Adrenocortical H295R Cells Research Articles

Transcriptional regulation of human ferredoxin reductase through an intronic enhancer in steroidogenic cells

Ferredoxin reductase (FDXR, also known as adrenodoxin reductase) is a mitochondrial flavoprotein that transfers electrons from NADPH to mitochondrial cytochrome P450 enzymes, mediating the function of an iron–sulfur cluster protein, ferredoxin. FDXR functions in various metabolic processes including steroidogenesis. It is well known that multiple steroidogenic enzymes are regulated by a transcription factor steroidogenic factor-1 (SF-1, also known as Ad4BP). Previously, we have shown that SF-1 transduction causes human mesenchymal stem cell differentiation into steroidogenic cells. Genome-wide analysis of differentiated cells, using a combination of DNA microarray and promoter tiling array analyses, showed that FDXR is a novel SF-1 target gene. In this study, the transcriptional regulatory mechanism of FDXR was examined in steroidogenic cells. A chromatin immunoprecipitation assay revealed that a novel SF-1 binding region was located within intron 2 of the human FDXR gene. Luciferase reporter assays showed that FDXR transcription was activated through the novel SF-1 binding site within intron 2. Endogenous SF-1 knockdown in human adrenocortical H295R and KGN cells decreased FDXR expression. In H295R cells, strong binding of two histone markers of active enhancers, histones H3K27ac and H3K4me2, were detected near the SF-1 binding site within intron 2. Furthermore, the binding of these histone markers was decreased concurrent with SF-1 knockdown in H295R cells. These results indicated that abundant FDXR expression in these steroidogenic cells was maintained through SF-1 binding to the intronic enhancer of the FDXR gene.

1α,25-Dihydroxyvitamin D3 inhibits the human H295R cell proliferation by cell cycle arrest: A model for a protective role of vitamin D receptor against adrenocortical cancer

Using the human H295R adrenocortical carcinoma cell line as a model, we analyzed the role of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3)]–vitamin D receptor (VDR) axis in the growth of adrenocortical cancer (ACC). The presence of VDR in various adrenocortical tissues, including ACC, was also investigated. DNA synthesis was evaluated by [3H]thymidine cell incorporation after treatment with 1α,25(OH)2D3 at increasing doses. The effect of 1α,25(OH)2D3 on cell cycle and apoptosis was analyzed with a flow cytometer. Cyclin-dependent kinase 4 (CDK4) expression, a molecular marker of G1-S cell cycle transition phase, was evaluated in cells treated with 1α,25(OH)2D3 before and after VDR gene silencing. 1α,25(OH)2D3 treatment inhibited cell proliferation by 20% at a dose of 1nM, in parallel with steroid secretion decrease. A cell cycle arrest in G1, with no change in apoptotic cell proportion, was observed after 10nM 1α,25(OH)2D3 cell exposure. CDK4 activation was reduced by 10nM 1α,25(OH)2D3 but was not affected by 1α,25(OH)2D3 after VDR gene silencing. Expression of VDR mRNA was lower in ACC than in benign adrenocortical tumors. VDR immunostaining was evident in benign tumors but it was weak in ACC tissues. ConclusionsSlightly supra-physiological concentrations of 1α,25(OH)2D3 have a moderate anti-proliferative effect on H295R cells. Anti-proliferative effect was due to cell cycle arrest in G1 phase, without inducing apoptosis. The low mRNA expression levels at qRT-PCR as well as the weak immunohistochemical expression of VDR in ACC, suggests a protective role of VDR against malignant adrenocortical growth.

Abstract C134: EN3356, a novel CYP17A inhibitor for the treatment of castration resistant prostate cancer.

