Molecular cloning and functional analysis of the human Na(+)/H(+) exchanger NHE3 promoter.
Na(+)/H(+) exchanger (NHE) isoforms NHE2 and NHE3, colocalized to the brush border membrane of the epithelial cells, exhibit differences in their pattern of tissue expression and regulation by various molecular signals. To investigate the mechanisms involved in regulation of NHE3 gene expression, the human NHE3 promoter region was cloned and characterized. Primer extension experiments located the transcription start site to a position 116 nucleotides upstream from the translation start codon. The 5'-flanking region lacked a CCAAT box but contained a TATA-like sequence. Nucleotide sequencing of the 5'-flanking region revealed the presence of a number of cis elements including Sp1, AP-2, MZF-1, CdxA, Cdx-2, steroid and nonsteroid hormone receptor half sites, and a phorbol 12-myristate 13-acetate-response element. Transient transfection experiments using C2/bbe cell line defined a maximal promoter activity in -95/+5 region. The regulatory response elements clustered within this region include a potential transcription factor IID (TF IID), a CACCC, two Sp1, and two AP-2 motifs. Deletion of a fragment containing the AP-2 and Sp1 motifs resulted in a drastic decrease in promoter activity. In gel mobility shift assays, an oligonucleotide spanning from -78 to -56 bp bound a recombinant AP-2, and the corresponding binding activity in nuclear extracts was supershifted with anti-AP2alpha antibody. Our studies suggest that the NHE3 expression is regulated by a combination of cis elements and their cognate transcription factors that include the AP-2 and Sp1 family members.
Malakooti
Jaleh
J
Department of Medicine, Section of Digestive and Liver Diseases, University of Illinois at Chicago and Chicago Veterans Affairs Westside Division, Chicago, Illinois 60612, USA. malakoot@uic.edu
Memark
V C
VC
Dudeja
Pradeep K
PK
Ramaswamy
Krishnamurthy
K
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004847:Epithelial Cells; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006801:Humans; D007422:Intestines; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D050656:Transcription Factor AP-2; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000494:pharmacology; D019473:Transcription Factors, TFII / Q000494:pharmacology; D014162:Transfection
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C000613385:SLC9A3 protein, human; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D008156:Luciferases; D013755:Tetradecanoylphorbol Acetate