Beclometasone but not fluticasone modulates PDGFD expression in the H295R adrenal cell line.

BackgroundAdrenal suppression is a clinically concerning side effect of inhaled corticosteroid (ICS) treatment in patients with asthma. Increased susceptibility to ICS-induced adrenal suppression has previously been identified in those with the rs591118 polymorphism in Platelet Derived Growth Factor D (PDGFD). The mechanism underpinning this relationship is not known.MethodsH295R cells were genotyped for rs591118 using a validated Taqman PCR allelic discrimination assay. H295R cell viability was determined after treatment with beclometasone and fluticasone (range 0-330 μM). Cortisol was measured in cell culture medium using competitive enzyme immunoassay.ResultsPDGFD protein expression in H295R cells was confirmed using Western blotting. When ACTH and forskolin were added to H295R cells, a reduction in PDGFD expression was seen which was then restored by incubation with prochloraz, a known inhibitor of steroidogenesis.A dose-dependent, decrease in PDGFD expression was observed with beclometasone (over a 24 h incubation period) but not with beclometasone incubations beyond 24 hour nor with fluticasone (at 24 or 48 hours).ConclusionsH295R cells express PDGFD protein which can be modulated by incubation with steroidogenesis agonists and antagonists and additionally with exogenous beclometasone.

Transcriptomic Response Dynamics of Human Primary and Immortalized Adrenocortical Cells to Steroidogenic Stimuli

Adrenal steroid hormone production is a dynamic process stimulated by adrenocorticotropic hormone (ACTH) and angiotensin II (AngII). These ligands initialize a rapid and robust gene expression response required for steroidogenesis. Here, we compare the predominant human immortalized cell line model, H295R cell, with primary cultures of adult adrenocortical cells derived from human kidney donors. We performed temporally resolved RNA-seq on primary cells stimulated with either ACTH or AngII at multiple time points. The magnitude of the expression dynamics elicited by ACTH was greater than AngII in primary cells. This is likely due to the larger population of adrenocortical cells that are responsive to ACTH. The dynamics of stimulus-induced expression in H295R cells are mostly recapitulated in primary cells. However, there are some expression responses in primary cells absent in H295R cells. These data are a resource for the endocrine community and will help researchers determine whether H295R is an appropriate model for the specific aspect of steroidogenesis that they are studying.

Mesenchymal Stem Cell-Conditioned Media Regulate Steroidogenesis and Inhibit Androgen Secretion in a PCOS Cell Model via BMP-2

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women. Previous studies have demonstrated the therapeutic efficacy of human bone marrow mesenchymal stem cells (BM-hMSCs) for PCOS; however, the regulatory mechanism remains unknown. Bone morphogenetic proteins (BMPs) secreted by BM-hMSCs may underlie the therapeutic effect of these cells on PCOS, based on the ability of BMPs to modulate androgen production and alter steroidogenesis pathway enzymes. In this study, we analyze the effect of BMP-2 on androgen production and steroidogenic pathway enzymes in H295R cells as a human PCOS in vitro cell model. In H295R cells, BMP-2 significantly suppressed cell proliferation, androgen production, and expression of androgen-synthesizing genes, as well as inflammatory gene expression. Furthermore, H295R cells treated with the BM-hMSCs secretome in the presence of neutralizing BMP-2 antibody or with BMP-2 gene knockdown showed augmented expression of androgen-producing genes. Taken together, these results indicate that BMP-2 is a key player mediating the favorable effects of the BM-hMSCs secretome in a human PCOS cell model. BMP-2 overexpression could increase the efficacy of BM-hMSC-based therapy, serving as a novel stem cell therapy for patients with intractable PCOS.

