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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.

Vascular calcification resulting from hyperphosphatemia is a major determinant of mortality in chronic kidney disease (CKD). Vascular calcification is driven by aldosterone-sensitive osteogenic transformation of vascular smooth muscle cells (VSMCs). We show that even in absence of exogenous aldosterone, silencing and pharmacological inhibition (spironolactone, eplerenone) of the mineralocorticoid receptor (MR) ameliorated phosphate-induced osteo-/chondrogenic transformation of primary human aortic smooth muscle cells (HAoSMCs). High phosphate concentrations up-regulated aldosterone synthase (CYP11B2) expression in HAoSMCs. Silencing and deficiency of CYP11B2 in VSMCs ameliorated phosphate-induced osteogenic reprogramming and calcification. Phosphate treatment was followed by nuclear export of APEX1, a CYP11B2 transcriptional repressor. APEX1 silencing up-regulated CYP11B2 expression and stimulated osteo-/chondrogenic transformation. APEX1 overexpression blunted the phosphate-induced osteo-/chondrogenic transformation and calcification of HAoSMCs. Cyp11b2 expression was higher in aortic tissue of hyperphosphatemic klotho-hypomorphic (kl/kl) mice than in wild-type mice. In adrenalectomized kl/kl mice, spironolactone treatment still significantly ameliorated aortic osteoinductive reprogramming. Our findings suggest that VSMCs express aldosterone synthase, which is up-regulated by phosphate-induced disruption of APEX1-dependent gene suppression. Vascular CYP11B2 may contribute to stimulation of VSMCs osteo-/chondrogenic transformation during hyperphosphatemia.

BackgroundNuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor regulating a plethora of detoxifying enzymes and antioxidant genes involved in drug metabolism and defence against oxidative stress. The glucocorticoid receptor (GR) is a ligand-induced transcription factor involved in the regulation of energy supply for metabolic needs to cope with various stressors. GR activity is controlled by glucocorticoids, which are synthesized in the adrenal glands and regenerated mainly in the liver from inactive cortisone by 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1).Methods and Principal FindingsUsing transfected HEK-293 cells and hepatic H4IIE cells we show that glucocorticoids, activated by 11β-HSD1 and acting through GR, suppress the Nrf2-dependent antioxidant response. The expression of the marker genes NQO1, HMOX1 and GST2A was suppressed upon treatment of 11β-HSD1 expressing cells with cortisone, an effect that was reversed by 11β-HSD1 inhibitors. Furthermore, our results demonstrate that elevated glucocorticoids lowered the ability of cells to detoxify H2O2. Moreover, a comparison of gene expression in male and female rats revealed an opposite sexual dimorphism with an inverse relationship between 11β-HSD1 and Nrf2 target gene expression.ConclusionsThe results demonstrate a suppression of the cellular antioxidant defence capacity by glucocorticoids and suggest that elevated 11β-HSD1 activity may lead to impaired Nrf2-dependent antioxidant response. The gender-specific differences in hepatic expression levels of 11β-HSD1 and Nrf2 target genes and the impact of pharmacological inhibition of 11β-HSD1 on improving cellular capacity to cope with oxidative stress warrants further studies in vivo.

3,4-Methylenedioxymethamphetamine (MDMA, ‘ecstasy') and methylphenidate are widely used psychoactive substances. MDMA primarily enhances serotonergic neurotransmission, and methylphenidate increases dopamine but has no serotonergic effects. Both drugs also increase norepinephrine, resulting in sympathomimetic properties. Here we studied the effects of MDMA and methylphenidate on 24-hour plasma steroid profiles. 16 healthy subjects (8 men, 8 women) were treated with single doses of MDMA (125 mg), methylphenidate (60 mg), MDMA + methylphenidate, and placebo on 4 separate days using a cross-over study design. Cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycorticosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, and testosterone were repeatedly measured up to 24 h using liquid chromatography-tandem mass spectroscopy. MDMA significantly increased the plasma concentrations of cortisol, corticosterone, 11-dehydrocorticosterone, and 11-deoxycorticosterone and also tended to moderately increase aldosterone levels compared with placebo. MDMA also increased the sum of cortisol + cortisone and the cortisol/cortisone ratio, consistent with an increase in glucocorticoid production. MDMA did not alter the levels of cortisone, DHEA, DHEAS, androstenedione, or testosterone. Methylphenidate did not affect any of the steroid concentrations, and it did not change the effects of MDMA on circulating steroids. In summary, the serotonin releaser MDMA has acute effects on circulating steroids. These effects are not observed after stimulation of the dopamine and norepinephrine systems with methylphenidate. The present findings support the view that serotonin rather than dopamine and norepinephrine mediates the acute pharmacologically induced stimulation of the hypothalamic-pituitary-adrenal axis in the absence of other stressors.

Lysergic acid diethylamide (LSD) is a serotonin 5-hydroxytryptamine-2A (5-HT 2A ) receptor agonist that is used recreationally worldwide. Interest in LSD research in humans waned after the 1970s, but the use of LSD in psychiatric research and practice has recently gained increasing attention. LSD produces pronounced acute psychedelic effects, but its influence on plasma steroid levels over time have not yet been characterized in humans. The effects of LSD (200µg) or placebo on plasma steroid levels were investigated in 16 healthy subjects using a randomized, double-blind, placebo-controlled cross-over study design. Plasma concentrationtime profiles were determined for 15 steroids using liquid-chromatography tandem massspectrometry. LSD increased plasma concentrations of the glucocorticoids cortisol, cortisone, corticosterone, and 11-dehydrocorticosterone compared with placebo. The mean maximum concentration of LSD was reached at 1.7h. Mean peak psychedelic effects were reached at 2.4h, with significant alterations in mental state from 0.5h to >10h. Mean maximal concentrations of cortisol and corticosterone were reached at 2.5h and 1.9h, and significant elevations were observed 1.5-6h and 1-3h after drug administration, respectively. LSD also significantly increased plasma concentrations of the androgen dehydroepiandrosterone but not other androgens, progestogens, or mineralocorticoids compared with placebo. A close relationship was found between plasma LSD concentrations and changes in plasma cortisol and corticosterone and the psychotropic response to LSD, and no clockwise hysteresis was observed. In conclusion, LSD produces significant acute effects on circulating steroids, especially glucocorticoids. LSD-induced changes in circulating glucocorticoids were associated with plasma LSD concentrations over time and showed no acute pharmacological tolerance.

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