Steroidogenesis In Adrenocortical Cells Research Articles

Disruptive effects of plasticizers bisphenol A, F, and S on steroidogenesis of adrenocortical cells.

Endocrine disrupting chemicals (EDCs) are known to interfere with endocrine homeostasis. Their impact on the adrenal cortex and steroidogenesis has not yet been sufficiently elucidated. This applies in particular to the ubiquitously available bisphenols A (BPA), F (BPF), and S (BPS). NCI-H295R adrenocortical cells were exposed to different concentrations (1nM-1mM) of BPA, BPF, BPS, and an equimolar mixture of them (BPmix). After 72 hours, 15 endogenous steroids were measured using LC-MS/MS. Ratios of substrate and product of CYP-regulated steps were calculated to identify most influenced steps of steroidogenesis. mRNA expression of steroidogenic enzymes was determined by real-time PCR. Cell viability remained unaffected at bisphenol concentrations lower than 250 µM. All tested bisphenols and their combination led to extensive alterations in the quantified steroid levels. The most profound fold changes (FC) in steroid concentrations after exposure to BPA (>10µM) were seen for androstenedione, e.g. a 0.37±0.11-fold decrease at 25µM (p≤0.0001) compared to vehicle-treated controls. For BPF, levels of 17-hydroxyprogesterone were significantly increased by 25µM (FC 2.57±0.49, p≤0.001) and 50µM (FC 2.65±0.61, p≤0.0001). BPS treatment led to a dose-dependent decrease of 11-deoxycorticosterone at >1µM (e.g. FC 0.24±0.14, p≤0.0001 at 10µM). However, when combining all three bisphenols, additive effects were detected: e.g. 11-deoxycortisosterone was decreased at doses >10µM (FC 0.27±0.04, p≤0.0001, at 25µM), whereas 21-deoxycortisol was increased by 2.92±0.20 (p≤0.01) at 10µM, and by 3.21±0.45 (p≤0.001) at 50µM. While every measured androgen (DHEA, DHEAS, androstenedione, testosterone, DHT) was lowered in all experiments, estradiol levels were significantly increased by BPA, BPF, BPS, and BPmix (e.g. FC 3.60±0.54, p≤0.0001 at 100µM BPF). Calculated substrate-product ratios indicated an inhibition of CYP17A1-, and CYP21A2 mediated conversions, whereas CYP11B1 and CYP19A1 showed higher activity in the presence of bisphenols. Based on these findings, most relevant mRNA expression of CYP genes were analysed. mRNA levels of StAR, CYP11B1, and CYP17A1 were significantly increased by BPF, BPS, and BPmix. In cell culture, bisphenols interfere with steroidogenesis at non-cytotoxic levels, leading to compound-specific patterns of significantly altered hormone levels. These results justify and call for additional in-vivo studies to evaluate effects of EDCs on adrenal gland functionality.

New insights into signal transduction pathways in adrenal steroidogenesis: role of mitochondrial fusion, lipid mediators, and MAPK phosphatases.

Hormone-receptor signal transduction has been extensively studied in adrenal gland. Zona glomerulosa and fasciculata cells are responsible for glucocorticoid and mineralocorticoid synthesis by adrenocorticotropin (ACTH) and angiotensin II (Ang II) stimulation, respectively. Since the rate-limiting step in steroidogenesis occurs in the mitochondria, these organelles are key players in the process. The maintenance of functional mitochondria depends on mitochondrial dynamics, which involves at least two opposite events, i.e., mitochondrial fusion and fission. This review presents state-of-the-art data on the role of mitochondrial fusion proteins, such as mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), in Ang II-stimulated steroidogenesis in adrenocortical cells. Both proteins are upregulated by Ang II, and Mfn2 is strictly necessary for adrenal steroid synthesis. The signaling cascades of steroidogenic hormones involve an increase in several lipidic metabolites such as arachidonic acid (AA). In turn, AA metabolization renders several eicosanoids released to the extracellular medium able to bind membrane receptors. This report discusses OXER1, an oxoeicosanoid receptor which has recently arisen as a novel participant in adrenocortical hormone-stimulated steroidogenesis through its activation by AA-derived 5-oxo-ETE. This work also intends to broaden knowledge of phospho/dephosphorylation relevance in adrenocortical cells, particularly MAP kinase phosphatases (MKPs) role in steroidogenesis. At least three MKPs participate in steroid production and processes such as the cellular cycle, either directly or by means of MAP kinase regulation. To sum up, this review discusses the emerging role of mitochondrial fusion proteins, OXER1 and MKPs in the regulation of steroid synthesis in adrenal cortex cells.

