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Evidence that Cdx2 regulation of goblet cell specific gene expression is redox‐sensitive
Author(s) -
Khan T A,
Gaskins H R
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1085
Subject(s) - transactivation , cdx2 , promoter , mutant , luciferase , biology , microbiology and biotechnology , transcription factor , enterocyte , gene expression , gene , biochemistry , homeobox , transfection , small intestine
We are examining the extent to which intestinal epithelial cell differentiation into goblet cells versus enterocytes is a consequence of redox‐sensitive transcriptional regulatory pathways. Sequence alignment of Cdx2, an intestinal specific transcription factor, revealed the presence of two cysteines C131 and C165 in the regulatory region, evolutionarily conserved only among higher amniotes. To determine if these cysteines confer redox sensitivity, they were mutated to serines, creating single mutants Cdx2/C131S and Cdx2/C165S and the double mutant Cdx2/C131S/C165S. Cdx2 responsive promoters of two goblet cell specific genes, trefoil factor 3 ( TFF3 ) and intestinal mucin 2 ( MUC2 ) were studied using luciferase as a reporter in the intestinal epithelial LS174T cell line. Transactivation of the two promoters using mutants showed a significant decrease ( P < 0.05) in luciferase activity as compared to wild type Cdx2 suggesting the involvement of the two cysteines in transactivation. The redox sensitivity of Cdx2 was also analyzed by adding buthionine sulphoximine (50 μM), an oxidizing agent, which enhanced Cdx2 transactivation of the TFF3 and MUC2 promoters. The DNA binding activity of mutant Cdx2s was similar to the wild type Cdx2 in a mobility shift assay. However, in contrast to goblet cell specific promoters, preliminary data with an enterocyte specific promoter, sucrase isomaltase, showed similar transactivation by wild type and mutant Cdx2s. Together, these results indicate that transactivation of goblet cell specific genes by Cdx2 may be affected by the redox environment to a greater extent than enterocyte‐specific genes. This work was supported by the NIH (R01 DK061568).