Role of the c/ebp proteins in hepatic and vascular-smooth-muscle
transcription of the human nhe-1 gene.
Kolyada, Alexey Y., Conrado A. Johns, and Nicolaos E. Madias.
Department of Medicine, Tufts University School of Medicine, and
Division of Nephrology, New England Medical Center, Boston,
Massachusetts 02111
APStracts 2:0220C, 1995.
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 (NHE-1) is important for gene transcription in cells
of hepatic origin (HepG2) 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 HepG2 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, co-transfection experiments of NHE-1 0.25-kb promoter/CAT
construct and C/EBP expression vectors showed that C/EBP[alpha] and
C/EBP[delta] are transactivators of the NHE-1 proximal promoter in
HepG2 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 NHE
-1 gene.
Received 21 December 1994; accepted in final form 26 May 1995.
APS Manuscript Number C730-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 8 June 1995.