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.

Regulation of human (adrenal) androgen biosynthesis-New insights from novel throughput technology studies

Androgens are precursors for sex steroids and are predominantly produced in the human gonads and the adrenal cortex. They are important for intrauterine and postnatal sexual development and human reproduction. Although human androgen biosynthesis has been extensively studied in the past, exact mechanisms underlying the regulation of androgen production in health and disease remain vague. Here, the knowledge on human androgen biosynthesis and regulation is reviewed with a special focus on human adrenal androgen production and the hyperandrogenic disorder of polycystic ovary syndrome (PCOS). Since human androgen regulation is highly specific without a good animal model, most studies are performed on patients harboring inborn errors of androgen biosynthesis, on human biomaterials and human (tumor) cell models. In the past, most studies used a candidate gene approach while newer studies use high throughput technologies to identify novel regulators of androgen biosynthesis. Using genome wide association studies on cohorts of patients, novel PCOS candidate genes have been recently described. Variant 2 of the DENND1A gene was found overexpressed in PCOS theca cells and confirmed to enhance androgen production. Transcriptome profiling of dissected adrenal zones established a role for BMP4 in androgen synthesis. Similarly, transcriptome analysis of human adrenal NCI-H295 cells identified novel regulators of androgen production. Kinase p38α (MAPK14) was found to phosphorylate CYP17 for enhanced 17,20 lyase activity and RARB and ANGPTL1 were detected in novel networks regulating androgens. The discovery of novel players for androgen biosynthesis is of clinical significance as it provides targets for diagnostic and therapeutic use.

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.

Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability

Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.

Matrix metalloproteinase (MMP)-2 and MMP-9 activity and localization during ventral prostate atrophy and regrowth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modulação de proteínas regulatórias e juncionais da barreira hematotesticular em células de Sertoli humanas (HSec) expostas ao Monobutil ftalato (MBP) e ao Bisfenl A (BPA)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Theca cells and the regulation of ovarian androgen production

Theca cells are essential for female reproduction being the source of androgens that are precursors for follicular oestrogen synthesis and also signal through androgen receptors (AR) in the ovary and elsewhere. Theca cells arise from mesenchymal cells around the secondary follicle stage. Their recruitment, proliferation and cytodifferentiation are influenced, directly or indirectly, by paracrine signals from granulosa cells and oocyte although uncertainty remains over which are the critically important signals at particular stages. In a reciprocal manner, theca cells secrete factors that influence granulosa cell proliferation and differentiation at different follicle stages. Differentiated theca interna cells acquire responsiveness to luteinizing hormone (LH) and other endocrine signals and express components of the steroidogenic machinery required for androgen biosynthesis. They also express insulin-like peptide 3 (INSL3) and its receptor (RXFP2), levels of which increase during bovine antral follicle development. INSL3 signaling may play a role in promoting androgen biosynthesis since knockdown of either INSL3 or its receptor (RXFP2) in bovine theca cells inhibits androgen biosynthesis while exogenous INSL3 can raise androgen secretion. Bone morphogenetic proteins (BMPs) of thecal or granulosal origin suppress thecal production of both INSL3 and androgen. Inhibin, produced in greatest amounts by granulosa cells of preovulatory follicles, reverses these BMP actions. Thus, BMP-induced inhibition of thecal androgen production may be mediated by reduced INSL3-RXFP2 signaling. Activins also inhibit androgen production in an inhibin-reversible manner and recent evidence in sheep indicates that theca cells synthesize and secrete activin, implying an autocrine role in suppressing androgen biosynthesis in smaller follicles, akin to that envisaged for BMPs.

Regulation of androgen biosynthesis - A short review and preliminary results from the hyperandrogenic starvation NCI-H295R cell model

Regulation of androgen production is poorly understood. Adrenarche is the physiologic event in mid-childhood when the adrenal zona reticularis starts to produce androgens through specific expression of genes for enzymes and cofactors necessary for androgen synthesis. Similarly, expression and activities of same genes and products are deregulated in hyperandrogenic disorders such as the polycystic ovary syndrome (PCOS). Numerous studies revealed involvement of several signaling pathways stimulated through G-protein coupled receptors or growth factors transmitting their effects through cAMP- or non-cAMP-dependent signaling. Overall a complex network regulates androgen synthesis targeting involved genes and proteins at the transcriptional and post-translational levels. Newest players in the field are the DENND1A gene identified in PCOS patients and the MAPK14 which is the kinase phosphorylating CYP17 for enhanced lyase activity. Next generation sequencing studies of PCOS patients and transcriptome analysis of androgen producing tissues or cell models provide newer tools to identify modulators of androgen synthesis.

Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (−/−) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (−/−) mice and minimizes Cyp 4a12 expression and arachidonate ω-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (−/−) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 ω-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

Background: Melatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation. Methods: Twenty-four adult Wistar rats, 60 days old (+/-250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL + 95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle + melatonin [ 100 mu g/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a. m. Results: Melatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated. Conclusions: We suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation.

Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

Pires-Oliveira M, Maragno AL, Parreiras-E-Silva LT, Chiavegatti T, Gomes MD, Godinho RO. Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo. J Appl Physiol 108: 266-273, 2010. First published November 19, 2009; doi:10.1152/japplphysiol.00490.2009.-Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.

Intrinsic regional differences in androgen receptors and dihydrotestosterone metabolism in human preadipocytes

Androgens play an important role in regulating the central obesity that is a strong risk factor for cardiovascular disease and insulin resistance. This study confirms that androgen receptors are present in subcultured human preadipocytes, with androgen receptor gene expression and saturable specific dihydrotestosterone binding, dissociation constant 1.02 - 2.56 nM and maximal binding capacity 30.8 - 55.7 fmol/mg protein. There was an intrinsic regional difference in androgen receptor complement, with more androgen receptors in visceral than in subcutaneous preadipocytes. Dihydrotestosterone was metabolised by human preadipocytes, with more androstanediol produced by subcutaneous than visceral preadipocytes. While dihydrotestosterone metabolism was insufficient to explain the regional variation in androgen binding, both of these differences would reduce the androgen responsiveness of the subcutaneous preadipocytes compared with visceral preadipocytes. There were no gender differences in androgen binding or metabolism. While the direct effects of androgens on human PAS remain uncertain, these regional differences suggest that AR-mediated regulation of certain PA functions influences adipose tissue distribution.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.

Bone morphogenetic proteins (BMP)-4,-6, and-7 potently suppress basal and luteinizing hormone-induced androgen production by bovine theca interna cells in primary culture: Could ovarian hyperandrogenic dysfunction be caused by a defect in thecal BMP signaling?

We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and - 7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, - 6, and - 7 potently suppress both basal ( P < 0.0001; respective IC50 values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced ( P < 0.0001; respective IC50 values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3 beta-hydroxysteroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment ( P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and - 7, respectively, and the observation that both antagonists enhanced ( P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP- 4 and - 7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.