Lyubomir G. Nashev

Hexose-6-phosphate dehydrogenase determines the reaction direction of 11β-hydroxysteroid dehydrogenase type 1 as an oxoreductase

The impact of hexose‐6‐phosphate dehydrogenase (H6PDH) on 11β‐hydroxysteroid dehydrogenase (11β‐HSD) type 1 activity was investigated upon coexpression in HEK‐293 cells. Confocal microscopy analysis indicated colocalisation of both enzymes at the lumenal side of the endoplasmic reticulum (ER) membrane. Functional analysis in intact cells revealed fivefold stimulation of 11β‐HSD1 oxoreductase activity and sixfold decrease of dehydrogenase activity upon coexpression with H6PDH, without changing kinetic parameters in cell lysates. Thus, H6PDH directly determines the reaction direction of 11β‐HSD1 in intact cells as an oxoreductase without changing intrinsic catalytic properties of 11β‐HSD1 by regenerating NADPH in the ER‐lumen.

Organotins Disrupt the 11β-Hydroxysteroid Dehydrogenase Type 2-Dependent Local Inactivation of Glucocorticoids

Organotins, important environmental pollutants widely used in agricultural and industrial applications, accumulate in the food chain and induce imposex in several marine species as well as neurotoxic and immunotoxic effects in higher animals. Reduced birth weight and thymus involution, observed upon exposure to organotins, can also be caused by excessive glucocorticoid levels. We now demonstrate that organotins efficiently inhibit 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), converting active 11β-hydroxyglucocorticoids into inactive 11-ketoglucocorticoids, but not 11β-HSD1, which catalyzes the reverse reaction. Di- and tributyltin as well as di- and triphenyltin inhibited recombinant and endogenous 11β-HSD2 in lysates and intact cells with IC50 values between 500 nM and 3 μM. Dithiothreitol protected 11β-HSD2 from organotin-dependent inhibition, indicating that organotins act by binding to one or more cysteines. Mutational analysis and 3-D structural modeling revealed several important interactions of cysteines in 11β-HSD2. Cys90, Cys228, and Cys264 were essential for enzymatic stability and catalytic activity, suggesting that disruption of such interactions by organotins leads to inhibition of 11β-HSD2. Enhanced glucocorticoid concentrations due to disruption of 11β-HSD2 function may contribute to the observed organotin-dependent toxicity in some glucocorticoid-sensitive tissues such as thymus and placenta.

Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells

BackgroundMicroglia, the resident macrophage-like cells in the brain, regulate innate immune responses in the CNS to protect neurons. However, excessive activation of microglia contributes to neurodegenerative diseases. Corticosteroids are potent modulators of inflammation and mediate their effects by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Here, the coordinated activities of GR and MR on the modulation of the nuclear factor-κB (NF-κB) pathway in murine BV-2 microglial cells were studied.MethodsBV-2 cells were treated with different corticosteroids in the presence or absence of MR and GR antagonists. The impact of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) was determined by incubating cells with 11-dehydrocorticosterone, with or without selective inhibitors. Expression of interleukin-6 (IL-6), tumor necrosis factor receptor 2 (TNFR2), and 11β-HSD1 mRNA was analyzed by RT-PCR and IL-6 protein expression by ELISA. NF-κB activation and translocation upon treatment with various corticosteroids were visualized by western blotting, immunofluorescence microscopy, and translocation assays.ResultsGR and MR differentially regulate NF-κB activation and neuroinflammatory parameters in BV-2 cells. By converting inactive 11-dehydrocorticosterone to active corticosterone, 11β-HSD1 essentially modulates the coordinated action of GR and MR. Biphasic effects were observed for 11-dehydrocorticosterone and corticosterone, with an MR-dependent potentiation of IL-6 and tumor necrosis factor-α (TNF-α) expression and NF-κB activation at low/moderate concentrations and a GR-dependent suppression at high concentrations. The respective effects were confirmed using the MR ligand aldosterone and the antagonist spironolactone as well as the GR ligand dexamethasone and the antagonist RU-486. NF-κB activation could be blocked by spironolactone and the inhibitor of NF-κB translocation Cay-10512. Moreover, an increased expression of TNFR2 was observed upon treatment with 11-dehydrocorticosterone and aldosterone, which was reversed by 11β-HSD1 inhibitors and/or spironolactone and Cay-10512.ConclusionsA tightly coordinated GR and MR activity regulates the NF-κB pathway and the control of inflammatory mediators in microglia cells. The balance of GR and MR activity is locally modulated by the action of 11β-HSD1, which is upregulated by pro-inflammatory mediators and may represent an important feedback mechanism involved in resolution of inflammation.

