Human Adrenal H295R Cells Research Articles

Essential oil metabolites can regulate adrenal androgen production by inhibition of CYP17A1 activities

Endocrine‐disrupting chemicals (EDCs) can affect human steroid metabolism. Previous clinical case reports have shown that some Essential Oils (EOs) like lavender oil and tea tree oil may act as potential EDCs and are linked to prepubertal gynecomastia in boys and premature thelarche in girls due to regular exposure to lavender based fragrances among Hispanic population. These studies suggested role of EOs in steroid metabolism in humans. We have screened a range of EO metabolites for effects on androgen production by CYP17A1.For preliminary screening, human adrenal NCI H295R cells were treated with 10 µM of test compounds for 24 hours. The test compounds had been extracted and purified from natural resources and are found as major components in EOs. For CYP17A1 activity, the conversion of radiolabelled substrate, 17‐Hydroxy‐pregnenolone to Dehydroepiandrosterone was determined using tritiated water release assay.Eucalyptol, Dihydro‐β‐Ionone, (‐)‐α‐pinene were extracted from eucalyptus, rose and pine resin. Out of about 50 test compounds, eucalyptol, Dihydro‐β‐Ionone & (‐)‐α‐pinene showed 20% to 40% inhibition of DHEA production. Rest of the compounds showed either no or low inhibition. Some compounds were also tested for effects on CYP19A1 (aromatase) activity where upto 30% inhibition was observed.EOs are often used in various beauty and hygiene products as they have few known side‐effects. However, prolonged exposure to these products may result in steroid imbalance. Due to their anti‐androgenic activity, these compounds should be studied further as chemical leads for the treatment of hyperandrogenic disorders such as Prostate cancer and Polycystic ovary syndrome.

Comparative proteomic analysis to identify the novel target gene of angiotensin II in adrenocortical H295R cells.

Angiotensin II (Ang II) is a well-known peptide that maintains the balance of electrolytes in the higher vertebrates. Ang II stimulation in the adrenal gland induces the synthesis of mineralocorticoids, mainly aldosterone, through the up-regulation of aldosterone synthase (CYP11B2) gene expression. Additionally, it has been reported that Ang II activates multiple signaling pathways such as mitogen-activated protein kinase (MAPK) and Ca2+ signaling. Although Ang II has various effects on the cellular signaling in the adrenal cells, its biological significance, except for the aldosterone synthesis, is still unclear. In this study, we attempted to search the novel target gene(s) of Ang II in the human adrenal H295R cells using a proteomic approach combined with stable isotopic labeling using amino acid in cell culture (SILAC). Interestingly, we found that Ang II stimulation elevated the expression of phosphofructokinase type platelet (PFKP) in both protein and mRNA levels. Moreover, transactivation of PFKP by Ang II was dependent on extracellular-signal-regulated kinase (ERK) 1/2 activation. Finally, we observed that Ang II treatment facilitated glucose uptake in the H295R cells. Taken together, we here identified PFKP as a novel target gene of Ang II, indicating that Ang II not only stimulates steroidogenesis but also affects glucose metabolism.

