DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines

CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.     

Transcriptional activation of the MUC2 gene by p53

MUC2 is one of the major components of mucins that provide a protective barrier between epithelial surfaces and the gut lumen. We investigated possible alterations of MUC2 gene expression by p53 and p21(Sdi1/Waf1/Cip1) in a human colon cancer cell line, DLD-1, establishing subclones in which a tetracycline-regulatable promoter controls exogenous p53 and p21 expression. MUC2 mRNA more significantly increased in response to p53 than to p21. Unexpectedly, MUC2 expression was also induced in human osteosarcoma cells, U-2OS and Saos-2, by exogenous p53. We next performed a reporter assay to test the direct regulation of MUC2 gene expression by p53. Deletion and mutagenesis of the MUC2 promoter region showed that it contains two sites for transactivation by p53. Furthermore, an electrophoretic mobility shift assay indicated that p53 binds to those elements. We analyzed MUC2 expression in other cell types possessing a functional p53 after exposure to various forms of stress. In MCF7 breast cancer and A427 lung cancer cells, MUC2 expression was increased along with the endogenous p53 level by actinomycin D, UVC, and x-ray, but not in RERF-LC-MS lung cancer cells carrying a mutated p53. These results suggest that p53 directly activates the MUC2 gene in many cell types.     

The human intestinal cell lines Caco-2 and LS174T as models to study cell-type specific mucin expression

Mucin expression was studied during proliferation and differentiation of the enterocyte-like Caco-2 and goblet cell-like LS174T cell lines. Caco-2 cells express mRNAs of MUC1, MUC3, MUC4 and MUC5A/C whereas MUC2 and MUC6 mRNAs are virtually absent. Furthermore, MUC3 mRNA is expressed in a differentiation dependent manner, as is the case for enterocytes. Concomitantly MUC3 protein precursor (550 kDa) was detected in Caco-2 cells. In LS174T cells mucin mRNAs of MUC1, MUC2 and MUC6 are constitutively expressed at high levels, whereas MUC3, MUC4 and MUC5A/C mRNAs are present at low levels. At the protein level LS174T cells express the goblet cell specific mucin protein precursors MUC2, MUC5A/C and MUC6 with apparent molecular masses of about 600 kDa, 470/500 kDa and 400 kDa respectively. MUC3 protein is not detectable. Furthermore, human gallbladder mucin protein (470 kDa precursor), of which the gene has not yet been identified, is expressed in LS174T cells. In addition, synthesis and secretion of the goblet cell specific mature MUC2, MUC5A/C and human gallbladder mucin was demonstrated in LS174T cells. It is concluded that Caco-2 and LS174T cell lines provide excellentin vitro models to elucidate the cell-type specific mechanisms responsible for mucin expression.Abbreviations SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - DMEM Dulbecco's modified Eagle's medium - EMEM Eagle's minimum essential medium - Endo-H endo--N-acetylglucosaminidase H - HGBM human gallbladder mucin - dpc days past confluence - PBS phosphate buffered saline     

IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo

Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-alpha and IL-1beta levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells.     

E-selectin and ICAM-1 are incorporated into detergent-insoluble membrane domains following clustering in endothelial cells

The homeobox gene Cdx1 is a regulator of intestinal epithelial cell proliferation and differentiation. Using a transfection approach, we showed here that the oncogenic activation of the beta-catenin pathway stimulates the endogenous expression of the Cdx1 mRNA as well as the activity of the Cdx1 promoter in cancer cells of the human colon. Reciprocally, the paralogue homeobox gene Cdx2 exerts an inhibitory effect on the basal and on the beta-catenin-stimulated activity of the Cdx1 promoter. The inhibitory effect of CDX2 requires the intact homeodomain. It is not dependent on canonical CDX binding sites in the Cdx1 promoter nor on the cis-elements specifically targeted by the beta-catenin/Tcf complex. We conclude that the oncogenically activated beta-catenin and CDX2 have opposite and independent effects on the Cdx1 homeobox gene.     

Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter

Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.     

Differential regulation of CDX1 and CDX2 gene expression by deficiency in methyl group donors

The CDX2 and CDX1 homeobox genes have respectively a tumour suppressor and proliferative role in the intestinal epithelium. We analyzed DNA methylation and histones modifications associated with CDX2 and CDX1 promoters in two human colon cancer cell lines expressing differentially these genes, Caco2/TC7 [CDX2 positive-CDX1 negative] and HT29 [CDX2 negative-CDX1 negative] cells. Chromatin immunoprecipitation experiments indicated that CDX2 and CDX1 gene expression correlated with a histone modifications pattern characterizing active chromatin (H3K4 trimethylated and H3 acetylated). Bisulfite DNA sequencing and methylation-specific PCR showed that CDX2 and CDX1 promoters display no methylation in HT29 cells even though both genes are not expressed. In contrast, the CDX1 promoter is methylated in Caco2/TC7. DNA demethylation by 5aza-dC or the combination of 5aza-dC plus SAHA, an inhibitor of histone deacetylases, restored CDX1 expression in Caco2/TC7 cells but these treatments were inefficient on both CDX2 and CDX1 in HT29 cells. Thus, in colon cancer cells the changes in chromatin conformation are heterogeneous and repression of CDX2 and CDX1 in HT29 cells is not due to epigenetic mechanisms. In vivo, dietary deprivation of methyl groups in rats upregulated CDX1 mRNA and downregulated to a lesser extent CDX2 mRNA expression. Moreover, methyl group deprivation downregulated CDX2 protein by changing its phosphorylation pattern. The changes in CDX2 and CDX1 expression determined by methyl group deprivation may constitute one of the mechanisms sustaining the protective role attributed to folate in colon cancer.