Genetic, biochemical, and clinical studies of patients with A328V or R213C mutations in 11betaHSD2 causing apparent mineralocorticoid excess. (17/232)

Apparent mineralocorticoid excess is a recessively inherited hypertensive syndrome caused by mutations in the 11beta-hydroxysteroid dehydrogenase type 2 gene, which encodes the enzyme normally responsible for converting cortisol to inactive cortisone. Failure to convert cortisol to cortisone in mineralocorticoid-sensitive tissues permits cortisol to bind to and activate mineralocorticoid receptors, causing hypervolemic hypertension. Typically, these patients have increased ratios of cortisol to cortisone and of 5alpha- to 5beta-cortisol metabolites in serum and urine. We have studied 3 patients in 2 families with severe, apparent mineralocorticoid excess and other family members in terms of their genetic, biochemical, and clinical parameters, as well as normal controls. Two brothers were homozygous for an A328V mutation and the third patient was homozygous for an R213C mutation in the 11beta-hydroxysteroid dehydrogenase type 2 gene; both mutations caused a marked reduction in the activity of the encoded enzymes in transfection assays. The steroid profiles of the 7 heterozygotes and 2 other family members studied were completely normal. The results of a novel assay used to distinguish 5alpha- and 5beta-tetrahydrometabolites suggest that 5beta-reductase activity is reduced or inhibited in apparent mineralocorticoid excess. In 1 patient undergoing renal dialysis for chronic renal insufficiency, direct control of salt and water balance completely corrected the hypertension, emphasizing the importance of mineralocorticoid action in this syndrome.  (+info)

Inhibition of IMCD 11 beta-hydroxysteroid dehydrogenase type 2 by low pH and acute acid loading. (18/232)

Mineralocorticoid receptors in the inner medullary collecting duct (IMCD) are protected from glucocorticoid binding by an enzyme, 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2). To study the role of 11 beta-HSD2 in acid-base homeostasis, 11 beta-HSD2 activity was measured in rat IMCD-enriched cell suspensions. Homogenates of cell suspensions were incubated in buffers ranging in pH from 6.00 to 8.15 in the presence of 1 microCi of 3H-corticosterone (CS) and 400 microM NAD+. Enzyme activity was expressed as the amount of 3H-CS converted to 3H-11-dehydrocorticosterone (DHCS). IMCD 11 beta-HSD2 activity at pH 6.5 was 49% of activity at pH 7.5; 22.5 versus 11.0 fmol/microgram of protein per h. Experiments also were performed on intact cell suspensions at pH 7.5 and 6.5. There was a 42% inhibition in the IMCD cell suspension conversion rate of 3H-CS to 3H-11-DHCS at pH 6.5; 13.1 versus 7.6 fmol/microgram per h (P < 0.005). In cell suspensions at pH 7.5, 1-day acid loading caused a 26% inhibition in conversion rate, 13.2 versus 9.9 fmol/microgram per h (P < 0.05), when compared with controls. These results suggest that during acute metabolic acidosis, IMCD 11 beta-HSD2 is inhibited and may allow access to the mineralocorticoid receptors by glucocorticoids.  (+info)

The N-terminal anchor sequences of 11beta-hydroxysteroid dehydrogenases determine their orientation in the endoplasmic reticulum membrane. (19/232)

11beta-Hydroxysteroid dehydrogenase enzymes (11beta- HSD) regulate the ratio of active endogenous glucocorticoids to their inactive keto-metabolites, thereby controlling the access of glucocorticoids to their cognate receptors. In this study, the topology and intracellular localization of 11beta-HSD1 and 11beta-HSD2 have been analyzed by immunohistochemistry and protease protection assays of in vitro transcription/translation products. 11beta-HSD constructs, tagged with the FLAG epitope, were transiently expressed in HEK-293 cells. The enzymatic characteristics of tagged and native enzymes were indistinguishable. Fluorescence microscopy demonstrated the localization of both 11beta-HSD1 and 11beta-HSD2 exclusively to the endoplasmic reticulum (ER) membrane. To examine the orientation of tagged 11beta-HSD enzymes within the ER membrane, we stained selectively permeabilized HEK-293 cells with anti-FLAG antibody. Immunohistochemistry revealed that the N terminus of 11beta-HSD1 is cytoplasmic, and the catalytic domain containing the C terminus is protruding into the ER lumen. In contrast, the N terminus of 11beta-HSD2 is lumenal, and the catalytic domain is facing the cytoplasm. Chimeric proteins where the N-terminal anchor sequences of 11beta-HSD1 and 11beta-HSD2 were exchanged adopted inverted orientation in the ER membrane. However, both chimeric proteins were not catalytically active. Furthermore, mutation of a tyrosine motif to alanine in the transmembrane segment of 11beta-HSD1 significantly reduced V(max). The subcellular localization of 11beta-HSD1 was not affected by mutations of the tyrosine motif or of a di-lysine motif in the N terminus. However, residue Lys(5), but not Lys(6), turned out to be critical for the topology of 11beta-HSD1. Mutation of Lys(5) to Ser inverted the orientation of 11beta-HSD1 in the ER membrane without loss of catalytic activity. Our results emphasize the importance of the N-terminal transmembrane segments of 11beta-HSD enzymes for their proper function and demonstrate that they are sufficient to determine their orientation in the ER membrane.  (+info)