HIV Drug Efavirenz Inhibits CYP21A2 Activity with Possible Clinical Implications

Background: The HIV drugs lopinavir and ritonavir have recently been reported to cause transient adrenal insufficiency in preterm newborns. We, therefore, considered HIV drugs as a cause of transiently elevated 17-hydroxyprogesterone (17OHP) levels in a neonatal screening test for congenital adrenal hyperplasia in a preterm girl exposed to zidovudine, efavirenz, tenofovir, and emtricitabine. Objective: So far, HIV drugs have not been tested for their effect on steroidogenesis and the steroidogenic enzyme activity of CYP21A2 specifically in an in vitro system. Methods: We tested the effect of efavirenz, tenofovir, emtricitabine, and zidovudine on steroidogenesis of human adrenal H295R cells. Cells were treated with the drugs at different concentrations including concentrations in therapeutic use. The effect on CYP21A2 activity was assessed by testing the conversion of radiolabeled 17OHP to 11-deoxycortisol. Cell viability was tested by an MTT assay. In addition, recombinant human CYP21A2 protein was used to assess direct drug effects on CYP21A2 activity. Results: We observed significantly decreased CYP21A2 activity in both in vitro testing systems after treatment with efavirenz at therapeutic concentrations. Moreover, efavirenz affected cell viability. By contrast, the other test drugs did not affect steroidogenesis. Follow-up of our patient revealed elevated 17OHP and androgen levels during the first weeks of life, but values normalized spontaneously. Genetic testing for CYP21A2 mutations was negative. Thus, it remains unsettled whether the transient 17OHP elevation in this baby was due to a drug effect. Conclusion: The HIV drug efavirenz inhibits CYP21A2 activity in vitro through direct interaction with enzyme catalysis at therapeutic concentrations. This may have clinical implications for HIV treatment in children and adults. However, so far, clinical data are scarce, and further studies are needed to be able to draw clinical conclusions.

Endocrine disruption by several aniline derivatives and related mechanisms in a human adrenal H295R cell line and adult male zebrafish

Aniline and aniline derivatives have been widely used in the production of pesticides, pharmaceuticals, cosmetic, dyes, rubber, and adhesives products. These chemicals can easily be released into the environment through industrial and municipal discharges or as degradation byproducts. Several studies have suggested that aniline and some of its derivatives could cause reproductive toxicity in aquatic organisms. However, knowledge on the endocrine disruption potentials of these chemicals is limited only to aniline and associated mechanisms are rarely investigated. The objective of this study was to investigate the potential of major aniline derivatives, i.e., 3,4-dichloroaniline (3,4-DCA), 1-naphthylamine (1-NPA), and 4,4′-methylenedianiline (4,4′-MDA), to disrupt sex steroid production and other biological processes. For this purpose, the human adrenal H295R cell line and adult male zebrafish (Danio rerio) were used. In the H295R cell line, all tested aniline derivatives decreased testosterone (T) levels. Regulatory changes of several steroidogenic genes, i.e., down-regulation of StAR or CYP17 genes, and up-regulation of CYP19A, observed in the H295R cells could explain the sex hormone disruption. In male zebrafish, generally similar directions of changes, i.e., decreases in T levels and increased E2/T ratios, were observed. Again, down-regulation of key steroidogenic genes such as cyp17 or 3β-hsd, but slight up-regulation of cyp19a gene observed in the fish could explain the sex hormone changes. The results of our study demonstrate that all tested aniline derivatives could influence steroidogenesis and disrupt sex hormone balance toward reduced androgenicity. Consequences of anti-androgenicity following long-term exposure warrant further investigation.

Identification of the fungicide epoxiconazole by virtual screening and biological assessment as inhibitor of human 11β-hydroxylase and aldosterone synthase

Humans are constantly exposed to a multitude of environmental chemicals that may disturb endocrine functions. It is crucial to identify such chemicals and uncover their mode-of-action to avoid adverse health effects. 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) catalyze the formation of cortisol and aldosterone, respectively, in the adrenal cortex. Disruption of their synthesis by exogenous chemicals can contribute to cardio-metabolic diseases, chronic kidney disease, osteoporosis, and immune-related disorders. This study applied in silico screening and in vitro evaluation for the discovery of xenobiotics inhibiting CYP11B1 and CYP11B2. Several databases comprising environmentally relevant pollutants, chemicals in body care products, food additives and drugs were virtually screened using CYP11B1 and CYP11B2 pharmacophore models. A first round of biological testing used hamster cells overexpressing human CYP11B1 or CYP11B2 to analyze 25 selected virtual hits. Three compounds inhibited CYP11B1 and CYP11B2 with IC50 values below 3 μM. The most potent inhibitor was epoxiconazole (IC50 value of 623 nM for CYP11B1 and 113 nM for CYP11B2, respectively); flurprimidol and ancymidol were moderate inhibitors. In a second round, these three compounds were tested in human adrenal H295R cells endogenously expressing CYP11B1 and CYP11B2, confirming the potent inhibition by epoxiconazole and the more moderate effects by flurprimidol and ancymidol. Thus, the in silico screening, prioritization of chemicals for initial biological tests and use of H295R cells to provide initial mechanistic information is a promising strategy to identify potential endocrine disruptors inhibiting corticosteroid synthesis. A critical assessment of human exposure levels and in vivo evaluation of potential corticosteroid disrupting effects by epoxiconazole is required.