Why is 11β-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen?: Physiological relevance of the membrane topology of 11β-HSD1

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is essential for the local activation of glucocorticoid receptors (GR). Unlike unliganded cytoplasmic GR, 11beta-HSD1 is an endoplasmic reticulum (ER)-membrane protein with lumenal orientation. Cortisone might gain direct access to 11beta-HSD1 by free diffusion across membranes, indirectly via intracellular binding proteins or, alternatively, by insertion into membranes. Membranous cortisol, formed by 11beta-HSD1 at the ER-lumenal side, might then activate cytoplasmic GR or bind to ER-lumenal secretory proteins. Compartmentalization of 11beta-HSD1 is important for its regulation by hexose-6-phosphate dehydrogenase (H6PDH), which regenerates cofactor NADPH in the ER lumen and stimulates oxoreductase activity. ER-lumenal orientation of 11beta-HSD1 is also essential for the metabolism of the alternative substrate 7-ketocholesterol (7KC), a major cholesterol oxidation product found in atherosclerotic plaques and taken up from processed cholesterol-rich food. An 11beta-HSD1 mutant adopting cytoplasmic orientation efficiently catalyzed the oxoreduction of cortisone but not 7KC, indicating access to cortisone from both sides of the ER-membrane but to 7KC only from the lumenal side. These aspects may be relevant for understanding the physiological role of 11beta-HSD1 and for developing therapeutic interventions to control glucocorticoid reactivation.

11β-Hydroxysteroid dehydrogenase 1 reductase activity is dependent on a high ratio of NADPH/NADP+ and is stimulated by extracellular glucose

To assess the impact of the NADPH/NADP(+) ratio and the influence of extracellular glucose on 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity, we applied microsomal preparations and intact HEK-293 cells expressing 11beta-HSD1 in the presence or absence of hexose-6-phosphate dehydrogenase (H6PDH). A NADPH/NADP(+) ratio of ten or higher was required for efficient microsomal 11beta-HSD1 reductase activity. Measurements in intact cells suggested that the ER-luminal NADPH concentration is highly sensitive to fluctuating extracellular glucose levels. Lowering glucose in the culture medium dose-dependently decreased 11beta-HSD1 reductase activity and diminished the cortisol/cortisone ratio measured after 24h of incubation. Coexpression with H6PDH potentiated 11beta-HSD1 reductase activity at high glucose. This effect was significantly decreased at low glucose, with concomitantly increased 11beta-HSD1 dehydrogenase activity. In contrast, 11beta-HSD1 reductase activity in H4IIE liver cells and in 3T3-L1 adipocytes was less sensitive to changes in the medium. 11beta-HSD1 dehydrogenase activity was observed in H4IIE cells only at subphysiological glucose levels, indicating a highly efficient supply of substrate for H6PDH and NADPH generation in the ER-lumen. Our results suggest significant cell type-specific differences in ER-luminal NADPH generation that might allow a fine-tuned regulation of glucocorticoid action.

Direct protein-protein interaction of 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen

Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11beta-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17beta-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11beta-HSD1 but not with the related enzymes 11beta-HSD2 and 17beta-HSD2, suggesting a specific interaction between H6PDH and 11beta-HSD1. The use of the 11beta-HSD1/11beta-HSD2 chimera indicates that the N-terminal 39 residues of 11beta-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11beta-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11beta-HSD1. The protein-protein interaction and the involvement of the N-terminus of 11beta-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11beta-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11beta-HSD1 and H6PDH involves a direct physical interaction of the two proteins.

The UV-filter benzophenone-1 inhibits 17β-hydroxysteroid dehydrogenase type 3: Virtual screening as a strategy to identify potential endocrine disrupting chemicals

The prevalence of male reproductive disorders and testicular cancer is steadily increasing. Because the exposure to chemicals disrupting natural hormone action has been associated with these diseases, it is important to identify endocrine disrupting chemicals (EDCs) and their targets of action. Here, a 3D-structural database that can be applied for virtual screening approaches to facilitate the identification of EDCs was constructed. The database was screened using pharmacophores of 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), which catalyzes the last step of testosterone synthesis in testicular Leydig cells and plays an essential role during male sexual development. Among other chemicals, benzophenone (BP) UV-filters were predicted as potential 17beta-HSD3 inhibitors. Biological analyses revealed (2,4-dihydroxyphenyl)-phenylmethanone (also known as benzophenone-1, BP-1) as an inhibitor of human 17beta-HSD3 (IC(50) 1.05microM). BP-1 also efficiently blocked conversion of androstenedione to testosterone by mouse and rat 17beta-HSD3 in whole-organ enzyme assays. Moreover, BP-1 antagonized the testosterone-dependent activation of androgen receptors (IC(50) 5.7microM), suggesting synergistic anti-androgenic effects of BP-1 by preventing testosterone formation and blocking receptor activation. In addition, analyses of several commonly used UV-filters on estrogen- and androgen-metabolizing 17beta-HSD enzymes revealed 3-benzylidene camphor (3-BC) and 4-methylbenzylidene camphor (4-MBC) as low micromolar 17beta-HSD2 inhibitors. In conclusion, screening of virtual chemical structure libraries can facilitate the identification of compounds interfering with hormone action. The potential disruption of 17beta-HSD enzyme function by the UV-filters BP-1, 3-BC and 4-MBC requires further investigation and should be considered for safety assessment of these chemicals.

The glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 has broad substrate specificity: Physiological and toxicological considerations

The primary function of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is to catalyze the conversion of inactive to active glucocorticoid hormones and to modulate local glucocorticoid-dependent gene expression. Thereby 11beta-HSD1 plays a key role in the regulation of metabolic functions and in the adaptation of the organism to energy requiring situations. Importantly, elevated 11beta-HSD1 activity has been associated with metabolic disorders, and recent investigations with rodent models of obesity and type 2 diabetes provided evidence for beneficial effects of 11beta-HSD1 inhibitors, making this enzyme a promising therapeutic target. Several earlier and recent studies, mainly performed in vitro, revealed a relatively broad substrate spectrum of 11beta-HSD1 and suggested that this enzyme has additional functions in the metabolism of some neurosteroids (7-oxy- and 11-oxyandrogens and -progestins) and 7-oxysterols, as well as in the detoxification of various xenobiotics that contain reactive carbonyl groups. While there are many studies on the effect of inhibitors on cortisone reduction and circulating glucocorticoid levels and on the transcriptional regulation of 11beta-HSD1 in obesity and diabetes, only few address the so-called alternative functions of this enzyme. We review recent progress on the biochemical characterization of 11beta-HSD1, with a focus on cofactor and substrate specificity and on possible alternative functions of this enzyme.

Discovery of Nonsteroidal 17β-Hydroxysteroid Dehydrogenase 1 Inhibitors by Pharmacophore-Based Screening of Virtual Compound Libraries

17Beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) plays a pivotal role in the local synthesis of the most potent estrogen estradiol. Its expression is a prognostic marker for the outcome of patients with breast cancer and inhibition of 17beta-HSD1 is currently under consideration for breast cancer prevention and treatment. We aimed to identify nonsteroidal 17beta-HSD1 inhibitor scaffolds by virtual screening with pharmacophore models built from crystal structures containing steroidal compounds. The most promising model was validated by comparing predicted and experimentally determined inhibitory activities of several flavonoids. Subsequently, a virtual library of nonsteroidal compounds was screened against the 3D pharmacophore. Analysis of 14 selected compounds yielded four that inhibited the activity of human 17beta-HSD1 (IC 50 below 50 microM). Specificity assessment of identified 17beta-HSD1 inhibitors emphasized the importance of including related short-chain dehydrogenase/reductase (SDR) members to analyze off-target effects. Compound 29 displayed at least 10-fold selectivity over the related SDR enzymes tested.

Hexose-6-phosphate Dehydrogenase Modulates 11β-Hydroxysteroid Dehydrogenase Type 1-Dependent Metabolism of 7-keto- and 7β-hydroxy-neurosteroids

Background The role of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in the regulation of energy metabolism and immune system by locally reactivating glucocorticoids has been extensively studied. Experiments determining initial rates of enzyme activity revealed that 11β-HSD1 can catalyze both the reductase and the dehydrogenase reaction in cell lysates, whereas it predominantly catalyzes the reduction of cortisone to cortisol in intact cells that also express hexose-6-phosphate dehydrogenase (H6PDH), which provides cofactor NADPH. Besides its role in glucocorticoid metabolism, there is evidence that 11β-HSD1 is involved in the metabolism of 7-keto- and 7-hydroxy-steroids; however the impact of H6PDH on this alternative function of 11β-HSD1 has not been assessed. Methodology We investigated the 11β-HSD1-dependent metabolism of the neurosteroids 7-keto-, 7α-hydroxy- and 7β-hydroxy-dehydroepiandrosterone (DHEA) and 7-keto- and 7β-hydroxy-pregnenolone, respectively, in the absence or presence of H6PDH in intact cells. 3D-structural modeling was applied to study the binding of ligands in 11β-HSD1. Principal Findings We demonstrated that 11β-HSD1 functions in a reversible way and efficiently catalyzed the interconversion of these 7-keto- and 7-hydroxy-neurosteroids in intact cells. In the presence of H6PDH, 11β-HSD1 predominantly converted 7-keto-DHEA and 7-ketopregnenolone into their corresponding 7β-hydroxy metabolites, indicating a role for H6PDH and 11β-HSD1 in the local generation of 7β-hydroxy-neurosteroids. 3D-structural modeling offered an explanation for the preferred formation of 7β-hydroxy-neurosteroids. Conclusions Our results from experiments determining the steady state concentrations of glucocorticoids or 7-oxygenated neurosteroids suggested that the equilibrium between cortisone and cortisol and between 7-keto- and 7-hydroxy-neurosteroids is regulated by 11β-HSD1 and greatly depends on the coexpression with H6PDH. Thus, the impact of H6PDH on 11β-HSD1 activity has to be considered for understanding both glucocorticoid and neurosteroid action in different tissues.