Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11ß-hydroxysteroid dehydrogenase activities in primary cell cultures.

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

The Role of microRNA in the Regulation of Cortisol Metabolism in the Adipose Tissue in the Course of Obesity.

The similarity of the clinical picture of metabolic syndrome and hypercortisolemia supports the hypothesis that obesity may be associated with impaired expression of genes related to cortisol action and metabolism in adipose tissue. The expression of genes encoding the glucocorticoid receptor alpha (GR), cortisol metabolizing enzymes (HSD11B1, HSD11B2, H6PDH), and adipokines, as well as selected microRNAs, was measured by real-time PCR in adipose tissue from 75 patients with obesity, 19 patients following metabolic surgery, and 25 normal-weight subjects. Cortisol levels were analyzed by LC-MS/MS in 30 pairs of tissues. The mRNA levels of all genes studied were significantly (p < 0.05) decreased in the visceral adipose tissue (VAT) of patients with obesity and normalized by weight loss. In the subcutaneous adipose tissue (SAT), GR and HSD11B2 were affected by this phenomenon. Negative correlations were observed between the mRNA levels of the investigated genes and selected miRNAs (hsa-miR-142-3p, hsa-miR-561, and hsa-miR-579). However, the observed changes did not translate into differences in tissue cortisol concentrations, although levels of this hormone in the SAT of patients with obesity correlated negatively with mRNA levels for adiponectin. In conclusion, although the expression of genes related to cortisol action and metabolism in adipose tissue is altered in obesity and miRNAs may be involved in this process, these changes do not affect tissue cortisol concentrations.

Phthalates cause a low-renin phenotype commonly found in premature infants with idiopathic neonatal hypertension.

Since the 1970s, when the initial reports of neonatal hypertension related to renal artery thromboembolism were published, other secondary causes of neonatal hypertension have been reported. Those infants with no identifiable cause of hypertension were labeled with a variety of terms. Herein, we describe such infants as having idiopathic neonatal hypertension (INH). Most, but not all, of these hypertensive infants were noted to have bronchopulmonary dysplasia (BPD). More recently, reports described common clinical characteristics seen in INH patients, whether or not they had BPD. This phenotype includes low plasma renin activity, presentation near 40 weeks postmenstrual age, and a favorable response to treatment with spironolactone. A small prospective study in INH patents showed evidence of mineralocorticoid receptor activation due to inhibition of 11ß-HSD2, the enzyme that converts cortisol to the less potent mineralocorticoid-cortisone. Meanwhile, phthalate metabolites have been shown to inhibit 11ß-HSD2 in human microsomes. Premature infants can come in contact with exceptionally large phthalate exposures, especially those infants with BPD. This work describes a common low-renin phenotype, commonly seen in patients categorized as having INH. Further, we review the evidence that hypertension in INH patients with the low-renin phenotype may be mediated by phthalate-associated inhibition of 11ß-HSD2. Lastly, we review the implications of these findings regarding identification, treatment, and prevention of the low-renin hypertension phenotype seen in premature infants categorized as having INH.

The effect of cold exposure on the levels of glucocorticoids, 11-hydroxysteroid dehydrogenase 2, and placental vascularization in a rat model.