Glycyrrhetinic acid-induced apoptosis in thymocytes: impact of 11beta-hydroxysteroid dehydrogenase inhibition. (20/232)

It has been proposed that glycyrrhetinic acid (GA) enhances endogenous glucocorticoid (GC) action by suppressing the metabolism of the steroid. We show here that marked involution of the thymus occurred within 24 h of a single intraperitoneal administration of GA in mice. Thymocytes from mice treated with GA exhibited DNA cleavage and mitochondrial transmembrane potential disruption, as demonstrated with agarose gel electrophoresis and flow cytometric analysis. Immunocytochemical staining revealed that CD4(+)CD8(+) double positive cells markedly decreased after GA treatment. In contrast to GA in vivo, GA in vitro did not induce apoptosis of cultured thymocytes. These findings suggest that the apoptosis-inducing effect of GA on thymocytes is due to its indirect action. Because GA has been known to inhibit 11beta-hydroxysteroid dehydrogenase (11beta-HSD), we measured the enzyme activity in major organs and endogenous corticosterone concentration after GA treatment. The results showed a significant decrease of 11beta-HSD activity (P < 0.0001) and an increase in serum corticosterone concentration (P < 0.005). We concluded that the inhibition of hepatic 11beta-HSD activity by GA has a serious effect on GC metabolism, which results in a significant elevation of systemic GC levels. Apoptosis of thymocytes occurred as a consequence of the elevation in the level of endogenous corticosterone.  (+info)

Mutants of 11beta-hydroxysteroid dehydrogenase (11-HSD2) with partial activity: improved correlations between genotype and biochemical phenotype in apparent mineralocorticoid excess. (21/232)

Mutations in the kidney isozyme of human 11-hydroxysteroid dehydrogenase (11-HSD2) cause apparent mineralocorticoid excess, an autosomal recessive form of familial hypertension. We studied 4 patients with AME, identifying 4 novel and 3 previously reported mutations in the HSD11B2 (HSD11K) gene. Point mutations causing amino acid substitutions were introduced into a pCMV5/11HSD2 expression construct and expressed in mammalian CHOP cells. Mutations L179R and R208H abolished activity in whole cells. Mutants S180F, A237V, and A328V had 19%, 72%, and 25%, respectively, of the activity of the wild-type enzyme in whole cells when cortisol was used as the substrate and 80%, 140%, and 55%, respectively, of wild-type activity when corticosterone was used as the substrate. However, these mutant proteins were only 0.6% to 5.7% as active as the wild-type enzyme in cell lysates, suggesting that these mutations alter stability of the enzyme. In regression analyses of all AME patients with published genotypes, several biochemical and clinical parameters were highly correlated with mutant enzymatic activity, demonstrated in whole cells, when cortisol was used as the substrate. These included the ratio of urinary cortisone to cortisol metabolites (R(2)=0.648, P<0.0001), age at presentation (R(2)=0.614, P<0.0001), and birth weight (R(2)=0.576, P=0.0004). Approximately 5% conversion of cortisol to cortisone is predicted in subjects with mutations that completely inactivate HSD11B2, suggesting that a low level of enzymatic activity is mediated by another enzyme, possibly 11-HSD1.  (+info)

11beta-hydroxysteroid dehydrogenase and corticosteroid action in lyon hypertensive rats. (22/232)

Adrenocorticosteroid activity in Lyon hypertensive (LH) and low blood pressure (LL) rat strains differ in several respects. Abnormal activity of 11beta-hydroxysteroid dehydrogenase enzymes (11beta-HSD1 and 11beta-HSD2), which interconvert corticosterone and inactive 11-dehydrocorticosterone, might contribute to the LH phenotype by regulating corticosteroid hormone access to receptors. 11beta-HSD2 (expressed in kidney but not liver) prevents endogenous glucocorticoids from binding to mineralocorticoid receptors. 11beta-HSD1 (expressed in liver and kidney) favors active glucocorticoid formation from 11-dehydrocorticosterone. 11beta-HSD properties in LH and LL have been compared by several approaches: (1) 11betaHSD activities have been measured in vitro as corticosterone dehydrogenation and in vivo as interconversion of injected cortisol and cortisone; (2) the effects of cortisol and cortisone on urine electrolytes and volume have been measured; and (3) 11beta-HSD mRNA expression has been measured by in situ hybridization. 11beta-HSD2 enzyme activities in LH and LL rats were similar and urinary cortisone:cortisol ratios were not different after cortisol injection. Cortisol caused a natriuresis and kaliuresis in both strains, with a slightly reduced response in LH rats. Renal 11beta-HSD2 mRNA expression was slightly lower in LH rats. 11beta-HSD1 was less active in LH than LL rats: enzyme activities were lower in tissue extracts; urinary cortisone:cortisol was lower in LL rats after cortisone injections; cortisone increased urine volume in LL but not LH rats; and mRNA levels tended to be lower in LH tissues. We conclude that 11beta-HSD1 is impaired in LH rats. The LH phenotype of heavier adrenals, raised corticosterone, and reduced thymus weight is similar to that described for 11beta-HSD1 knockout mice.  (+info)

Regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene expression by LH and interleukin-1beta in cultured rat granulosa cells. (23/232)

Granulosa cells from preovulatory follicles show increased expression of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) at the time of ovulation. As ovulation may be an inflammatory process, this may be a mechanism of local enhancement of the activity of anti-inflammatory glucocorticoids. In this study, we examined direct effects of LH, the proinflammatory cytokine, interleukin-1beta (IL-1beta), and pharmacological activators of protein kinase A (PKA) (forskolin and dibutyryl (db) cAMP) and PKC (LH-releasing hormone and phorbol 12-myristate 13-acetate (PMA)) signalling on the expression of 11betaHSD1 mRNA in vitro. Granulosa cells from immature female rat ovaries were cultured (pretreatment) in serum-free medium 199 containing recombinant human (rh) FSH (1 ng/ml) for 48 h to induce responsiveness to LH. Cell monolayers were then washed and cultured (test treatment) for a further 12 h in the presence of rhLH (0-100 ng/ml), IL-1beta (0-50 ng/ml), or both. Total RNA was extracted from granulosa cell monolayers and taken for quantitative ribonuclease protection analysis of 11betaHSD1 mRNA. The low level of 11betaHSD1 mRNA detectable in unstimulated (control) cultures was increased approximately twofold by the 48-h pretreatment with rhFSH. Subsequent exposure to rhLH (1-100 ng/ml) for a further 12 h dose-dependently increased 11betaHSD1 mRNA expression by an additional two- to threefold. Forskolin (10 microM), db-cAMP (2 mM), LH-releasing hormone (LHRH; 1 microM) and PMA (200 nM) were also stimulatory. IL-1beta (0.05-50 ng/ml) stimulated 11betaHSD1 mRNA expression in a dose-related manner, both in the absence and in the presence of rhLH (3 ng/ml). The interaction between IL-1beta (5 ng/ml) and rhLH (3 ng/ml) was additive. Co-treatment with a 50-fold excess of IL-1 receptor antagonist fully reversed the action of IL-1beta. We conclude that 11betaHSD1 mRNA expression in functionally mature granulosa cells is directly stimulated by gonadotrophins and IL-1beta in vitro, potentially involving post-receptor signalling via PKA- and PKC-mediated pathways. Thus both LH and IL-1beta may serve physiological roles in the upregulation of 11betaHSD1 gene expression by granulosa cells in ovulatory follicles.  (+info)

Regulation of 11beta-hydroxysteroid dehydrogenase isoforms and glucocorticoid receptor gene expression in the rat uterus. (24/232)

Glucocorticoids are known to have diverse effects on the uterus, generally believed to be mediated by the glucocorticoid receptor (GR). To date, two isoforms of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been identified, namely 11betaHSD1 and 11betaHSD2, which interconvert active and inactive glucocorticoids and regulate local levels of hormones available to the GR in target tissues. The aim of the present study was to examine the uterine expression of 11betaHSD and GR mRNA. The interplay of these parameters is probably an important factor in determining actions of glucocorticoids on the uterus. Using Northern analysis we investigated the uterine expression of 11betaHSD1, 11betaHSD2 and GR mRNA in relation to serum levels of sex steroid hormones and uterine progesterone receptor mRNA expression in an animal model. Immature female rats were treated with 10 IU pregnant mare serum gonadotrophin (PMSG) followed by 10 IU human chorionic gonadotrophin (hCG) 48 h afterwards, and then killed at 0, 3, 6, 9, 12 and 24 h and 5 days after the hCG injection. Expression of both 11betaHSD1 and 11betaHSD2 mRNA in total uterine RNA was found to be up-regulated by more than 50% at 48 h after PMSG injection when oestradiol levels were also high. Following hCG treatment the expression of 11betaHSD1 and 11betaHSD2 further increased to reach maximal levels at 24 and 12 h respectively. GR mRNA expression was down-regulated by more than 50% by PMSG but gradually recovered after hCG injection. The results show that mRNA expression of 11betaHSD1, 11betaHSD2 and GR in the uterus is developmentally regulated, suggesting that these key determinants of glucocorticoid action may play an important role in uterine function.  (+info)