Previously, we reported that IFN-gamma and TNF-alpha downregulate the expression of the human Na(+)/H(+) exchanger (NHE)3 gene by modulating Sp1/Sp3 transcription factors in C2BBe1 cells. It is reported that butyrate inhibits IFN-gamma and TNF-alpha signaling pathways. In this study, we have investigated the effect of sodium butyrate (NaB) and IFN-gamma/TNF-alpha on human NHE3 promoter activity. In transient transfection studies, NaB (5 mM) led to 10-fold stimulation of NHE3 promoter activity after incubation for 24 h. With 5'-deletion analysis, the NaB-responsive region was mapped to the NHE3 core promoter, bp -95 to + 5, which we had shown previously to confer responsiveness to IFN-gamma/TNF-alpha. The stimulatory effect of NaB on the NHE3 promoter was reduced by 60% in the presence of IFN-gamma/TNF-alpha. Mutually, the repressive effect of these cytokines was attenuated by NaB. Knockdown of Sp1 and Sp3 expression with small interfering RNA (siRNA) resulted in a significant resistance to NaB effects. NaB treatment showed no effect on Sp1 and Sp3 protein expression as assessed by Western blot analyses. Gel mobility shift assays with nuclear proteins from NaB-treated cells showed enhanced binding of Sp1 and Sp3 to the NHE3 promoter. The phosphatase inhibitor okadaic acid (200 nM) blocked the stimulatory effect of NaB on the NHE3 promoter. NaB effects on the NHE3 promoter were significantly attenuated by protein phosphatase (PP)1alpha- and PP2Aalpha-specific siRNA transfection. Our data suggest that the differential regulation of NHE3 gene expression by NaB and IFN-gamma/TNF-alpha is mediated through alternative pathways that converge on Sp1/Sp3.