Sublethal Hyperthermia Transiently Disrupts Cortisol Steroidogenesis in Adrenocortical Cells.

Primary aldosteronism is the most common cause of secondary hypertension. The first-line treatment adrenalectomy resects adrenal nodules and adjacent normal tissue, limiting suitability to those who present with unilateral disease. Use of thermal ablation represents an emerging approach as a possible minimally invasive therapy for unilateral and bilateral disease, to target and disrupt hypersecreting aldosterone-producing adenomas, while preserving adjacent normal adrenal cortex. To determine the extent of damage to adrenal cells upon exposure to hyperthermia, the steroidogenic adrenocortical cell lines H295R and HAC15 were treated with hyperthermia at temperatures between 37 and 50°C with the effects of hyperthermia on steroidogenesis evaluated following stimulation with forskolin and ANGII. Cell death, protein/mRNA expression of steroidogenic enzymes and damage markers (HSP70/90), and steroid secretion were analyzed immediately and 7 days after treatment. Following treatment with hyperthermia, 42°C and 45°C did not induce cell death and were deemed sublethal doses while ≥50°C caused excess cell death in adrenal cells. Sublethal hyperthermia (45°C) caused a significant reduction in cortisol secretion immediately following treatment while differentially affecting the expression of various steroidogenic enzymes, although recovery of steroidogenesis was evident 7 days after treatment. As such, sublethal hyperthermia, which occurs in the transitional zone during thermal ablation induces a short-lived, unsustained inhibition of cortisol steroidogenesis in adrenocortical cells in vitro.

Adrenocortical Gap Junctions and Their Functions.

Adrenal cortical steroidogenesis and proliferation are thought to be modulated by gap junction-mediated direct cell–cell communication of regulatory molecules between cells. Such communication is regulated by the number of gap junction channels between contacting cells, the rate at which information flows between these channels, and the rate of channel turnover. Knowledge of the factors regulating gap junction-mediated communication and the turnover process are critical to an understanding of adrenal cortical cell functions, including development, hormonal response to adrenocorticotropin, and neoplastic dedifferentiation. Here, we review what is known about gap junctions in the adrenal gland, with particular attention to their role in adrenocortical cell steroidogenesis and proliferation. Information and insight gained from electrophysiological, molecular biological, and imaging (immunocytochemical, freeze fracture, transmission electron microscopic, and live cell) techniques will be provided.

Tumor Necrosis Factor-α Regulates Glucocorticoid Synthesis in the Adrenal Glands of Trypanosoma cruzi Acutely-Infected Mice. The Role of TNF-R1

Adrenal steroidogenesis is under a complex regulation involving extrinsic and intrinsic adrenal factors. TNF-α is an inflammatory cytokine produced in response to tissue injury and several other stimuli. We have previously demonstrated that TNF-R1 knockout (TNF-R1−/−) mice have a dysregulated synthesis of glucocorticoids (GCs) during Trypanosoma cruzi acute infection. Since TNF-α may influence GCs production, not only through the hypothalamus-pituitary axis, but also at the adrenal level, we now investigated the role of this cytokine on the adrenal GCs production. Wild type (WT) and TNF-R1−/− mice undergoing acute infection (Tc-WT and Tc-TNF-R1−/− groups), displayed adrenal hyperplasia together with increased GCs levels. Notably, systemic ACTH remained unchanged in Tc-WT and Tc-TNF-R1−/− compared with uninfected mice, suggesting some degree of ACTH-independence of GCs synthesis. TNF-α expression was increased within the adrenal gland from both infected mouse groups, with Tc-WT mice showing an augmented TNF-R1 expression. Tc-WT mice showed increased levels of P-p38 and P-ERK compared to uninfected WT animals, whereas Tc-TNF-R1−/− mice had increased p38 and JNK phosphorylation respect to Tc-WT mice. Strikingly, adrenal NF-κB and AP-1 activation during infection was blunted in Tc-TNF-R1−/− mice. The accumulation of mRNAs for steroidogenic acute regulatory protein and cytochrome P450 were significantly increased in both Tc-WT and Tc-TNF-R1−/− mice; being much more augmented in the latter group, which also had remarkably increased GCs levels. TNF-α emerges as a potent modulator of steroidogenesis in adrenocortical cells during T. cruzi infection in which MAPK pathways, NF-κB and AP-1 seem to play a role in the adrenal synthesis of pro-inflammatory cytokines and enzymes regulating GCs synthesis. These results suggest the existence of an intrinsic immune-adrenal interaction involved in the dysregulated synthesis of GCs during murine Chagas disease.

