Tumor necrosis factor alpha and phorbol 12-myristate-13-acetate down-regulate human 11beta-hydroxysteroid dehydrogenase type 2 through p50/p50 NF-kappaB homodimers and Egr-1.

The 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) regulates access of 11beta-hydroxyglucocorticoids to the mineralocorticoid receptor by reducing the hydroxyl group of these steroids at position 11. Previous cell culture studies revealed that tumor necrosis factor-alpha (TNF-alpha) down-regulates 11beta-HSD2 activity. Here, we demonstrate that transgenic mice overexpressing TNF-alpha have decreased mRNA abundance and activity of 11beta-HSD2 in kidney tissue, indicating that this effect may occur also in vivo. The analysis of the transcriptional regulation of 11beta-HSD2 by TNF-alpha and phorbol 12-myristate-13-acetate (PMA) with in vivo genomic footprinting in human colon SW620 cells revealed stimulus-dependent protein-DNA interactions in three promoter regions, kappaB1, Sp1/Egr-1I, and Sp1/Egr-1II. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated the relevance of NF-kappaB binding to kappaB1 and of Egr-1 binding to Sp1/Egr-1 sites for the PMA and TNF-alpha effect. We observed a temporal switch of binding to kappaB1 site from active p65/p50 heterodimers to inactive p50/p50 homodimers. TNF-alpha or PMA treatment for 24 h resulted in accumulation of p50 and decrease of p65 nuclear proteins. Overexpression of p50 inhibited HSD11B2 promoter activity and overexpression of Egr-1 inhibited transactivation of the HSD11B2 promoter by p65/p50. In conclusion, TNF-alpha and PMA down-regulate expression and activity of 11beta-HSD2 most likely by a coordinate binding of p50/p50 and Egr-1 to the HSD11B2 promoter.

Hypoxia causes down-regulation of 11 beta-hydroxysteroid dehydrogenase type 2 by induction of Egr-1.

Hypoxia causes several renal tubular dysfunctions, including abnormal handling of potassium and sodium and increased blood pressure. Therefore, we investigated the impact of hypoxia on 11beta-hydroxysteroid dehydrogenase (11beta-HSD2) enzyme, a crucial prereceptor gatekeeper for renal glucocorticosteroid-mediated mineralocorticoid action. The effect of hypoxia was assessed in vitro by incubating LLC-PK1 cells with antimycin A, an inhibitor of mitochondrial oxidative phosphorylation. Antimycin A induced a dose- and time-dependent reduction of 11beta-HSD2 activity. The early growth response gene, Egr-1, a gene known to be stimulated by hypoxia was investigated because of a potential Egr-1 binding site in the promoter region of 11beta-HSD2. Antimycin A induced Egr-1 protein and Egr-1-regulated luciferase gene expression. This induction was prevented with the MAPKK inhibitor PD 98059. Overexpression of Egr-1 reduced endogenous 11beta-HSD2 activity in LLC-PK1 cells, indicating that MAPK ERK is involved in the regulation of 11beta-HSD2 in vitro. In vivo experiments in rats revealed that Egr-1 protein increases, whereas 11beta-HSD2 mRNA decreases, in kidney tissue after unilateral renal ischemia and in humans the renal activity of 11beta-HSD2 as assessed by the urinary ratio of (tetrahydrocortisol+5alpha-tetrahydrocortisol)/tetrahydrocortisone declined when volunteers were exposed to hypoxemia at high altitude up to 7000 m. Thus, hypoxia decreases 11beta-HSD2 transcription and activity by inducing Egr-1 in vivo and in vitro. This mechanism might account for enhanced renal sodium retention and hypertension associated with hypoxic conditions.

Tumor necrosis factor-alpha upregulates 11beta-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-beta in HepG2 cells.

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11beta-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-alpha increases 11beta-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11beta-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-alpha, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-alpha-induced transcription of the 11beta-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-alpha acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-alpha in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPbeta-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPalpha, but also C/EBPbeta, in basal and only of C/EBPbeta in the TNF-alpha-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPbeta on the proximal HSD11B1 promoter upon TNF-alpha treatment. In conclusion, C/EBPalpha and C/EBPbeta control basal transcription, and TNF-alpha upregulates 11beta-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPbeta to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-alpha-mediated 11beta-HSD1 regulation, and that TNF-alpha stimulates enzyme activity in vivo.

Dehydroepiandrosterone inhibits the amplification of glucocorticoid action in adipose tissue.

Dehydroepiandrosterone (DHEA) exerts beneficial effects on blood glucose levels and insulin sensitivity in obese rodents and humans, resembling the effects of peroxisome proliferator-activated receptor-gamma (PPARgamma) ligands and opposing those of glucocorticoids; however, the underlying mechanisms remain unclear. Glucocorticoids are reactivated locally by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which is currently considered as a promising target for the treatment of obesity and diabetes. Using differentiated 3T3-L1 adipocytes, we show that DHEA causes downregulation of 11beta-HSD1 and dose-dependent reduction of its oxoreductase activity. The effects of DHEA were comparable with those of the PPARgamma agonist rosiglitazone but not additive. Furthermore, DHEA reduced the expression of hexose-6-phosphate dehydrogenase, which stimulates the oxoreductase activity of 11beta-HSD1. These findings were confirmed in white adipose tissue and in liver from DHEA-treated C57BL/6J mice. Analysis of the transcription factors involved in the DHEA-dependent regulation of 11beta-HSD1 expression revealed a switch in CCAAT/enhancer-binding protein (C/EBP) expression. C/EBPalpha, a potent activator of 11beta-HSD1 gene transcription, was downregulated in 3T3-L1 adipocytes and in liver and adipose tissue of DHEA-treated mice, whereas C/EBPbeta and C/EBPdelta, attenuating the effect of C/EBPalpha, were unchanged or elevated. Our results further suggest a protective effect of DHEA on adipose tissue by upregulating PPARalpha and downregulating leptin, thereby contributing to the reduced expression of 11beta-HSD1. In summary, we provide evidence that some of the anti-diabetic effects of DHEA may be caused through inhibition of the local amplification of glucocorticoids by 11beta-HSD1 in adipose tissue.

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

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.