Alternative pathway androgen biosynthesis and human fetal female virilization.

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography-tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography-mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.

Regulation of aldosterone synthase by activator transcription factor/cAMP response element-binding protein family members.

Aldosterone synthesis is regulated by angiotensin II (Ang II) and K(+) acting in the adrenal zona glomerulosa, in part through the regulation of aldosterone synthase (CYP11B2). Here, we analyzed the role of cAMP response element (CRE)-binding proteins (CREBs) in the regulation of CYP11B2. Expression analysis of activator transcription factor (ATF)/CREB family members, namely the ATF1 and ATF2, the CREB, and the CRE modulator, in H295R cells and normal human adrenal tissue was performed using quantitative real-time PCR. Ang II-induced phosphorylation of ATF/CREB members was analyzed by Western blot analysis, and their subsequent binding to the CYP11B2 promoter using chromatin immunoprecipitation assay. Aldosterone production and CYP11B2 expression were measured in small interfering RNA-transfected cells to knockdown the expression of ATF/CREB members. CYP11B2 promoter activity was measured in H295R cells cotransfected with NURR1 (NR4A2) alone or with constitutively active vectors for ATF/CREB members. Ang II induced phosphorylation of ATF1, ATF2, and CRE modulator in a time-dependent manner. Based on chromatin immunoprecipitation analysis, there was an increased association of these proteins with the CYP11B2 promoter after Ang II and K(+) treatment. Phosphorylated ATF/CREB members also bound the CYP11B2 promoter. Knockdown of ATF/CREB members reduced Ang II and K(+) induction of adrenal cell CYP11B2 mRNA expression and aldosterone production. The constitutively active ATF/CREB vectors increased the promoter activity of CYP11B2 and had a synergistic effect with NURR1. In summary, these results suggest that ATF/CREB and NGFI-B family members play a crucial role in the transcriptional regulation of CYP11B2 and adrenal cell capacity to produce aldosterone.

The role of TASK1 in aldosterone production and its expression in normal adrenal and aldosterone-producing adenomas.

OBJECTIVES: Aldosterone production in the adrenal glomerulosa is mainly regulated by angiotensin II and K+. Adrenal glomerulosa cells are uniquely sensitive to extracellular K+. Genetic deletion of subunits of K+-selective leak-channels (KCNK), TASK1 and/or TASK3, in mice generates animals with hyperaldosteronism and histological changes in the adrenal cortex. Herein, we studied the expression of TASK1 in human adrenocortical cells, as well as its role in aldosterone production in H295R cells. DESIGN: TASK1 expression was investigated by comparative microarray analysis of aldosterone-producing adenomas (APA) and normal adrenals (NAs). The effects of TASK1 knockdown by siRNA transfection were investigated in H295R cells. Fluo-4 fluorescent measurements of intracellular Ca2+ and pharmacological inhibition of Ca2+ -dependent calmodulin kinases (CaMK) were performed to better define the effects of TASK1 on Ca2+ signalling pathways. RESULTS: Microarray analysis of APA and NA showed similar expression of TASK1 between these two groups. However, in APA, NA and H295R cells the expression of TASK1 was predominant when compared with other KCNK family members. Knockdown of TASK1 (with siRNA) induced the expression of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2), and also stimulated pregnenolone and aldosterone production. Cells transfected with siTASK1 had increased intracellular Ca2+, leading to activation of CaMK and increased expression of CYP11B2. CONCLUSIONS: Our study reveals the predominant expression of TASK1 over other KCNK family genes in the human adrenal cortex. Herein, we also described the role of TASK1 in the regulation of human aldosterone production through regulation of intracellular Ca2+ and CaMK signalling pathways.

Role of angiotensin II-induced rapid response genes in the regulation of enzymes needed for aldosterone synthesis.