Effects of food restriction on steroidogenesis in dispersed adrenocortical cells from Yarrow’s Spiny Lizard (Sceloporus jarrovii)

Changes in energy balance can lead to functional alterations at all levels of the hypothalamic–pituitary–adrenal (HPA) axis. However, relatively little is known about how energy balance affects functional properties of adrenocortical cells themselves. We investigated effects of restricted food intake on sensitivity to ACTH and rates of steroidogenesis in adrenocortical cells isolated from growing female and male Yarrow’s Spiny Lizards (Sceloporus jarrovii). At the end of the feeding regimen, we assayed acute (3h) progesterone (P4), corticosterone (B), and aldosterone (ALDO) production in response to ACTH in dispersed adrenocortical cells. Food restriction depressed growth rate by about 50% in both males and females but did not alter baseline plasma B measured at 10weeks in either sex. At the cellular level, food restriction had the following effects: (1) increased basal B production in both sexes and basal ALDO production in males, (2) increased net maximal rates of production of P4, B, and ALDO in response to ACTH, and (3) no overall effect on adrenocortical cellular sensitivity to ACTH. There were modest sex differences: overall rates of P4 production were 46% greater in cells from females than from males, and in response to food restriction, the net maximal rate of ALDO production was 50% greater in cells from males than from females. Our results demonstrate that food restriction in S. jarrovii increases adrenocortical cellular rates of steroid production without affecting overall cellular sensitivity to ACTH.

Effect of chronic administration of nicotine or cocaine on steroidogenesis in rat adrenocortical cells

Rats were implanted subcutaneously with osmotic mini-pumps containing either 0.9% NaCl, nicotine (1.5 or 4.5 mg kg-1 day-1), or cocaine (30 mg kg-1 day-1), for 14 days. Neither nicotine nor cocaine treatment significantly altered the maximal rate of steroidogenesis in adrenocortical cell preparations from the animals. However, pretreatment with cocaine increased the sensitivity of the preparation to stimulation by ACTH, the ED50 was 5 pM compared with 10 pM from control animals. Addition of nicotine or cocaine at concentrations up to 100 microM to adrenal cell suspensions from naive rats did not stimulate steroidogenesis or increase the sensitivity of cells to ACTH stimulation. These results suggest that the primary chronic effect of nicotine on steroidogenesis is exerted at the level of the hypothalamus and/or pituitary and not directly on adrenocortical cells. On the contrary, pretreatment with cocaine causes persistent changes in adrenocortical cells.

Upregulation of TLR2 and TLR4 in the human adrenocortical cells differentially modulates adrenal steroidogenesis

Rapid activation of adrenal steroid release plays a pivotal role in an organism's first line of defense during sepsis. Adrenal gland function is often suppressed in critically ill patients and negatively impacts the overall survival rate. Increasingly, experimental and clinical evidence suggests that Toll-like receptors (TLRs), components of the innate immune system, play a key role in the mediation of systemic responses to invading pathogens during sepsis. In the present study, we aimed to elucidate the effect of TLR2, TLR4 and CD14 upregulation on adrenocortical cell steroidogenesis. We found that TLR4 and CD14 but not TLR2 overexpression in NCI-H295R cells inhibited basal and acute cortisol and aldosterone production. This effect could be partially explained by reduced expression of enzymes involved in the synthesis of latter steroids – CYP11B1 and CYP11B2. Together, these data suggest that TLR upregulation in the steroid producing cells may be involved in the adrenal gland dysfunction during sepsis.

