Abstract
Dysregulation of tight junctions (TJs) is often associated with human diseases including carcinogenesis and recent studies support role of TJ integral proteins in the regulation of Epithelial-to-Mesenchymal Transition (EMT). In this regard, expression of claudin-1, a key constituent of TJs, is highly increased in colon cancer and is causally associated with the tumor growth and progression. However, mechanism/s underlying regulation of claudin-1 expression in intestinal epithelial cells remains poorly understood. In our studies, we have identified putative binding sites for intestinal transcription factors Cdx1, -2 and GATA4 in the 5′-flanking region of the claudin-1 gene. Our further studies using full length and/or deletion mutant constructs in two different human colon cancer cell lines, SW480 and HCT116, showed key role of Cdx1, Cdx2 and GATA4 in the regulation of claudin-1 mRNA expression. However, overexpression of Cdx2 had the most potent effect upon claudin-1 mRNA expression and promoter activity. Also, in colon cancer patient samples, we observed a significant and parallel correlation between claudin-1 and Cdx2 expressions. Chromatin immunoprecipitation (ChIP) assay confirmed the Cdx2 binding with claudin-1 promoter in vivo. Using Cdx2 deletion mutant constructs, we further mapped the Cdx2 C-terminus domain to be important in the regulation of claudin-1 promoter activity. Interestingly, co-expression of activated β-catenin further induced the Cdx2-dependent upregulation of claudin-1 promoter activity while expression of the dominant negative (dn)-TCF-4 abrogated this activation. Taken together, we conclude that homeodomain transcription factors Cdx1, Cdx2 and GATA4 regulate claudin-1 gene expression in human colon cancer cells. Moreover, a functional crosstalk between Wnt-signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins and GATA-proteins is demonstrated in the regulation of claudin-1 promoter-activation.
Highlights
Tight junctions (TJs), the most apical cell-cell adhesion, help regulate the polarity and differentiated state of the epithelial cells
To examine whether Cdx1, Cdx2 and/or GATA4 help regulate colonic claudin-1 expression, we determined the effect of overexpression of above transcription factors upon endogenous claudin-1 expression
Quantitative RT-PCR showed similar induction of claudin-1 mRNA expression upon overexpression of all the above transcription factors and once again this induction was maximum in cells over-expressing Cdx2 (Figure 1C)
Summary
Tight junctions (TJs), the most apical cell-cell adhesion, help regulate the polarity and differentiated state of the epithelial cells. Disruption of cell-cell junctions along with concomitant changes in the expression of associated proteins helps induce invasion and metastatic progression in cancer. The claudin family of proteins is integral to the structure and function of tight junctions, and altered expression of claudin family members; in a tissue specific manner, has been detected in multiple cancers. Studies including ours have demonstrated that claudin-1 expression is dysregulated in a variety of cancers including colorectal cancer [3,5]. It is further worthy here to note that in colon cancer, dysregulated claudin-1 expression associates with the tumor progression and metastasis [5]. Claudin-1 is a target of b-catenin/Tcf signaling, a key regulator of colonic homeostasis and neoplastic growth/progression [6]. Details of the transcriptional regulation of colonic claudin-1 expression are not clearly understood
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