Role of C/EBP proteins in hepatic and vascular smooth muscle transcription of human NHE1 gene.
We have recently shown that regulatory element D (nucleotides -239 to -215) of the 0.25-kb promoter of the human growth factor-activatable Na+/H+ exchanger (NHE1) is important for gene transcription in cells of hepatic origin (Hep G2) and vascular smooth muscle origin (VSM A7r5). This element contains a sequence (nucleotides -230 to -222) with complete homology to the C/EBP binding site. We now demonstrate that nucleotide substitution mutations disrupting this C/EBP site suppressed transcription in Hep G2 cells, VSM A7r5 cells, and Sprague-Dawley VSM cells in primary culture. These mutations abolished the binding of rat liver nuclear activities as well as transcription factors C/EBP alpha, C/EBP beta, and C/EBP delta expressed in COS-1 cell lysates to element D. Anti-C/EBP antibodies supershifted DNA-protein complexes formed between hepatic nuclear activities or C/EBP proteins expressed in COS-1 cell lysates and regulatory element D. Finally, cotransfection experiments of NHE1 0.25-kb promoter-chloramphenicol acetyltransferase (CAT) construct and C/EBP expression vectors showed that C/EBP alpha and C/EBP delta are transactivators of the NHE1 proximal promoter in Hep G2 and VSM A7r5 cells. These results indicate that members of the C/EBP family of transcription factors are involved in the regulation of hepatic and vascular smooth muscle transcription of the human NHE1 gene.
Kolyada
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AY
Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts, USA.
Johns
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CA
Madias
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NE
D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D005809:Genes, Regulator; D006801:Humans; D008099:Liver / Q000502:physiology*; D008297:Male; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics*; D014158:Transcription, Genetic / Q000502:physiology*; D015533:Transcriptional Activation
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D015335:Molecular Probes; D009687:Nuclear Proteins; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1