OBJECTIVE: Cold exposure (CE) before birth is one of the initial stressors that may impact mammalian pregnancy, changing placental and fetal development and affecting the health of the offspring. While glucocorticoids (GCs) participate in the body's response to the stress of CE, the specific mechanisms of their action are unclear. This study aims to determine the effect of CE stress on the placenta and to test whether stress, caused by cold exposure in pregnancy impairs fetal development by changing placental angiogenesis via excessive GC expression. MATERIALS AND METHODS: CE rat model was created by exposing 30 SD rats to cold preconception, or during the first, second, and third weeks of pregnancy. Serum cortisol and soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels, physiological index changes (food intake, body weight change and blood pressure), and pregnancy outcomes (fetal rat weight, number of live fetal rats, and placental weight) were collected at baseline and at different time points after the conception. Protein expression levels of 11 ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), glucocorticoid receptor, vascular endothelial growth factor A (VEGF-A), placental growth factor (PIGF), and sFlt-1 in placental tissues were measured by western blotting. Cytokeratin (CK) and laminin (LN) in trophoblasts, and α-actin in vascular smooth muscle of the spiral arteries of pregnant rats after the systemic cold treatment were assessed by immunofluorescence and visualized by fluorescent microscopy. To test the effect of 11ß-HSD2 levels on the placental recasting, human first-trimester extravillous trophoblast cells (HTR8/SVneo) underwent knockdown using specific 11ß-HSD2 siRNA constructs.  Expression levels of 11ß-HSD2 were analyzed by quantitative real-time PCR (qPCR) and into HTR8 cells, and the expression levels of the 11ß-HSD2 gene in each group were measured using qPCR. Cell migration and invasion was assessed by Transwell migration assay, and sFlt-1 levels in HTR8 cells were measured by ELISA. RESULTS: CE pre-conception led to consistently increasing serum corticosterone and sFlt-1 levels throughout pregnancy, and persistently increased diastolic blood pressure (DBP) in rat CE model compared to control animals. CE during the second week of gestation (Gp.3) was associated with significantly lower placental weight (p=0.0003). Cold exposure in the third week (Gp.4) was associated with significantly (p=0.001) lower fetal weight. CE pre-conception was associated with significantly decreased placental levels of 11ß-HSD2, glucocorticoid receptor, VEGF-A, PIGF, and sFlt-1 proteins and α-actin compared to the control group. Silencing 11ß-HSD2 by siRNA led to reduced cell migrations and invasion, and markedly increased expression levels of sFlt-1 in HTR8/SVneo cells (p<0.05). CONCLUSIONS: Pre-conception cold exposure and during early pregnancy leads to increased GCs levels and impaired placental 11ß-HSD2 activity. We suggest that the subsequent 11ß-HSD2-induced increase in the sFlt-1expression during early pregnancy may affect placental vascular remodeling and change placental morphological structure and function.

Selenium attenuates the cadmium-induced placenta glucocorticoid barrier damage by up-regulating the expression of specificity protein 1.

Cadmium (Cd) is an environmental pollutant and pregnant women are especially susceptible to the effects of exposure to Cd. Our previous study found Cd can be accumulated in the placenta and causes fetal growth restriction (FGR) through damage the placental glucocorticoid barrier. Selenium (Se), as an essential micronutrient, can allivate Cd-induced toxicity. In this study, we aim to explore the protective mechanism of Se against Cd-induced the placental glucocorticoid barrier damage and FGR. Pregnant Sprague Dawley (SD) rats were exposed to CdCl2 (1 mg/kg/day) and Na2 SeO3 (0.1-0.2-0.3 mg/kg/day) by gavage from gestational day (GD) 0 to GD 19. The results showed that reduced fetal weight, increased corticosterone concentrations in the maternal and fetal serum, and impaired placental labyrinth layer blood vessel development, appeared in pregnant rats after Cd exposure and improved after treated with Se. In cell experiments, we confirmed that Se reduces Cd-induced apoptosis. Moreover, Se can abolish Cd-induced 11ß-HSD2 and specificity protein 1 (Sp1) decreasing in vivo and vitro. In human JEG-3 cells, the knockdown of Sp1 expression by small interfering RNA can suppressed the protective effect of Se on Cd-induced 11ß-HSD2 decreasing. In general, our results demonstrated that Se is resistant to Cd-induced FGR through upregulating the placenta barrier via activation of the transcription factor Sp1.

Glucocorticoid Production in Lymphoid Organs: Acute Effects of Lipopolysaccharide in Neonatal and Adult Mice.

Glucocorticoids (GCs) are critical modulators of the immune system. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating GC levels and is stimulated by endotoxins. Lymphoid organs also produce GCs; however, it is not known how lymphoid GC levels are regulated in response to endotoxins. We assessed whether an acute challenge of lipopolysaccharide (LPS) increases lymphoid levels of progesterone and GCs, and expression of steroidogenic enzymes and key HPA axis components (eg, corticotropin-releasing hormone [CRH], adrenocorticotropic hormone [ACTH]). We administered LPS (50 µg/kg intraperitoneally) or vehicle control to male and female C57BL/6J neonatal (postnatal day [PND] 5) and adult (PND90) mice and collected blood, bone marrow, thymus, and spleen 4 hours later. We measured progesterone, 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone via liquid chromatography-tandem mass spectrometry. We measured gene expression of key steroidogenic enzymes (Cyp11b1, Hsd11b1, and Hsd11b2) and HPA axis components (Crh, Crhr1, Pomc, and Mc2r) via quantitative polymerase chain reaction. At PND5, LPS induced greater increases in steroid levels in lymphoid organs than in blood. In contrast, at PND90, LPS induced greater increases in steroid levels in blood than in lymphoid organs. Steroidogenic enzyme transcripts were present in all lymphoid organs, and LPS altered steroidogenic enzyme expression predominantly in the spleen. Lastly, we detected transcripts of key HPA axis components in all lymphoid organs, and there was an effect of LPS in the spleen. Taken together, these data suggest that LPS regulates GC production by lymphoid organs, similar to its effects on the adrenal glands, and the effects of LPS might be mediated by local expression of CRH and ACTH.

