Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency.

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

Modulation of human CYP19A1 activity by mutant NADPH P450 oxidoreductase.

Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated Km of 126 nm for androstenedione and a Vmax of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.

Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis.

Androgens are essential for sexual development and reproduction. However, androgen regulation in health and disease is poorly understood. We showed that human adrenocortical H295R cells grown under starvation conditions acquire a hyperandrogenic steroid profile with changes in steroid metabolizing enzymes HSD3B2 and CYP17A1 essential for androgen production. Here we studied the regulatory mechanisms underlying androgen production in starved H295R cells. Microarray expression profiling of normal versus starved H295R cells revealed fourteen differentially expressed genes; HSD3B2, HSD3B1, CYP21A2, RARB, ASS1, CFI, ASCL1 and ENC1 play a role in steroid and energy metabolism and ANGPTL1, PLK2, DUSP6, DUSP10 and FREM2 are involved in signal transduction. We discovered two new gene networks around RARB and ANGPTL1, and show how they regulate androgen biosynthesis. Transcription factor RARB stimulated the promoters of genes involved in androgen production (StAR, CYP17A1 and HSD3B2) and enhanced androstenedione production. For HSD3B2 regulation RARB worked in cooperation with Nur77. Secretory protein ANGPTL1 modulated CYP17A1 and DUSP6 expression by inducing ERK1/2 phosphorylation. By contrast, our studies revealed no evidence for hormones or cell cycle involvement in regulating androgen biosynthesis. In summary, these studies establish a firm role for RARB and ANGPTL1 in the regulation of androgen production in H295R cells.

Characterization of a novel CYP19A1 (aromatase) R192H mutation causing virilization of a 46,XX newborn, undervirilization of the 46,XY brother, but no virilization of the mother during pregnancies.

BACKGROUND: P450 aromatase (CYP19A1) is essential for the biosynthesis of estrogens from androgen precursors. Mutations in the coding region of CYP19A1 lead to autosomal recessive aromatase deficiency. To date over 20 subjects have been reported with aromatase deficiency which may manifest during fetal life with maternal virilization and virilization of the external genitalia of a female fetus due to low aromatase activity in the steroid metabolizing fetal-placental unit and thus high androgen levels. During infancy, girls often have ovarian cysts and thereafter fail to enter puberty showing signs of variable degree of androgen excess. Moreover, impact on growth, skeletal maturation and other metabolic parameters is seen in both sexes. OBJECTIVE AND HYPOTHESIS: We found a novel homozygous CYP19A1 mutation in a 46,XX girl who was born at term to consanguineous parents. Although the mother did not virilize during pregnancy, the baby was found to have a complex genital anomaly at birth (enlarged genital tubercle, fusion of labioscrotal folds) with elevated androgens at birth, normalizing thereafter. Presence of 46,XX karyotype and female internal genital organs (uterus, vagina) together with biochemical findings and follow-up showing regression of clitoral hypertrophy, as well as elevated FSH suggested aromatase deficiency. Interestingly, her older brother presented with mild hypospadias and bilateral cryptorchidism and was found to carry the same homozygous CYP19A1 mutation. To confirm the clinical diagnosis, genetic, functional and computational studies were performed. METHODS AND RESULTS: Genetic analysis revealed a homozygous R192H mutation in the CYP19A1 gene. This novel mutation was characterized for its enzymatic activity (Km, Vmax) in a cell model and found to have markedly reduced catalytic activity when compared to wild-type aromatase; thus explaining the phenotype. Computational studies suggest that R192H disrupts the substrate access channel in CYP19A1 that may affect binding of substrates and exit of catalytic products. CONCLUSION: R192H is a novel CYP19A1 mutation which causes a severe phenotype of aromatase deficiency in a 46,XX newborn and maybe hypospadias and cryptorchidism in a 46,XY, but no maternal androgen excess during pregnancy.

Differential regulation of human 3ß-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model.

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3ß-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

Role of AMP-activated protein kinase on steroid hormone biosynthesis in adrenal NCI-H295R cells.