Angiotensin II-Induced Expression of Brain-Derived Neurotrophic Factor in Human and Rat Adrenocortical Cells

Angiotensin II (Ang II) is a major regulator of steroidogenesis in adrenocortical cells, and is also an effective inducer of cytokine and growth factor synthesis in several cell types. In microarray analysis of H295R human adrenocortical cells, the mRNA of brain-derived neurotrophic factor (BDNF), a neurotrophin widely expressed in the nervous system, was one of the most up-regulated genes by Ang II. The aim of the present study was the analysis of the Ang II-induced BDNF expression and BDNF-induced effects in adrenocortical cells. Real-time PCR studies have shown that BDNF is expressed in H295R and rat adrenal glomerulosa cells. In H295R cells, the kinetics of Ang II-induced BDNF expression was faster than that of aldosterone synthase (CYP11B2). Inhibition of calmodulin kinase by KN93 did not significantly affect the Ang II-induced stimulation of BDNF expression, suggesting that it occurs by a different mechanism from the CYP11B2-response. Ang II also caused candesartan-sensitive, type-1 Ang II receptor-mediated stimulation of BDNF gene expression in primary rat glomerulosa cells. In rat adrenal cortex, BDNF protein was localized to the subcapsular region. Ang II increased BDNF protein levels both in human and rat cells, and BDNF secretion of H295R cells. Ang II also increased type-1 Ang II receptor-mediated BDNF expression in vivo in furosemide-treated rats. In rat glomerulosa cells, BDNF induced tropomyosin-related kinase B receptor-mediated stimulation of EGR1 and TrkB expression. These data demonstrate that Ang II stimulates BDNF expression in human and rat adrenocortical cells, and BDNF may have a local regulatory function in adrenal glomerulosa cells.

Possible Contribution of 2-Aminoethoxydiphenyl-borate-sensitive Ca2+ Mobilization to Adrenocorticotropin-induced Glucocorticoid Synthesis in Rat Adrenocortical Cells

Cytoplasmic calcium ([Ca(2+)](i)) provided through voltage-dependent Ca(2+) channels (VDCC) plays an important role in adrenocorticotropin (ACTH)-induced steroidogenesis in adrenocortical cells. To identify alternative mechanisms for [Ca(2+)](i) supply, we investigated the 2-aminoethoxydiphenyl borate (2APB)-sensitive pathway as one of the possible signaling pathways involved in [Ca(2+)](i) supply for ACTH-induced steroidogenesis. In monolayers of cultured rat adrenal fasciculate and reticularis cells, ACTH at 10(-11) M stimulated corticosterone synthesis without increasing intracellular cAMP, and corticosterone synthesis was decreased by 10 microM 2APB by 51.8% (6.71 +/- 0.97 vs. 3.23 +/- 0.05 ng/mL/4 hours; p<0.05). Furthermore, 2APB significantly decreased the 10(-11) M ACTH-stimulated [Ca(2+)](i). ACTH increased the intracellular inositol-1,4,5-trisphosphate (IP3) content with a peak at 10(-13) M ACTH, which illustrates the possibility that ACTH activates IP3/diacylglycerol- dependent protein kinase C signal transduction. However, the difference in ACTH concentrations between that responsible for the IP3 increase and steroidogenesis without elevated cAMP, suggest a hypothesis that IP3 is not required for steroidogenesis, but does involve an unknown messenger, which stimulates the release of Ca(2+) from the ER or the subsequent store-operated Ca(2+) entry (SOCE). The pregnenolone concentration in the culture medium was increased by ACTH, which was significantly suppressed by 2APB, showing that the 2APB-sensitive Ca(2+) supply affects cholesterol transport into the mitochondrial membrane via steroidogenic acute regulatory protein. Therefore, the SOCE may contribute to ACTH-induced steroidogenesis in the mitochondrial region. In conclusion, the [Ca(2+)](i) used for steroidogenesis may be derived from a 2APB-sensitive pathway and via VDCCs, particularly at physiological concentrations of ACTH. We suggest that ACTH receptors activate steroidogenesis via inositol triphosphate, or an unknown downstream messenger, which could be inhibited by 2APB.