High glucose stimulates expression of aldosterone synthase (CYP11B2) and secretion of aldosterone in human adrenal cells.

Aldosterone synthase is the key rate‐limiting enzyme in adrenal aldosterone production, and induction of its gene (CYP11B2) results in the progression of hypertension. As hypertension is a frequent complication among patients with diabetes, we set out to elucidate the link between diabetes mellitus and hypertension. We examined the effects of high glucose on CYP11B2 expression and aldosterone production using human adrenal H295R cells and a stable H295R cell line expressing a CYP11B2 5′‐flanking region/luciferase cDNA chimeric construct. d‐glucose (d‐glu), but not its enantiomer l‐glucose, dose dependently induced CYP11B2 transcription and mRNA expression. A high concentration (450 mg·dL−1) of d‐glu time dependently induced CYP11B2 transcription and mRNA expression. Moreover, high glucose stimulated secretion of aldosterone into the media. Transient transfection studies using deletion mutants/nerve growth factor‐induced clone B (NGFIB) response element 1 (NBRE‐1) point mutant of CYP11B2 5′‐flanking region revealed that the NBRE‐1 element, known to be activated by transcription factors NGFIB and NURR1, was responsible for the high glucose‐mediated effect. High glucose also induced the mRNA expression of these transcription factors, especially that of NURR1, but NURR1 knockdown using its siRNA did not affect high glucose‐induced CYP11B2 mRNA expression. Taken together, it is speculated that high glucose may induce CYP11B2 transcription via the NBRE‐1 element in its 5′‐flanking region, resulting in the increase in aldosterone production although high glucose‐induced NURR1 is not directly involved in the effect. Additionally, glucose metabolism and calcium channels were found to be involved in the high glucose effect. Our observations suggest one possible explanation for the high incidence of hypertension in patients with diabetes.

Combined transcriptome and metabolome analyses of metformin effects reveal novel links between metabolic networks in steroidogenic systems

Metformin is an antidiabetic drug, which inhibits mitochondrial respiratory-chain-complex I and thereby seems to affect the cellular metabolism in many ways. It is also used for the treatment of the polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. In addition, metformin possesses antineoplastic properties. Although metformin promotes insulin-sensitivity and ameliorates reproductive abnormalities in PCOS, its exact mechanisms of action remain elusive. Therefore, we studied the transcriptome and the metabolome of metformin in human adrenal H295R cells. Microarray analysis revealed changes in 693 genes after metformin treatment. Using high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS-NMR), we determined 38 intracellular metabolites. With bioinformatic tools we created an integrated pathway analysis to understand different intracellular processes targeted by metformin. Combined metabolomics and transcriptomics data analysis showed that metformin affects a broad range of cellular processes centered on the mitochondrium. Data confirmed several known effects of metformin on glucose and androgen metabolism, which had been identified in clinical and basic studies previously. But more importantly, novel links between the energy metabolism, sex steroid biosynthesis, the cell cycle and the immune system were identified. These omics studies shed light on a complex interplay between metabolic pathways in steroidogenic systems.

