Sp1 and Sp3 mediate NHE2 gene transcription in the intestinal epithelial cells.
Our previous studies have identified a minimal Sp1-driven promoter region (nt -36/+116) directing NHE2 expression in mouse renal epithelial cells. However, this minimal promoter region was not sufficient to support active transcription of NHE2 gene in the intestinal epithelial cells, suggesting the need for additional upstream regulatory elements. In the present study, we used nontransformed rat intestinal epithelial (RIE) cells as a model to identify the minimal promoter region and transcription factors necessary for the basal transcription of rat NHE2 gene in the intestinal epithelial cells. We identified a region within the rat NHE2 gene promoter located within nt -67/-43 upstream of transcription initiation site as indispensable for the promoter function in intestinal epithelial cells. Mutations at nt -56/-51 not only abolished the DNA-protein interaction in this region, but also completely abolished NHE2 gene promoter activity in RIE cells. Supershift assays revealed that Sp1 and Sp3 interact with this promoter region, but, contrary to the minimal promoter indispensable for renal expression of NHE2, both transcription factors expressed individually in Drosophila SL2 cells activated rat NHE2 gene promoter. Moreover, Sp1 was a weaker transactivator and when coexpressed in SL2 cells it reduced Sp3-mediated NHE2 basal promoter activity. Furthermore, DNase I footprinting confirmed that nt -58/-51 is protected by nuclear protein from RIE cells. We conclude that the mechanism of basal control of rat NHE2 gene promoter activity is different in the renal and intestinal epithelium, with Sp3 being the major transcriptional activator of NHE2 gene transcription in the intestinal epithelial cells.
Hua
Ping
P
Department of Pediatrics, Steele Memorial Children's Research Center, 1501 N. Campbell Ave., Tucson, AZ 85724, USA.
Xu
Hua
H
Uno
Jennifer K
JK
Lipko
Maciej A
MA
Dong
Jiali
J
Kiela
Pawel R
PR
Ghishan
Fayez K
FK
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004331:Drosophila melanogaster; D024202:Electrophoretic Mobility Shift Assay; D007413:Intestinal Mucosa / Q000502:physiology*; D011401:Promoter Regions, Genetic; D051381:Rats; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis*; D016329:Sp1 Transcription Factor / Q000502:physiology*; D051705:Sp3 Transcription Factor / Q000502:physiology*
C513110:Slc9a2 protein, rat; D017923:Sodium-Hydrogen Exchangers; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D051705:Sp3 Transcription Factor