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.

Fig. 1.

Effect of epidermal growth factor (EGF) on the intestinal Na⁺/H⁺ exchanger NHE8 expression in rats. Brush-border membrane vesicles (BBMVs) were isolated from the jejunal (A) or ileal (B) mucosa of control (CT) rats and EGF rats. BBM protein (30 μg) was loaded on SDS-PAGE gels, and immunoblots were performed. Rat NHE8 antibody and β-actin antibody were used to detect NHE8 and β-actin, respectively. The expression of NHE8 protein is calculated by the density of NHE8 band over that of β-actin band. Bar chart shows the NHE8 protein expression indicated as means ± SE in the sum of 4 independent experiments. *P ≤ 0.01 for control groups vs. EGF groups. Inset: corresponding Western blot image.

Fig. 2.

Effect of EGF on the intestinal NHE8 mRNA expression in rats. RNAs were isolated from the ileal mucosa of control rats or EGF rats and used for real-time PCR. NHE8 mRNA and TATA box binding protein (TBP) mRNA were amplified with rat-specific NHE8 and TBP primers. The changes in NHE8 gene expression is analyzed by the comparative cycle threshold (Ct) method. Data are means ± SE from total 18 rats (9 for EGF group, 9 for control group). *P ≤ 0.01 for control group vs. EGF group.

Fig. 3.

Effect of EGF on the endogenous NHE8 mRNA expression in human intestinal epithelial (Caco-2) cells. Caco-2 cells were cultured in normal medium or EGF-containing medium for 18 h before harvest. RNAs were isolated from these cells and were used for RT-PCR. Real-time PCR was performed with human NHE8 or TBP primers in separate reactions. Results are means ± SE from 3 to 5 separate experiments. *P < 0.01 for control vs. EGF treatment.

Fig. 4.

Effect of EGF on human NHE8 gene promoter activity. Cells were cotransfected with pGL3 basic (pGL3b) or human NHE8 promoter constructs (pGL3b/-671, pGL3b/-89, pGL3b/-32) and pRL-CMV. EGF was applied 18 h before harvesting cells for measuring promoter activities. Promoter reporter assay was performed 40 h after transfection. Promoter activity is shown as a relative activity that is a ratio of firefly luciferase activity driven by NHE8 promoter over Renela luciferase activity driven by CMV promoter. The degree of inhibition is shown as the ratio of luciferase activity in EGF-treated cells over luciferase activity in vehicle-treated cells. Results are means ± SE from 6 separate experiments. *P < 0.01 for control vs. EGF treatment.

Fig. 5.

Effect of EGF on DNA/protein interaction at the proximal promoter region of the human NHE8 gene. A: identification of DNA-protein interaction on the basal promoter region of the human NHE8 gene by gel mobility shift assays (GMSAs). Nuclear proteins were isolated from EGF-treated (EGF) and non-EGF-treated (CT) Caco-2 cells. DNA oligos containing GC box region on the minimal promoter region of the human NHE8 gene were end labeled with [³²P]ATP and used as a probe for GMSAs. Results shown are representative of 3 separate experiments. B: identification of the transcriptional factor involving in EGF regulation. DNA Oligos were end-labeled with [³²P]ATP and used as a probe in GMSAs. Nuclear protein was isolated from non-EGF-treated (CT) and EGF-treated (EGF) cells, and GMSAs were performed as indicated in the materials and methods. Sp1 and Sp3 antibodies (4 μg/binding) were used for supershift experiments. The exposure time on supershift GMSAs was longer for EGF-treated cells due to these cells having weaker protein-DNA interaction. SS, supershift DNA-protein interaction complex.

Fig. 6.

Signaling pathway studies of EGF regulation of hNHE8 gene expression. Caco-2 cells were transfected with promoter construct pGL3/-32 bp and pretreated with various inhibitors for 2 h before EGF was added. Relative change is shown as the ratio of luciferase activity in EGF-treated cells to luciferase activity in vehicle-treated cells in the presence or absence of various inhibitors. Results are means ± SE from 4 independent experiments. *P < 0.03 for EGF-, SB-, H7-treated cells vs. others.

Fig. 7.

EGF regulation on intestinal NHE8. In the intestinal epithelial cells, EGF binds to EGF receptor (EGFR) and activates mitogen-activated protein kinase (MAPK) pathway. Activated ERK reduces Sp3 binding at the NHE8 promoter region. This ultimately results in the reduced NHE8 gene expression in the intestinal epithelial cells.