An in Vitro triple screen model for human mineralocorticoid receptor activity.

The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron and other tissues. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. We developed an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussia luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45 nM and 0.30 nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6 nM and 56.0 nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20 nM (CV1-MRLuc) to ∼2000 nM, while the EC50 for aldosterone remained unchanged. The addition of 18ß-glycyrrhetinic acid (18ß-GA), a HSD11B2 inhibitor, restored the potency of cortisol back to ∼70 nM in CV1-hMRLuc-HSD11B2 cells. Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts.

Prenatal psychological distress and 11ß-HSD2 gene expression in human placentas: Systematic review and meta-analysis.

BACKGROUND: The placenta acts as a buffer to regulate the degree of fetal exposure to maternal cortisol through the 11-Beta Hydroxysteroid Dehydrogenase isoenzyme type 2 (11-ß HSD2) enzyme. We conducted a systematic review and meta-analysis to assess the effect of prenatal psychological distress (PPD) on placental 11-ß HSD2 gene expression and explore the related mechanistic pathways involved in fetal neurodevelopment. METHODS: We searched PubMed, Embase, Scopus, APA PsycInfo®, and ProQuest Dissertations for observational studies assessing the association between PPD and 11-ß HSD2 expression in human placentas. Adjusted regression coefficients (ß) and corresponding 95% confidence intervals (CIs) were pooled based on three contextual PPD exposure groups: prenatal depression, anxiety symptoms, and perceived stress. RESULTS: Of 3159 retrieved records, sixteen longitudinal studies involving 1869 participants across seven countries were included. Overall, exposure to PPD disorders showed weak negative associations with the placental 11-ß HSD2 gene expression as follows: prenatal depression (ß -0.01, 95% CI 0.05-0.02, I2=0%), anxiety symptoms (ß -0.02, 95% CI 0.06-0.01, I2=0%), and perceived stress (ß -0.01 95% CI 0.06-0.04, I2=62.8%). Third-trimester PPD exposure was more frequently associated with lower placental 11-ß HSD2 levels. PPD and placental 11-ß HSD2 were associated with changes in cortisol reactivity and the development of adverse health outcomes in mothers and children. Female-offspring were more vulnerable to PPD exposures. CONCLUSION: The study presents evidence of a modest role of prenatal psychological distress in regulating placental 11-ß HSD2 gene expression. Future prospective cohorts utilizing larger sample sizes or advanced statistical methods to enhance the detection of small effect sizes should be planned. Additionally, controlling for key predictors such as the mother's ethnicity, trimester of PPD exposure, mode of delivery, and infant sex is crucial for valid exploration of PPD effects on fetal programming.

Thiram, an inhibitor of 11ß-hydroxysteroid dehydrogenase type 2, enhances the inhibitory effects of hydrocortisone in the treatment of osteosarcoma through Wnt/ß-catenin pathway.

BACKGROUND: The anti-osteosarcoma effects of hydrocortisone and thiram, an inhibitor of type 2 11ß-hydroxysteroid dehydrogenase (11HSD2), have not been reported. The purpose of this study was to investigate the effects of hydrocortisone alone or the combination of hydrocortisone with thiram on osteosarcoma and the molecular mechanism, and determine whether they can be as new therapeutic agents for osteosarcoma. METHODS: Normal bone cells and osteosarcoma cells were treated with hydrocortisone or thiram alone or in combination. The cell proliferation, migration, cell cycle and apoptosis were detected by using CCK8 assay, wound healing assay, and flow cytometry, respectively. An osteosarcoma mouse model was established. The effect of drugs on osteosarcoma in vivo was assessed by measuring tumor volume. Transcriptome sequencing, bioinformatics analysis, RT-qPCR, Western blotting (WB), enzymelinked immunosorbent assay (ELISA) and siRNA transfection were performed to determine the molecular mechanisms. RESULTS: Hydrocortisone inhibited the proliferation and migration, and induced apoptosis and cell cycle arrest of osteosarcoma cells in vitro. Hydrocortisone also reduced the volume of osteosarcoma in mice in vivo. Mechanistically, hydrocortisone decreased the levels of Wnt/ß-catenin pathway-associated proteins, and induced the expression of glucocorticoid receptor α (GCR), CCAAT enhancer-binding protein ß (C/EBP-beta) and 11HSD2, resulting in a hydrocortisone resistance loop. Thiram inhibited the activity of the 11HSD2 enzyme, the combination of thiram and hydrocortisone further enhanced the inhibition of osteosarcoma through Wnt/ß-catenin pathway. CONCLUSIONS: Hydrocortisone inhibits osteosarcoma through the Wnt/ß-catenin pathway. Thiram inhibits 11HSD2 enzyme activity, reducing hydrocortisone inactivation and promoting the effect of hydrocortisone through the same pathway.