Decreased 11ß-Hydroxysteroid Dehydrogenase Type 2 Expression in the Kidney May Contribute to Nicotine/Smoking-Induced Blood Pressure Elevation in Mice.

[Figure: see text].

11-Ketotestosterone is a major androgen produced in porcine adrenal glands and testes.

Porcine steroid hormone profiles have some unique characteristics. We previously studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs). To investigate porcine steroidogenesis, we induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. Using cAMP, adenovirus-mediated introduction of steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP) induced the differentiation of PSPA cells into sex steroid-producing cells. Introducing SF-1/Ad4BP also induced the aldo-keto reductase 1C1 (AKR1C1) gene. Porcine AKR1C1 had 17ß-hydroxysteroid dehydrogenase activity, which converts androstenedione and 11-ketoandrostenedione into testosterone (T) and 11-ketotestosteorne (11KT). Furthermore, differentiated cells expressed hydroxysteroid 11ß-dehydrogenase 2 (HSD11B2) and produced 11KT. HSD11B2 was expressed in testicular Leydig cells and the adrenal cortex. 11KT was present in the plasma of both immature male and female pigs, with slightly higher levels in the male pigs. T levels were much higher in the male pigs. It is noteworthy that in the female pigs, the 11KT levels were >10-fold higher than the T levels. However, castration altered the 11KT and T plasma profiles in the male pigs to near those of the females. 11KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11KT is produced in porcine adrenal glands and testes, and may regulate cardiovascular functions through eNOS expression.

Influence of labor on direct and indirect determinants of placental 11beta-hydroxysteroid dehydrogenase activity.

PURPOSE: Labor is a complex process involving multiple para-, auto- and endocrine cascades. The interaction of cortisol, corticotropin-releasing hormone (CRH) and progesterone is essential. The action of cortisol on the human feto-placental unit is regulated by 11beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2/HSD11B2) that converts cortisol into inactive cortisone. The majority of studies on the assessment of placental 11ß-HSD2 function determined indirect activity parameters. It remains elusive if indirect measurements correlate with enzymatic function and if these parameters are affected by potential confounders (e.g., mode of delivery). Thus, we compared determinants of indirect 11ß-HSD2 tissue activity with its direct enzymatic turnover rate in placental samples from spontaneous births and cesarean (C)-sections. METHODS: Using LC-MS/MS, we determined CRH, cortisol, cortisone, progesterone and 17-hydroxy(OH)-progesterone in human term placentas (spontaneous birth vs. C-section, n = 5 each) and measured the enzymatic glucocorticoid conversion rates in placental microsomes. Expression of HSD11B1, 2 and CRH was determined via qRT-PCR in the same samples. RESULTS: Cortisol-cortisone ratio correlated with direct microsomal enzymatic turnover. While this observation seemed independent of sampling site, a strong influence of mode of delivery on tissue steroids was observed. The mRNA expression of HSD11B2 correlated with indirect and direct cortisol turnover rates in C-section placentas only. In contrast to C-sections, CRH, cortisol and cortisone levels were significantly increased in placental samples following spontaneous birth. CONCLUSION: Labor involves a series of complex hormonal processes including activation of placental CRH and glucocorticoid metabolism. This has to be taken into account when selecting human cohorts for comparative analysis of placental steroids.

Tributyltin and triphenyltin induce 11ß-hydroxysteroid dehydrogenase 2 expression and activity through activation of retinoid X receptor α.