Regulation of human androgen biosynthesis is poorly understood. However, detailed knowledge is needed to eventually solve disorders with androgen dysbalance. We showed that starvation growth conditions shift steroidogenesis of human adrenal NCI-H295R cells towards androgen production attributable to decreased HSD3B2 expression and activity and increased CYP17A1 phosphorylation and 17,20-lyase activity. Generally, starvation induces stress and energy deprivation that need to be counteracted to maintain proper cell functions. AMP-activated protein kinase (AMPK) is a master energy sensor that regulates cellular energy balance. AMPK regulates steroidogenesis in the gonad. Therefore, we investigated whether AMPK is also a regulator of adrenal steroidogenesis. We hypothesized that starvation uses AMPK signaling to enhance androgen production in NCI-H295R cells. We found that AMPK subunits are expressed in NCI-H295 cells, normal adrenal tissue and human as well as pig ovary cells. Starvation growth conditions decreased phosphorylation, but not activity of AMPK in NCI-H295 cells. In contrast, the AMPK activator 5-aminoimidazole-4-carboxamide (AICAR) increased AMPKα phosphorylation and increased CYP17A1-17,20 lyase activity. Compound C (an AMPK inhibitor), directly inhibited CYP17A1 activities and can therefore not be used for AMPK signaling studies in steroidogenesis. HSD3B2 activity was neither altered by AICAR nor compound C. Starvation did not affect mitochondrial respiratory chain function in NCI-H295R cells suggesting that there is no indirect energy effect on AMPK through this avenue. In summary, starvation-mediated increase of androgen production in NCI-H295 cells does not seem to be mediated by AMPK signaling. But AMPK activation can enhance androgen production through a specific increase in CYP17A1-17,20 lyase activity.

Metformin inhibits human androgen production by regulating steroidogenic enzymes HSD3B2 and CYP17A1 and complex I activity of the respiratory chain.

Metformin is treatment of choice for the metabolic consequences seen in polycystic ovary syndrome for its insulin-sensitizing and androgen-lowering properties. Yet, the mechanism of action remains unclear. Two potential targets for metformin regulating steroid and glucose metabolism are AMP-activated protein kinase (AMPK) signaling and the complex I of the mitochondrial respiratory chain. Androgen biosynthesis requires steroid enzymes 17α-Hydroxylase/17,20 lyase (CYP17A1) and 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2), which are overexpressed in ovarian cells of polycystic ovary syndrome women. Therefore, we aimed to understand how metformin modulates androgen production using NCI-H295R cells as an established model of steroidogenesis. Similar to in vivo situation, metformin inhibited androgen production in NCI cells by decreasing HSD3B2 expression and CYP17A1 and HSD3B2 activities. The effect of metformin on androgen production was dose dependent and subject to the presence of organic cation transporters, establishing an important role of organic cation transporters for metformin's action. Metformin did not affect AMPK, ERK1/2, or atypical protein kinase C signaling. By contrast, metformin inhibited complex I of the respiratory chain in mitochondria. Similar to metformin, direct inhibition of complex I by rotenone also inhibited HSD3B2 activity. In conclusion, metformin inhibits androgen production by mechanisms targeting HSD3B2 and CYP17-lyase. This regulation involves inhibition of mitochondrial complex I but appears to be independent of AMPK signaling.

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.

Pioglitazone inhibits androgen production in NCI-H295R cells by regulating gene expression of CYP17 and HSD3B2.

Thiazolidinediones (TZDs) such as pioglitazone and rosiglitazone are widely used as insulin sensitizers in the treatment of type 2 diabetes. In diabetic women with polycystic ovary syndrome, treatment with pioglitazone or rosiglitazone improves insulin resistance and hyperandrogenism, but the mechanism by which TZDs down-regulate androgen production is unknown. Androgens are synthesized in the human gonads as well as the adrenals. We studied the regulation of androgen production by analyzing the effect of pioglitazone and rosiglitazone on steroidogenesis in human adrenal NCI-H295R cells, an established in vitro model of steroidogenesis of the human adrenal cortex. Both TZDs changed the steroid profile of the NCI-H295R cells and inhibited the activities of P450c17 and 3betaHSDII, key enzymes of androgen biosynthesis. Pioglitazone but not rosiglitazone inhibited the expression of the CYP17 and HSD3B2 genes. Likewise, pioglitazone repressed basal and 8-bromo-cAMP-stimulated activities of CYP17 and HSD3B2 promoter reporters in NCI-H295R cells. However, pioglitazone did not change the activity of a cAMP-responsive luciferase reporter, indicating that it does not influence cAMP/protein kinase A/cAMP response element-binding protein pathway signaling. Although peroxisome proliferator-activated receptor gamma (PPARgamma) is the nuclear receptor for TZDs, suppression of PPARgamma by small interfering RNA technique did not alter the inhibitory effect of pioglitazone on CYP17 and HSD3B2 expression, suggesting that the action of pioglitazone is independent of PPARgamma. On the other hand, treatment of NCI-H295R cells with mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) enhanced promoter activity and expression of CYP17. This effect was reversed by pioglitazone treatment, indicating that the MEK/ERK signaling pathway plays a role in regulating androgen biosynthesis by pioglitazone.

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3beta-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities.

The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3beta-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the 'androgenic' steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.