Extracellular Acidosis Stimulates NHE2 Expression through Activation of Transcription Factor Egr-1 in the Intestinal Epithelial Cells

Na+/H+ exchangers (NHEs) play important roles in regulating internal pH (pHi), cell volume and neutral Na+ absorption in the human intestine. Earlier studies have shown that low extracellular pH (pHe) and metabolic acidosis increases the expression and function of NHE1-3 genes. However, transcriptional mechanisms involved remained unknown. Therefore, we investigated the molecular mechanisms underlying acid-induced NHE2 expression in C2BBe1 and SK-CO15 intestinal epithelial cells. Assessing total RNA and protein by RT-PCR and Western blot analysis, respectively, displayed significant increases in the NHE2 mRNA and protein levels in cells exposed to acidic media (pH 6.5 and 6.7) compared to normal medium. Acid treatment was also associated with a significant enhancement in NHE2 transport activity. Quantification of the heterogeneous nuclear RNA indicated that the rate of NHE2 transcription was increased in response to acid. Furthermore, acid caused a significant increase in NHE2 promoter activity confirming transcriptional upregulation. Through functional and mutational studies the acid-response element was mapped to a 15-nucleotide GC-rich sequence at bp −337 to −323 upstream from the transcription start site. We previously identified this element as an overlapping Egr-1/Sp1/Egr-1 motif that was essential for the NHE2 upregulation by mitogen-induced transcription factor Egr-1. Cells exposed to acid exhibited a temporal increase in Egr-1 mRNA and protein expression. These events were followed by Egr-1 nuclear accumulation, as detected by immunofluorescence microscopy, and potentiated its in vitro and in vivo interaction with the NHE2 promoter. Disruption of ESE motif and knockdown of Egr-1 expression by targeted small interfering RNA abrogated the acid-induced NHE2 transcriptional activity. These data indicate that the acid-dependent NHE2 stimulation is implemented by transcriptional upregulation of NHE2 via acid-induced Egr-1 in the intestinal epithelial cells.