Abstract Androgens are a major driver of prostate cancer growth. As prostate cancer progresses, it can escape standard androgen deprivation therapy by relying on extra-testicular CYP17 enzyme activity for androgen synthesis. EN3356 is a novel non-steroidal CYP17 inhibitor that reduces androgen synthesis and is designed to treat castration-resistant prostate cancer. EN3356 provides potent inhibition of CYP17 lyase activity in rat and human testicular microsomes, and in human adrenocortical carcinoma H295R cells it also inhibits testosterone synthesis potently. In contrast to abiraterone, EN3356 is selective for inhibiting testosterone synthesis over cortisol synthesis. This finding suggests that EN3356 may not require co-administration of prednisone to mitigate mineralocorticoid excess. In pre-clinical studies, EN3356 exhibited high oral bioavailability. EN3356 inhibited serum testosterone levels in the rat and caused a robust decrease of seminal vesicle and prostate weights following 14-days of oral administration. GLP safety studies were completed in two preclinical species and identified a high safety margin. A combination Phase I/II study in metastatic castration-resistant prostate cancer is currently under initiation in the US and is anticipated to start enrolling subjects in the fourth quarter of 2013. Based on the potent inhibitory activity of EN3356 against CYP17 lyase in vitro, selective inhibition of testosterone synthesis versus cortisol synthesis, and robust weight reduction of androgen sensitive organs in vivo, EN3356 has the potential to be a selective and efficacious therapy for the treatment of castration-resistant prostate cancer. Disclosure: Funding for this research was provided by Endo Pharmaceuticals Inc. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C134. Citation Format: Nicholas J. Laping, Dhanalakshmi Sivanandhan, Raghava Reddy Kethiri, Purushottam M. Dewang, Ajith Vijayendra, Mohd Zainuddin, Ramesh Mullangi, Niranjan Rao, Aranapakam M. Venkatesan, Sanjeeva Reddy, Sandeep Gupta, Roger A. Smith. EN3356, a novel CYP17A inhibitor for the treatment of castration resistant prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C134.

QRFP induces aldosterone production via PKC and T-type calcium channel-mediated pathways in human adrenocortical cells: evidence for a novel role of GPR103

Hormonal regulation of adrenal function occurs primarily through activation of GPCRs. GPCRs are central to many of the body's endocrine and neurotransmitter pathways. Recently, it was shown that activation of GPR103 by its ligand QRFP induced feeding, locomotor activity, and metabolic rate, and QRFP is bioactive in adipose tissue of obese individuals. Given that the adrenal gland is a pivotal organ for energy balance and homeostasis, we hypothesized that GPR103 and QRFP are involved in steroidogenic responses. Using qRT-PCR and immunohistochemistry, we mapped both GPR103 and QRFP in human fetal and adult adrenal gland as well as rat adrenals. Both were primarily localized in the adrenal cortex but not in the medulla. Activation of GPR103 in human adrenocortical H295R cells led to a decrease in forskolin-increased cAMP and an increase of intracellular Ca(2+) levels. In addition, treatment of H295R cells with QRFP induced aldosterone and cortisol secretion as measured by ELISA. These increases were accompanied by increased expression and activity of StAR, CYB11B1, and CYP11B2 as assessed by qRT-PCR and luciferase reporter assay, respectively. Using specific inhibitors, we also demonstrated that aldosterone induction involves MAPK, PKC, and/or T-type Ca(2+) channel-dependent pathways. These novel data demonstrate that QRFP induces adrenal steroidogenesis in vitro by regulating key steroidogenic enzymes involving MAPK/PKC and Ca(2+) signaling pathways.

Protein kinase C-induced activin A switches adrenocortical steroidogenesis to aldosterone by suppressing CYP17A1 expression

Functional zonation of the adrenal cortex is a consequence of the zone-specific expression of P450c17 (CYP17A1) and its cofactors. Activin and inhibin peptides are differentially produced within the zones of the adrenal cortex and have been implicated in steroidogenic control. In this study, we investigated whether activin and inhibin can function as intermediates in functional zonation of the human adrenal cortex. Activin A suppressed CYP17A1 expression and P450c17 function in adrenocortical cell lines as well as in primary adrenal cell cultures. Inhibin βA-subunit mRNA and activin A protein levels were found to be increased up to 1,900-fold and 49-fold, respectively, after protein kinase C (PKC) stimulation through PMA or angiotensin II in H295R adrenocortical carcinoma cells. This was confirmed in HAC15 cells and for PMA in primary adrenal cell cultures. Both PMA and Ang II decreased CYP17A1 expression in the adrenocortical cell lines, whereas PMA concurrently suppressed CYP17A1 levels in the primary cultures. Inhibition of activin signaling during PKC stimulation through silencing of the inhibin βA-subunit or blocking of the activin type I receptor opposed the PMA-induced downregulation of CYP17A1 expression and P450c17 function. In contrast, PKA stimulation through adrenocorticotrophin or forskolin increased expression of the inhibin α-subunit and betaglycan, both of which are antagonists of activin action. These data indicate that activin A acts as a PKC-induced paracrine factor involved in the suppression of CYP17A1 in the zona glomerulosa and can thereby contribute to functional adrenocortical zonation.