Regulation of distal tubule sodium transport: mechanisms and roles in homeostasis and pathophysiology.

Regulated Na+ transport in the distal nephron is of fundamental importance to fluid and electrolyte homeostasis. Further upstream, Na+ is the principal driver of secondary active transport of numerous organic and inorganic solutes. In the distal nephron, Na+ continues to play a central role in controlling the body levels and concentrations of a more select group of ions, including K+, Ca++, Mg++, Cl-, and HCO3-, as well as water. Also, of paramount importance are transport mechanisms aimed at controlling the total level of Na+ itself in the body, as well as its concentrations in intracellular and extracellular compartments. Over the last several decades, the transporters involved in moving Na+ in the distal nephron, and directly or indirectly coupling its movement to that of other ions have been identified, and their interrelationships brought into focus. Just as importantly, the signaling systems and their components-kinases, ubiquitin ligases, phosphatases, transcription factors, and others-have also been identified and many of their actions elucidated. This review will touch on selected aspects of ion transport regulation, and its impact on fluid and electrolyte homeostasis. A particular focus will be on emerging evidence for site-specific regulation of the epithelial sodium channel (ENaC) and its role in both Na+ and K+ homeostasis. In this context, the critical regulatory roles of aldosterone, the mineralocorticoid receptor (MR), and the kinases SGK1 and mTORC2 will be highlighted. This includes a discussion of the newly established concept that local K+ concentrations are involved in the reciprocal regulation of Na+-Cl- cotransporter (NCC) and ENaC activity to adjust renal K+ secretion to dietary intake.

Sufentanil alleviates pre-eclampsia via silencing microRNA-24-3p to target 11ß-Hydroxysteroid dehydrogenase type 2.

Pre-eclampsia (PE) is a prevalent pregnancy disease characterized by insufficient trophoblast cell migration (HTR8/SVneo). Consequently, accelerating trophoblast cell proliferation might ameliorate PE. This study assessed the effects and molecular mechanisms of Sufentanil (SUF) on HTR8/SVneo cells proliferation. HTR8/SVneo cells and PE clinical samples were used. Peripheral blood was collected from PE patients' samples, and microRNA (miR)-24-3p and 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) was analyzed in the blood and cells. HTR8/SVneo cells were treated with varying SUF concentrations or transfected with miR-24-3p mimics/inhibitors, or HSD11B2 elevation vector. CCK-8, colony formation, transwell, and flow cytometry assays were then carried out. Association of miR-24 - 3p with HSD11B2 was investigated. PE animal model was constructed using Wistar rats to verify SUF's role on PE in vivo. According to the results, SUF boosted HTR8/SVneo cell proliferation, and inhibited miR-24-3p to accelerate HSD11B2. MiR-24-3p was increased in PE, while HSD11B2 was inhibited, and miR-24-3p targeted HSD11B2. HSD11B2 reversed miR-24-3p's repression on HTR/SVneo cell advancement. SUF restrained PE's progression in vivo and in vitro via mediating the miR-24-3p/HSD11B2 axis. In conclusion, SUF enhances HSD11B2 via repressing miR-24-3p, thereby suppressing PE's progression. The study provides an insight into the possibility of using SUF as a novel therapeutic target for PE, which acts via combining with miR-24-3p.

Rank- and sex-specific differences in the neuroendocrine regulation of glucocorticoids in a wild group-living fish.