Exposure to the environmental pollutants organotins is of toxicological concern for the marine ecosystem and sensitive human populations, including pregnant women and their unborn children. Using a placenta cell model, we investigated whether organotins at nanomolar concentrations affect the expression and activity of 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). 11ß-HSD2 represents a placental barrier controlling access of maternal glucocorticoids to the fetus. The organotins tributyltin (TBT) and triphenyltin (TPT) induced 11ß-HSD2 expression and activity in JEG-3 placenta cells, an effect confirmed at the mRNA level in primary human trophoblast cells. Inhibition/knock-down of retinoid X receptor alpha (RXRα) in JEG-3 cells reduced the effect of organotins on 11ß-HSD2 activity, mRNA and protein levels, revealing involvement of RXRα. Experiments using RNA and protein synthesis inhibitors indicated that the effect of organotins on 11ß-HSD2 expression was direct and caused by increased transcription. Induction of placental 11ß-HSD2 activity by TBT, TPT and other endocrine disrupting chemicals acting as RXRα agonists may affect placental barrier function by altering the expression of glucocorticoid-dependent genes and resulting in decreased availability of active glucocorticoids for the fetus, disturbing development and increasing the risk for metabolic and cardiovascular complications in later life.

Development of prednisone resistance in naïve canine lymphoma: Longitudinal evaluation of NR3C1α, ABCB1, and 11ß-HSD mRNA expression.

Prednisone resistance develops rapidly and essentially universally when dogs with lymphoma are treated with corticosteroids. We investigated naturally occurring mechanisms of prednisone resistance in seven dogs with naïve multicentric lymphoma, treated with oral prednisone; four dogs were administered concurrent cytotoxic chemotherapy. Expression of NR3C1α, ABCB1 (formerly MDR1), 11ß-HSD1, and 11ß-HSD2 mRNA was evaluated in neoplastic lymph nodes by real-time RT-PCR. Changes of expression levels at diagnosis and at time of clinical resistance to prednisone were compared longitudinally using a Wilcoxon signed-rank test. Clinical resistance to prednisone was observed after a median of 68 days (range: 7-348 days) after initiation of treatment. Relative to pretreatment samples, prednisone resistance was associated with decreased NR3C1α expression in biopsies of all dogs with high-grade lymphoma (six dogs, p=.031); one dog with indolent T-zone lymphoma had increased expression of NR3C1α. Resistance was not consistently associated with changes in ABCB1, 11ß-HSD1, or 11ß-HSD2 expression. Decreased expression of the glucocorticoid receptor (NR3C1α) may play a role in conferring resistance to prednisone in dogs with lymphoma. Results do not indicate a broad role for changes in expression of ABCB1, 11ß-HSD1, and 11ß-HSD2 in the emergence of prednisone resistance in lymphoma-bearing dogs.

Cortisol regulation of Na+, K+-ATPase ß1 subunit transcription via the pre-receptor 11ß-hydroxysteroid dehydrogenase 1-like (11ß-Hsd1L) in gills of hypothermal freshwater milkfish, Chanos chanos.

Hypothermal stress changes the balance of osmoregulation by affecting Na+, K+-ATPase (Na-K-ATPase) activity or inducing modulation to epithelium permeability in fish. Meanwhile, cellular concentrations of cortisol can be modulated by the pre-receptor enzymes 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-Hsd1 and 2). In fish, increasing levels of exogenous cortisol stimulate Na+ uptake via specific interaction with cortisol. This study investigated cortisol effects on expression of Na-K-ATPase subunit proteins and activity in gills of milkfish under hypothermal stress and revealed that the plasma cortisol contents as well as gill 11ß-hsd1l and na-k-atpase ß1 mRNA abundance were decreased in fresh water (FW) milkfish. Meanwhile, in the seawater (SW) milkfish, the plasma cortisol contents and gill 11ß-hsd1l and na-k-atpase ß1 mRNA abundance was increased under hypothermal stress. On the other hand, the abundance of 11ß-hsd2 mRNA increased in both FW and SW. In addition, 11ß-hsd1l expression increased in FW milkfish but decreased in SW milkfish after cortisol injection. Accordingly, the results that gill Na-K-ATPase activity of FW milkfish was affected by environmental temperatures as well as cortisol-dependent Na-K-ATPase ß1-subunit levels might be due to increased expression of 11ß-hsd1l that elevated intracellular cortisol contents. In hypothermal SW milkfish, decreasing abundance of Na-K-ATPase ß1 protein due to reduced expression of 11ß-hsd1l was found after cortisol injection. Thus, under hypothermal stress, 11ß-HSD1L in FW milkfish gills was used to modulate cortisol and the following effects on increasing the transcription of Na-K-ATPase ß1 protein.