Incorporation of 14C-cholesterol in human adrenal corticocarcinoma H295R cell line and online-radiodetection of produced 14C-steroid hormone metabolites

This study demonstrates the addition of 14C-cholesterol to the human cell line H295R will in-situ form radiolabeled steroid hormones allowing for new mechanistic and metabolic insights. The aim of the present study was to in-situ radiolabel steroid hormones from cell line-incorporated 14C-cholesterol using the OECD guideline 456, H295R steroidogenesis in-vitro assay. Radiodetection of the steroid metabolites of the steroidogenic pathway allows for an improved understanding of the various enzymatic mechanisms involved without necessarily being dependent on quantification. Generated radiolabeled steroids were analyzed using HPLC hyphenated with a Flow Scintillation Analyzer (FSA). H295R cells were incubated with radiolabeled cholesterol and cell media were collected and prepared by solid phase extraction and analyzed with HPLC-FSA. For successful radiolabeling of the steroids in the steroidogenesis of H295R cells, radioactive cholesterol may potentially only need to be added just before the cells are incubated for 72h in well plates. Based on the obtained HPLC-FSA chromatograms, and confirmation of the observations by studies in the literature, a qualitative time profile for the production of steroid hormones was estimated. Multiple radiolabeled steroid hormones were identified by means of analytical standards and UV (ultraviolet) co-chromatography, though the elucidation of multiple metabolites remains unresolved. Although online radiodetection proved to suffer from suboptimal sensitivity, the concept of radiolabeling the steroidogenesis in H295R cells with 14C-cholesterol and detecting the radiolabeled steroid hormones online was proved and may assist in further toxicological studies.

Resveratrol inhibits androgen production of human adrenocortical H295R cells by lowering CYP17 and CYP21 expression and activities.

Resveratrol, a natural compound found in grapes, became very popular for its suggested protective effects against aging. It was reported to have similar positive effects on the human metabolism as caloric restriction. Recently, positive effects of resveratrol on steroid biosynthesis in cell systems and in humans suffering from polycystic ovary syndrome have also been reported, but the exact mechanism of this action remains unknown. Sirtuins seem targeted by resveratrol to mediate its action on energy homeostasis. In this study, we investigated the mechanisms of action of resveratrol on steroidogenesis in human adrenal H295R cells. Resveratrol was found to inhibit protein expression and enzyme activities of CYP17 and CYP21. It did not alter CYP17 and CYP21 mRNA expression, nor protein degradation. Only SIRT3 mRNA expression was found to be altered by resveratrol, but SIRT1, 3 and 5 overexpression did not result in a change in the steroid profile of H295R cells, indicating that resveratrol may not engage sirtuins to modulate steroid production. Previous studies showed that starvation leads to a hyperandrogenic steroid profile in H295R cells through inhibition of PKB/Akt signaling, and that resveratrol inhibits steroidogenesis of rat ovarian theca cells via the PKB/Akt pathway. Therefore, the effect of resveratrol on PKB/Akt signaling was tested in H295R cells and was found to be decreased under starvation growth conditions, but not under normal growth conditions. Overall, these properties of action together with recent clinical findings make resveratrol a candidate for the treatment of hyperandrogenic disorders such as PCOS.

Steroid profiling in H295R cells to identify chemicals potentially disrupting the production of adrenal steroids.

The validated OECD test guideline 456 based on human adrenal H295R cells promotes measurement of testosterone and estradiol production as read-out to identify potential endocrine disrupting chemicals. This study aimed to establish optimal conditions for using H295R cells to detect chemicals interfering with the production of key adrenal steroids. H295R cells' supernatants were characterized by liquid chromatography-mass spectrometry (LC-MS)-based steroid profiling, and the influence of experimental conditions including time and serum content was assessed. Steroid profiles were determined before and after incubation with reference compounds and chemicals to be tested for potential disruption of adrenal steroidogenesis. The H295R cells cultivated according to the OECD test guideline produced progestins, glucocorticoids, mineralocorticoids and adrenal androgens but only very low amounts of testosterone. However, testosterone contained in Nu-serum was metabolized during the 48h incubation. Thus, inclusion of positive and negative controls and a steroid profile of the complete medium prior to the experiment (t=0h) was necessary to characterize H295R cells' steroid production and indicate alterations caused by exposure to chemicals. Among the tested chemicals, octyl methoxycinnamate and acetyl tributylcitrate resembled the corticosteroid induction pattern of the positive control torcetrapib. Gene expression analysis revealed that octyl methoxycinnamate and acetyl tributylcitrate enhanced CYP11B2 expression, although less pronounced than torcetrapib. Further experiments need to assess the toxicological relevance of octyl methoxycinnamate- and acetyl tributylcitrate-induced corticosteroid production. In conclusion, the extended profiling and appropriate controls allow detecting chemicals that act on steroidogenesis and provide initial mechanistic evidence for prioritizing chemicals for further investigations.