Individuals that live in groups experience different challenges based on their social rank and sex. Glucocorticoids have a well-established role in coordinating responses to challenges and glucocorticoid levels often vary between ranks and sexes. However, the neuroendocrine mechanisms regulating glucocorticoid dynamics in wild groups are poorly understood, making it difficult to determine the functional consequences of differences in glucocorticoid levels. Therefore, we observed wild social groups of a cooperatively breeding fish (Neolamprologus pulcher) and evaluated how scale cortisol content (an emerging method to evaluate cortisol dynamics in fishes) and expression of glucocorticoid-related genes varied across group members. Scale cortisol was detectable in ~50% of dominant males (7/17) and females (7/15)-but not in any subordinates (0/16)-suggesting that glucocorticoid levels were higher in dominants. However, the apparent behavioural and neuroendocrine factors regulating cortisol levels varied between dominant sexes. In dominant females, higher cortisol was associated with greater rates of territory defense and increased expression of corticotropin-releasing factor in the preoptic and hypothalamic regions of the brain, but these patterns were not observed in dominant males. Additionally, transcriptional differences in the liver suggest that dominant sexes may use different mechanisms to cope with elevated cortisol levels. While dominant females appeared to reduce the relative sensitivity of their liver to cortisol (fewer corticosteroid receptor transcripts), dominant males appeared to increase hepatic cortisol breakdown (more catabolic enzyme transcripts). Overall, our results offer valuable insights on the mechanisms regulating rank- and sex-based glucocorticoid dynamics, as well as the potential functional outcomes of these differences.

Detection of Urinary Exosomal HSD11B2 mRNA Expression: A Useful Novel Tool for the Diagnostic Approach of Dysfunctional 11ß-HSD2-Related Hypertension.

Objective: Apparent mineralocorticoid excess (AME) is an autosomal recessive disorder caused by the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) enzyme deficiency, traditionally assessed by measuring either the urinary cortisol metabolites ratio (tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone, THF+5αTHF/THE) or the urinary cortisol/cortisone (F/E) ratio. Exosomal mRNA is an emerging diagnostic tool due to its stability in body fluids and its biological regulatory function. It is unknown whether urinary exosomal HSD11B2 mRNA is related to steroid ratio or the HSD11B2 662 C>G genotype (corresponding to a 221 A>G substitution) in patients with AME and essential hypertension (EH). Aim of the Study: To detect and quantify HSD11B2 mRNA from urinary exosomes in samples from family members affected by AME and EH, and to evaluate the relationship between exosomal HSD11B2 mRNA, steroid ratio, 662C>G genotype, and hypertension. Methods: In this observational case-control study, urinary steroid ratios and biochemical parameters were measured. Urinary exosomes were extracted from urine and exosomal HSD11B2 mRNA was quantified by Droplet Digital PCR (ddPCR). B2M (ß-2 microglobulin) gene was selected as the reference housekeeping gene. Results: Among family members affected by AME, exosomal urinary HSD11B2 mRNA expression was strictly related to genotypes. The two homozygous mutant probands showed the highest HSD11B2 mRNA levels (median 169, range 118-220 copies/µl) that progressively decreased in 221 AG heterozygous with hypertension (108, range 92-124 copies/µl), 221 AG heterozygous normotensives (23.35, range 8-38.7 copies/µl), and wild-type 221 AA subjects (5.5, range 4.5-14 copies/µl). Heterozygous hypertensive subjects had more HSD11B2 mRNA than heterozygous normotensive subjects. The F/E urinary ratio correlated with HSD11B2 mRNA copy number (p < 0.05); HSD11B2 mRNA strongly decreased while THF+5αTHF/THE increased in the two probands after therapy. In the AME family, HSD11B2 copy number correlated with both F/E and THF+5αTHF/THE ratios, whereas in EH patients, a high F/E ratio reflected a reduced HSD11B2 mRNA expression. Conclusions: HSD11B2 mRNA is detectable and quantifiable in urinary exosomes; its expression varies according to the 662 C>G genotype with the highest levels in homozygous mutant subjects. The HSD11B2 mRNA overexpression in AME could be due to a compensatory mechanism of the enzyme impairment. Exosomal mRNA is a useful tool to investigate HSD11B2 dysregulation in hypertension.

Maternal stress, placental 11ß-hydroxysteroid dehydrogenase type 2, and infant HPA axis development in humans: Psychosocial and physiological pathways.

