Type 2 11beta-hydroxysteroid dehydrogenase activity in human ovarian cancer

In the ovary cortisol-cortisone inter-conversion is catalyzed by the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD). Its role in carcinomas of human ovary is unknown. The majority of ovarian cancers are derived from ovarian surface epithelium and the inflammation caused by successive ovulation seems to a play a role in the development of cancer. Cortisol is known to act as anti-inflammatory agent and its metabolism by type 1 and type 11beta-HSD may control the inflammatory action by cortisol in ovary. We undertook this study to investigate type 2 11beta-HSD activity which functions exclusively oxidative direction, in normal ovarian tissue compared to ovarian epithelial cancer. Ovarian tissue was obtained from patients undergoing hysterectomy for both benign and malignant disease. Tissue was placed immediately on dry ice and subsequently transferred to a freezer where they were maintained at -70 degrees C. NAD dependent 11beta-HSD activity was then determined in this tissue. T-test was performed to determine statistical significance. Mean type 2 enzyme activity was 0.87 +/- 1.65 pmol/min g tissue in normal ovarian tissue versus a mean enzyme activity of 2.96 +/- 1.37 pmol/mim g tissue in from cancer specimens. This difference was statistically significant with a p-value of 0.03. Type 2 1beta-HSD activity in ovarian cancer specimens was significantly higher than enzyme activity measured in normal post-menopausal ovarian tissue. Decreased cortisol levels due type 2 1beta-HSD activity may play a role neoplastic transformation as well as tumor proliferation in ovarian cancer by eliminating anti-inflammatory action of cortisol.     

Calcium inhibits human placental 11beta-hydroxysteroid dehydrogenase type 2 activity

The effect of Ca2+ on the conversion of cortisol to its inert metabolite cortisone, the reaction catalyzed by the microsomal enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), was investigated in human placental microsomes. Placental microsomal 11beta-HSD2 activity, as determined by the rate of conversion of cortisol to cortisone, was inhibited up to 50% by increasing free Ca2+ concentrations from 22 to 268 nM. The Ca2+-induced inhibition was reversible since chelation of endogenous Ca2+ with EGTA increased 11beta-HSD2 activity up to 200%. Ca2+ decreased the maximal velocity (Vmax) of the 11beta-HSD2 catalyzed conversion of cortisol to cortisone without altering the Km of 11beta-HSD2 for cortisol, indicating that Ca2+ modulates the catalytic efficiency rather than the substrate binding of 11beta-HSD2. Moreover, the Ca2+-induced inhibition does not appear to involve altered cofactor (NAD+) binding since the inhibition of microsomal 11beta-HSD2 activity by a sub-maximal concentration of free Ca2+ was not overcome by increasing the concentration of NAD+. These findings in the microsomes were then extended to an intact cell system, JEG-3 cells, an established model for human placental trophoblasts. In these cells, an increase in cytosolic free Ca2+ concentration ([Ca2+]i) elicited by a known physiological stimulus, PGF(2alpha), was accompanied by a 40% decrease in the level of 11beta-HSD2 activity. Furthermore, the PGF(2alpha)-induced inhibition of 11beta-HSD2 activity was abrogated when increases in [Ca2+]i were blocked with the intracellular Ca2+ chelator, BAPTA. Collectively, these results demonstrate for the first time that Ca2+ inhibits human placental 11beta-HSD2 activity by a post-translational mechanism not involving substrate or cofactor binding.     

Inhibition of 17 beta-hydroxysteroid dehydrogenase activity in human endometrium by adrenal androgens

The effect of dehydroepiandrosterone sulphate (DHA-S) and its metabolites dehydroepiandrosterone (DHA) and 5-androstene-3 beta, 17 beta-diol (ADIOL) on the activity of 17 beta-hydroxysteroid dehydrogenase in human endometrial tissue was investigated by an isotope ratio technique. The apparent KM for oestradiol was 1.59 X 10(-6) M. All three androgens inhibited the metabolism of oestradiol and the apparent Ki values were: ADIOL, 2.05 X 10(-6) M; DHA-S and DHA, 1.59 X 10(-6) M. However, ADIOL acted by direct competition with oestradiol for the active enzyme site whereas inhibition by DHA and its sulphate was non-competitive. DHA-S and DHA were more potent inhibitors of oestradiol metabolism than was ADIOL. These results support the hypothesis that adrenal androgens could be involved in the development of endometrial hyperplasia and adenocarcinoma. Inhibition of oestradiol metabolism could increase the concentration of oestradiol in endometrial tissue and if unopposed by progesterone, e.g. after the menopause or in subjects with ovulatory defects, could stimulate abnormal endometrial growth.     

11 beta-hydroxysteroid dehydrogenase activity in the mammary gland

Levels of 11 beta-hydroxysteroid dehydrogenase activity in mammary gland homogenates from pregnant and lactating Sprague-Dawley rats were determined by incubation with [3H]corticosterone under standard conditions, followed by thin-layer chromatography of incubated media. Enzyme activity was high in virgin and pregnant rats, but fell soon after parturition, suggesting a possible role for this enzyme in the co-ordinate regulation of glucocorticoid effects on milk protein synthesis.     

