Silencing of GSTP1, a prostate cancer prognostic gene, by the estrogen receptor-ß and endothelial nitric oxide synthase complex.

We recently identified in prostate tumors (PCa) a transcriptional prognostic signature comprising a significant number of genes differentially regulated in patients with worse clinical outcome. Induction of up-regulated genes was due to chromatin remodeling by a combinatorial complex between estrogen receptor (ER)-ß and endothelial nitric oxide synthase (eNOS). Here we show that this complex can also repress transcription of prognostic genes that are down-regulated in PCa, such as the glutathione transferase gene GSTP1. Silencing of GSTP1 is a common early event in prostate carcinogenesis, frequently caused by promoter hypermethylation. We validated loss of glutathione transferase (GST) P1-1 expression in vivo, in tissue microarrays from a retrospective cohort of patients, and correlated it with decreased disease-specific survival. Furthermore, we show that in PCa cultured cells ERß/eNOS causes GSTP1 repression by being recruited at estrogen responsive elements in the gene promoter with consequential remodeling of local chromatin. Treatment with ERß antagonist or its natural ligand 5α-androstane-3ß,17ß-diol, eNOS inhibitors or ERß small interference RNA abrogated the binding and reversed GSTP1 silencing, demonstrating the direct involvement of the complex. In vitro, GSTP1 silencing by ERß/eNOS was specific for cells from patients with worse clinical outcome where it appeared the sole mechanism regulating GSTP1 expression because no promoter hypermethylation was present. However, in vivo chromatin immunoprecipitation assays on fresh PCa tissues demonstrated that silencing by ERß/eNOS can coexist with promoter hypermethylation. Our findings reveal that the ERß/eNOS complex can exert transcriptional repression and suggest that this may represent an epigenetic event favoring inactivation of the GSTP1 locus by methylation. Moreover, abrogation of ERß/eNOS function by 3ß-adiol emphasizes the significance of circulating or locally produced sex steroid hormones or their metabolites in PCa biology with relevant clinical/therapeutic implications.

Anti-anxiety, cognitive, and steroid biosynthetic effects of an isoflavone-based dietary supplement are gonad and sex-dependent in rats.

Isoflavone-rich diets are associated with reduced menopausal symptoms and lowered risk of cancers of reproductive tissues. Isoflavones may mimic some effects of estrogen by binding to estrogen receptors, and/or altering steroid availability. Despite their potential health benefits, neither the effects, nor mechanisms, of isoflavones are well understood. We hypothesized that isoflavones would alter behavior and physiology of rats in sex and/or gonad-dependent manner. An isoflavone-based, commercially-available, dietary supplement was administered via subcutaneous implantation to female and male, intact and gonadectomized Long-Evans rats. Affective (elevated plus-maze), cognitive (water-maze), and reproductive (sexual) behavior was examined. Weights of reproductive structures were measured, as an index of trophic effects. Steroid levels in circulation and brain regions associated with behavioral measures were evaluated by radioimmunoassay. The supplement increased anti-anxiety behavior of intact, but not gonadectomized, rats. The supplement enhanced visual-spatial performance of all rats, but this effect was most evident among proestrous female rats, which had the poorest spatial performance. There were neither effects of the supplement on sexual behavior, mass of reproductive tissues, nor plasma steroid levels. The supplement increased levels of 5α-androstane,17ß-diol-3α-diol (3α-diol) in the hippocampus (but not other brain regions) of gonadectomized females. Thus, the supplement altered anxiety and cognitive behavior and brain production of steroids; however, the anti-anxiety effects were limited to rats with an intact reproductive axis and effects on cognitive performance and neurosteriodogenesis were most evident among intact and gonadectomized, female rats respectively.

An alternate pathway for androgen regulation of brain function: activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol.

The complexity of gonadal steroid hormone actions is reflected in their broad and diverse effects on a host of integrated systems including reproductive physiology, sexual behavior, stress responses, immune function, cognition, and neural protection. Understanding the specific contributions of androgens and estrogens in neurons that mediate these important biological processes is central to the study of neuroendocrinology. Of particular interest in recent years has been the biological role of androgen metabolites. The goal of this review is to highlight recent data delineating the specific brain targets for the dihydrotestosterone metabolite, 5alpha-androstane, 3beta,17beta-diol (3beta-Diol). Studies using both in vitro and in vivo approaches provide compelling evidence that 3beta-Diol is an important modulator of the stress response mediated by the hypothalmo-pituitary-adrenal axis. Furthermore, the actions of 3beta-Diol are mediated by estrogen receptors, and not androgen receptors, often through a canonical estrogen response element in the promoter of a given target gene. These novel findings compel us to re-evaluate the interpretation of past studies and the design of future experiments aimed at elucidating the specific effects of androgen receptor signaling pathways.

