Identifying the location of epidermal growth factor-responsive element involved in the regulation of type IIb sodium-phosphate cotransporter expression in porcine intestinal epithelial cells

Phosphate is an important mineral nutrient for both human and animals in growth and physiological functions; thus, much effort in the past has been made to clarify the mechanisms governing its absorption. Previous studies have found that epidermal growth factor (EGF) inhibits phosphate absorption in human intestinal cells via modulating the interaction of transcriptional factor c-myb with sodium-phosphate cotransporter (NaPi-IIb) gene promoter. This finding provoked our interest in determining the effect of EGF on NaPi-IIb gene expression in intestinal cells of pigs and the location of EGF-responsive element in the gene promoter. Using quantitative PCR, it was observed that EGF significantly reduced NaPi-IIb gene expression in porcine intestinal epithelial IPEC-J2 cells. Transfection with a series of constructs that contain different lengths of the 5-flanking promoter region of the NaPi-IIb gene manifested that EGF-responsive element is located in the -1200 to -800 region. Further, c-myb was extracted from the cell nucleus of IPEC cells that were exposed to EGF or not via immunoprecipitation. The electrophoretic mobility shift assay showed a specific binding of transcription factor c-myb to labelled probes encompassing DNA sequence from -1092 to -1085 (-TCCAGTTG-). This protein-DNA complex was decreased with cells exposed to EGF and abrogated when c-myb was pre-incubated with excessive unlabelled competitive probes. Results from mutagenesis studies demonstrated that the c-myb-binding site is the EGF-responsive element involved in the regulation of NaPi-IIb expression. Identifying the location of EGF-responsive element contributes to understanding mechanisms underlying EGF down-regulated NaPi-IIb gene expression and provides a foundation for further investigating EGF-regulatory functions in phosphate absorption in pig intestine.