Extramitochondrial OPA1 and adrenocortical function

We have previously described that silencing of the mitochondrial protein OPA1 enhances mitochondrial Ca2+ signaling and aldosterone production in H295R adrenocortical cells. Since extramitochondrial OPA1 (emOPA1) was reported to facilitate cAMP-induced lipolysis, we hypothesized that emOPA1, via the enhanced hydrolysis of cholesterol esters, augments aldosterone production in H295R cells. A few OPA1 immunopositive spots were detected in ∼40% of the cells. In cell fractionation studies OPA1/COX IV (mitochondrial marker) ratio in the post-mitochondrial fractions was an order of magnitude higher than that in the mitochondrial fraction. The ratio of long to short OPA1 isoforms was lower in post-mitochondrial than in mitochondrial fractions. Knockdown of OPA1 failed to reduce db-cAMP-induced phosphorylation of hormone-sensitive lipase (HSL), Ca2+ signaling and aldosterone secretion. In conclusion, OPA1 could be detected in the post-mitochondrial fractions, nevertheless, OPA1 did not interfere with the cAMP – PKA – HSL mediated activation of aldosterone secretion.

Silencing Mutated β-Catenin Inhibits Cell Proliferation and Stimulates Apoptosis in the Adrenocortical Cancer Cell Line H295R

ContextAdrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic options. Activating β-catenin somatic mutations are found in ACC and have been associated with a poor clinical outcome. In fact, activation of the Wnt/β-catenin signaling pathway seems to play a major role in ACC aggressiveness, and might, thus, represent a promising therapeutic target.ObjectiveSimilar to patient tumor specimen the H295 cell line derived from an ACC harbors a natural activating β-catenin mutation. We herein assess the in vitro and in vivo effect of β-catenin inactivation using a doxycyclin (dox) inducible shRNA plasmid in H295R adrenocortical cancer cells line (clone named shβ).ResultsFollowing dox treatment a profound reduction in β-catenin expression was detectable in shβ clones in comparison to control clones (Ctr). Accordingly, we observed a decrease in Wnt/βcatenin-dependent luciferase reporter activity as well as a decreased expression of AXIN2 representing an endogenous β-catenin target gene. Concomitantly, β-catenin silencing resulted in a decreased cell proliferation, cell cycle alterations with cell accumulation in the G1 phase and increased apoptosis in vitro. In vivo, on established tumor xenografts in athymic nude mice, 9 days of β-catenin silencing resulted in a significant reduction of CTNNB1 and AXIN2 expression. Moreover, continous β-catenin silencing, starting 3 days after tumor cell inoculation, was associated with a complete absence of tumor growth in the shβ group while tumors were present in all animals of the control group.ConclusionIn summary, these experiments provide evidences that Wnt/β-catenin pathway inhibition in ACC is a promising therapeutic target.

Pregnane glycosides interfere with steroidogenic enzymes to down‐regulate corticosteroid production in human adrenocortical H295R cells

A group of bioactive steroidal glycosides (pregnanes) with anorectic activity in animals was isolated from several genera of milkweeds including Hoodia and Asclepias. In this study, we investigated the effects, structure-activity relationships, and mechanism of action of pregnane glycosides on steroidogenesis in human adrenocortical H295R cells. Administration of pregnane glycosides for 24 h suppressed the basal and forskolin-stimulated release of androstenedione, corticosterone, and cortisone from H295R cells. The conversion of progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to either androstenedione or 11-deoxycortisol was most strongly affected, with 12-cinnamoyl-, benzoyl-, and tigloyl-containing pregnanes showing the highest activity. Incubation of pregnane glycosides for 24 h had no effect on mRNA transcripts of CYP11A1, CYP21A1, CYP11B1 cytochrome enzymes and steroidogenic acute regulatory protein (StaR) protein, yet resulted in twofold decrease in HSD3B1 mRNA levels. At the same time, pregnane glycosides had no effect on the CYP1, 2, or 3 drug and steroid metabolism enzymes and showed weak Na(+) /K(+) ATPase and glucocorticoid receptor binding. Taken together, these data suggest that pregnane glycosides specifically suppress steroidogenesis through strong inhibition of 11β-hydroxylase and steroid 17-alpha-monooxygenase, and weak inhibition of cytochrome P450 side chain cleavage enzyme and 21β-hydroxylase, but not 3β-hydroxysteroid dehydrogenase/isomerase.

