Molecular mechanism of rat NHE3 gene promoter regulation by sodium butyrate.
Sodium butyrate (NaB) stimulates sodium and water absorption by inducing colonic Na(+)/H(+) exchange. NaB induces Na(+)/H(+) exchanger (NHE)3 activity and protein and mRNA expression both in vivo and in vitro. Our previously published observations indicated that this induction is Ser/Thr kinase dependent and that NaB-responsive elements were localized within -320/-34 bp of the rat NHE3 promoter. Here we further delineate the mechanism of NaB-mediated NHE3 gene transcription. Transient and stable transfection of Caco-2 cells with NHE3 gene reporter constructs identified Sp binding site SpB at position -58/-55 nt as critical for NaB-mediated induction. Gel mobility shift (GMSA) and DNA affinity precipitation assays indicated NaB-induced binding of Sp3 and decreased binding of Sp1 to SpB element. While no changes in expression of Sp1 or Sp3 were noted, NaB induced phosphorylation of Sp1 and acetylation of Sp3. Sp3 was a more potent inducer of NHE3 gene transcription, which suggested that change in balance, favoring binding of Sp3 to the SpB site, would result in significant increase in NHE3 promoter activity. Small interfering RNA studies in Caco-2 cells and data from NaB-treated SL2 cells used as a reconstitution model confirmed this hypothesis. In addition to the SpB site, which played a permissive role, an upstream novel butyrate response element located at -196/-175 nt was necessary for maximal induction. GMSA identified a protein-DNA complex with a -196/-175 nt probe; this interaction was not affected by NaB treatment, thus suggesting that in response to NaB Sp3 binding to site SpB precedes and results in recruitment of the putative factor to this upstream site.
Kiela
Pawel R
PR
Dept. of Pediatrics, Steele Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724, USA.
Kuscuoglu
Nesrin
N
Midura
Anna J
AJ
Midura-Kiela
Monica T
MT
Larmonier
Claire B
CB
Lipko
Maciej
M
Ghishan
Fayez K
FK
D000107:Acetylation; D000818:Animals; D000930:Antidiarrheals / Q000494:pharmacology*; D020148:Butyric Acid / Q000494:pharmacology*; D018938:Caco-2 Cells; D004247:DNA / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism; D049410:Luciferases, Renilla; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D015854:Up-Regulation
D000930:Antidiarrheals; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C000613385:SLC9A3 protein, human; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D020148:Butyric Acid; D051705:Sp3 Transcription Factor; D004247:DNA; D049410:Luciferases, Renilla