Partitioning of 5alpha-dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol activated pathways for stimulating human prostate cancer LNCaP cell proliferation.

The growth and development of the prostate gland are regulated by androgens. Despite our understanding of molecular actions of 5alpha-dihydrotestosterone (5alpha-DHT) in the prostate through the trans-activation of the androgen receptor (AR), comprehensive analysis of androgen responsive genes (ARGs) has just been started. Moreover, expression changes induced by the androgen effects of 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), a metabolite of 5alpha-DHT through the action of 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), remain undefined. We demonstrated that both 5alpha-DHT and 3alpha-diol stimulated similar levels of androgen sensitive human prostate cancer LNCaP cell proliferation. However, consistent with the fact that 3alpha-diol has low affinity toward the AR, 3alpha-diol did not elicit the same levels of AR trans-activation activity as that of 5alpha-DHT. Since LNCaP cells respond to androgen stimulation by transcriptionally activating the AR downstream genes, gene expression patterns between 0 and 48 h following 3alpha-diol and 5alpha-DHT stimulation were analyzed using cDNA-based membrane arrays to define the temporal regulation of ARGs. Array analysis identified 217 and 219 androgen-modulated genes in at least one time point following 3alpha-diol and 5alpha-DHTstimulation, respectively, including key regulators of cell proliferation. Only a subset of these genes (143) was regulated by both androgens. These data suggest that 3alpha-diol exerts androgenic effects independent of the action of 5alpha-DHT in steroid target tissues. Accordingly, 3alpha-diol might activate cell proliferation cascades independent of AR pathway in the prostate.

Penile development is initiated in the tammar wallaby pouch young during the period when 5alpha-androstane-3alpha,17beta-diol is secreted by the testes.

Virilization of the urogenital tract is under the control of testicular androgens in all mammals. In tammar young, prostate differentiation begins between d 20 and d 40 under the control of the testicular androgen 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol), but uncertainties exist about the control of penile development. We performed longitudinal studies up to d 150 of pouch life to define normal penile development and the effects of androgen administration and castration. In control animals the male phallus was longer than the female phallus by d 48. Closure of the urethra in males begins around d 60 and continues to at least d 150. Administration of supraphysiological doses of testosterone to females caused penile development equivalent to that of the male and also induced partial closure of the urethral groove by d 150. Castration of male pouch young at d 25 prevented penile development, whereas the penis in males castrated at d 40, 80, or 120 had partial closure of the urethral groove. Administration of 5alpha-adiol to females from d 20-40 also caused partial closure of the urethral groove and some growth of the phallus at d 150, whereas 5alpha-adiol treatment from d 40-80 or 80-120 caused some penile growth but had little effect on urethral development. These findings, together with the fact that we found no sex differences in plasma levels of testosterone, dihydrotestosterone, 5alpha-adiol, dehydroepiandrosterone, or androstenedione from d 51-227, clearly indicate that the action of 5alpha-adiol between d 20 and 40 imprints later differentiation of the male penis.

Unsolved problems in male physiology: studies in a marsupial.

Testicular androgens induce formation of the male urogenital tract in all mammals. In marsupials male development occurs after birth and over a prolonged period. For example, in the tammar wallaby virilization of the Wolffian ducts begins by day 20, prostate formation begins about day 25, and phallic development starts after day 80 of pouch life. Between days 20 and 40 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in tammar testes and secreted into plasma. Administration of 5alpha-adiol to pouch young females induces urogenital sinus virilization by day 40 and formation of a mature male prostate and phallus by day 150. 5alpha-Adiol is synthesized in pouch young testes by two pathways, one involving testosterone and dihydrotestosterone and the other 5alpha-pregnane-3alpha,17alpha-diol-20-one and androsterone as intermediates, both utilizing steroid 5alpha-reductase. In target tissues 5alpha-adiol acts via the androgen receptor after conversion to dihydrotestosterone but may have other actions as well. Whether 5alpha-adiol plays a role in male development in placental mammals is uncertain.

Prostate formation in a marsupial is mediated by the testicular androgen 5 alpha-androstane-3 alpha,17 beta-diol.

