Zinc finger transcription factor Egr-1 is involved in stimulation of NHE2 gene expression by phorbol 12-myristate 13-acetate.
The apical membrane Na(+)/H(+) exchanger isoforms NHE2 and NHE3 are involved in transepithelial Na(+) absorption in the intestine. However, they exhibit differences in their pattern of tissue expression and regulation of their activity by various molecular signals. To study the mechanisms involved in the transcriptional regulation of these genes, we characterized cis-acting elements within the human NHE2 promoter that regulate NHE2 promoter expression in C2BBe1 cells. A small DNA region (-85/+249) was involved in the regulation of basal transcriptional activity of the NHE2 promoter as determined by transient transfection assays. RT-PCR analysis showed that NHE2 mRNA was upregulated in response to phorbol 12-myristate 13-acetate (PMA). Results from actinomycin D-treated cells indicated that the regulation of the NHE2 gene by PMA occurs in part at the transcriptional level. Furthermore, PMA treatment led to a 100% increase in promoter activity through elements located on the -415/+249 DNA fragment. A PMA-induced nuclear factor that bound to the NHE2 promoter was identified as the transcription factor Egr-1. We identified two PMA response elements in the -415/+1 promoter region that bind to Sp1 and Sp3 in untreated nuclear extracts and to Egr-1 in PMA-treated nuclear extracts. In cotransfection experiments, Egr-1 was able to transactivate the NHE2 promoter. Our data indicate that Egr-1 may play a key role in regulated expression of the human NHE2 gene.
Malakooti
Jaleh
J
Dept. of Medicine, Section of Digestive and Liver Diseases, University of Illinois, Chicago, IL 60612, USA. malakoot@uic.edu
Sandoval
Ricardo
R
Memark
Vanchad C
VC
Dudeja
Pradeep K
PK
Ramaswamy
Krishnamurthy
K
D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics*; D051766:Early Growth Response Protein 1; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D007202:Indicators and Reagents; D010957:Plasmids; D012313:RNA; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics; D051705:Sp3 Transcription Factor; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D007202:Indicators and Reagents; C034477:RNA primers; D012333:RNA, Messenger; C081517:SLC9A2 protein, human; C497121:SP3 protein, human; D017923:Sodium-Hydrogen Exchangers; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D012313:RNA; D013755:Tetradecanoylphorbol Acetate