Expression and function of OXE receptor, an eicosanoid receptor, in steroidogenic cells

Hormonal regulation of steroidogenesis involves arachidonic acid (AA) metabolism through the 5-lipoxygenase pathway. One of the products, 5-hydroperoxy-eicosatetraenoic acid (5-HpETE), acts as a modulator of the activity of the steroidogenic acute regulatory (StAR) protein promoter. Besides, an oxoeicosanoid receptor of the leukotriene receptor family named OXE-R is a membrane protein with high affinity and response to 5-HpETE, among other AA derivatives. The aim of our work was to elucidate whether this receptor may be involved in steroidogenesis. RT-PCR and western blot analysis demonstrated the presence of the mRNA and protein of the receptor in human H295R adrenocortical cells. The treatment of H295R or MA-10 cells (murine Leydig cell line) with 8Br-cAMP together with docosahexaenoic acid (DHA, an antagonist of the receptor) partially reduced StAR induction and steroidogenesis. On the contrary, 5-oxo-ETE – the prototypical agonist, with higher affinity and potency on the receptor – increased cAMP-dependent steroid production, StAR mRNA and protein levels. These results lead us to conclude that AA might modulate StAR induction and steroidogenesis, at least in part, through 5-HpETE production and activation of a membrane receptor, such as the OXE-R.

Acid Ceramidase (ASAH1) Represses Steroidogenic Factor 1-Dependent Gene Transcription in H295R Human Adrenocortical Cells by Binding to the Receptor

Adrenocorticotropin (ACTH) signaling increases glucocorticoid production by promoting the interaction of transcription factors and coactivator proteins with the promoter of steroidogenic genes. The nuclear receptor steroidogenic factor 1 (SF-1) is essential for steroidogenic gene transcription. Sphingosine (SPH) is a ligand for SF-1. Moreover, suppression of expression of acid ceramidase (ASAH1), an enzyme that produces SPH, increases the transcription of multiple steroidogenic genes. Given that SF-1 is a nuclear protein, we sought to define the molecular mechanisms by which ASAH1 regulates SF-1 function. We show that ASAH1 is localized in the nuclei of H295R adrenocortical cells and that cyclic AMP (cAMP) signaling promotes nuclear sphingolipid metabolism in an ASAH1-dependent manner. ASAH1 suppresses SF-1 activity by directly interacting with the receptor. Chromatin immunoprecipitation (ChIP) assays revealed that ASAH1 is recruited to the promoter of various SF-1 target genes and that ASAH1 and SF-1 colocalize on the same promoter region of the CYP17A1 and steroidogenic acute regulatory protein (StAR) genes. Taken together, these results demonstrate that ASAH1 is a novel coregulatory protein that represses SF-1 function by directly binding to the receptor on SF-1 target gene promoters and identify a key role for nuclear lipid metabolism in regulating gene transcription.

Circulating Very-Low-Density Lipoprotein from Subjects with Impaired Glucose Tolerance Accelerates Adrenocortical Cortisol and Aldosterone Synthesis

Apart from their role in cardiovascular homeostasis and immunomodulation, aldosterone and cortisol are also implicated in the pathogenesis of insulin resistance and type 2 diabetes mellitus (T2DM). Furthermore, glycoxidative modifications of lipoproteins are increasingly recognized as an etiological factor for increased cardiovascular morbidity and mortality in prediabetic individuals. The causative relationship between in vivo lipoprotein modifications and steroidogenesis in subjects with impaired glucose tolerance (IGT), however, is not well defined. Therefore, we aimed to investigate the impact of in vivo modified lipoproteins on aldosterone and cortisol release from human adrenocortical H295R cells. Following an oral glucose tolerance test, 20 individuals with normal glucose tolerance (NGT) and 20 IGT subjects were randomly selected from the ongoing PRAEDIAS prevention study in our department. Cells were incubated for 24 h with lipoproteins isolated from NGT and IGT individuals and aldosterone and cortisol release was measured in the supernatants. VLDL induced a greater stimulating effect on adrenocortical aldosterone and cortisol release compared to HDL and LDL. Moreover, IGT-VLDL evoked a significantly higher effect (p<0.05) on hormone release than NGT-VLDL. Incubation of cells with in vitro modified lipoproteins and specific pharmacological inhibitors suggests that VLDL presumably recruits ERK1/2 as one of the downstream effectors of Jak-2. In summary, in vivo modified VLDL are able to promote prediabetic hormonal dysregulation by modulating adrenocortical steroidogenesis via Jak-2-ERK dependent pathway.

