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. 2002 Apr 26;277(17):14647-56.
doi: 10.1074/jbc.M111549200. Epub 2002 Feb 15.

In vivo footprinting of the human 11beta-hydroxysteroid dehydrogenase type 2 promoter: evidence for cell-specific regulation by Sp1 and Sp3

Andrea R Nawrocki  1 , Christopher E Goldring, Radina M Kostadinova, Felix J Frey, Brigitte M Frey
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In vivo footprinting of the human 11beta-hydroxysteroid dehydrogenase type 2 promoter: evidence for cell-specific regulation by Sp1 and Sp3

Andrea R Nawrocki et al. J Biol Chem. .
Free article

Abstract

11beta-Hydroxysteroid dehydrogenase type 2 is selectively expressed in aldosterone _target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11beta-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 11beta-hydroxysteroid dehydrogenase type 2.

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