Exposure to an Extremely-Low-Frequency Magnetic Field Stimulates Adrenal Steroidogenesis via Inhibition of Phosphodiesterase Activity in a Mouse Adrenal Cell Line.

Extremely low-frequency magnetic fields (ELF-MFs) are generated by power lines and household electrical devices. In the last several decades, some evidence has shown an association between ELF-MF exposure and depression and/or anxiety in epidemiological and animal studies. The mechanism underlying ELF-MF-induced depression is considered to involve adrenal steroidogenesis, which is triggered by ELF-MF exposure. However, how ELF-MFs stimulate adrenal steroidogenesis is controversial. In the current study, we investigated the effect of ELF-MF exposure on the mouse adrenal cortex-derived Y-1 cell line and the human adrenal cortex-derived H295R cell line to clarify whether the ELF-MF stimulates adrenal steroidogenesis directly. ELF-MF exposure was found to significantly stimulate adrenal steroidogenesis (p < 0.01–0.05) and the expression of adrenal steroid synthetic enzymes (p < 0.05) in Y-1 cells, but the effect was weak in H295R cells. Y-1 cells exposed to an ELF-MF showed significant decreases in phosphodiesterase activity (p < 0.05) and intracellular Ca2+ concentration (p < 0.01) and significant increases in intracellular cyclic adenosine monophosphate (cAMP) concentration (p < 0.001–0.05) and cAMP response element-binding protein phosphorylation (p < 0.05). The increase in cAMP was not inhibited by treatment with NF449, an inhibitor of the Gs alpha subunit of G protein. Our results suggest that ELF-MF exposure stimulates adrenal steroidogenesis via an increase in intracellular cAMP caused by the inhibition of phosphodiesterase activity in Y-1 cells. The same mechanism may trigger the increase in adrenal steroid secretion in mice observed in our previous study.

Computational analysis of liquid chromatography-tandem mass spectrometric steroid profiling in NCI H295R cells following angiotensin II, forskolin and abiraterone treatment

Adrenal steroid hormones, which regulate a plethora of physiological functions, are produced via tightly controlled pathways. Investigations of these pathways, based on experimental data, can be facilitated by computational modeling for calculations of metabolic rate alterations. We therefore used a model system, based on mass balance and mass reaction equations, to kinetically evaluate adrenal steroidogenesis in human adrenal cortex-derived NCI H295R cells. For this purpose a panel of 10 steroids was measured by liquid chromatographic-tandem mass spectrometry. Time-dependent changes in cell incubate concentrations of steroids – including cortisol, aldosterone, dehydroepiandrosterone and their precursors – were measured after incubation with angiotensin II, forskolin and abiraterone. Model parameters were estimated based on experimental data using weighted least square fitting. Time-dependent angiotensin II- and forskolin-induced changes were observed for incubate concentrations of precursor steroids with peaks that preceded maximal increases in aldosterone and cortisol. Inhibition of 17-alpha-hydroxylase/17,20-lyase with abiraterone resulted in increases in upstream precursor steroids and decreases in downstream products. Derived model parameters, including rate constants of enzymatic processes, appropriately quantified observed and expected changes in metabolic pathways at multiple conversion steps. Our data demonstrate limitations of single time point measurements and the importance of assessing pathway dynamics in studies of adrenal cortical cell line steroidogenesis. Our analysis provides a framework for evaluation of steroidogenesis in adrenal cortical cell culture systems and demonstrates that computational modeling-derived estimates of kinetic parameters are an effective tool for describing perturbations in associated metabolic pathways.