Glycyrrhizic acid suppresses type 2 11 beta-hydroxysteroid dehydrogenase expression in vivo

Licorice-derivatives such as glycyrrhizic acid (GA) competitively inhibit 11β-hydroxysteroid dehydrogenase(11β-HSD) type 2 (11-HSD2) enzymatic activity, and chronic clinical use often results in pseudoaldosteronism. Since the effect of GA on 11-HSD2 expression remains unknown, we undertook in vivo and in vitro studies. Male Wistar rats were given 30, 60 or 120 mg/kg of GA twice a day for 2 weeks. Plasma corticosterone was decreased in those given the 120 mg dose, while urinary corticosterone excretion was increased in those given the 30 and 60 mg doses but decreased in those given 120 mg GA. NAD⁺-dependent dehydrogenase activity in kidney microsomal fraction was decreased in animals receiving doses of 60 and 120 mg GA. The 11-HSD2 protein and mRNA levels were decreased in those given 120 mg GA. In contrast, in vitro studies using mouse kidney M1 cells revealed that 24 h treatment with glycyrrhetinic acid did not affect the 11-HSD2 mRNA expression levels. Thus, in addition to its role as a competitive inhibitor of 11-HSD2, the chronic high dose of GA suppresses mRNA and protein expression of 11-HSD2 possibly via indirect mechanisms. These effects may explain the prolonged symptoms after cessation of GA administration in some pseudoaldosteronism patients.     

Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1

The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.     

Novel non-steroidal inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates glucocorticoid action at the pre-receptor stage by converting cortisone to cortisol. 11β-HSD1 is selectively expressed in many tissues including the liver and adipose tissue where metabolic events are important. Metabolic syndrome relates to a number of metabolic abnormalities and currently has a prevalence of >20% in adult Americans. 11β-HSD1 inhibitors are being investigated by many major pharmaceutical companies for type 2 diabetes and other abnormalities associated with metabolic syndrome. In this area of intense interest a number of structural types of 11β-HSD1 inhibitor have been identified. It is important to have an array of structural types as the physicochemical properties of the compounds will determine tissue distribution, HPA effects, and ultimately clinical utility. Here we report the discovery and synthesis of three structurally different series of novel 11β-HSD1 inhibitors that inhibit human 11β-HSD1 in the low micromolar range. Docking studies with 1–3 into the crystal structure of human 11β-HSD1 reveal how the molecules may interact with the enzyme and cofactor and give further scope for structure based drug design in the optimisation of these series.     

Epithelial and adipose cells isolated from mammary glands of pregnant and lactating rats differ in 11 beta-hydroxysteroid dehydrogenase activity

Both adipose and epithelial cells isolated from the mammary glands of pregnant and lactating rats show 11 beta-hydroxysteroid dehydrogenase (11-HSD) activity, as measured by conversion of corticosterone to 11-dehydrocorticosterone. Activity in adipose cells from pregnant rats is 3-fold higher than in lactating rats. Epithelial cells from pregnant rats show one-twentieth of the activity of adipose cells, and activity is lower still in epithelial cells from lactating rats. Explants incubated for 48 h extensively metabolized corticosterone to 11-dehydrocorticosterone, and to a much lesser extent to a second unknown metabolite which is found in tissue extracts but not conditioned medium. Mammary gland 11-HSD may thus constitute one of the physiological mechanisms preventing premature milk production in response to glucocorticoids.     

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.     

Mild adrenal 3 beta-hydroxysteroid dehydrogenase deficiency with hyperaldosteronism

The patient was admitted to our hospital at 19 and again at 22-yr of age for hirsutism and hypertension. Her baseline and ACTH-stimulated plasma 17-hydroxy pregnenolone, dehydroepiandrosterone and dehydroepiandrosterone sulfate were increased whereas plasma 17-hydroxy progesterone and androstenedione were normal and responded poorly to ACTH. Plasma deoxycorticosterone, corticosterone and cortisol baseline levels were normal, and they responded normally to ACTH. The plasma aldosterone concentration (PAC) was always high and responded well to ACTH, angiotensin III and furosemide-upright stimulation. However, plasma renin activity (PRA) was normal or slightly high, and responded normally to furosemide-upright stimulation and fluorohydrocortisone suppression. Dexamethasone (2 mg/day) for 1-2 weeks suppressed the androgens, cortisol and corticosterone levels. PRA and PAC were suppressed temporally, but PRA returned to normal and PAC to be a high level after 2 weeks of dexamethasone administration. Blood pressure was also reduced temporally but returned to a high level after 2 weeks of dexamethasone. These results indicate that primary aldosteronism and dexamethasone-suppressible hyperaldosteronism were not likely to be present, and unknown aldosterone stimulating factors which potentiated the action of endogenous angiotensin II or ACTH might be responsible for the hyperaldosteronism in this patient. We conclude that this patient had a mild and non-salt losing 3 beta-HSD deficiency in the zona reticularis with normal fasciculata and high glomerulosa function.     

