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

Abstract

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.

Epidermal Growth Factor, through Alleviating Oxidative Stress, Protect IPEC-J2 Cells from Lipopolysaccharides-Induced Apoptosis

“…Hence, we used LPS, a major integral component of the outer membrane of Gram-negative bacteria, which can induce cell injury [ 11 , 12 , 13 , 14 ], to establish a cell injury model to investigate the protective effects of EGF on oxidative injury and apoptosis in IPEC-J2 cells. In the present study, we first evaluated the toxicity of EGF and LPS in IPEC-J2 cells; the results showed that EGF at 100 ng/mL can significantly increase cell growth, which is consistent with our previous study, wherein EGF at 100 ng/mL inhibited NaPi-IIb expression [ 27 ], and LPS at 1.0 μg/mL caused a dramatic decrease of cell viability. It also showed that cells cultured with EGF for 24 h had higher cell growth rates, and cells cultured with LPS for 24 h had lower cell growth rates.…”

Mechanisms of Epidermal Growth Factor Effect on Animal Intestinal Phosphate Absorption: A Review

“…The study in rat and human CACO2 cells from Xu et al ( 24 ) showed that EGF significantly inhibited the expression of NaPi-IIb gene. Consistent with Xu et al ( 24 ), our previous study also found that EGF downregulated NaPi-IIb expression in IPEC-J2 cells ( 26 ), indicating the loss of active transcellular transport of Pi in the small intestine. This suggested that, under normal conditions, EGF inhibited the active transport of Pi.…”

“…The EGF response region was located in the promoter between −784 and −729 base pair (bp) of the promoter of human, and the downregulation of promoter function is mediated by EGF-activated protein kinase C/protein kinase A PKC/PKA and MAPK pathways ( 25 ). Previous work in our laboratory showed that the EGF response region was located in the −1,092 to −1,085 bp region (5′-TCCAGTTG-3′) in porcine intestinal epithelial cells, IPEC-J2 ( 26 ). Further studies showed that EGF downregulated the expression of NaPi-IIb in IPEC-J2 cells by activating signaling molecules such as EGFR, PKA, PKC, P38, extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK) ( 68 ).…”