in LS5 cells was confirmed further by using labeled AP-1 wild-type (WT) and mutated (Mut.) oligonucleotide probes and 200-fold molar excess of cold probes as competitors (WT 200× cold AP-1 and Mut. 200× cold AP-1, respectively). Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions

10 Figure 9 AP-1 components c-Jun and Fra-1 are involved in the nuclear protein complex. (A) The nuclear fractions of cells (LSC2, LS5, MC1, and M22) were subjected to SDS–polyacrylamide gel electrophoresis and then immunoblotted with antibodies directed against AP-1 components (c-Jun, Fra-1, c-Fos, Fra-2, and Jun D). (B) Nuclear protein complex binding to the AP-1 site was disrupted by anti–galectin-3, anti–c-Jun, and anti–Fra-1 antibodies. Nuclear protein (5 ug) from cell line LS5 was incubated with anti–galectin-3 monoclonal antibody (anti–Gal-3), anti–c-Jun antibody, or anti–Fra-1 antibody and DNA binding reactions were performed using an AP-1 3′-end biotin-labeled double-stranded oligonucleotide probe as described in Figure 8. Normal rat IgG was added to the reaction as control. Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions

11 Figure 10 Nuclear galectin-3 is associated with the MUC2 promoter at the AP-1 site. (A) The association of galectin-3, c-Jun, and Fra-1 with the MUC2 promoter was confirmed in LS5 cells. Chromatin immunoprecipitation was performed using anti–galectin-3, anti–c-Jun, or anti–Fra-1 antibodies, and IgG as the negative control. The MUC2 promoter was amplified by PCR using flanking primers described in the Materials and Methods section. (B) Galectin-3 association with AP-1 components c-Jun and Fra-1 was confirmed by chromatin immunoprecipitation–immunoblot. LS5 cells were fixed, lysed, sonicated, and incubated with either anti–galectin-3, anti–c-Jun, or anti–Fra-1 antibodies, and IgG as the negative control. The samples were pulled down by salmon sperm DNA/protein A agarose and immunoblotted with antibodies to Fra-1, c-Jun, or galectin-3. Gastroenterology  , DOI: ( /j.gastro ) Copyright © 2005 American Gastroenterological Association Terms and Conditions


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