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Am J Physiol Cell Physiol 269: C1408-C1416, 1995;
0363-6143/95 $5.00
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AJP - Cell Physiology, Vol 269, Issue 6 C1408-C1416, Copyright © 1995 by American Physiological Society

ARTICLES

Role of C/EBP proteins in hepatic and vascular smooth muscle transcription of human NHE1 gene

A. Y. Kolyada, C. A. Johns and N. E. Madias
Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts, USA.

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.


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