Role for Germline Mutations and a Rare Coding Single Nucleotide Polymorphism Within the KCNJ5 Potassium Channel in a Large Cohort of Sporadic Cases of Primary Aldosteronism

Primary aldosteronism (autonomous aldosterone production with suppressed renin) plays an important pathophysiological role in what has been previously labeled as essential hypertension. Besides the recently described germline mutations in the KCNJ5 potassium channel associated with familial primary aldosteronism, somatic mutations in the same channel have been identified within aldosterone-producing adenomas. In this study, we have resequenced the flanking and coding region of KCNJ5 in peripheral blood DNA from 251 white subjects with primary aldosteronism to look for rare variants that might be important for the pathophysiology of sporadic primary aldosteronism. We have identified 3 heterozygous missense mutations (R52H, E246K, and G247R) in the cohort and found that 12 (5% of the cohort) were carriers for the rare nonsynonymous single nucleotide polymorphism rs7102584 causing E282Q substitution of KCNJ5. By expressing the channels in Xenopus oocytes and human adrenal H295R cells, we have shown that the R52H, E246K, and E282Q substitutions are functional, but the G247R mutation is indistinguishable from wild type. Although the functional substitutions are remote from the selectivity filter, they affect the inward-rectification, the ability of the KCNJ5 channels to conduct Na(+) currents and ATII-induced aldosterone release from the H295R cell line. Together these data suggest that germline variation in the KCNJ5 gene has a role to play in the common sporadic form as well as the much rarer syndromic forms of primary aldosteronism.

Angiotensin II receptor blockers differentially affect CYP11B2 expression in human adrenal H295R cells

We generated a stable H295R cell line expressing aldosterone synthase gene (CYP11B2) promoter/luciferase chimeric reporter construct that is highly sensitive to angiotensin II (AII) and potassium, and defined AII receptor blocker (ARB) effects. In the presence of AII, all ARBs suppressed AII-induced CYP11B2 transcription. However, telmisartan alone increased CYP11B2 transcription in the absence of AII. Telmisartan dose-dependently increased CYP11B2 transcription/mRNA expression and aldosterone secretion. Experiments using CYP11B2 promoter mutants indicated that the Ad5 element was responsible. Among transcription factors involved in the element, telmisartan significantly induced NGFIB/NURR1 expression. KN-93, a CaMK inhibitor, abrogated the telmisartan-mediated increase of CYP11B2 transcription/mRNA expression and NURR1 mRNA expression, but not NGFIB mRNA expression. NURR1 over-expression significantly augmented the telmisartan-mediated CYP11B2 transcription, while high-dose olmesartan did not affect it. Taken together, telmisartan may stimulate CYP11B2 transcription via NGFIB and the CaMK-mediated induction of NURR1 that activates the Ad5 element, independent of AII type 1 receptor.

Abstract 2049: A novel, potent, Cyp17 inhibitor with favorable pharmacokinetics and oral bioavailability for potential treatment of human castration resistant prostate cancer (CRPC).

Abstract Cytochrome P450 17α-hydroxylase/C17,20-lyase (CYP17) is a key enzyme in androgen bio-synthesis in adrenals and testes. It has been validated clinically as an effective target for CRPC through successful clinical trials with abiraterone acetate. Abiraterone acetate is a selective, potent inhibitor of Cyp17, and was recently approved as a treatment for CRPC. We have designed a novel Cyp17 inhibitor, BMN680, with improved pharmacokinetic (PK) profile and oral bioavailability while maintaining potent Cyp17 inhibition. BMN680 inhibits Cyp17 lyase activity with IC50 of 0.8 nM and reduces testosterone production in human adrenal cancer cell line H295R cells with IC50 of 3.87 nM. in vitro metabolic stability studies indicate that BMN680 has a half-life of 5-hr in human liver microsome and 8-hr half-life in human hepatocytes. Animal PK profiling in rats and monkeys demonstrated BMN680 has in vivo half-life of &amp;gt;4 hr and oral bioavailability of &amp;gt;30%. Single oral dosing of BMN680 at 10 mg/kg in normal, intact, Cynomolgus monkeys resulted in suppression of plasma testosterone level for up to 24 hour. Pharmacodynamic (PD) effect of single and multi-day oral dosing of BMN680 in monkey will be presented. In human liver microsome Cyp panel screen, BMN680 exhibited IC50 greater than 1.5 uM for all Cyp enzymes tested including 1A2, 2C9, 2C19, 2D6, 3A4, indicating low potential of drug-drug interaction. In conclusion, BMN680 is a potent Cyp17 inhibitor with low interaction with Cyp450 isoforms, and desirable PK and oral bioavailability. BMN680 can effectively suppress testosterone production in vivo, and will be further evaluated in preclinical development. Citation Format: Ying Feng, Bing Wang, Daniel Chu, Leonard Post, Yuqiao Shen. A novel, potent, Cyp17 inhibitor with favorable pharmacokinetics and oral bioavailability for potential treatment of human castration resistant prostate cancer (CRPC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2049. doi:10.1158/1538-7445.AM2013-2049