Expression of human kidney 11beta-hydroxysteroid dehydrogenase (11-HSD2) in bacteria

The kidney isozyme of 11beta-hydroxysteroid dehydrogenase (11-HSD2) protects the mineralocorticoid receptor from spurious activation by glucocorticoids. To explore structure-function relationships, human 11-HSD2 cDNA was subcloned into the bacterial expression vector, pET25b. E. coli transformed with wild-type cDNA produced active enzyme that retained biochemical characteristics of the native protein. The addition of 6 histidine residues to the C-terminus of the wild-type enzyme (11-HSD2/His) increased activity 2-fold. Whereas wild-type activity was almost completely sedimented following 100,000g centrifugation, 10-30% of total activity of 11-HSD2/His remained in the supernatant. The 11-HSD2 isozyme normally contains three N-terminal hydrophobic domains. Mutant 11-HSD2/His possessing a single hydrophobic domain retained partial activity, but elimination of all domains inactivated the enzyme. Thus, the N-terminal hydrophobic domains are essential for complete activity of 11-HSD2 but association with an intact cell membrane is not.     

11beta-hydroxysteroid dehydrogenase functions reversibly as an oxidoreductase in the rat hippocampus in vivo

The localization in the brain and metabolism of 3H-labeled corticosterone (B) and 11-dehydrocorticosterone (A) of high specific radioactivity was determined after stereotaxic injection into the hippocampus of anesthetized rats. [3H]B was cleared very rapidly with, on average, only about 7% being recovered after 5 min and 0.5% after 30 min. Most of this 3H-radioactivity was localized in the area surrounding the site of injection with little diffusion to adjacent areas. These findings make it possible to compare the short term metabolism of [3H]A and [3H]B in different lobes of the hippocampus in the same animal and establish their local equilibrium point in vivo. Under these conditions, about 5% conversion of each steroid to the other was observed in contrast to the situation in cultured hippocampal cells where 11beta-hydroxysteroid dehydrogenase (11-HSD) has been shown by others to act primarily as a reductase catalyzing the conversion of A to B. This method can also be used to study the effect of inhibitors such as 11alpha-hydroxyprogesterone, applied locally in the brain, on the metabolism of corticosteroids. The rate of conversion [3H]B or [3H]A to their dihydro- and tetrahydro-derivatives capable of modulating the GABAa receptor in the hippocampus was much lower than their interconversion. Thus, factors which influence the direction of the 11-HSD catalyzed reaction are important in regulating not only salt appetite and blood pressure but also the levels of neuroactive metabolites of corticosterone.     

Effect of cellular differentiation on 11beta-hydroxysteroid dehydrogenase activity in the intestine.

11beta-Hydroxysteroid dehydrogenase (11betaHSD) converts endogenous glucocorticoids to their biologically inactive 11-dehydro derivatives and is therefore able to determine, at least in part, the biological action of glucocorticoids. Type 1 11betaHSD has both oxidase and reductase activities interconverting corticosterone and 11-dehydrocorticosterone, whereas type 2 11betaHSD has only oxidase activity converting corticosterone to 11-dehydrocorticosterone. Since 11betaHSD expression is regulated during development and by hormones in a tissue-specific manner and since glucocorticoids play an important role in postnatal intestinal maturation, we investigated the role of corticosteroids and cytodifferentiation in the regulation of intestinal 11betaHSD. Using rat intestinal organ cultures and epithelial cell lines derived from rat small intestine (IEC-6, IEC-18) and from human colon adenocarcinoma (Caco-2, HT-29), we analyzed the effect of corticosteroids and cytodifferentiation on 11betaHSD. Screening of the clonal cell lines showed that Caco-2 cells expressed by far the greatest 11betaHSD2 oxidase activity, lower activity was observed in HT-29 cells, and lowest activity was seen in IEC cells. Treatment with dexamethasone (50 nM) increased the activity of 11betaHSD2 in IEC-6 cells (+59%) and HT-29 cells (+31%), whereas aldosterone (50 nM) stimulated 11betaHSD2 in IEC-6 cells only (+31%). Caco-2 cells and IEC-18 cells did not respond to corticosteroids. Growth of IEC-6 cells on Matrigel, treatment of HT-29 cells with butyrate, and postconfluency of Caco-2 cells increased not only the markers of cytodifferentiation, such as alkaline phosphatase and sucrase, but also the activity of 11betaHSD2 in all of these cell lines (IEC-6, +96%; HT-29, +139%; Caco-2, +95%). Addition of corticosteroids to these more differentiated cell cultures did not enhance 11betaHSD2 activity. In intestinal organ cultures of suckling rat small intestine, dexamethasone and aldosterone stimulated 11betaHSD by more than 300%. We conclude that corticosteroids markedly and differentially regulate intestinal 11betaHSD2 and that cytodifferentiation of intestinal epithelial cells is associated with upregulation of 11betaHSD2 activity that is independent of corticosteroids.