Galectin-3 Modulates MUC2 Mucin Expression in Human Colon Cancer Cells at the Level of Transcription via AP-1 Activation

Abstract

Galectin-3 and MUC2 intestinal mucin each have been correlated with the malignant behavior of colon cancer cells. Galectin-3 modulates expression of MUC2 protein, but the specific regulatory mechanisms are unknown. This study sought to determine how galectin-3 increases MUC2 expression. Galectin-3 levels in human colon cancer cells of high and low metastatic ability were manipulated via expression of galectin-3 complementary DNA in sense or antisense orientation. Galectin-3 and MUC2 protein expression were determined by Western analysis and immunocytochemistry. Transient transfections of promoter reporter constructs were used to monitor MUC2 transcription and AP-1 activity. Electrophoretic mobility shift assays, site-directed mutagenesis, and chromatin immunoprecipitation were used to monitor the participation of AP-1 in MUC2 transcription. Alterations in galectin-3 levels correlated with both MUC2 protein expression and transcription. By using MUC2 promoter constructs of different lengths, galectin-3 responsiveness was found between 1500 and 2186 bp upstream of the translation start site, a region that contains 1 consensus AP-1 binding site. AP-1 activity paralleled MUC2 transcription in the different cell lines. Mutation in the AP-1 site markedly decreased MUC2 promoter activity, and MUC2 transcription was inhibited by cotransfection with a dominant-negative AP-1 vector. Electrophoretic mobility shift assays, co-immunoprecipitation, and chromatin immunoprecipitation analyses suggested an association between galectin-3, c-Jun, and Fra-1 in forming a complex at the AP-1 site on the MUC2 promoter. Galectin-3 up-regulation of MUC2 transcription occurs at the level of transcription through AP-1 activation. This may have important implications for understanding the role of galectin-3 and MUC2 in colon cancer metastasis.