Human adrenal cells that express both 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) contribute to adrenal androstenedione production

Androstenedione is one of several weak androgens produced in the human adrenal gland. 3β-Hydroxysteroid dehydrogenase type 2 (HSD3B2) and cytochrome b5 (CYB5A) are both required for androstenedione production. However, previous studies demonstrated the expression of HSD3B2 within the zona glomerulosa (ZG) and fasciculata (ZF) but low levels in the zona reticularis. In contrast, CYB5A expression increases in the zona reticularis (ZR) in human adrenal glands. Although their colocalization has been reported in gonadal theca and Leydig cells this has not been studied in the human adrenal. Therefore, we immonolocalized HSD3B2 and CYB5A in normal human adrenal glands and first demonstrated their co-expression in the cortical cells located at the border between the ZF and ZR in normal human adrenal. Results of in vitro studies using the human adrenal H295R cells treated with the HSD3B2 inhibitor, trilostane, also demonstrated a markedly decreased androstenedione production. Decreasing CYB5A mRNA using its corresponding siRNA also resulted in significant inhibition of androstenedione production in the H295R cells. These findings together indicate that there are a group of cells co-expressing HSD3B2 and CYB5A with hybrid features of both ZF and ZR in human adrenal cortex, and these hybrid cortical cells may play an important role in androstenedione production in human adrenal gland.

Impact of Differential P450c17 Phosphorylation by cAMP Stimulation and by Starvation Conditions on Enzyme Activities and Androgen Production in NCI-H295R Cells

CYP17A1 plays a pivotal role in the biosynthesis of androgens in the adrenals and the gonads. Although this enzyme catalyzes two different reactions on one single active site, its specific activities are regulated independently. Although the 17alpha-hydroxylase activity is rather constant and regulated by gene expression, the 17,20-lyase activity varies significantly with the amount of cofactors or by protein phosphorylation. cAMP increases CYP17A1 expression, P450c17 phosphorylation, and androgen production. However, the exact mechanism(s) and the specific regulators of CYP17A1 remain unknown. Therefore, we studied the regulation of adrenal androgen biosynthesis in human adrenal H295R cells focusing on CYP17A1. We analyzed androgen production and P450c17 activities in H295R cells grown under normal and serum-free conditions and/or after stimulation with 8-bromoadenosine-cAMP. H295R cells grown in starvation medium produced more androgens and had decreased HSD3B2 expression and activity but increased P450c17-17,20-lyase activity and serine phosphorylation. Although starvation increased serine phosphorylation of P450c17 specifically, cAMP stimulation enhanced threonine phosphorylation exclusively. Time-course experiments revealed that a short cAMP stimulation augmented threonine phosphorylation of P450c17 but did not increase 17,20-lyase activity. By contrast, long cAMP stimulation increased androgen production through increased P450c17 activities by enhancing CYP17A1 gene expression. We conclude that serum withdrawal shifts steroidogenesis of H295R cells towards androgen production, providing a suitable model for detailed studies of androgen regulation. In addition, our study shows that starvation and cAMP stimulation regulate P450c17 phosphorylation differentially and that an increase in P450c17 phosphorylation does not necessarily lead to enhanced enzyme activity and androgen production.