INTRODUCTION: Prenatal stress is known to influence fetal hypothalamic-pituitary-adrenal axis (HPA axis) development. Placental 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) is a central gene in this pathway, but little is known about what influences its functioning. We assess how maternal distress influences HSD11B2 functioning, and how HSD11B2 in turn, is associated with infant HPA axis development. METHODS: Data come from 24 mother-infant dyads on the Galápagos Islands. Using adjusted linear regression models, we assess the effects of maternal psychosocial (stress and depressive symptoms, measured by the Perceived Stress Scale and the Patient Health Questionnaire-8, respectively) and physiological (HPA axis dysregulation) distress during pregnancy on HSD11B2 methylation and expression and then test how these HSD11B2 measures influence infant HPA axis development. RESULTS: Maternal HPA axis dysregulation during pregnancy is associated with lower placental HSD11B2 expression, which is associated with an exaggerated cortisol reactivity in infants. Sex-specific analyses revealed that maternal depressive symptoms may influence the functioning of placental HSD11B2 differently in girls (n = 11, 46%) than in boys (n = 13, 54%), though the sample size was small. DISCUSSION: These results support a disrupted adaptive framework, in which the ability to upregulate HSD11B2 expression in response to acute stress diminishes as maternal stress becomes chronic. In this model, chronic stress may exhaust the protective mechanism of HSD11B2, leaving the infant vulnerable to high levels of maternal cortisol, which could injure the fetal HPA axis and disrupt long-term neurobehavioral and metabolic development. While larger studies will be needed to confirm these findings, this study offers exploratory results on the effects of maternal distress on both HSD11B2 methylation and expression and the effect of HSD11B2 on offspring HPA axis development.

Sp1 participates in the cadmium-induced imbalance of the placental glucocorticoid barrier by suppressing 11ß-HSD2 expression.

Cadmium (Cd) is widely present in the environment as a heavy metal poison. Prenatal Cd exposure can damage the placental glucocorticoid barrier, leading to foetal growth restriction (FGR), but the molecular mechanism is unknown. We aimed to study the effects of prenatal Cd exposure on 11ß-HSD2 and its possible involvement in Cd induced damage in the placental glucocorticoid barrier. Pregnant rats were treated with CdCl2 (1.0 mg/kg/day) by gavage from gestational day (GD) 9-19. Maternal exposure to Cd increased the FGR rate of the offspring, and the levels of corticosterone in the placenta, maternal and foetal serum. Further in vitro experiments with placenta or JEG3 cells indicated that Cd was able to decrease 11ß-HSD2 and Sp1 expression in trophoblast cells but did not affect 11ß-HSD1. Additionally, decreased p300 and Sp1 enrichment at the 11ß-HSD2 promoter region was observed in the cells treated with Cd. Decreasing or increasing Sp1 expression accordingly inhibited or promoted the expression of 11ß-HSD2 and further decreased or increased p300 and Sp1 enrichment at the 11ß-HSD2 promoter region. In conclusion, Cd inhibits the expression of 11ß-HSD2 by affecting the binding of p300 to 11ß-HSD2 via a decrease in Sp1 expression, which damages the placental glucocorticoid barrier and exposes the foetus to excessive glucocorticoids, resulting in FGR. These findings reveal a possible underlying molecular mechanism by which Cd exposure leads to FGR.

Cortisol and Cortisone in Early Childhood in Very-Low-Birthweight Infants and Term-Born Infants.

INTRODUCTION: Besides programming of the hypothalamic-pituitary-adrenal (HPA) axis, changes in the activity of 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) could contribute to the later metabolic and cardiovascular consequences of preterm birth. OBJECTIVE: We compared serum cortisol, cortisone, and cortisol/cortisone ratio in early childhood in very-low-birthweight (VLBW) infants and term appropriate for gestational age (AGA) born infants. METHODS: We included 41 VLBW infants, participating in the randomized controlled Neonatal Insulin Replacement Therapy in Europe trial, and 64 term AGA-born infants. Cortisol and cortisone were measured in blood samples taken at 6 months and 2 years corrected age (VLBW children) and at 3 months and 1 and 2 years (term children). At 2 years of (corrected) age (HDL) cholesterol, triglycerides, glucose, and insulin were also measured. RESULTS: During the first 2 years of life, cortisol/cortisone ratio is higher in VLBW children compared to term children. In the total group of children, cortisol/cortisone ratio is positively related to triglycerides at 2 years of (corrected) age. In VLBW children, over the first 2 years of life both cortisol and cortisone are higher in the early-insulin group compared to the standard care group. CONCLUSIONS: In VLBW infants, lower 11ß-HSD2 activity probably contributes to the long-term metabolic and cardiovascular risks. In VLBW infants, early insulin treatment could affect programming of the HPA axis, resulting in higher cortisol and cortisone levels during early childhood.

