Biochemical and Biophysical Research Communications
Functional crosstalk between Wnt signaling and Cdx-related transcriptional activation in the regulation of the claudin-2 promoter activity
Section snippets
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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Authors contributed equally to this work.