Transcriptional inhibition of intestinal NHE8 expression by glucocorticoids involves Pax5

Abstract

Sodium/hydrogen exchangers (NHEs) are a family of proteins that transport sodium ions into the cells by moving protons out of the cells. They play a major role in sodium absorption, cell volume regulation, and intracellular pH regulation. Three out of nine identified NHEs (NHE2, NHE3, and NHE8) are expressed on the apical membrane of intestinal epithelial cells. Glucocorticoids have been found to regulate NHE3 function in the intestine, but it is unknown if they have a similar function on NHE8 expression. Interestingly, high glucocorticoid levels in the intestine coincide chronologically with the change from high expression of NHE8 to high expression of NHE3. Studies were performed to explore the role of glucocorticoids on NHE8 expression during intestinal maturation. Brush-border membrane vesicles were isolated from intestinal epithelia, and Western blotting was performed to determine NHE8 protein expression of suckling male rats treated with methylpredisolone. 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 transcription factors involved in glucocorticoid-mediated regulation. Our results showed that the expression of NHE8 mRNA and protein was decreased in glucocorticoid-treated rats and human intestinal epithelial cells (Caco-2). The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by glucocorticoid treatment. GMSAs suggested that the reduction in promoter activity in the presence of glucocorticoids was due to enhanced transcription factor Pax5 binding on the NHE8 proximal promoter region. In conclusion, this study showed that glucocorticoids inhibit NHE8 gene expression by increasing Pax5 binding on NHE8 gene promoter, suggesting an important role for Pax5 during intestinal maturation.

Fig. 1.

Effect of methylprednisolone on the intestinal NHE8 protein expression in rats. Brush-border membrane vesicles (BBMVs) were isolated from the intestinal mucosa of control or methylprednisolone-treated rats. BBMV protein (30 μg) was loaded on SDS-PAGE and immunoblotted. Rat NHE8 antibody and β-actin antibody were used to detect NHE8 and β-actin, respectively. The expression of NHE8 protein was calculated by dividing the optical density of the NHE8 band over that of the β-actin band. Bars show the NHE8 protein expression indicated as means ± SE in the sum of 4 independent experiments. *P ≤ 0.01 for control (CT) groups vs. methylprednisolone (MP) groups. Inset: the corresponding Western blot image. A: effect of methylprednisolone on NHE8 protein expression in rat jejunum. B: effect of methylprednisolone on NHE8 protein expression in rat ileum.

Fig. 2.

Effect of methylprednisolone on the intestinal NHE8 mRNA expression in rats. RNA was isolated from the intestinal mucosa of control (CT) or methylprednisolone-treated (MP) rats and used for Real-Time PCR. NHE8 mRNA and TATA binding protein (TBP) mRNA were amplified with rat-specific NHE8 and TBP primers. The changes in NHE8 gene expression were analyzed by the comparative cycle threshold (C_t) method. Data are means ± SE from a total 18 rats (9 for the methylprednisolone group, 9 for the control group). *P ≤ 0.01 for the control group vs. the methylprednisolone group. Left: effect of methylprednisolone on NHE8 mRNA expression in rat jejunum. Right: effect of methylprednisolone on NHE8 mRNA expression in rat ileum.

Fig. 3.

Effect of dexamethasone (Dex) on the endogenous NHE8 expression in human intestinal epithelial cells (Caco-2). Caco-2 cells were cultured in standard medium or dexamethasone-containing medium (100 ng/ml) for 18 h before harvest. RNA was isolated from these cells and used for RT-PCR. Real-Time PCR was performed with human NHE8 or TBP primers in separate reactions. Total protein was prepared from cells and used for Western blot. Results are means ± SE from 3–5 separate experiments. *P < 0.01 for control vs. dexamethasone treatment. A: effect of dexamethasone on NHE8 mRNA expression in Caco-2 cells. B: effect of dexamethasone on NHE8 protein expression in Caco-2 cells.

