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

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Phosphate is an important mineral element for human and animals, participating in a variety of biological processes, such as ATP synthesis, acid/base regulation and the formation of bone and nucleotides (Xu et al., 2005; Fang et al., 2012). Phosphate absorption is dependent on type IIb sodium‐phosphate cotransporters (NaPi‐IIb) located in the apical membranes of enterocytes (Xu et al., 2005). As protein abundance of NaPi‐IIb in enterocyte membranes is under the regulation of various factors, including hormones, cytokines, dietary nutritions (e.g. vitamin D3, calcium and phosphate) and local acid–base status, the phosphate absorption is easily impacted (Stauber et al., 2005; Xu et al., 2005; Kido et al., 2013; Huber et al., 2015). In modern pig farming, several types of phosphorus compounds are extensively used as important feed additive to meet the need in phosphorus, while, in many cases, pig presents a relatively low absorptivity in phosphorus, resulting in the growth retardation, the economic loss in feeding and the environmental phosphorus contamination (Kemme et al., 1997; Walsh et al., 2012). Thus, it is required to further elucidate the mechanisms governing phosphorus absorption.
Early study has found that epidermal growth factor (EGF) can inhibit expression of NaPi‐IIb in weaned and suckling rats (Arar et al., 1999). Later, it was also uncovered that EGF can down‐regulate NaPi‐IIb expression by suppressing the binding of c‐myb to promoter region of NaPi‐IIb gene in human intestinal epithelial Caco‐2 cells (Xu et al., 2003a). Epidermal growth factor is a 53‐amino acids peptide that is mainly produced by duodenum and salivary gland and widely existed in serum, breast milk, urine and semen. Epidermal growth factor binds to its receptor, inducing the autophosphorylation of specific tyrosine residues on the receptor (Li et al., 2013). These phosphorylated amino acids serve as docking sites to recruit signalling molecules that regulate intracellular signalling networks, such as those mediated by MAPK, PKC and Akt; therefore, EGF is involved in the regulation of various biological functions (Li et al., 2013). c‐myb, belonged to Myb family, is a transcription factor responsible for controlling expression of a wide array of genes implicated in multiple physiological and pathological processes, like transactivation of cytosolic alanine aminotransferase, differentiation of colon epithelial cells and vascular smooth muscle cells, neutrophil maturation, autophagy and tumorigenesis (Ramsay et al., 2005; Anemaet et al., 2010; Lee et al., 2016; Li et al., 2016; Jin et al., 2016; Shikatani et al., 2016). As transcription factor binding to gene promoter is a major approach to regulate the gene transcription, it is easy to understand that EGF regulating the binding of c‐myb to NaPi‐IIb gene promoter can influence its expression in human intestinal cells.
Currently, it is uncertain about whether EGF has the ability to control NaPi‐IIb expression by regulation of c‐myb in pig intestinal epithelial cells, although our previous study has identified that NaPi‐IIb gene sequence in pig shares high similarity with that in human (Zhifeng et al., 2012). In this study, quantitative PCR (qPCR) was conducted to identify the effect of EGF on NaPi‐IIb gene expression. Further, promoter truncation experiment, protein‐DNA binding assay and site‐directed mutagenesis were performed to identify the promoter sequence essential for c‐myb banding and EGF regulating NaPi‐IIb gene expression. This research provides a theoretical basis for study of the transcriptional regulation of the NaPi‐IIb gene by EGF.
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