Somatic Mutations of GNA11 and GNAQ in CTNNB1-Mutant Aldosterone-Producing Adenomas Increases Aldosterone and Aldosterone Synthase (CYP11B2)

Most aldosterone-producing adenomas (APA) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell-clusters of normal adrenals suggests the existence of co-driver mutations which influence the development or phenotype of APAs. Gain-of-function mutations in both CTNNB1 and GNA11 were found by whole exome sequencing in 3 of 41 APAs from a UK/Irish cohort. Targeted sequencing for exon 3 mutations of CTNNB1 and p.Gln209 mutations of either GNA11 or closely homologous GNAQ confirmed these and 7 further double mutant APAs in this discovery cohort. The presence of GNA11/Q p.Gln209 mutations in CTNNB1 mutant APAs were replicated in 2 cohorts from France (n=14) and Sweden (n=3). In total, 16 (59%) of the 27 CTNNB1 mutant APAs investigated had a mutation at p.Gln209 of GNA11 (n=11) or GNAQ (n=5). Interestingly, CTNNB1-mutant APAs were more commonly present in women (23/27), and of these, those with GNA11/Q mutations were all women except for a pubertal boy. To also note, 9 of 10 of the UK/Irish double mutant APAs in the discovery cohort presented in puberty, pregnancy, or menopause. Mutation of p.Gln209, or homologous p.Gln in GNAS, GNA12-14, impair hydrogen bonds between G-protein α and β subunits. Transfection of H295R cells, an immortalised adrenocortical cell line heterozygous for the p.Ser45Pro mutation of CTNNB1 but wild-type for GNA11-14/Q/S, by each of the GNA11/Q mutations increased aldosterone secretion and CYP11B2 expression (encoding aldosterone synthase) by 1.93-6.1-fold and 8.0-9.8-fold respectively, compared to vector or wild-type -transfected cells. In ZG, GNA11/Q mediate the aldosterone response to angiotensin II, via stimulation of intracellular Ca2+ release by inositol trisphosphate. In the mutant-transfected cells, the stimulatory effect of angiotensin II 10 nM was retained. In order to determine whether the p.Gln209 mutations stimulate aldosterone production even in the absence of CTNNB1 activation, the transfections of H295R cells were repeated after either 24-h treatment with the CTNNB1 inhibitor, ICG-001, or silencing of CTNNB1 using the ONTARGETplus SMARTpool SiRNAs (Dharmacon). Both interventions reduced the aldosterone production relative to vehicle/control-treated cells; however neither ICG-001 nor silencing of CTNNB1 blunted the fold-increase in aldosterone secretion seen in mutant-transfected cells compared to wild-type. In summary, we report the discovery of gain-of-function mutations of the G-protein, GNA11, or its close homologue, GNAQ, in multiple APAs which majority presented during periods of high LH/HCG. To date, the mutation is always residue p.Gln209, and associated with a gain-of-function mutation of CTNNB1. These GNA11/Q p.Gln209 mutations increase aldosterone and CYP11B2 production both in the presence and in the absence of CTNNB1 activation.

Cytotoxic Effect of Progesterone, Tamoxifen and Their Combination in Experimental Cell Models of Human Adrenocortical Cancer

Progesterone (Pg) and estrogen (E) receptors (PgRs and ERs) are expressed in normal and neoplastic adrenal cortex, but their role is not fully understood. In literature, Pg demonstrated cytotoxic activity on AdrenoCortical Carcinoma (ACC) cells, while tamoxifen is cytotoxic in NCI-H295R cells. Here, we demonstrated that in ACC cell models, ERs were expressed in NCI-H295R cells with a prevalence of ER-β over the ER-α.Metastasis-derived MUC-1 and ACC115m cells displayed a very weak ER-α/β signal, while PgR cells were expressed, although at low level. Accordingly, these latter were resistant to the SERM tamoxifen and scarcely sensitive to Pg, as we observed a lower potency compared to NCI-H295R cells in cytotoxicity (IC50: MUC-1 cells: 67.58 µM (95%CI: 63.22–73.04), ACC115m cells: 51.76 µM (95%CI: 46.45–57.67) and cell proliferation rate. Exposure of NCI-H295R cells to tamoxifen induced cytotoxicity (IC50: 5.43 µM (95%CI: 5.18–5.69 µM) mainly involving ER-β, as their nuclear localization increased after tamoxifen: Δ A.U. treated vs untreated: 12 h: +27.04% (p < 0.01); 24 h: +36.46% (p < 0.0001). This effect involved the SF-1 protein reduction: Pg: −36.34 ± 9.26%; tamoxifen: −46.25 ± 15.68% (p < 0.01). Finally, in a cohort of 36 ACC samples, immunohistochemistry showed undetectable/low level of ERs, while PgR demonstrated a higher expression. In conclusion, ACC experimental cell models expressed PgR and low levels of ER in line with data obtained in patient tissues, thus limiting the possibility of a clinical approach targeting ER. Interestingly, Pg exerted cytotoxicity also in metastatic ACC cells, although with low potency.

