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J. Biol. Chem., Vol. 276, Issue 49, 45530-45538, December 7, 2001
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From the Mouse As4.1 cells, obtained after
transgene-targeted oncogenesis to induce neoplasia in renal renin
expressing cells, express high levels of renin mRNA from their
endogenous Ren-1c gene. We have previously
identified a 242-base pair enhancer (coordinates
Critical Roles of a Cyclic AMP Responsive Element and an E-box in
Regulation of Mouse Renin Gene Expression*
§,¶,
,,**,,,, and§§
Department of Molecular and Cellular
Biology, Roswell Park Cancer Institute, Buffalo, New York 14263 and the
Departments of Internal Medicine and Physiology & Biophysics,
University of Iowa College of Medicine, Iowa City, Iowa 52242
2866 to 2625
relative to the CAP site) upstream of the mouse
Ren-1c gene. This enhancer, in combination with
the proximal promoter (117 to +6), activates transcription nearly 2 orders of magnitude in an orientation independent fashion. To further
delimit sequences necessary for transcriptional activation, renin
promoter-luciferase reporter gene constructs containing selected
regions of the Ren-1c enhancer were analyzed
after transfection into As4.1 cells. These results demonstrate that
several regions are required for full enhancer activity. Sequences from
2699 to 2672, which are critical for the enhancer activity, contain
a cyclic AMP responsive element (CRE) and an E-box. Electrophoretic
mobility shift assays demonstrated that transcription factors
CREB/CREM and USF1/USF2 in As4.1 cell nuclear extracts bind to
oligonucleotides containing the Ren-1c CRE and
E-box, respectively. These two elements are capable of synergistically
activating transcription from the Ren-1c
promoter. Moreover, mutation of either the CRE or E-box results in
almost complete loss of enhancer activity, suggesting the critical roles these two elements play in regulating mouse
Ren-1c gene expression. Although the
Ren-1c gene contains a CRE, its
expression is not induced by cAMP in As4.1 cells. This appears to
reflect constitutive activation of protein kinase A in As4.1 cells
since treatment with the protein kinase A inhibitor, H-89, caused a
significant reduction in Ren-1c gene expression
and this reduction is mediated through the CRE at 2699 to
2688.
*
This work was supported in part by National Institute of
Health Grants HL48459 and CA16056 (to K. W. G.) and HL48058,
HL61446, and HL55006 (to C. D. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Southern Australia Dental Service, Somerton
Park Dental Complex, Adelaide, Australia.
§§
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Biology, Roswell Park Cancer Institute, Elm and Carlton
Sts., Buffalo, NY 14263-0001. Tel.: 716-845-4572; Fax: 716-845-8169;
E-mail: gross@acsu.buffalo.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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