Epidermal growth factor inhibits intestinal NHE8 expression via reducing its basal transcription

Hua Xu, Bo Zhang, Jing Li, Huacong Chen, James Tooley, Fayez K Ghishan

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume299
Issue number1
DOIs
StatePublished - Jul 2010

Fingerprint

Epidermal Growth Factor
Caco-2 Cells
Sp3 Transcription Factor
Gene Expression
Messenger RNA
Sodium-Hydrogen Antiporter
Membranes
Gene Expression Regulation
Electrophoretic Mobility Shift Assay
Intestinal Mucosa
Microvilli
Cell Size
Genetic Promoter Regions
Human Activities
Small Intestine
Transfection
Real-Time Polymerase Chain Reaction
Proteins
Western Blotting
Gels

Keywords

  • Caco-2 cells
  • Intestine
  • Sodium/hydrogen exchangers
  • Sp3

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

Epidermal growth factor inhibits intestinal NHE8 expression via reducing its basal transcription. / Xu, Hua; Zhang, Bo; Li, Jing; Chen, Huacong; Tooley, James; Ghishan, Fayez K.

In: American Journal of Physiology - Cell Physiology, Vol. 299, No. 1, 07.2010.

Research output: Contribution to journalArticle

@article{84cf76107e15428b8a324788e2139de3,
title = "Epidermal growth factor inhibits intestinal NHE8 expression via reducing its basal transcription",
abstract = "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.",
keywords = "Caco-2 cells, Intestine, Sodium/hydrogen exchangers, Sp3",
author = "Hua Xu and Bo Zhang and Jing Li and Huacong Chen and James Tooley and Ghishan, {Fayez K}",
year = "2010",
month = "7",
doi = "10.1152/ajpcell.00081.2010",
language = "English (US)",
volume = "299",
journal = "American Journal of Physiology",
issn = "0363-6143",
publisher = "American Physiological Society",
number = "1",

}

TY - JOUR

T1 - Epidermal growth factor inhibits intestinal NHE8 expression via reducing its basal transcription

AU - Xu, Hua

AU - Zhang, Bo

AU - Li, Jing

AU - Chen, Huacong

AU - Tooley, James

AU - Ghishan, Fayez K

PY - 2010/7

Y1 - 2010/7

N2 - 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.

AB - 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.

KW - Caco-2 cells

KW - Intestine

KW - Sodium/hydrogen exchangers

KW - Sp3

UR - http://www.scopus.com/inward/record.url?scp=77953777889&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77953777889&partnerID=8YFLogxK

U2 - 10.1152/ajpcell.00081.2010

DO - 10.1152/ajpcell.00081.2010

M3 - Article

C2 - 20375273

AN - SCOPUS:77953777889

VL - 299

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6143

IS - 1

ER -