Cortisol and 11 beta-hydroxysteroid dehydrogenase type 2 as potential determinants of renal citrate excretion in healthy children.

BACKGROUND: In patients with Cushing disease, renal citrate excretion is reduced. A low urinary citrate concentration is a risk factor for nephrolithiasis. Since higher acid loading is one major determinant of reduced citrate excretion, we aimed to examine whether glucocorticoids still within the physiological range may already impact on urinary citrate excretion independently of acid-base status. METHODS: Overall, 132 healthy prepubertal participants of the DOrtmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study who had collected two successive 24-h urine samples (at 1 and 2 years) before the start of their pubertal growth spurt were included in the study. Net acid excretion capacity (NAEC), urinary potential renal acid load (PRAL), creatinine, calcium, and various cortisol metabolites were measured in all samples. Glucocorticoid quantification was done by GC-MS and radioimmunoassay. RESULTS: In regression models multivariable-adjusted for 24-h urinary PRAL, NAEC, creatinine and calcium, urinary free cortisol (UFF), 6ß-hydroxycortisol, and 20α-dihydrocortisol showed significant inverse relationships (P ≤ 0.02) with 24-h renal citrate output. By contrast, the estimate of renal 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), i.e., the ratio of urinary free cortisone/UFF, associated positively with urinary citrate (P = 0.04). CONCLUSIONS: In line with studies in hypercortisolic state, even moderately high cortisol levels in healthy children, still within the physiological range, may negatively impact on the kidney's citrate excretion. Besides, a higher 11ß-HSD2 activity, favoring cortisol inactivation, is paralleled by an increased citrate excretion.

Maternal high-fructose intake increases circulating corticosterone levels via decreased adrenal corticosterone clearance in adult offspring.

Global fructose consumption is on the rise; however, maternal high-fructose intake may have adverse effects on offspring. We previously demonstrated that excessive fructose intake by rat dams altered steroidogenic gene transcription in the hippocampus of offspring. Herein, we examined how maternal high-fructose intake influences the regulation of adrenal glucocorticoid levels in offspring. Rat dams received 20% fructose solution during gestation and lactation. After weaning, the offspring were provided normal water. Maternal high-fructose intake did not alter mRNA expression levels of adrenal corticosterone-synthesizing and corticosterone-inactivating proteins or the circulating adrenocorticotropic hormone levels of offspring at postnatal day (PD) 21; however, it increased circulating corticosterone levels and decreased mRNA and protein levels of adrenal 5α-reductase type 1 and 11ß-hydroxysteroid dehydrogenase type 2 in offspring at PD160. Furthermore, maternal high-fructose intake enhanced DNA methylation of the adrenal 5α-reductase 1 promoter region in PD160 offspring. Thus, maternal high-fructose intake was found to affect adrenal steroid hormone clearance in adult offspring - at least in part - through epigenetic mechanisms.

cAMP/PKA/EGR1 signaling mediates the molecular mechanism of ethanol-induced inhibition of placental 11ß-HSD2 expression.

