Transcriptional regulation of the human Na+/H+ exchanger NHE3 by serotonin in intestinal epithelial cells

Abstract

Serotonin (5-HT) decreases NHE2 and NHE3 activities under acute conditions in human intestinal epithelial cells. Here, we have investigated the effects of 5-HT on expression of the human NHE3 gene and the mechanisms underlying its transcriptional regulation in differentiated C2BBe1 cells. Treatment of the human intestinal epithelial cell line, C2BBe1, with 5-HT (20 microM) resulted in a significant decrease in NHE3 mRNA and protein expression. In transient transfection studies, 5-HT repressed the NHE3 promoter activity by approximately 55%. The repression of the NHE3 promoter activity in response to 5-HT was accompanied by reduced DNA-binding activity of transcription factors Sp1 and Sp3 to the NHE3 promoter without alteration in their nuclear levels. Pharmacological inhibitors of protein kinase C reversed the inhibitory effect of 5-HT on the promoter activity. Our data indicate that 5-HT suppresses the transcriptional activity of the NHE3 promoter and this effect may be mediated by PKCalpha and modulation of DNA-binding affinities of Sp1 and Sp3.

Fig. 1

Dose- and time-dependent effects of serotonin on the expression of the NHE3 mRNA in C2BBe1 cells. Differentiated serum-starved C2BBe1 cells treated with different doses of 5-HT for 4 h (A), or with 20 μM concentration for 0, 2, 4, 6, 8 and 24 h; total RNA was extracted and subjected to RT-PCR (B). GAPDH was used as an internal control. Effect of serotonin on the NHE3 protein levels examined by Western blot analysis (C), differentiated serum-starved cells were treated with serotonin for the indicated time period and total cell proteins were prepared and separated (100 μg/lane) by 10% SDS-polyacrylamide gel electrophoresis. NHE3 protein was detected using anti-NHE3 goat polyclonal antibody. The blot was re-probed with tubulin antibody. Densitometric analysis of each experiment is shown on the right panels. Data are presented as intensity of the experimental sample relative to the GAPDH intensity in the corresponding sample. The activity in the control was set at 100. Results were obtained from three separate experiments (mean ± S.D.). * P<0.05 compared to control.

Fig. 2

Functional analysis of the NHE3 promoter by luciferase assays and identification of serotonin-responsive region. Promoter construct p-1507/+131 was transiently transfected into differentiated C2BBe1 cells (A). The effect of indicated doses of serotonin on NHE3 promoter activity was determined by luciferase assays. The luciferase activities are presented relative to the normalized activity of the promoter-less pGL2-Basic. 5′deletion NHE3 promoter constructs were transiently transfected into C2BBe1 cells and treated with or without serotonin (20 μM) for 16 h (B). Values shown are mean ± S.E. obtained from three different experiments performed in triplicate assays on different days. Statistical differences from the control values were determined by Student’s t test (‡ P = 0.007; * P = 0.006; ** P = 0.0002; *** P = 0.0001).

Fig. 3

Effect of 5-HT on the DNA-binding activity of nuclear proteins to the NHE3 core promoter region. Nucleotide sequence of the NHE3 core promoter region (bp −95 to −5) is shown and probes used for GMSAs are indicated (A). DNA-binding of nuclear proteins from untreated and serotonin treated cells to the probes A and B (B). Detailed analysis of DNA-binding characteristics of probe A and nuclear proteins from untreated and 5-HT treated cells (C). Lanes 1–5 and 6–9 show complex formation between probe A and nuclear proteins from untreated or serotonin-treated cells, respectively. The binding specificity of these complexes was examined by competition assays where excess unlabeled Sp1-specific probe was used (lane 5). The identities of the proteins present in these complexes were established by supershift assays and are indicated by slow migrating supershift bands (SS). Effects of serotonin on the nuclear expression of Sp1 and Sp3 in C2BBe1 cells (D). Nuclear proteins (20 μg/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and Western blots were performed. Sp1 and Sp3 were detected using Sp1 and Sp3 antibodies, respectively. The blots were re-probed with actin antibody. C, control; Ab, antibody; NP, nuclear proteins.

Fig. 4

Effects of protein kinase inhibitors on the serotonin-mediated repression of the NHE3 core promoter activity. Differentiated C2BBe1 cells were transiently transfected with the p-95/+5. After serum-starvation, cell were treated without (control) or with the chelerythrine chloride (CC) (A), GÖ6976 (B), or Rp-8-Br-cAMP (C), for 1 h prior to further incubation in the presence or absence of 5-HT for 16 h. The luciferase activities are presented as percentage of the control (n=3, mean ± S.E.). ‡, *** P < 0.0005; * P = 0.49.