Phosphodiesterase 11A (PDE11A) Gene Defects in Patients with ACTH-Independent Macronodular Adrenal Hyperplasia (AIMAH): Functional Variants May Contribute to Genetic Susceptibility of Bilateral Adrenal Tumors

Phosphodiesterases (PDEs) are key regulatory enzymes of intracellular cAMP levels. PDE11A function has been linked to predisposition to adrenocortical tumors. The aim of the study was to study the PDE11A gene in a large cohort of patients with ACTH-independent macronodular adrenal hyperplasia (AIMAH) and in control subjects. The PDE11A entire coding region was sequenced in 46 patients with AIMAH and 192 controls. Two variants found in AIMAH patients were transiently expressed in HEK 293 and adrenocortical H295R cells for further functional studies. The frequency of all PDE11A variants was significantly higher among patients with AIMAH (28%) compared to controls (7.2%) (P = 5 × 10(-5)). Transfection of the two PDE11A variants found in AIMAH patients only (D609N or M878V) showed that cAMP levels were higher, after forskolin stimulation, in cells transfected with the PDE11A mutants, compared to cells transfected with the wild-type PDE11A in HEK 293 cells (P < 0.05). Moreover, transfection with mutants PDE11A increased transcriptional activity of a cAMP-response element reporter construct compared to wild-type PDE11A in HEK 293 cells (P < 0.0004 for D609N and P < 0.003 for M878V) and in the adrenocortical H295R cells (P < 0.05 for D609N and M878V). In addition, analysis of cAMP levels in intact living culture cells by fluorescence resonance energy transfer probes showed increased cAMP in forskolin-treated cells transfected with PDE11A variants compared with wild-type PDE11A (P < 0.05). We conclude that PDE11A genetic variants may increase predisposition to AIMAH.

Inactivation of PRKARIA or PRKAR2B Increases Cell Proliferation and Decreases Apoptosis, Delineating Distinct Molecular Mechanisms in Adrenocortical Human H295R Cell Line

Inactivation of PRKARIA or PRKAR2B Increases Cell Proliferation and Decreases Apoptosis, Delineating Distinct Molecular Mechanisms in Adrenocortical Human H295R Cell Line

Efficacy of the novel dual PI3-kinase/mTOR inhibitor NVP-BEZ235 in a preclinical model of adrenocortical carcinoma

Adrenocortical cancer is a rare malignancy for which current pharmacological therapies are still insufficient. We tested the effect of a novel PI3 kinase – mammalian target of rapamycin dual inhibitor (NVP-BEZ235) on proliferation of the H295R adrenocortical cancer cell line in vitro and grown as xenografts in immunodeficient mice. NVP-BEZ235 was able to significantly inhibit phosporylation of Akt kinase and S6 ribosomal protein in H295R cells and to significantly reduce their proliferation in vitro and xenograft growth in vivo. The drug also induced activation of Erk phosphorylation, which could be inhibited by simultaneous treatment with the Erk inhibitor FR180204. This latter drug synergized with NVP-BEZ235 in the inhibition of H295R proliferation in vitro. Our data suggest that dual PI3K/mTOR inhibitors may represent a useful pharmacological tool in the therapy of advanced adrenocortical cancer and that simultaneous inhibition of both Erk and PI3K – mTOR pathways may be required to obtain a higher antiproliferative effect in this type of tumor.

Comprehensive DNA methylation analysis of benign and malignant adrenocortical tumors