It is known that inhibiting 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) expression in the placenta can cause fetal over-exposure to maternal glucocorticoids and induce intrauterine growth restriction (IUGR); these effects ultimately increase the risk of adult chronic diseases. This study aimed to investigate the molecular mechanism of the prenatal ethanol exposure (PEE)-induced inhibition of placental 11ß-HSD2 expression. Pregnant Wistar rats were intragastrically administered ethanol (4 g/kg/d) from gestational days 9 to 20. The levels of maternal and fetal serum corticosterone and placental 11ß-HSD2-related gene expression were analyzed. Furthermore, we investigated the mechanism of reduced placental 11ß-HSD2 expression induced by ethanol treatment (15-60 mM) in HTR-8/SVneo cells. In vivo, PEE decreased fetal body weights and increased maternal and fetal serum corticosterone and early growth response factor 1 (EGR1) expression levels. Moreover, histone modification changes (decreased acetylation and increased di-methylation of H3K9) to the HSD11B2 promoter and lower 11ß-HSD2 expression levels were observed. In vitro, ethanol decreased cAMP/PKA signaling and 11ß-HSD2 expression and increased EGR1 expression in a concentration-dependent manner. A cAMP agonist and EGR1 siRNA reversed the ethanol-induced inhibition of 11ß-HSD2 expression. Together, PEE reduced placental 11ß-HSD2 expression, and the underlying mechanism is associated with ethanol-induced histone modification changes to the HSD11B2 promoter through the cAMP/PKA/EGR1 pathway.

nAChRs-ERK1/2-Egr-1 signaling participates in the developmental toxicity of nicotine by epigenetically down-regulating placental 11ß-HSD2.

Impaired placental 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) activity which inactivates maternal glucocorticoids is associated with poor fetal growth and a higher risk of chronic diseases in adulthood. This study aimed to elucidate the epigenetically regulatory mechanism of nicotine on placental 11ß-HSD2 expression. Pregnant Wistar rats were administered 1.0 mg/kg nicotine subcutaneously twice a day from gestational day 9 to 20. The results showed that prenatal nicotine exposure increased corticosterone levels in the placenta and fetal serum, disrupted placental morphology and endocrine function, and reduced fetal bodyweight. Meanwhile, histone modification abnormalities (decreased acetylation and increased di-methylation of histone 3 Lysine 9) on the HSD11B2 promoter and lower-expression of 11ß-HSD2 were observed. Furthermore, the expression of nicotinic acetylcholine receptor (nAChR) α4/ß2, the phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and Ets-like protein-1 (Elk-1), and the expression of early growth response-1 (Egr-1) were increased in the nicotine groups. In human BeWo cells, nicotine decreased 11ß-HSD2 expression, increased nAChRα9 expression, and activated ERK1/2/Elk-1/Egr-1 signaling in the concentration (0.1-10 µM)-dependent manner. Antagonism of nAChRs, inhibition of ERK1/2 and Egr-1 knockdown by siRNA were able to block/abrogate the effects of nicotine on histone modification and expression of 11ß-HSD2. Taken together, nicotine can impair placental structure and function, and induce fetal developmental toxicity. The underlying mechanism involves histone modifications and down-regulation of 11ß-HSD2 through nAChRs/ERK1/2/Elk-1/Egr-1 signaling, which increases active glucocorticoids levels in the placenta and fetus, and eventually inhibits the fetal development.

The in vitro metabolism of 11ß-hydroxyprogesterone and 11-ketoprogesterone to 11-ketodihydrotestosterone in the backdoor pathway.

Increased circulating 11ß-hydroxyprogesterone (11OHP4), biosynthesised in the human adrenal, is associated with 21-hydroxylase deficiency in congenital adrenal hyperplasia. 17α-hydroxyprogesterone levels are also increased, with the steroid's metabolism to dihydrotestosterone in the backdoor pathway contributing to hyperandrogenic clinical conditions. In this study we investigated the in vitro biosynthesis and downstream metabolism of 11OHP4. Both cytochrome P450 11ß-hydroxylase and aldosterone synthase catalyse the biosynthesis of 11OHP4 from progesterone (P4) which is converted to 11-ketoprogesterone (11KP4) by 11ß-hydroxysteroid dehydrogenase type 2, while type 1 readily catalysed the reverse reaction. We showed in HEK-293 cells that these C11-oxy C21 steroids were metabolised by steroidogenic enzymes in the backdoor pathway-5α-reductase (SRD5A) and 3α-hydroxysteroid type 3 (AKR1C2) converted 11OHP4 to 5α-pregnan-11ß-ol,3,20-dione and 5α-pregnan-3α,11ß-diol-20-one, while 11KP4 was converted to 5α-pregnan-3,11,20-trione and 5α-pregnan-3α-ol-11,20-dione (alfaxalone), respectively. Cytochrome P450 17α-hydroxylase/17,20-lyase catalysed the hydroxylase and lyase reaction to produce the C11-oxy C19 steroids demonstrated in the conversion of alfaxalone to 11-oxy steroids demonstrated in the conversion of alfaxalone to 11ketoandrosterone. In LNCaP cells, a prostate cancer cell model endogenously expressing the relevant enzymes, 11OHP4 and 11KP4 were metabolised to the potent androgen, 11-ketodihydrotestosterone (11KDHT), thus suggesting the C11-oxy C21 steroids contribute to the pool of validating the in vitro biosynthesis of C11-oxy C19 steroids from C11-oxy C21 steroids. The in vitro reduction of 11KP4 at C3 and C5 by AKR1C2 and SRD5A has confirmed the metabolic route of the urinary metabolite, 3α,20α-dihydroxy-5ß-pregnan-11-one. Although our assays have demonstrated the conversion of 11OHP4 and 11KP4 by steroidogenic enzymes in the backdoor pathway yielding 11KDHT, thus suggesting the C11-oxy C21 steroids contribute to the pool of potent androgens, the in vivo confirmation of this metabolic route remains challenging.