Sex-specific regulation of stress-induced fetal glucocorticoid surge by the mouse placenta.

Antenatal stress increases the prevalence of diseases in later life, which shows a strong sex-specific effect. However, the underlying mechanisms remain unknown. Maternal glucocorticoids can be elevated by stress and are potential candidates to mediate the effects of stress on the offspring sex-specifically. A comprehensive evaluation of dynamic maternal and placental mechanisms modulating fetal glucocorticoid exposure upon maternal stress was long overdue. Here, we addressed this gap in knowledge by investigating sex-specific responses to midgestational stress in mice. We observed increased levels of maternal corticosterone, the main glucocorticoid in rodents, along with higher corticosteroid-binding globulin levels at midgestation in C57Bl/6 dams exposed to sound stress. This resulted in elevated corticosterone in female fetuses, whereas male offspring were unaffected. We identified that increased placental expression of the glucocorticoid-inactivating enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2; Hsd11b2 gene) and ATP-binding cassette transporters, which mediate glucocorticoid efflux toward maternal circulation, protect male offspring from maternal glucocorticoid surges. We generated mice with an Hsd11b2 placental-specific disruption (Hsd11b2PKO) and observed moderately elevated corticosterone levels in offspring, along with increased body weight. Subsequently, we assessed downstream glucocorticoid receptors and observed a sex-specific differential modulation of placental Tsc22d3 expression, which encodes the glucocorticoid-induced leucine zipper protein in response to stress. Taken together, our observations highlight the existence of unique and well-orchestrated mechanisms that control glucocorticoid transfer, exposure, and metabolism in the mouse placenta, pinpointing toward the existence of sex-specific fetal glucocorticoid exposure windows during gestation in mice.

Mimicry and well known genetic friends: molecular diagnosis in an Iranian cohort of suspected Bartter syndrome and proposition of an algorithm for clinical differential diagnosis.

BACKGROUND: Bartter Syndrome is a rare, genetically heterogeneous, mainly autosomal recessively inherited condition characterized by hypochloremic hypokalemic metabolic alkalosis. Mutations in several genes encoding for ion channels localizing to the renal tubules including SLC12A1, KCNJ1, BSND, CLCNKA, CLCNKB, MAGED2 and CASR have been identified as underlying molecular cause. No genetically defined cases have been described in the Iranian population to date. Like for other rare genetic disorders, implementation of Next Generation Sequencing (NGS) technologies has greatly facilitated genetic diagnostics and counseling over the last years. In this study, we describe the clinical, biochemical and genetic characteristics of patients from 15 Iranian families with a clinical diagnosis of Bartter Syndrome. RESULTS: Age range of patients included in this study was 3 months to 6 years and all patients showed hypokalemic metabolic alkalosis. 3 patients additionally displayed hypercalciuria, with evidence of nephrocalcinosis in one case. Screening by Whole Exome Sequencing (WES) and long range PCR revealed that 12/17 patients (70%) had a deletion of the entire CLCNKB gene that was previously identified as the most common cause of Bartter Syndrome in other populations. 4/17 individuals (approximately 25% of cases) were found to suffer in fact from pseudo-Bartter syndrome resulting from congenital chloride diarrhea due to a novel homozygous mutation in the SLC26A3 gene, Pendred syndrome due to a known homozygous mutation in SLC26A4, Cystic Fibrosis (CF) due to a novel mutation in CFTR and apparent mineralocorticoid excess syndrome due to a novel homozygous loss of function mutation in HSD11B2 gene. 1 case (5%) remained unsolved. CONCLUSIONS: Our findings demonstrate deletion of CLCNKB is the most common cause of Bartter syndrome in Iranian patients and we show that age of onset of clinical symptoms as well as clinical features amongst those patients are variable. Further, using WES we were able to prove that nearly 1/4 patients in fact suffered from Pseudo-Bartter Syndrome, reversing the initial clinical diagnosis with important impact on the subsequent treatment and clinical follow up pathway. Finally, we propose an algorithm for clinical differential diagnosis of Bartter Syndrome.

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.

Characterisation of the cancer-associated glucocorticoid system: key role of 11ß-hydroxysteroid dehydrogenase type 2.