Development of the male urogenital tract in mammals is mediated by testicular androgens. It has been tacitly assumed that testosterone acts through its intracellular metabolite dihydrotestosterone (DHT) to mediate this process, but levels of these androgens are not sexually dimorphic in plasma at the time of prostate development. Here we show that the 3 alpha-reduced derivative of DHT, 5 alpha-androstane-3 alpha,17 beta-diol (5 alpha-adiol), is formed in testes of tammar wallaby pouch young and is higher in male than in female plasma in this species during early sexual differentiation. Administration of 5 alpha-adiol caused formation of prostatic buds in female wallaby pouch young, and in tissue minces of urogenital sinus and urogenital tubercle radioactive 5 alpha-adiol was converted to DHT, suggesting that circulating 5 alpha-adiol acts through DHT in target tissues. We conclude that circulating 5 alpha-adiol is a key hormone in male development.

Role of androgens in female-pattern androgenetic alopecia, either alone or associated with other symptoms of hyperandrogenism.

The roles of androgen hypersecretion, in situ enzyme activity, and androgen receptors in androgenetic alopecia in women are still a matter of debate. We studied 187 women with alopecia, which we graded I, II, or III, according to Ludwig's classification, and 21 healthy control women. All participants were subjected to full basal and 1 h post-beta-1-24 corticotropin stimulation endocrine profiles. Abnormal hormone profiles were observed in 67% of the patients with alopecia alone (group A, n = 110) and in 84% of the patients with alopecia plus other symptoms of hyperandrogenism including acne, hirsutism, and menstrual cycle disturbances (group B, n = 77). Mean serum 5alpha-androstane-3alpha,17beta-diol glucuronide (3alpha-AdiolG) levels in all three patient groups (6.50+/-4.10, 8.90+/-5.80, and 14.70+/-8.90 nmol/l, respectively) correlated with the grade of alopecia (I-III) and were significantly higher than in the control group (4.80+/-2.05 nmol/l, P < 0.005). Mean serum sex hormone-binding globulin (SHBG) levels were inversely correlated with the grade of alopecia (I-III) and were significantly lower in all three patient groups (50.55+/-23.50, 40.00+/-17.65, and 38.80+/-14.10 nmol/l, respectively) than in the control group (61.15+/-17.65 nmol/l, P < 0.05). Mean serum levels of delta4-androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and 3alpha-AdiolG were higher in group B than in group A, and higher in group A than in the control group. The significant correlations found between adrenal secretion - either positive (with 3alpha-AdiolG levels and the body mass index) or negative (with SHBG levels) - might reflect the important contribution of secretory and metabolic components in the development of alopecia, the severity of which has been shown to be very closely related to observed levels of two of these parameters (3alpha-AdiolG and SHBG).

[Reduction of 5alpha-dehydrotestosterone in sex steroid target cells--catabolism or directed biotransformation into hormones?]. / Vosstanovlenie 5al'fa-digidrotestosterona v kletkakh-misheniakh polovykh steroidov--katabolizm ili napravlennaia biotransformatsiia v gormony?

Based on analysis of the published experimental data, we propose a hypothetical mechanism of action of two androgens, 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), which are produced when 5 alpha-dihydrotestosterone (DHT) is reduced in sex steroids target cells. The mode of action of these diols as regulatory factors assumes that the androgens possess autocrine, paracrine, and endocrine action that differs from the action of DHT in animals and man and do not merely represent catabolites or metabolites capable of controlling the level of true androgenic hormone DHT. It is proposed that both diols should be classified as parahormones.

Developmental patterns of serum 3 alpha-androstanediol glucuronide.

3 alpha-androstanediol glucuronide (3 alpha diolG) is a marker of peripheral tissue androgen metabolism. There are no previous data regarding complete paediatric reference ranges for 3 alpha diolG. In order to obtain reference values for 3 alpha diolG we have measured serum levels of 3 alpha diolG in 283 healthy children and adolescents, 146 boys and 137 girls, age 1 month to 20 years and 28 adults. A non-extraction, solid phase radioimmunoassay employing a polyclonal antiserum that is specific for 3 alpha diolG was used to measure serum 3 alpha diolG levels (intra assay variation 5.1-10.1%, inter assay variation 2.7-9.0%). There was a strong sex and age dependence (r = 0.8; p < 0.0001) of 3 alpha diolG levels throughout childhood and adolescence with males showing significantly higher levels of the androgen than females (p < 0.05). 3 alpha diolG serum levels (nmol/l +/- SD) correlated significantly with pubertal stage (p < 0.01). Interestingly, in 35 children with CAH serum 3 alpha diolG levels correlated well with clinical and metabolic status, i.e. 17OHP serum levels. In summary, we have established percentile curves for 3 alpha diolG levels in healthy children and adolescents. We hypothesize that on the basis of our reference values the single measurement of serum 3 alpha diolG could serve as a means to determine androgen status in children with disorders of puberty and sexual development.

