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J. Biol. Chem., Vol. 277, Issue 17, 14647-14656, April 26, 2002
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From the Division of Nephrology and Hypertension, Departments of
Internal Medicine and Clinical Research, Freiburgstrasse 15, University
Hospital of Berne, CH-3010 Berne, Switzerland
11
In Vivo Footprinting of the Human
11
-Hydroxysteroid Dehydrogenase Type 2 Promoter
EVIDENCE FOR CELL-SPECIFIC REGULATION BY Sp1 AND Sp3*
,
-Hydroxysteroid dehydrogenase type 2 is
selectively expressed in aldosterone target tissues, where it confers
aldosterone selectivity for the mineralocorticoid receptor by
inactivating 11-hydroxyglucocorticoids with a high affinity for the
mineralocorticoid receptor. The present investigation aimed to
elucidate the mechanisms accounting for the rigorous control of the
HSD11B2 gene in humans. Using dimethyl sulfate
in vivo footprinting via ligation-mediated PCR, we
identified potentially important regions for HSD11B2
regulation in human cell lines: two GC-rich regions in the first exon
(I and II) and two upstream elements (III and IV). The footprints suggest a correlation between the extent of in vivo protein
occupancy at three of these regions (I, II, and III) and the rate of
HSD11B2 transcription in cells with high (SW620),
intermediate (HCD, MCF-7, and HK-2), or low HSD11B2
mRNA levels (SUT). Moreover, gel shift assays with nuclear extracts
from these cell lines revealed that decreased HSD11B2
expression is related to a decreased binding activity with
oligonucleotides containing the putative regulatory elements. Antibody
supershifts identified the majority of the components of the binding
complexes as the transcription factors Sp1 and Sp3. Finally,
transient transfections with various deletion mutant reporters define
positive regulatory elements that might account for basal and selective
expression of 11-hydroxysteroid dehydrogenase type 2.
*
This work was supported by Swiss National Foundation for
Scientific Research Grant 31.061505.00.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.
To whom correspondence should be addressed. Tel.: 41-31-632-9476;
Fax: 41-31-632-9444; E-mail: andrea.nawrocki@dkf2.unibe.ch.
§
Present address: Department of Pharmacology, University of
Liverpool, 70 Pembroke Place, Liverpool L69 3BX, United Kingdom.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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