Recent advances in the study of 11ß-Hydroxysteroid dehydrogenase type 2 (11ß-HSD2)Inhibitors.

11ß-Hydroxysteroid dehydrogenase (11ß-HSD), which interconverts hormonally active cortisol and inactive cortisone in multiple human tissues, has two distinct isoforms named 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) and 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2). 11ß-HSD2 is an NAD+-dependent oxidase which lowers cortisol by converting it to cortisone while 11ß-HSD1 mainly catalyzes the reduction which converts cortisone into cortisol. Selective inhibition of 11ß-HSD2 is generally detrimental to health because the accumulation of cortisol can cause metabolic symptoms such as apparent mineralocorticoid excess (AME), fetal developmental defects and lower testosterone levels in males. There has been some advances on the study of 11ß-HSD2 inhibitors and we think it necessary to make a summary of the characteristics and inhibiting properties of latest 11ß-HSD2 inhibitors. As another review on 11ß-HSD2 inhibitors has been issued on 2011 (see review (Ma et al., 2011)), this mini-review concerns advances during the last 5 years.

Invited Commentary: An Ingenious Approach to Examining the Relationship Between Maternal Stress and Offspring Health?

The potentially deleterious effects on offspring health of excess maternal stress in pregnancy are important to understand-both whether observed associations are causal and through what mechanisms their effects may exert an influence. In this issue of the Journal, Räikkönen et al. (Am J Epidemiol. 2012;000(0):000-000) provide an ingenious test of a potential pathway through which maternal stress may influence offspring development. Licorice consumption is known to disrupt the ability of the placental enzyme 11ß-hydroxysteroid dehydrogenase type 2 to inactivate cortisol before it reaches the fetus, leading to higher levels of cortisol exposure. Higher levels of cortisol exposure are also hypothesized to underlie the mechanism through which maternal stress may disrupt fetal development. Thus licorice consumption may serve, in some ways, to mimic maternal stress. The authors report associations between heavy licorice consumption during pregnancy and a wide range of offspring outcomes, including changes in pubertal timing, intelligence quotient, and mental health. In our view, these results should be considered preliminary; more work needs to be completed to determine the relationship of prenatal licorice consumption to these outcomes. Nonetheless, these intriguing and suggestive results demonstrate that this line of work should be given high priority, and they set the stage for additional research moving forward.

Maternal Licorice Consumption During Pregnancy and Pubertal, Cognitive, and Psychiatric Outcomes in Children.

