11␤-Hydroxysteroid dehydrogenase enzymes (11␤-HSDSequence alignment and molecular modeling of human 11␤-HSD1 1 and human 11␤-HSD2, using the known three-dimensional structures of human dihydropteridine reductase and Streptomyces hydrogenans 20␤-HSD as templates, indicated that the structures of these members of the short chain dehydrogenase reductase family of proteins are very similar, despite only 18% sequence identity between their entire sequences (1). Although both 11␤-HSD enzymes control the conversion of biologically active glucocorticoids (cortisol in humans and corticosterone in rats and mice) to their inactive 11-keto forms (cortisone and 11-dehydrocorticosterone), there are important functional differences such as cofactor specificity, substrate affinity, or direction of the reaction. The isoform 11␤-HSD1 is expressed in a wide array of tissues, with highest levels in the liver, from where it was purified originally (2). It catalyzes both the oxidation and reduction of glucocorticoids but acts predominantly as an oxidoreductase, thereby increasing the concentration of active glucocorticoids (3-8). Studies on the purified protein demonstrated glycosylation and existence of a disulfide bond, suggesting that the bulk of 11␤-HSD1 is oriented to the ER lumen (9). By converting 11-keto-into 11␤-hydroxyglucocorticoids, 11␤-HSD1 plays an important role in the glucocorticosteroid receptor-mediated anti-inflammatory response of glucocorticoids (10). Mice deficient in 11␤-HSD1 were found to resist hyperglycemia provoked by obesity or stress (11). Other investigators provided evidence that 11␤-HSD1 plays a role in detoxification processes (12) and in the reductive metabolism of xenobiotics (13).The isoform 11␤-HSD2 is expressed at high levels in mineralocorticoid target cells such as the renal collecting duct cells (14 -17). 11␤-HSD2 catalyzes exclusively the dehydrogenation of 11␤-hydroxyglucocorticoids, utilizes NAD ϩ as a cofactor, and has a nanomolar K m for glucocorticoids (14 -18). By inactivating biologically active glucocorticoids before they occupy mineralocorticoid receptors (MR), 11␤-HSD2 confers aldosterone selectivity for the MR (19,20). In the syndrome of apparent mineralocorticoid excess (21-23) or in mice lacking 11␤-HSD2 (24), deficiency of 11␤-HSD2 allows glucocorticoids to bind to the MR in the distal tubule, leading to sodium retention, hypokalemia, and severe hypertension. Reports on the intracellular localization of 11␤-HSD2 are controversial. Whereas 11␤-HSD2 has been reported to be a microsomal enzyme (15,25) with exclusive localization to the ER membrane and the protein facing the cytoplasm (26 -28), evidence for nuclear localization was also presented (29 -34).To understand the differences in the physiological functions of 11␤-HSD1 and 11␤-HSD2, it is of great interest to know the exact topology and intracellular localization of these enzymes. Therefore, we evaluated the role of the N-terminal anchor sequences of 11␤-HSD1 and 11␤-HSD2 on their topology, in-

The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) is selectively expressed in aldosterone target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11β-hydroxyglucocorticoids. Variable activity of 11βHSD2 is relevant for blood pressure control and hypertension. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for the rigorous control of expression of the gene encoding 11βHSD2, HSD11B2. CpG islands covering the promoter and exon 1 of HSD11B2 were found to be densely methylated in tissues and cell lines with low expression but not those with high expression of HSD11B2. Demethylation induced by 5-aza-2′-deoxycytidine and procainamide enhanced the transcription and activity of the 11βHSD2 enzyme in human cells in vitro and in rats in vivo. Methylation of HSD11B2 promoter-luciferase constructs decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including those for Sp1/Sp3, Arnt, and nuclear factor 1 (NF1) diminished their DNA-binding activity. Herein NF1 was identified as a strong HSD11B2 stimulatory factor. The effect of NF1 was dependent on the position of CpGs and the combination of CpGs methylated. A methylated-CpG-binding protein complex 1 transcriptional repression interacted directly with the methylated HSD11B2 promoter. These results indicate a role for DNA methylation in HSD11B2 gene repression and suggest an epigenetic mechanism affecting this gene causally linked with hypertension.

Accelerated vascular calcification is a severe complication of chronic kidney disease contributing to high morbidity and mortality in patients undergoing renal replacement therapy. Sodium thiosulfate is increasingly used for the treatment of soft tissue calcifications in calciphylaxis. Therefore, we determined whether it also prevents development of vascular calcifications in chronic kidney disease. We found that uremic rats treated by thiosulfate had no histological evidence of calcification in the aortic wall whereas almost three-fourths of untreated uremic rats showed aortic calcification. Urinary calcium excretion was elevated and the calcium content of aortic, heart, and renal tissue was significantly reduced in the thiosulfate-treated compared to non-treated animals. Sodium thiosulfate treatment transiently lowered plasma ionized calcium and induced metabolic acidosis. It also lowered bone strength in the treated animals compared to their normal controls. Hence, sodium thiosulfate prevented vascular calcifications in uremic rats, likely by enhancing acid- and/or chelation-induced urinary calcium loss. The negative impact on rat bone integrity necessitates a careful risk-benefit analysis before sodium thiosulfate can be used in individual human patients.

Endogenously released or exogenously administered glucocorticosteroids are relevant hormones for controlling inflammation. Only 11β-hydroxy glucocorticosteroids, but not 11-keto glucocorticosteroids, activate glucocorticoid receptors. Since we found that glomerular mesangial cells (GMC) express 11β-hydroxysteroid dehydrogenase 1 (11β-OHSD1), which interconverts 11-keto glucocorticosteroids into 11β-hydroxy glucocorticosteroids (cortisone/cortisol shuttle), we explored whether 11β-OHSD1 determines the antiinflammatory effect of glucocorticosteroids. GMC exposed to interleukin (IL)-1β or tumor necrosis factor α (TNF-α) release group II phospholipase A2 (PLA2), a key enzyme producing inflammatory mediators. 11β-hydroxy glucocorticosteroids inhibited cytokine-induced transcription and release of PLA2 through a glucocorticoid receptor–dependent mechanism. This inhibition was enhanced by inhibiting 11β-OHSD1. Interestingly, 11-keto glucocorticosteroids decreased cytokine-induced PLA2 release as well, a finding abrogated by inhibiting 11β-OHSD1. Stimulating GMC with IL-1β or TNF-α increased expression and reductase activity of 11β-OHSD1. Similarly, this IL-1β– and TNF-α–induced formation of active 11β-hydroxy glucocorticosteroids from inert 11-keto glucocorticosteroids by the 11β-OHSD1 was shown in the Kiki cell line that expresses the stably transfected bacterial β-galactosidase gene under the control of a glucocorticosteroids response element. Thus, we conclude that 11β-OHSD1 controls access of 11β-hydroxy glucocorticosteroids and 11-keto glucocorticosteroids to glucocorticoid receptors and thus determines the anti-inflammatory effect of glucocorticosteroids. IL-1β and TNF-α upregulate specifically the reductase activity of 11β-OHSD1 and counterbalance by that mechanism their own proinflammatory effect.