Inhibition of Adrenocortical Carcinoma Cell Proliferation by Steroidogenic Factor-1 Inverse Agonists

ABSTRACT Context: Transcription factor steroidogenic factor-1 (SF-1) plays a pivotal role in the control of adrenocortical cell steroidogenesis and proliferation. SF-1 amplification and overexpression are found in most cases of childhood adrenocortical tumors (ACTs). Objective: Our objective was to investigate the effect of SF-1 inverse agonists of the alkyloxyphenol and isoquinolinone classes on the proliferation of human adrenocortical cell lines expressing SF-1 (H295R), in conditions of basal and increased SF-1 expression, or negative for SF-1 expression (SW-13). Main Outcome Measures: Proliferation assays, immunoblots, flow cytometric analyses, steroid hormone assays, and real-time quantitative PCR were used. Results: SF-1 inhibitors of the alkyloxyphenol class displayed a dose-dependent inhibitory effect on both SF-1-positive and -negative ACT cells, whereas SF-1 inverse agonists of the isoquinolinone class selectively inhibited cell proliferation elicited by SF-1 overexpression. These drugs also inhibited stimulated steroid hormone secretion and CYP21 and CYP17 mRNA expression. Conclusion: SF-1 inhibitors may represent a useful tool in the chemotherapy of ACTs.

Inhibition of Adrenocortical Carcinoma Cell Proliferation by Steroidogenic Factor-1 Inverse Agonists

Transcription factor steroidogenic factor-1 (SF-1) plays a pivotal role in the control of adrenocortical cell steroidogenesis and proliferation. SF-1 amplification and overexpression are found in most cases of childhood adrenocortical tumors (ACTs). Our objective was to investigate the effect of SF-1 inverse agonists of the alkyloxyphenol and isoquinolinone classes on the proliferation of human adrenocortical cell lines expressing SF-1 (H295R), in conditions of basal and increased SF-1 expression, or negative for SF-1 expression (SW-13). Proliferation assays, immunoblots, flow cytometric analyses, steroid hormone assays, and reverse transcription quantitative PCR were used. SF-1 inhibitors of the alkyloxyphenol class displayed a dose-dependent inhibitory effect on both SF-1-positive and -negative ACT cells, whereas SF-1 inverse agonists of the isoquinolinone class selectively inhibited cell proliferation elicited by SF-1 overexpression. These drugs also inhibited stimulated steroid hormone secretion and CYP21 and CYP17 mRNA expression. SF-1 inhibitors may represent a useful tool in the chemotherapy of ACTs.

Cholesterol sulphate affects production of steroid hormones by reducing steroidogenic acute regulatory protein level in adrenocortical cells

Steroidogenic acute regulatory (StAR) protein plays a crucial role in the intramitochondrial movement of cholesterol, where P450 side chain cleavage enzyme resides. Cholesterol sulphate (CS), which is present ubiquitously in mammalian tissues, is not only a precursor of sulphated adrenal steroids but also an inhibitor of cholesterol biosynthesis. This study was designed to examine the biological roles of CS in steroidogenesis in adrenocortical cells. Human adrenocortical carcinoma H295R cells were cultured with various amounts of CS. To evaluate steroid hormone synthesis, pregnenolone production in cells was assayed. The amount of pregnenolone produced by H295R cells in culture medium, to which over 50 mug/ml CS was added, was significantly (P<0.05) decreased compared with that produced by control cells. Western blot analysis was performed to determine StAR protein level using whole cell extracts from cells. StAR protein level decreased when the concentration of CS in the medium was 50 mug/ml, whereas the level of glyceraldehyde-3-phosphate dehydrogenase did not change. To examine the mechanism by which StAR gene expression is controlled, we performed RT-PCR and measured promoter activity in cells transfected with pGL(2) StAR reporter constructs. StAR mRNA level and promoter activity were decreased in cells. The decrease in StAR protein level is a result of the low StAR gene expression level. In conclusion, CS affects the production of steroid hormones by reducing StAR protein level in adrenocortical cells.

