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Regulation of 11 beta-hydroxysteroid dehydrogenase type 2 in health and disease states.

English title Regulation of 11 beta-hydroxysteroid dehydrogenase type 2 in health and disease states.
Applicant Frey Felix Julius
Number 102153
Funding scheme Project funding
Research institution Respiratory Medicine Department Universitätsklinik Inselspital
Institution of higher education University of Berne - BE
Main discipline Clinical Endocrinology
Start/End 01.10.2003 - 30.09.2008
Approved amount 771'465.00
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All Disciplines (2)

Discipline
Clinical Endocrinology
Clinical Pathophysiology

Keywords (6)

Sodium; hypertension; cortisol; 11 beta-hydroxysteroid; dehydrogenase; salt-sensitivity

Lay Summary (English)

Lead
Lay summary
The 11b-hydroxysteroid dehydrogenase type 2 enzyme (11b-HSD2) is a cytoplasmic NADH-dependent short chain alcohol dehydrogenase catalyzing the conversion of active C-11-hydroxylated corticosteroids (cortisol,corticosterone) into their inactive C-11-keto-metabolites (cortisone, 11-dehydrocorticosterone). This enzyme exhibits a remarkable cell-specific constitutive expression in mineralocorticoid target tissues, such as epithelial cells from the colon or the cortical collecting tubule, where its primary function is to protect the nonselective mineralocorticoid receptor (MR) from promiscuous activation by 11b-hydroxyglucocorticoids.Mutations in the exons of the 11b-HSD2 gene cause the apparent mineralocorticoid excess syndrome, a rare form of salt-sensitive hypertension. An intermediary phenotype of this syndrome is an increased urinary ratio of cortisol/cortisone and of their corresponding tetrahydrometabolites, reflecting an apparent low activity of 11b-HSD2. A slightly reduced activity of the 11b-HSD2 enzyme is associated with salt-sensitive increase in blood pressure and polymorphic markers in the gene in normal volunteers and patients with essential hypertension.Furthermore, associations between microsatellites or variants of the11b-HSD2 gene and the occurrence of end stage renal failure (ESRD) have been observed in african americans and caucasians. All these findings suggest that a variable activity of 11b-HSD2 is relevant for blood pressure control. The frequency of mutated exons in the 11b-HSD2 gene is extremely low. Thus other mechanisms accounting for the variable 11b-HSD2 enzyme activity have to be considered.
We hypothesize now that interindividual differences in the activity of11b-HSD2 are attributable to changes in the non-coding region of the gene.The transcriptional activity of 11b-HSD2 appears to be highly dependent on the interaction between defined DNA regions and DNA-binding proteins in the promoter region, as recently shown by us by using in vivo footprinting, a technology which allows a view on a gene-regulatory region during transcription in the intact chromosomal environment. Therefore, the main focus of this grant proposal is the promoter of the 11b-HSD2 gene.The specific aims are as follows:
(1) To define the DNA region and the transcription factors accounting for the TNF-a-induced transcriptional inhibition of 11b-HSD2, by performing(i) expression studies using promoter constructs containing different lengths of the 5’-flanking region of the 11b-HSD2 gene, (ii) DNA mobility shift assays (EMSA) and (iii) in vivo footprinting. Furthermore, we propose to establish whether the TNF-a induced down-regulation of 11b-HSD2 is present in vivo, investigations to be performed in transgenic mice overexpressing TNF-a. In vitro TNF-a-mediated inhibition of 11b-HSD2 by a MEK/ERK pathway was previously shown in our lab. TNF-a is chosen as a model endobiotic given its steadily increasing relevance as a cardiovascular/metabolic risk factor.
(2) To find variants of the promoter sequence, and if present to define their biological relevance. For that purpose (i) single strand conformational polymorphism of PCR products from the upstream region (550bp) will be performed in about 1000 subjects with hypertension, ESRD, diabetes with normal renal function or healthy volunteers, with or without salt-sensitivity; (ii) variants will be sequenced, and functional relevance assessed by EMSA and expression studies; (iii) urinary cortisol/cortisone metabolites will be determined by GC-MS, as a measure of 11b-HSD2 activity.
(3) To establish whether the reduced transcription of renal 11b-HSD2 in offspring of low protein diet pregnancies is functionally relevant, whereby function will be assessed in rats by determining (i) the pattern of urinary corticosterone metabolites with GC-MS and (ii) salt-sensitivity of blood pressure with telemetry. If functional changes are observed, we hypothesize, that a partial silencing of the 11b-HSD2 promoter by methylation accounts for this epigenetic effect.
(4) To proof that CpG methylation of the promoter occurs and is functionally relevant. Therefore we shall (i) demonstrate that the 5 ’-untranslated region comprises CpG islands and that the degree of methylation affects the expression 11b-HSD2 in vitro, and, (ii) provide evidence that the methylation status is relevant for the apparent expression of 11b-HSD2 and salt-sensitivity in vivo by administering two different inhibitors of DNA methyltransferase, 5-aza-2’deoxycytidine and procainamide to rats and humans.
Rationale: The basic hypothesis of the studies proposed is that changes at the level of the promoter of 11b-HSD2 confers glucocorticoid-mediated enhanced mineralocorticoid effects. The selection of the models to be studied is guided by clinical situations generally considered to be of practical relevance, for which we have preliminary results, patients available for investigations and technical competence from previous studies.
Direct link to Lay Summary Last update: 21.02.2013

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Associated projects

Number Title Start Funding scheme
138670 Regulation of 11 betahydroxysteroid dehydrogenase type 2 in health and disease states 01.10.2011 Project funding
122135 11 beta-hydrosysteroid dehydrogenases in renal allograft donors and patients with nonalcoholic fatty liver disease or nonalcoholic steatohepatitis 01.10.2008 Project funding
61505 Regulation of 11 beta-hydroxysteroid dehydrogenases in health anddisease states. 01.10.2000 Project funding

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