Earlier puberty, especially in girls, is associated with physical and mental disorders. Prenatal glucocorticoid exposure influences the timing of puberty in animal models, but the human relevance of those findings is unknown. We studied whether voluntary consumption of licorice, which contains glycyrrhizin (a potent inhibitor of placental 11ß-hydroxysteroid dehydrogenase type 2, the "barrier" to maternal glucocorticoids), by pregnant women was associated with pubertal maturation (height, weight, body mass index for age, difference between current and expected adult height, Tanner staging, score on the Pubertal Development Scale), neuroendocrine function (diurnal salivary cortisol, dexamethasone suppression), cognition (neuropsychological tests), and psychiatric problems (as measured by the Child Behavior Checklist) in their offspring. The children were born in 1998 in Helsinki, Finland, and examined during 2009-2011 (mean age = 12.5 (standard deviation (SD), 0.4) years; n = 378). Girls exposed to high maternal glycyrrhizin consumption (≥500 mg/week) were taller (mean difference (MD) = 0.4 SD, 95% confidence interval (CI): 0.1, 0.8), were heavier (MD = 0.6 SD, 95% CI: 0.2, 1.9), and had higher body mass index for age (MD = 0.6 SD, 95% CI: 0.2, 0.9). They were also 0.5 standard deviations (95% CI: 0.2, 0.8) closer to adult height and reported more advanced pubertal development (P < 0.04). Girls and boys exposed to high maternal glycyrrhizin consumption scored 7 (95% CI: 3.1, 11.2) points lower on tests of intelligence quotient, had poorer memory (P < 0.04), and had 3.3-fold (95% CI: 1.4, 7.7) higher odds of attention deficit/hyperactivity disorder problems compared with children whose mothers consumed little to no glycyrrhizin (≤249 mg/week). No differences in cortisol levels were found. Licorice consumption during pregnancy may be associated with harm for the developing offspring.

Apparent mineralocorticoid excess and the long term treatment of genetic hypertension.

Apparent mineralocorticoid excess (AME) is a genetic disorder causing severe hypertension, hypokalemia, and hyporeninemic hypoaldosteronism owing to deficient 11 beta-hydroxysteroid dehydrogenase type-2 (11ßHSD2) enzyme activity. The 11ßHSD2 enzyme confers mineralocorticoid receptor specificity for aldosterone by converting cortisol to its inactive metabolite, cortisone and inactivating the cortisol-mineralocorticoid receptor complex. The 20year follow-up of a consanguineous Iranian family with three sibs affected with AME shows the successes and pitfalls of medical therapy with spironolactone. The three sibs, (female, male, female) were diagnosed at the ages of 14, 11, and 4 years, respectively. At diagnosis, hypertensive retinopathy and left ventricular hypertrophy were present in the eldest female and retinopathy was noted in the male sib. Spironolactone treatment resulted in decreased blood pressure and rise in serum potassium levels. The older female, age 36, developed reduced left ventricular function with mitral and tricuspid regurgitation and renal failure after her second pregnancy. She was treated with renal transplantation resulting in cure of AME with decreased blood pressure and weaning from antihypertensives. Her younger sibs, age 34 and 26, do not have end organ damage. Early and vigilant treatment improves morbidity in patients with AME. Mineralocorticoid receptor antagonists normalize blood pressure, correct hypokalemia and reduce hypertensive end-organ damage in patients with AME. Low dose dexamethasone can be considered, though the response may be variable. Future directions of therapy include selective mineralocorticoid antagonists.

Variants of HSD11B2 gene in hypertensive disorders of pregnancy.

INTRODUCTION: One of the hypotheses concerning the etiology of gestational hypertension (GH) and pre-eclampsia (PE) assumes that they develop as a result of placenta malfunctioning at the early stage of pregnancy. Placental dysfunction is also associated with the decreased activity of 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), which in normal pregnancy protects the fetus from the excess of maternal cortisol. OBJECTIVE: The aim of the study was to analyze the sequence of HSD11B2, a gene that encodes 11ß-HSD2, searching for mutations and haplotypes associated with the increased risk of GH or PE. Those may serve as potential genetic markers of GH and PE. METHODS: The study was performed in case-control structure and included pregnant women (in third trimester) diagnosed with: GH, PE or being normotensive (control group). The research comprised DNA sequencing of HSD11B2, followed by restriction analysis (PCR-RFLP). The linkage disequilibrium analysis and haplotype-based case-control analysis were performed. RESULTS: Six sequence variations were observed. Four mutations were indicated in the coding region of HSD11B2 and the other two in 3'-UTR. Two SNPs: c.468C > A and c.534G > A were found to be in total disequilibrium. CONCLUSIONS: High variability in HSD11B2 sequence was indicated in the study population, but the relevance of observed SNPs to GH or PE development was not confirmed.