BACKGROUND: Recent studies have shown that production of cortisol not only takes place in several non-adrenal peripheral tissues such as epithelial cells but, also, the local inter-conversion between cortisone and cortisol is regulated by the 11ß-hydroxysteroid dehydrogenases (11ß-HSDs). However, little is known about the activity of this non-adrenal glucocorticoid system in cancers. METHODS: The presence of a functioning glucocorticoid system was assessed in human skin squamous cell carcinoma (SCC) and melanoma and further, in 16 epithelial cell lines from 8 different tissue types using ELISA, western blotting and immunofluorescence. 11ß-HSD2 was inhibited both pharmacologically and by siRNA technology. Naïve CD8+ T cells were used to test the paracrine effects of cancer-derived cortisol on the immune system in vitro. Functional assays included cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical data of 11ß-HSD expression were generated using tissue microarrays of 40 cases of human SCCs as well as a database featuring 315 cancer cases from 15 different tissues. RESULTS: We show that cortisol production is a common feature of malignant cells and has paracrine functions. Cortisol production correlated with the magnitude of glucocorticoid receptor (GR)-dependent inhibition of tumour-specific CD8+ T cells in vitro. 11ß-HSDs were detectable in human skin SCCs and melanoma. Analyses of publicly available protein expression data of 11ß-HSDs demonstrated that 11ß-HSD1 and -HSD2 were dysregulated in the majority (73%) of malignancies. Pharmacological manipulation of 11ß-HSD2 activity by 18ß-glycyrrhetinic acid (GA) and silencing by specific siRNAs modulated the bioavailability of cortisol. Cortisol also acted in an autocrine manner and promoted cell invasion in vitro and cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical analyses using tissue microarrays showed that expression of 11ß-HSD2 was significantly reduced in human SCCs of the skin. CONCLUSIONS: The results demonstrate evidence of a cancer-associated glucocorticoid system and show for the first time, the functional significance of cancer-derived cortisol in tumour progression.

A repressive role of enhancer of zeste homolog 2 in 11ß-hydroxysteroid dehydrogenase type 2 expression in the human placenta.

The expression of 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which acts as a placental glucocorticoid barrier, is silenced in cytotrophoblasts but substantially up-regulated during syncytialization. However, the repressive mechanism of 11ß-HSD2 expression before syncytialization and how this repression is lifted during syncytialization remain mostly unresolved. Here we found that enhancer of zeste homolog 2 (EZH2) accounts for the silence of 11ß-HSD2 expression via trimethylation of histone H3 lysine 27 at the promoter of the 11ß-HSD2 gene. Further studies revealed that, upon syncytialization, human chorionic gonadotropin reduced the phosphorylation of retinoblastoma protein (pRB) via activation of the cAMP/PKA pathway, which sequesters E2F transcription factor 1 (E2F1), the transcription factor for EZH2 expression. As a result of inactivation of the pRB-E2F1-EZH2 pathway, the repressive marker trimethylation of histone H3 lysine 27 at the 11ß-HSD2 promoter is removed, which leads to the robust expression of 11ß-HSD2 during syncytialization.

The liquorice root derivative glycyrrhetinic acid can ameliorate ionoregulatory disturbance in rainbow trout (Oncorhynchus mykiss) abruptly exposed to ion-poor water.

To consider the idea that a dietary botanical supplement could act as an adaptogen in a teleost fish, the effect of a liquorice root derivative (18ß-glycyrrhetinic acid, 18ßGA) on rainbow trout following an acute ionoregulatory stressor was examined. Freshwater (FW) trout were fed a control or 18ßGA supplemented diet (0, 5, or 50µg 18ßGA/g diet) for 2weeks, then abruptly exposed to ion-poor water (IPW) for 24h. Following IPW exposure, muscle moisture content and serum cortisol levels elevated and serum [Na(+)] and/or [Cl(-)] reduced in control and 50µg/g 18ßGA-fed fish. However, these endpoints were unaltered in 5µg/g 18ßGA-fed fish. Gill tissue was investigated for potential mechanisms of 18ßGA action by examining mRNA abundance of genes encoding corticosteroid receptors (CRs), 11ß-hydroxysteroid dehydrogenase 2 (11ß-hsd2), and tight junction (TJ) proteins, as well as Na(+)-K(+)-ATPase and H(+)-ATPase activity, and mitochondrion-rich cell (MRC) morphometrics. Following IPW exposure, CR and 11ß-hsd2 mRNA, MRC fractional surface, Na(+)-K(+)-ATPase and H(+)-ATPase activity were unaltered or decreased in 50µg 18ßGA fish, as was mRNA encoding select TJ proteins. In contrast, 5µg 18ßGA-fed fish exhibited elevated 11ß-hsd2 and CR mRNA abundance versus 50µg 18ßGA-fed, and reduced MRC apical area as well as some differences in TJ protein mRNA abundance versus control fish. Data suggest that 18ßGA, at low levels, may be adaptogenic in trout and might help to ameliorate ionoregulatory perturbation following IPW exposure. This seems to occur, in part, through 18ßGA-induced alterations in the biochemistry and physiology of the gill.