Differential regulation and roles of urocortins in human adrenal H295R cells

Three urocortins (Ucns) are known as members of the corticotropin-releasing factor (CRF) family of peptides and serve as natural ligands for CRF receptors. Ucn1 and Ucn3 exhibit potent effects on the adrenal system via the CRF receptors. This study aimed to explore the regulation and roles of Ucns in the adrenal system using human adrenal carcinoma H295R cells, which express Ucn1, Ucn2, Ucn3, CRF receptor type 1 (CRF 1 receptor), and CRF receptor type 2a (CRF 2a receptor) mRNA. Forskolin, which stimulates adenylate cyclase and then increases intracellular cAMP production, was shown to transiently decrease Ucn1 and Ucn2 mRNA levels, but increase Ucns 1–3 mRNA levels in H295R cells. Steroidogenic acute regulatory protein, Cyp11β1, and Cyp11β2 mRNA levels, and both cortisol and aldosterone secretions were elevated by Ucn1. Cell viability was reduced by both Ucn1 and Ucn3 via the CRF 2 receptor in H295R cells. Ucn1 and Ucn3 increased the expression of the cAMP-response element binding protein and extracellular signal-related kinase (ERK) phosphorylations. The ERK and protein kinase A pathways were involved in Ucn3-decreased cell viability.

A biphasic effect of the fungicide prochloraz on aldosterone, but not cortisol, secretion in human adrenal H295R cells—Underlying mechanisms

The widely used imidazole fungicide prochloraz displays anti-androgenic effects partly via inhibition of testicular steroidogenesis and testosterone secretion. Adrenal steroidogenesis and hormone secretion may also be a target of this endocrine disruptor. Herein, we demonstrate a dose-dependent inhibition of cortisol secretion and a biphasic effect on aldosterone secretion, with a 2-fold stimulation at low concentrations and a strong inhibition at high concentrations, following prochloraz treatment (0–10μM) of human adrenocortical H295R cells. Analysis of the dose-dependent effects of prochloraz on the secretion of steroidogenic intermediates suggested that the observed effects on cortisol and aldosterone secretion might be mediated by inhibition of the steroidogenic steps catalysed by CYP17A1 and CYP21A2. The inhibition of CYP17A1 was reflected on the level of expression of steroidogenic genes as analysed by quantitative RT-PCR. In addition, analysis of enzyme activity showed a dose-dependent inhibitory effect of prochloraz on the activity of CYP17A1 and CYP21A2, but not CYP11B1. We have demonstrated specific effects of prochloraz on adrenal steroidogenic pathways and hormone secretion via inhibition of steroidogenic CYP enzymes. The disruption of adrenal hormone secretion may result in altered endocrine homeostasis and affect human health.

Neuropeptide Y modulates steroid production of human adrenal H295R cells through Y1 receptors

Neuropeptide Y (NPY) is abundantly expressed in the nervous system and acts on target cells through NPY receptors. The human adrenal cortex and adrenal tumors express NPY receptor subtype Y1, but its function is unknown. We studied Y1-mediated signaling, steroidogenesis and cell proliferation in human adrenal NCI-H295R cells. Radioactive ligand binding studies showed that H295R cells express Y1 receptor specifically. NPY treatment of H295R cells stimulated the MEK/ERK1/2 pathway, confirming that H295R cells express functional Y1 receptors. Studies of the effect of NPY and related peptide PYY on adrenal steroidogenesis revealed a decrease in 11-deoxycortisol production. RIA measurements of cortisol from cell culture medium confirmed this finding. Co-treatment with the Y1 antagonist BIBP2336 reversed the inhibitory effect of NPY on cortisol production proving specificity of this effect. At mRNA level, NPY decreased HSD3B2 and CYP21A2 expression. However NPY revealed no effect on cell proliferation. Our data show that NPY can directly regulate human adrenal cortisol production.