Serotoninergic control of prolactin secretion in prepubertal male rats.

The role of the serotoninergic system in the control of prolactin (PRL) secretion has been studied in prepubertal male rats. Serum PRL concentration was measured in 16-day-old male rats at different times after the administration of 5-hydroxytryptophan (5-HTP), a precursor of serotonin (5-HT) synthesis, alone or in combination with fluoxetine, a specific inhibitor of 5-HT uptake; DL-p-parachlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis; and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a selective agonist of 5-HT1A receptors. Also, serum PRL concentration and pituitary content were measured after 5-HTP administration in castrated males implanted with silastic capsules containing testosterone or 5 alpha-androstane-3 alpha,17 beta-diol (alpha-diol). We found that: the reduction in serotoninergic activity after PCPA administration did not modify serum PRL concentrations; the stimulatory effect of 5-HTP on PRL secretion was not observed before day 16; the effects of 5-HTP or 5-HTP and fluoxetine were similar in intact and orchidectomized males; a significant increase in PRL secretion took place after 8-OH-DPAT administration; the duration of the stimulatory effect of 5-HTP increased after alpha-diol treatment; and pituitary PRL content increased after 5-HTP injection in intact males and decreased in castrated males treated with testosterone or alpha-diol.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms regulating male sexual behavior in the rat: role of 3 alpha- and 3 beta-androstanediols.

To assess whether naturally occurring 5 alpha-androstanediols (5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol) play a role in the regulation of male sexual behavior in the rat, their capability to restore copulatory behavior in castrated animals was evaluated. Androstanediols were chronically administered either alone or in combination with 5 alpha-dihydrotestosterone (DHT) or with estradiol-17 beta (E2). Animals treated with testosterone (T), DHT, E2, and vehicle, either alone or in different combinations, served as controls. The occurrence of mounting, intromission, and ejaculation as well as detailed parameters of copulatory behavior were recorded twice per week for 3 weeks. At the end of treatments, the weights of sex accessory organs were also recorded. When 3 beta, 5 alpha-androstanediol (3 beta-diol; 500 micrograms/day) was administered in combination with DHT (300 micrograms/day), full copulatory behavior was restored in all subjects in a manner similar to that obtained with E2 plus DHT or T plus DHT combinations, thus indicating an estrogen-like behavioral effect of 3 beta-diol. Administration of 3 alpha, 5 alpha-androstanediol (3 alpha-diol; 500 micrograms/day) combined with DHT also restored sexual behavior, though to a lesser extent. When 3 alpha-diol (500 micrograms/day) was simultaneously administered with E2 (5 micrograms/day), the copulatory behavior of castrated animals was fully restored in a fashion similar to that observed after administration of DHT plus E2 and T plus E2 combinations, indicating a potent androgen-like effect of 3 alpha-diol.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens modulate growth hormone-releasing factor-induced GH release from bovine anterior pituitary cells in static culture.

Static primary cultures of bovine anterior pituitary (AP) cells were utilized to study the effect of sex steroids on basal growth hormone (GH) and GH-releasing hormone (GRF)-stimulated release of GH. The AP cells (5 x 10(5) cells/well) were allowed to attach for 72 hr and become confluent before treatments were imposed. Cells were incubated for an additional 24, 48 or 72 hr with either estradiol-17 beta (E2, 10(-11) to 10(-8) M), testosterone (T, 10(-8) to 10(-5) M), dihydrotestosterone (DHT, 10(-9) to 10(-6) M) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol, 10(-11) to 10(-8) M). Media were collected every 24 hr and GH concentrations determined by RIA. Incubation of calf AP cells with gonadal steroids did not affect (P > 0.05) basal GH released at 24, 48, or 72 hr. In another experiment, calf AP cells were incubated with the same concentrations of the steroids for 24 hr, media harvested, cells washed and challenged in serum-free media for 1 hr with bovine GRF 1-44-NH2 (10(-8) M). In non-steroid treated wells, GRF increased (P < 0.05) GH from 58 to 134 ng/ml. Incubation with E2 or 3 alpha-diol did not affect (P > 0.05) GRF-induced GH release; however, preincubation with T (10(-5) M) and DHT (10(-9), 10(-8) and 10(-7) M) increased (P < 0.05) GRF-induced GH release above control concentrations (195, 235, 190 and 185 ng/ml, respectively). At the doses tested, sex steroids did not affect basal release of GH, but androgens increased responsiveness of somatotropes to GRF.

Physiopathology of plasma androstanediol-glucuronide.