Adenovirus-delivered DKK3/WNT4 and Steroidogenesis in Primary Cultures of Adrenocortical Cells

The Wnt family molecules Dickkopf-3 (DKK3) and WNT4 are present at higher concentrations in the zona glomerulosa than in the rest of the adrenal cortex. In order to study direct effects of these proteins on adrenocortical cell function, we created adenoviruses encoding human DKK3 and WNT4. When added to cultured human adrenocortical cells, DKK3 inhibited aldosterone and cortisol biosynthesis, either alone or together with cyclic AMP. WNT4 increased steroidogenesis when added alone but decreased it in the presence of cyclic AMP. A control adenovirus encoding GFP had no effect. RNA was prepared from cultured cells and was assayed by real-time PCR. CYP11A1 (cholesterol side-chain cleavage enzyme), HSD3B2 (3beta-hydroxysteroid dehydrogenase type II), CYP17 (17 alpha-hydroxylase), CYP21 (21-hydroxylase) and CYP11B1 (11 beta-hydroxylase) mRNAs were all increased by cyclic AMP, whereas CYP11B2 (aldosterone synthase) was unaffected. DKK3 decreased cyclic AMP-stimulated CYP17. WNT4 increased both CYP17 and CYP21 in the absence of cyclic AMP. Both DKK3 and WNT4 increased the level of CYP11B2. These data show that these Wnt signaling molecules have multiple actions on steroidogenesis in adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels. The co-localization of DKK3 and WNT4 in the glomerulosa and their stimulation of CYP11B2 imply an action on glomerulosa-specific function.

Sesquiterpene lactone helenalin suppresses Leydig and adrenocortical cell steroidogenesis by inhibiting expression of the steroidogenic acute regulatory protein

Helenalin is a potent anti-inflammatory and anti-neoplastic agent isolated from several plant species of the Asteracea family. Here, we have investigated the effects of helenalin on steroidogenesis activated by adrenocorticotropic hormone (ACTH) and human chorionic gonadotropin (hCG) in primary cultures of rat adrenocortical and Leydig cells. Our findings demonstrate that helenalin inhibits both ACTH- and hCG-activated steroidogenesis in these cells. This effect was already evident after 2–3 h treatment with helenalin. In contrast, steroidogenesis from 22R-OHC, a cell-permeable form of cholesterol, was not inhibited by helenalin, suggesting that the expression of the steroidogenic acute regulartory (StAR) protein might be inhibited by this compound. Indeed, helenalin attenuated StAR protein expression activated by ACTH and hCG in adrenocortical and Leydig cells as assessed by PAGE/Western analyses. This inhibitory action of helenalin on steroidogenic cell functions indicates novel mechanisms of action of this compound which may be of potential therapeutic interest. However, it also poses safety concerns relating to possible negative side-effects on anabolism and systemic stress.

Th-P15:132 The stimulatory effects of low density lipoprotein on steroidogenesis in adrenocortical cells decline with in vitro oxidation and in vivo modification

Th-P15:132 The stimulatory effects of low density lipoprotein on steroidogenesis in adrenocortical cells decline with in vitro oxidation and in vivo modification

Janus Kinase 2 Signaling in the Angiotensin II‐Dependent Activation of StAR Expression

Angiotensin II (Ang II)‐stimulated aldosterone production in adrenocortical glomerulosa cells requires de novo expression of the steroidogenic acute regulatory protein (StAR). We previously reported that StAR mRNA levels and promoter–reporter gene activity in transiently transfected H295R human adrenocortical cells were stimulated by Ang II and the goals for the current study were to identify signaling pathways activated by Ang II that contribute to StAR transcriptional activation. Using StAR promoter–reporter gene activity and pharmacological inhibition of signaling pathways, we have shown that Ang II‐stimulated StAR transcription in H295R cells is dependent upon both influx of external Ca2 + and tyrosine kinase signaling and is enhanced by protein kinase C and mitogen‐activated protein kinase (ERK1/2) activation. In particular, Janus tyrosine kinase‐2 (Jak2) activation was increased with Ang‐II treatment of H295R cells and the select Jak2 inhibitor, AG490, blocked Ang II‐dependent Jak2 activation, StAR reporter gene activity, and steroid production. The Ang II‐dependent, but not (Bu)2cAMP‐dependent, induction of StAR mRNA was also blocked by AG490 and shown to be sensitive to cycloheximide treatment. Together our data support Jak2 as a novel pathway in the Ang II‐dependent activation of StAR expression and steroidogenesis in adrenocortical cells and indicate a requirement for ongoing protein synthesis in Ang II‐mediated StAR transcription.

