409 MOLECULAR MECHANISM OF RAT NHE3 PROMOTER REGULATION BY SODIUM BUTYRATE

SCFAs, and especially butyrate (NaB), stimulate sodium and water absorption by inducing colonic Na+/H+ exchange (NHE). NaB induces NHE3 activity, protein and mRNA expression both in vivo and in vitro. Our previously published observations indicated that this induction is PKA-dependent and NaB-responsive elements were localized within -320 /-34 bp of the NHE3 promoter. Here, we further delineate the mechanism of NaB-mediated NHE3 gene transcription. Transient transfection of Caco-2 cells with reporter constructs containing -320/+58 nt of the NHE gene promoter identified Sp binding site located at position -58-55 nt (SpB) as critical for NaB-mediated induction. Gel mobility shift assay (GMSA) with or without neutralizing antibodies for Sp1 or Sp3 indicated increased binding of Sp3 to SpB element, with no concomitant changes in Sp1 or Sp3 protein expression in the nuclei of NaB-treated Caco-2 cells. We also demonstrated that in SL-2 Drosophila cells transfected with HA-tagged Sp1 or Sp3 cDNA, Sp3 was a much stronger inducer of NHE3 gene transcription than Sp1, which suggests that even a small change in balance, favoring binding of Sp3 to SpB site might result in significant increase in NHE3 promoter activity. Also, siRNA experiments demonstrated that knock down of Sp3 expression in Caco-2 cells significantly blunted the response of NHE3 promoter to NaB. We have also found that p300 was not involved in the induction of NHE3 promoter by NaB, since its overexpression, expression a p300 mutant lacking the histone deacetylase domain, or overexpression of E1A oncogene remained without effect on NaB-stimulated NHE3 promoter activity. Surprisingly, a construct containing -118/+58 nt conferred only partial responsiveness to NaB. Similarly, -320/-33 nt of the NHE3 promoter placed upstream of a heterologous transcription start site was stimulated stronger that -118/-33 nt region suggesting an element upstream of the SpB site, crucial for a full response to NaB. This element was further narrowed down to a -196/-175 nt region. Although GMSA identified an unknown protein/DNA complex with -196/-175 nt probe, it's intensity was not affected by treatment of cells with NaB. This may suggest either post-translational modification leading to a higher transactivation potency of the putative factor, or a possibility that binding of Sp3 to SpB site has to precede and perhaps result in recruitment of the putative factor to site -196/-175.