The secretion, synthesis, and metabolism of cortisol and its downstream genes in the H-P-I axis of rare minnows (Gobiocypris rarus) are disrupted by acute waterborne cadmium exposure.

The H (hypothalamic)-P (pituitary)-I (interrenal) axis plays a critical role in the fish stress response and is regulated by several factors. Cadmium (Cd) is one of the most toxic heavy metals in the world, but its effects on the H-P-I axis of teleosts are largely unknown. Using rare minnow (Gobiocypris rarus) as an experimental animal, we found that Cd only disrupted the secretion and synthesis of cortisol. Neither hormones at the H or P level nor the expressions of their receptor genes (corticotropin-releasing hormone receptor (CRHR) and melanocortin receptor 2 (MC2R)) were affected. Steroidogenic acute regulator (StAR), CYP11A1 and CYP11B1, which encode the key enzymes in the cortisol synthesis pathway, were significantly up-regulated in the kidney (including the head kidney). The level of 11ß-HSD2, which is required for the conversion of cortisol to cortisone, was increased in the kidney, intestine, brain, and hepatopancreas, whereas the expression of 11ß-HSD1, which encodes the reverse conversion enzyme, was increased in the gill, kidney and almost unchanged in other tissues. The enzyme activity concentration of 11ß-HSD2 was increased in the kidney as well. The level of glucocorticoid receptor (GR) decreased in the intestine, gill and muscle, and the key GR regulator FK506 binding protein5 (FKBP5) was up-regulated in the GR-decreased tissues, whereas the level of nuclear receptor co-repressor 1 (NCoR1), another GR regulator remained almost unchanged. Thus, GR, FKBP5 and 11ß-HSD2 may be involved in Cd-induced cortisol disruption.

Glucocorticoid Sensitivity Is Highly Variable in Critically Ill Patients With Septic Shock and Is Associated With Disease Severity.

OBJECTIVES: To measure tissue glucocorticoid sensitivity in patients with septic shock and determine its relationship to standard measurements of adrenal function and of outcome. DESIGN: Prospective observational trial. SETTING: Teaching hospital ICU. SUBJECTS: Forty-one patients and 20 controls were studied. INTERVENTIONS: Glucocorticoid sensitivity was measured by in vitro suppression of cytokine production from lipopolysaccharide-stimulated leukocytes. MEASUREMENTS AND MAIN RESULTS: There was no significant difference between the groups in the relative suppression of cytokine production, although there was a greater range and variance in the patient data. Patients in the lowest quartile of glucocorticoid sensitivity had higher Acute Physiology and Chronic Health Evaluation II scores (25 [24-28] vs 20 [14-23]; p = 0.02) and a trend toward higher mortality (30% vs 0%; p = 0.2) compared to those in the highest. The mRNA expression of the ß variant of the glucocorticoid receptor and the 11-ß hydroxysteroid dehydrogenase 2 isozyme were significantly higher in patients compared to controls (8.6-fold, p = 0.002 and 10.1-fold, p = 0.0002, respectively). Changes in mRNA expression of these genes did not correlate with measurements of glucocorticoid sensitivity. CONCLUSIONS: Patients with septic shock and controls do not differ in their median glucocorticoid sensitivity. However, patients exhibited a greater variability in glucocorticoid responsiveness and had evidence of association between increased sickness sensitivity and reduced glucocorticoid sensitivity. Sensitivity to glucocorticoids did not appear to be mediated by changes in the expression of the ß variant of the glucocorticoid receptor or the 11-ß hydroxysteroid dehydrogenase 2 isozyme.