Janus Kinase 2 and Calcium Are Required for Angiotensin II-dependent Activation of Steroidogenic Acute Regulatory Protein Transcription in H295R Human Adrenocortical Cells

Angiotensin II- and K+-stimulated aldosterone production in the adrenocortical glomerulosa cells requires induction of the steroidogenic acute regulatory protein (StAR). While both agents activate Ca2+ signaling, the mechanisms leading to aldosterone synthesis are distinct, and the angiotensin II response cannot be mimicked by K+. We previously reported that StAR mRNA levels and promoter-reporter gene activity in transiently transfected H295R human adrenocortical cells were stimulated by angiotensin II but not by K+ treatment. The current study focused on identifying signaling pathways activated by angiotensin II that contribute to StAR transcriptional activation. We show that the angiotensin II-stimulated transcriptional activation of StAR was dependent upon influx of external calcium and requires protein kinase C activation. Furthermore we describe for the first time that the Janus tyrosine kinase family member, JAK2, was activated by angiotensin II treatment of H295R cells. Treatment of the cells with AG490, a selective inhibitor of JAK2, blocked JAK2 activation and StAR reporter gene activity and inhibited steroid production. Taken together these studies describe a novel pathway controlling StAR expression and steroidogenesis in adrenocortical cells.

Transforming growth factor beta1 inhibits aldosterone and cortisol production in the human adrenocortical cell line NCI-H295R through inhibition of CYP11B1 and CYP11B2 expression.

Transforming growth factor beta1 (TGFbeta1) has been shown to exert strong inhibitory effects on adrenocortical cell steroidogenesis. However, the molecular targets of TGFbeta1 in adrenocortical cells appear to differ between species. Here, we report the first characterization of the regulatory effects of TGFbeta1 on the steroidogenic functions of the human adrenocortical tumor cell line NCI-H295R. After treatment with 2 ng/ml TGFbeta1 for 24 h, basal production of corticosterone, cortisol and androstenedione was dramatically decreased. When TGFbeta1 was added simultaneously with forskolin, the production of cortisol and 11-hydroxyandrostenedione was decreased by 85% whereas that of deoxycortisol was increased. When TGFbeta1 was added simultaneously with angiotensin II, aldosterone production was reduced by 80%. We observed that TGFbeta1 strongly inhibits forskolin-induced steroid 11beta-hydroxylase activity and CYP11B1 mRNA levels, as well as angiotensin II-induced aldosterone synthase activity and CYP11B2 mRNA levels. CYP11B1 and CYP11B2 gene products thus appear as the major steroidogenic enzymes down-regulated by TGFbeta1 in the human adrenocortical tumor cell line NCI-H295R.

Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway.

The result of our previous study has shown that the K-ras mutant (pK568MRSV) transfected human adrenocortical cells can significantly increase cortisol production and independently cause cell transformation. The aim of this study is to investigate the effect of the active K-ras oncogene on the cortisol production in normal human adrenocortical cells. First we used isopropyl thiogalactoside to induce the inducible mutant K-ras expression plasmid, pK568MRSV, in the stable transfected human adrenocortical cells. The result showed that the increase of RasGTP levels in transfected cells was time-dependent after isopropyl thiogalactoside induction. Additionally, results from Western blot analysis revealed significant elevation in phosphorylation of c-Raf-1 and Mitogen-activated protein kinase. We also detected the levels of mRNA encoding Cholesterol side-chain cleavage enzyme (P450SCC), 17α-Hydroxylase/17,20-lyase (P450c17) and 3β-Hydroxysteroid dehydrogenase (3βHSD) were increased in human adrenocortical cells transfected with mutant K-ras after IPTG treatment. The increase of mRNA amount in P450scc P450c17 and 3βHSD and the elevation of cortisol level were inhibited with a pretreatment of PD098059, a specific extracellular signal-regulated kinase inhibitor. In our previous report, we proved that lovastatin, a pharmacological inhibitor of p21ras function, also reversed the increase of cortisol level in mutant K-ras stably transfected human adrenocortical cells. Taken together, these findings proved that the active mutant Ras enhanced not only cell proliferation but also steroidogenesis in steroidogenic phenotype cells by activating Raf-MEK-MAPK related signal transduction pathway. Therefore, we believe that K-ras mutants influence regulation of steroidogenesis in adrenocortical cells through RAF-MEK-MAPK pathway.British Journal of Cancer (2002) 87, 1000–1005. doi:10.1038/sj.bjc.6600589 www.bjcancer.com© 2002 Cancer Research UK