Investigation on Sex Hormone-Disruption Effects of Two Novel Brominated Flame Retardants (DBDPE and BTBPE) in Male Zebrafish (Danio rerio) and Two Human Cell Lines (H295R and MVLN)

Decabromodiphenyl ethane (DBDPE) and 1,2-bis(2,4,6-tribromo-phenoxy) ethane (BTBPE) are novel brominated flame retardants (NBFRs) and have been detected in variety of environment and biota. Although sex endocrine-disrupting potential has been suggested in experimental studies, their adverse effects on sex steroid hormones and underlying mechanisms are largely unclear. The purpose of the present study is to investigate the sex hormone-disrupting effects of two NBFRs using in vivo and in vitro models together. For this, male zebrafish (Danio rerio) along with human adrenocortical carcinoma (H295R) and breast carcinoma (MVLN) cell lines were employed. In male zebrafish, 14-day exposure to DBDPE significantly increased 17β-estradiol (E2) concentrations. Disruption of sex hormone regulation was also suggested after exposure to BTBPE, i.e., the increasing trend of E2 levels, E2/11-ketotestosterone (11-KT) ratio, and estrogen receptor-alpha (erα) and erβ gene expression levels. In H295R cells, an E2/T ratio showed an increasing trend by DBDPE exposure, but transcriptions of major genes in steroidogenesis pathway were not affected. Taken together, our observation implies that two NBFRs could cause the sex hormone disruption potential in male zebrafish and H295R cells but probably not through alteration of steroidogenesis pathway.

Antihypertensive treatment guided by genetics: PEARL-HT, the randomized proof-of-concept trial comparing rostafuroxin with losartan

We compared a standard antihypertensive losartan treatment with a pharmacogenomics-guided rostafuroxin treatment in never-treated Caucasian and Chinese patients with primary hypertension. Rostafuroxin is a digitoxigenin derivative that selectively disrupts the binding to the cSrc-SH2 domain of mutant α-adducin and of the ouabain-activated Na-K pump at 10–11 M. Of 902 patients screened, 172 were enrolled in Italy and 107 in Taiwan. After stratification for country and genetic background, patients were randomized to rostafuroxin or losartan, being the difference in the fall in office systolic blood pressure (OSBP) after 2-month treatment the primary endpoint. Three pharmacogenomic profiles (P) were examined, considering: P1, adding to the gene variants included in the subsequent P2, the variants detected by post-hoc analysis of a previous trial; P2, variants of genes encoding enzymes for endogenous ouabain (EO) synthesis (LSS and HSD3B1), EO transport (MDR1/ABCB1), adducin (ADD1 and ADD3); P3, variants of the LSS gene only. In Caucasians, the group differences (rostafuroxin 50 μg minus losartan 50 mg in OSBP mmHg) were significant both in P2 adjusted for genetic heterogeneity (P2a) and P3 LSS rs2254524 AA [9.8 (0.6–19.0), P = 0.038 and 13.4 (25.4–2.5), P = 0.031, respectively]. In human H295R cells transfected with LSS A and LSS C variants, the EO production was greater in the former (P = 0.038); this difference was abolished by rostafuroxin at 10–11 M. Chinese patients had a similar drop in OSBP to Caucasians with losartan but no change in OSBP with rostafuroxin. These results show that genetics may guide drug treatment for primary hypertension in Caucasians.

Microcystins and Microcystis aeruginosa PCC7806 extracts modulate steroidogenesis differentially in the human H295R adrenal model

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB−. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3β-hydroxysteroid dehydrogenase (3β-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3β-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB− had an opposite effect on 3β-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11β1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB− extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production.