Epidermal growth factor inhibits intestinal NHE8 expression via reducing its basal transcription.
Sodium/hydrogen exchangers (NHEs) play a major role in Na(+) absorption, cell volume regulation, and intracellular pH regulation. Of the nine identified mammalian NHEs, three (NHE2, NHE3, and NHE8) are localized on the apical membrane of epithelial cells in the small intestine and the kidney. Although the regulation of NHE2 and NHE3 expression has been extensively studied in the past decade, little is known about the regulation of NHE8 gene expression under physiological conditions. The current studies were performed to explore the role of epidermal growth factor (EGF) on NHE8 expression during intestinal maturation. Brush-border membrane vesicles (BBMV) were isolated from intestinal epithelia, and Western blot analysis was performed to determine NHE8 protein expression of sucking male rats treated with EGF. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcriptional factors involved in EGF-mediated regulation. Our results showed that intestinal NHE8 mRNA expression was decreased in EGF-treated rats and Caco-2 cells, and NHE8 protein abundance was also decreased in EGF-treated rats. The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by EGF treatment. This could be explained by reduced binding of transcription factor Sp3 on the NHE8 basal promoter region in the presence of EGF. Pretreatment with MEK1/2 inhibitor UO-126 could prevent EGF-mediated inhibition of NHE8 gene expression. In conclusion, this study showed that EGF inhibits NHE8 gene expression through reducing its basal transcription, suggesting an important role of EGF in regulating NHE expression during intestinal maturation.
Xu
Hua
H
University of Arizona Health Sciences Center, Tucson, Arizona 85724, USA.
Zhang
Bo
B
Li
Jing
J
Chen
Huacong
H
Tooley
James
J
Ghishan
Fayez K
FK
D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D018938:Caco-2 Cells; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D004815:Epidermal Growth Factor / Q000008:administration & dosage / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D008871:Microvilli / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D002070:Butadienes; D009570:Nitriles; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; C486775:SLC9A8 protein, human; C497121:SP3 protein, human; C502388:Slc9a8 protein, rat; D017923:Sodium-Hydrogen Exchangers; C113580:U 0126; D051705:Sp3 Transcription Factor; D004815:Epidermal Growth Factor; C482128:MAP2K2 protein, human; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human