Functional crosstalk between Wnt signaling and Cdx-related transcriptional activation in the regulation of the claudin-2 promoter activity

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Abstract

Assembly of tight junctions at the most apical part of the lateral cell membrane is a key event in the differentiation of polarized epithelial cells. Claudin-2, a transmembrane protein involved in tight junction strand formation, has turned out to play a crucial role for the paracellular barrier function by opening pores for small cations. Physiological and pathological variations of epithelial barrier function are accompanied by differential expression of tight junction proteins. Therefore, we characterized molecular mechanisms regulating claudin-2 gene expression. Genomic DNA containing the transcription start point of human claudin-2 was isolated and functionally characterized by reporter gene assays. Activity of the claudin-2 promoter was elevated in mouse mammary epithelial C57 cells expressing Wnt-1. LEF-1, a nuclear effector of the Wnt signaling pathway which is involved in the regulation of cell differentiation and polarization, was found to bind directly to the claudin-2 promoter as revealed by electrophoretic mobility shift assays. Expression of LEF-1 and β-catenin both enhanced claudin-2 promoter activity. This increase was reduced after mutation of LEF-1 binding sites within the claudin-2 promoter. Furthermore, claudin-2 promoter activity was found to be enhanced by the TCF-4/β-catenin transcription complex. Therefore, we conclude that gene expression mediated by the promoter of the human tight junction protein claudin-2 is regulated by factors involved in Wnt signaling. Moreover, a functional crosstalk between Wnt signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins could be demonstrated in the regulation of claudin-2 promoter-mediated gene expression.

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Materials and methods

Cell culture and chemicals. HEK293 cells were propagated in MEME with 10% FCS and antibiotics. Oligonucleotides were obtained from Metabion (Martinsried, Germany). Mouse mammary epithelial C57 cells expressing Wnt-1 [19] were maintained in DMEM with 10% FCS and antibiotics (penicillin, streptomycin, and G418). Both cell lines show no detectable amounts of endogenous claudin-2 expression.

Molecular cloning of genomic DNA. A 420 bp DNA fragment was amplified from human genomic DNA using the genome

Isolation of the human claudin-2 promoter

A 420 bp genomic DNA fragment encompassing the predicted transcription start point for human claudin-2 mRNA was isolated by genome walking (Fig. 1). Sequence analysis revealed potential binding sites for different factors acting in trans in promoter activation and regulation of gene expression including a CAAT-box and two binding sites for the nuclear effector of the Wnt signal transduction pathway (LEF/TCF, 5-WWCAAWGG-3[23], [24]). Furthermore, two binding sites for the caudal-related

The Wnt signaling pathway is involved in the regulation of claudin-2 gene expression

For claudin-2 a 420 bp DNA fragment was amplified from human genomic DNA which contains 263 bp of 5-flanking sequences from the translation start point and encompasses the transcription start point as deduced from a mouse claudin-2 cDNA (GenBank Accession No. AK004990). Within this sequence, two potential binding sites for LEF/TCF as nuclear factors of the Wnt-dependent signal transduction pathway were identified in close proximity. The importance of the Wnt signaling pathway for tight junction

Acknowledgements

We thank Anja Fromm, Sieglinde Lüderitz, and Susanna Schön for excellent technical assistance. We thank Dr. Jackie Papkoff for providing C57+/−Wnt expressing cells and Dr. Hans Clevers for providing the TCF-4 expression plasmid. This work was supported by the Deutsche Forschungsgemeinschaft (Schu 559/6).

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