Aldosterone is principally synthesized in the zona glomerulosa of the adrenal by a series of enzymatic reactions leading to the conversion of cholesterol to aldosterone. Angiotensin II (Ang II) is the major physiological regulator of aldosterone production acting acutely to stimulate aldosterone biosynthesis and chronically to increase the capacity of the adrenals to produce aldosterone. We previously defined eight transcription factors that are rapidly induced following Ang II treatment using three in vitro adrenocortical cell models. Herein, we investigated the function of these transcription factors in the regulation of the enzymes needed for aldosterone production. H295R adrenal cells were co-transfected with expression vectors for each transcription factor and promoter/reporter constructs prepared for genes encoding the enzymes needed for aldosterone production. NGFI-B family members induced promoter activity of 3-beta-hydroxysteroid-dehydrogenase type 2 (HSD3B2), 21-hydroxylase (CYP21A2), and aldosterone synthase (CYP11B2). The importance of NGFI-B in the regulation of CYP11B2 was also demonstrated by reduced CYP11B2 transcription in the presence of a dominant-negative-NGFI-B. A pharmacological approach was used to characterize the Ang II pathways regulating transcription of NGFI-B family genes. Transcription of NGFI-B members were decreased following inhibition of Ang II type 1 receptor (AT1R), protein kinase C (PKC), calcium/calmodulin-dependent kinases (CaMK), and Src tyrosine kinase (SRC). Taken together, these results suggest that Ang II binding to the AT1R increases activity of PKC, CaMK, and SRC, which act to increase expression of the family of NGFI-B genes as well as CYP11B2. Ang II induction of the NGFI-B family members represents an important pathway to increase the capacity of adrenal cells to produce aldosterone.

Angiotensin II regulation of adrenocortical gene transcription.

Angiotensin II (Ang II) is the key peptide hormone in the renin-angiotensin-aldosterone system (RAAS). Its ability to regulate levels of circulating aldosterone relies on actions on adrenal glomerulosa cells. Many of the Ang II effects on glomerulosa cells involve a precisely coordinated regulation of signaling cascades and gene expression. The development of genome-wide gene arrays has allowed the definition of transcriptome-wide effects of Ang II in adrenocortical cells. Analysis of the Ang II gene targets reveals broad effects on cellular gene expression, particularly the rapid induction of numerous transcription factors that may regulate long-term steroid metabolism and cell growth/proliferation. Herein we discuss the Ang II-induced genes in adrenocortical cells and review the progress in defining the role of these genes in zona glomerulosa function.

Activation of the ET-1/ETA pathway contributes to erectile dysfunction associated with mineralocorticoid hypertension.

INTRODUCTION: The cavernosal tissue is highly responsive to endothelin-1 (ET-1), and penile smooth muscle cells not only respond to but also synthesize ET-1. AIM: Considering that ET-1 is directly involved in end-organ damage in salt-sensitive forms of hypertension, we hypothesized that activation of the ET-1/ET(A) receptor pathway contributes to erectile dysfunction (ED) associated with mineralocorticoid hypertension. METHODS: Wistar rats were uninephrectomized and submitted to deoxycorticosterone acetate (DOCA)-salt treatment for 5 weeks. Control (Uni [uninephrectomized control]) animals were uninephrectomized and given tap water. Uni and DOCA-salt rats were simultaneously treated with vehicle or atrasentan (ET(A) receptor antagonist, 5 mg/Kg/day). Cavernosal reactivity to ET-1, phenylephrine (PE), ET(B) receptor agonist (IRL-1620) and electric field stimulation (EFS) were evaluated in vitro. Expression of ROCKalpha, ROCKbeta, myosin phosphatase target subunit 1 (MYPT-1), and extracellular signal-regulated kinase 1/2 (ERK 1/2) were evaluated by western blot analysis. ET-1 and ET(A) receptor mRNA expression was evaluated by real-time reverse-transcriptase polymerase chain reaction. Voltage-dependent increase in intracavernosal pressure/mean arterial pressure (ICP/MAP) was used to evaluate erectile function in vivo. MAIN OUTCOME MEASURE: ET(A) receptor blockade prevents DOCA-salt-associated ED. RESULTS: Cavernosal strips from DOCA-salt rats displayed augmented preproET-1 expression, increased contractile responses to ET-1 and decreased relaxation to IRL-1620. Contractile responses induced by EFS and PE were enhanced in cavernosal tissues from DOCA-salt hypertensive rats. These functional changes were associated with increased activation of the RhoA/Rho-kinase and ERK 1/2 pathways. Treatment of rats with atrasentan completely prevented changes in cavernosal reactivity in DOCA-salt rats and restored the decreased ICP/MAP, completely preventing ED in DOCA-salt rats. CONCLUSION: Activation of the ET-1/ET(A) pathway contributes to mineralocorticoid hypertension-associated ED. ET(A) receptor blockade may represent an alternative therapeutic approach for ED associated with salt-sensitive hypertension and in pathological conditions where increased levels of ET-1 are present.

Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells.

Angiotensin-II (Ang-II) regulates adrenal steroid production and gene transcription through several signaling pathways. Changes in gene transcription occur within minutes after Ang-II stimulation, causing an increase in aldosterone production and subsequent increase in the overall capacity to produce aldosterone. Our goal was to compare the Ang-II regulation of early gene expression and confirm the up-regulation of selected genes using quantitative real-time RT-PCR (qPCR) across three species, such as, human, bovine, and rat. Microarray analyses were performed using samples from control and Ang-II (10 nM)-treated (1 h) cells from human adrenocortical tumor cell line H295R, and primary adrenal glomerulosa cells from bovine and rat, applied respectively to human, bovine, and rat chips. qPCR was performed to confirm up-regulation of selected genes using mRNA. The microarray comparison revealed 18% similarity among the top 50 up-regulated genes, with human/rat, 20%; human/bovine, 36%; and rat/bovine, 26% similarity. The gene list generated by this comparison included: activating transcription factor 3, B-cell translocation gene (BTG2), Nuclear receptor subfamily 4, group A, member 1 (NR4A1), NR4A2, NR4A3, early growth response 1, v-fos FBJ murine osteosarcoma viral oncogene homolog (c-FOS), FOSB, and Jun family member B (JUNB). Pretreatment of H295R cells with cycloheximide had no effect on Ang-II induction of these genes, suggesting that they are direct targets of Ang-II signaling. The Ang-II gene targets have been defined in three different adrenocortical model systems. Several of the listed genes have previously been described as being key regulators of adrenocortical function. The presence of adrenal cell common genes in such distinct cell models strengthens the hypothesis that these genes are regulators of aldosterone production.

Mineralocorticoid receptor activation causes cerebral vessel remodeling and exacerbates the damage caused by cerebral ischemia.

Mineralocorticoid receptor antagonists protect against ischemic cerebrovascular disease; this appears to be caused by changes in cerebral vessel structure that would promote blood flow. Therefore, we hypothesized that mineralocorticoid receptor activation with deoxycorticosterone acetate would cause deleterious remodeling of the cerebral vasculature and exacerbate the damage caused by cerebral ischemia. Six-week-old male Wistar rats were treated with deoxycorticosterone acetate (200 mg/kg) for 6 weeks. At 12 weeks of age, the deoxycorticosterone acetate-treated rats had elevated systolic blood pressure compared with age-matched controls (157+/-5.9 versus 124+/-3.1 mm Hg deoxycorticosterone acetate versus control; P<0.05). The area of ischemic damage resulting from middle cerebral artery occlusion was greater in the deoxycorticosterone acetate-treated rats than control (63.5+/-3.72 versus 46.6+/-5.52% of the hemisphere infarcted, deoxycorticosterone acetate versus control; P<0.05). Middle cerebral artery structure was assessed using a pressurized arteriograph under calcium-free conditions. Over a range of intralumenal pressures, the lumen and ODs of the middle cerebral arteries were smaller in the deoxycorticosterone acetate-treated rats than the control rats (P<0.05). There was also an increase in the wall thickness and wall:lumen ratio in the vessels from deoxycorticosterone acetate-treated rats (P<0.05). The vessels from the deoxycorticosterone acetate-treated rats were stiffer than those from control rats as evidenced by a leftward shift in the stress/strain curve. These novel data suggest that mineralocorticoid receptor activation without salt loading and nephrectomy is sufficient to elicit deleterious effects on the cerebral vasculature that lead to inward hypertrophic remodeling and an increase in the ischemic damage in the event of a stroke.