The molecular pathogenesis of benign and malignant adrenocortical tumors (ACT) is incompletely clarified. The role of DNA methylation in adrenocortical tumorigenesis has not been analyzed in an unbiased, systematic fashion. Using the Infinium HumanMethylation27 BeadChip, the DNA methylation levels of 27,578 CpG sites were investigated in bisulfite-modified DNA from 6 normal adrenocortical tissue samples, 27 adrenocortical adenomas (ACA), and 15 adrenocortical carcinomas (ACC). Genes involved in cell cycle regulation, apoptosis, and transcriptional regulation of known or putative importance in the development of adrenal tumors showed significant and frequent hypermethylation. Such genes included CDKN2A, GATA4, BCL2, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. Comparing benign versus malignant ACT, a total of 212 CpG islands were identified as significantly hypermethylated in ACC. Gene expression studies of selected hypermethylated genes (CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, SCGB3A1/HIN1) in 6 normal and 16 neoplastic adrenocortical tissues (10 ACA and 6 ACC), displayed reduced gene expression in benign and malignant ACT versus normal adrenocortical tissue. Treatment with 5-aza-2'-deoxycytidine of adrenocortical cancer H-295R cells increased expression of the hypermethylated genes CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. In conclusion, the current study represents the first unbiased, quantitative, genome-wide study of adrenocortical tumor DNA methylation. Genes with altered DNA methylation patterns were identified of putative importance to benign and malignant adrenocortical tumor development.

H295R cells as a model for steroidogenic disruption: A broader perspective using simultaneous chemical analysis of 7 key steroid hormones

The effects of three model endocrine disruptors, prochloraz, ketoconazole and genistein on steroidogenesis were tested in the adrenocortical H295R cell line to demonstrate that a broader mechanistic understanding can be achieved in one assay by applying chemical analysis to the H295R assay. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone and 17β-estradiol) were analyzed using a novel and thoroughly validated GC–MS/MS method. In addition to the simultaneous quantification of 7 steroid hormones, the present method also negates the potential problems of cross-reactivity that can be encountered in some immunoassays. Although all 3 test compounds decrease the concentrations of the main sex steroids, the chemicals exerted different effects upstream in the pathway. Exposure to prochloraz resulted in increased hormone levels upstream of steroid 17 alpha-hydroxylase/17,20 lyase (P450c17) and decreases downstream. Ketoconazole inhibited the entire pathway, while exposure to genistein resulted in increased hormone levels upstream of 3-β-hydroxysteroid dehydrogenase (3β-HSD) and decreases downstream. The results demonstrate that chemical analysis combined with the H295R cell assay is an useful tool for studying the mechanisms by which endocrine disruptors interfere with the steroidogenic pathway.

Flavonoids exhibit diverse effects on CYP11B1 expression and cortisol synthesis

CYP11B1 catalyzes the final step of cortisol biosynthesis. The effects of flavonoids on transcriptional expression and enzyme activity of CYP11B1 were investigated using the human adrenocortical H295R cell model. All tested nonhydroxylated flavones including 3′,4′-dimethoxyflavone, α-naphthoflavone, and β-naphthoflavone upregulated CYP11B1 expression and cortisol production, whereas apigenin and quercetin exhibited potent cytotoxicity and CYP11B1 repression at high concentrations. Nonhydroxylated flavones stimulated CYP11B1-catalyzed cortisol formation at transcriptional level. Resveratrol increased endogenous and substrate-supported cortisol production like nonhydroxylated flavones tested, but it had no effect on CYP11B1 gene expression and enzyme activity. Resveratrol appeared to alter cortisol biosynthesis at an earlier step. The Ad5 element situated in the − 121/− 106 region was required for basal and flavone-induced CYP11B1 expression. Overexpression of COUP-TFI did not improve the responsiveness of Ad5 to nonhydroxylated flavones. Although COUP-TFI overexpression increased CYP11B1 and CYP11B2 promoter activation, its effect was not mediated through the common Ad5 element. Treating cells with PD98059 (a flavone-type MEK1 inhibitor) increased CYP11B1 promoter activity, but not involving ERK signaling because phosphorylation of ERK1/2 remained unvarying throughout the course of treatment. Likewise, AhR was not responsible for the CYP11B1-modulating effects of flavonoids because inconsistency with their effects on AhR activation. 3′,4′-dimethoxyflavone and 8-Br-cAMP additively activated CYP11B1 promoter activity. H-89 reduced 3′,4′-dimethoxyflavone-induced CYP11B1 promoter activation but to a lesser extent as compared to its inhibition on cAMP-induced transactivation. Our data suggest that constant exposure to nonhydroxylated flavones raises a potential risk of high basal and cAMP-induced cortisol synthesis in consequence of increased CYP11B1 expression.