Plasma androstanediol-glucuronide (ADG) is considered by many authors to be a highly reliable parameter of peripheral androgenicity. Recently, several authors have questioned the reliability of the ADG levels as a parameter of androgenicity. Our data obtained by continuous infusion experiments showed that in women the adrenal steroids, dehydroepiandrosterone sulfate, androstenedione and dehydroepiandrosterone are the major precursors of plasma ADG, accounting for almost the totality of circulating ADG. As expected, in view of its precursors, ADG levels decrease significantly with age. Dexamethasone causes a significant decrease of these levels, whereas in women with Addison's disease the levels are only 20% of normal levels; ovariectomy hardly influences ADG levels. Our data show that in women with moderate hirsutism, plasma ADG levels are no more often increased than the other androgens. In virilizing syndromes ADG levels are higher than expected from precursor levels, suggesting an increased 5 alpha-reductase activity. In hyperthyroidism as well as in euthyroid women with isolated suppressed thyroid stimulating hormone, ADG levels are increased without any sign of virilism. In men, ADG levels have testosterone as a major precursor, but the adrenals contribute to +/- 30% of ADG levels. After transdermal dihydrotestosterone gel, free androstanediol levels increased by a factor of 40, but ADG levels were only increased by a factor of 4, suggesting that the skin is not very effective in conjugating androstanediol. It is concluded that ADG levels in women reflect essentially adrenal precursor levels as well as 5 alpha-reductase activity in peripheral tissues inclusive of the liver.

Blood concentrations and biological effects of androstanediols at the onset of puberty in the female rat.

Androstanediols are the major products of the immature rat ovary. About two-thirds are present in blood as sulfates. One of the sulfate conjugates was identified as 5 alpha-androstane-3 beta,17 beta-diol-3-monosulfate. This steroid effectively inhibits postcastrational LH elevation in a concentration in which it is present in blood. Other androstanediol sulfates examined were without this activity. In the immature rat, levels of 3 alpha- and 3 beta-diol decrease sharply before the first ovulation. In Wistar rats, PMSG-treatment induced a similar decline in these steroids. When ovulation in PMSG-treated rats was blocked by pentobarbitone, the LH-surge and the decline of the 3 alpha- and 3 beta-diols was delayed. Thus, it seems that the decline in the blood concentration of the diols depends on the first LH surge.

The role of androgen metabolism in the control of androgen action in the rat prostate.

Recent results from a number of laboratories have led us to re-examine the role of 3 beta-androstanediol in the rat ventral prostate. Whereas previously 5 alpha-dihydrotestosterone and 3-beta androstanediol were thought to have distinctly separate effects on the prostate, we suggest that 3 beta-androstanediol serves only as an intermediate in the metabolism and removal of 5 alpha-dihydrotestosterone from the organ. In our view the action of androgens on the prostate are exerted exclusively through the binding of 5 alpha-dihydrotestosterone to the androgen receptor and its subsequent translocation to the nucleus. Differences in effects are related to the amount of 5 alpha-dihydrotestosterone available to the gland. 3 alpha-Hydroxysteroid dehydrogenase may play a critical role in modulating the level of 5 alpha-dihydrotestosterone available to the translocatable receptor.

Possible role of 5 alpha-androstane-3 alpha, 17 beta-diol in the control of follicle-stimulating hormone secretion in the immature female rat.

A possible role of 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol) in the control of FSH secretion was studied at various ages in ovariectomized rats. In the rat strain used, vaginal opening, coincident with first ovulation, generally occurs between 37 and 42 days of age. If 3 alpha-androstanediol alone was given as an ovarian substitute, an inhibitory effect on FSH release was evident with all three doses tested (50, 100, 300 microgram/100 g body wt) between 13 and 30 days of age; at 33-35 days of age only the 300 microgram dose caused some inhibition of FSH release. Results were more complex if 3 alpha-androstanediol was given in combined treatment with oestradiol and progesterone. Given with progesterone, 3 alpha-androstanediol showed a synergistic inhibitory action on FSH release between 20 and 30 days of age. However, when 3 alpha-androstanediol was combined with oestradiol a clear decrease in effect, as compared to the effect of oestradiol alone, was found between 20 and 30 days of age. Also the effect of combined oestradiol and progesterone treatment was greater than the effect of combined treatment with oestradiol, progesterone and 3 alpha-androstanediol. At all ages after day 20 none of the steroid combinations tested was capable of maintaining FSH levels in ovariectomized rats similar to those in intact rats. It is concluded that 3 alpha-androstanediol might play a role in the control of FSH secretion in the immature rat, but after day 20 the potentially inhibitory action of 3 alpha androstanediol on FSH secretion is limited in the presence of oestradiol.