Fig. 4.

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

Fig. 5.

Narrowing down the DNA region involved in dexamethasone regulation. Nuclear proteins were isolated from dexamethasone-treated (D) and nontreated (C) Caco-2 cells. Probes 1, 2, 3, and 4 for gel mobility shift assays (GMSAs) were made with [³²P]ATP end-labeled DNA oligos 1, 2, 3, and 4, respectively. The oligos span the proximal promoter region of the human NHE8 gene. Results shown are representative of 3 separate experiments.

Fig. 6.

Identification of the DNA sequences on the proximal promoter region of the human NHE8 gene involved in the dexamethasone regulation. A: identification of the DNA sequences involved in dexamethasone regulation. DNA oligo 3 was end-labeled with [³²P]ATP and used as a probe in GMSAs. Nuclear protein was isolated from Caco-2 cells, and GMSAs were performed as described in materials and methods. Mutant oligos (oligos M1, M2, M3, and M4) were used to identify the precise DNA sequences of the DNA-protein interaction. Bold letters indicate the mutation site at the promoter region. GMSA image indicates that cold probe and mutant probes (M1, M4) compete with the protein binding on the labeled oligo 3 probe, but mutant oligos (M2 and M3) could not compete with the protein binding on the labeled oligo 3 probe. B: functional characterization of DNA-protein interacting sequences at NHE8 promoter region. Wild-type (WT) promoter construct (pGL3b/−89) or mutant promoter constructs (pGL3b/−89M2 and pGL3b/−89M3) were cotransfected with pRL-CMV in Caco-2 cells. Promoter reporter assay was performed 18 h after dexamethasone treatment. Promoter activity was calculated as relative activity of firefly luciferase activity driven by NHE8 promoter to Renilla luciferase activity driven by CMV promoter. Fold changes were used to compare the promoter activity in the dexamethasone group with that of the control group. Results are means ± SE from 4 separate experiments. *P ≤ 0.05 for control treatment (CT) vs. dexamethasone treatment.

Fig. 7.

Identification of Pax5 interaction with NHE8 promoter in Caco-2 cells. A: GMSAs identification of the interacting protein at the NHE8 promoter region. Oligo 3 was end labeled with [³²P]ATP and used as a probe in GMSAs. Nuclear proteins were isolated from Caco-2 cells. Ap2 and Pax5 antibodies (4 μg/binding) were used for supershift experiments. GMSA indicated that Pax5 oligo competed with nuclear protein binding on the human NHE8 promoter and that Pax5 antibody (αPax5) partially blocked the interaction between Pax5 protein and the NHE8 promoter. In the same experiment, Ap2 antibody (αAp2) failed to compete with the DNA-protein interaction. B: Western blot detection of Pax5 expression in intestinal epithelial cells. Nuclear protein was extracted from Caco-2 cells and rat intestinal epithelial (RIE) cells, and 15 μg protein were used for Western blotting. Pax5 antibody (1:1,000 dilution) was used to detect Pax5 protein expression. Mouse spleen lysate was used as the positive control for Pax5 detection. C: PCR detection of Pax5 mRNA expression in Caco-2 cells. Total RNA was isolated from Caco-2 cells. RT-PCR was performed using human Pax5-specific primers. Primers F1–1236 and R1–1798 were used for the initial PCR. Primers F2–1308 and R2–1692 were used for nest PCR. Arrows indicate Pax5 PCR products. D: dexamethasone's effect on nuclear Pax5 abundance in Caco-2 cells. Nuclear protein was extracted from Caco-2 cells, and 15 μl protein extracts were used for Western blotting. Pax5 antibody and TBP antibody were used to detect Pax5 and TBP, respectively. The expression of Pax5 protein was calculated by the optical density of Pax5 band over that of TBP band. Bars show the Pax5 protein abundance in nuclear protein extracts. CT, nuclear protein from control cells; Dex, nuclear protein from dexamethasone-treated cells.