Mechanistic profiling of the cAMP-dependent steroidogenic pathway in the H295R endocrine disrupter screening system: New endpoints for toxicity testing

The need for implementation of effects on steroid synthesis and hormone processing in screening batteries of endocrine disruptive compounds is widely acknowledged. In this perspective, hormone profiling in the H295R adrenocortical cell system is extensively examined and recently OECD validated (TG 456) as a replacement of the minced testis assay. To further elucidate the complete mechanisms and endocrine responsiveness of this cell system, microarray-based gene expression profiling of the cAMP response pathway, one of the major pathways in steroidogenesis regulation, was examined in H295R cells. Next to the steroid synthesis pathway, a broader lipid metabolic pathway, including cholesterol uptake/biosynthesis, hormone metabolization and many hormone and nuclear receptors, are sensitive towards cAMP stimulation in this cell system. Moreover, these pathways were clearly dose and time responsive, indicating early regulation (10 h) of cholesterol uptake and mobilization genes and later expression (24–48 h) of cholesterol biosynthesis and steroid synthesis. Transcription network analysis suggested several important transcription factors that could be involved in regulation of the steroid hormone pathway, of which HNF4α, a broader lipid metabolism related transcription factor, might indicate some new transcription regulation patterns in this cell line. Overall we can conclude that the time dependent gene expression patterns of the strongly coordinated cholesterol supply and steroidogenesis pathways in the H295R cell system seem to reflect well the in vivo ACTH/cAMP signalling cascade in adrenal cells. Moreover, the completeness of the steroidogenic related pathways in terms of gene expression sensitivity, indicates the H295R cell line as a promising cell line in omics-based endocrine disruption screening.

The effect of OPA1 on mitochondrial Ca²⁺ signaling.

The dynamin-related GTPase protein OPA1, localized in the intermembrane space and tethered to the inner membrane of mitochondria, participates in the fusion of these organelles. Its mutation is the most prevalent cause of Autosomal Dominant Optic Atrophy. OPA1 controls the diameter of the junctions between the boundary part of the inner membrane and the membrane of cristae and reduces the diffusibility of cytochrome c through these junctions. We postulated that if significant Ca2+ uptake into the matrix occurs from the lumen of the cristae, reduced expression of OPA1 would increase the access of Ca2+ to the transporters in the crista membrane and thus would enhance Ca2+ uptake. In intact H295R adrenocortical and HeLa cells cytosolic Ca2+ signals evoked with K+ and histamine, respectively, were transferred into the mitochondria. The rate and amplitude of mitochondrial [Ca2+] rise (followed with confocal laser scanning microscopy and FRET measurements with fluorescent wide-field microscopy) were increased after knockdown of OPA1, as compared with cells transfected with control RNA or mitofusin1 siRNA. Ca2+ uptake was enhanced despite reduced mitochondrial membrane potential. In permeabilized cells the rate of Ca2+ uptake by depolarized mitochondria was also increased in OPA1-silenced cells. The participation of Na+/Ca2+ and Ca2+/H+ antiporters in this transport process is indicated by pharmacological data. Altogether, our observations reveal the significance of OPA1 in the control of mitochondrial Ca2+ metabolism.

Immediate-Early Transcriptional Response to Angiotensin II in Human Adrenocortical Cells

Angiotensin II binds to the angiotensin II receptors type 1 (AT1 receptors) in adrenocortical cells and triggers an intracellular signaling cascade leading to changes in the gene expression pattern. Here, we show that stimulation with angiotensin II induces the expression of biologically active early growth response (Egr)-1, a zinc finger transcription factor, in human H295R adrenocortical cells. Expression of a dominant-negative mutant of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression in angiotensin II-stimulated H295R cells, indicating that Ets-like protein-1 (Elk-1) or related ternary complex factors connect the intracellular signaling cascade elicited by activation of AT1 receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that angiotensin II stimulation increased the transcription activation potential of Elk-1. In addition, activator protein-1 transcriptional activity was significantly elevated in angiotensin II-treated H295R cells. Expression of c-Jun and c-Fos was increased as well as the transcription activation potential of c-Fos. Expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression in angiotensin II-stimulated adrenocortical cells, suggesting that the serum response element within the c-Fos promoter functions as an angiotensin II-response element. Expression of a dominant-negative mutant of c-Jun reduced activator protein-1 activity in angiotensin II-stimulated adrenocortical cells and reduced the up-regulation of c-Jun after angiotensin II stimulation. Thus, c-Jun regulates its own expression in adrenocortical cells. Together, the data show that angiotensin II stimulation activates the transcription factors Egr-1, Elk-1, c-Jun, and c-Fos in adrenocortical cells, leading to stimulus-dependent changes in the gene expression pattern.