Steroid Ligands, the Forgotten Triggers of Nuclear Receptor Action; Implications for Acquired Resistance to Endocrine Therapy.

PURPOSE: There is strong epidemiologic evidence indicating that estrogens may not be the sole steroid drivers of breast cancer. We hypothesize that abundant adrenal androgenic steroid precursors, acting via the androgen receptor (AR), promote an endocrine-resistant breast cancer phenotype. EXPERIMENTAL DESIGN: AR was evaluated in a primary breast cancer tissue microarray (n = 844). Androstenedione (4AD) levels were evaluated in serum samples (n = 42) from hormone receptor-positive, postmenopausal breast cancer. Levels of androgens, progesterone, and estradiol were quantified using LC/MS-MS in serum from age- and grade-matched recurrent and nonrecurrent patients (n = 6) before and after aromatase inhibitor (AI) therapy (>12 months). AR and estrogen receptor (ER) signaling pathway activities were analyzed in two independent AI-treated cohorts. RESULTS: AR protein expression was associated with favorable progression-free survival in the total population (Wilcoxon, P < 0.001). Pretherapy serum samples from breast cancer patients showed decreasing levels of 4AD with age only in the nonrecurrent group (P < 0.05). LC/MS-MS analysis of an AI-sensitive and AI-resistant cohort demonstrated the ability to detect altered levels of steroids in serum of patients before and after AI therapy. Transcriptional analysis showed an increased ratio of AR:ER signaling pathway activities in patients failing AI therapy (t test P < 0.05); furthermore, 4AD mediated gene changes associated with acquired AI resistance. CONCLUSIONS: This study highlights the importance of examining the therapeutic consequences of the steroid microenvironment and demonstrable receptor activation using indicative gene expression signatures.

Sex Differences in 11-Oxygenated Androgen Patterns Across Adulthood.

CONTEXT: The gonads are the major source of sex steroids during reproductive ages. The gonadal function declines abruptly in women and gradually in men. The adrenals produce 11-oxygenated androgens (11-oxyandrogens), which start rising during adrenarche. Following menopause, 11-oxyandrogens levels remain similar to reproductive ages. OBJECTIVE: To compare the circulating 11-oxyandrogen concentrations in men and women across adult ages. METHODS: We used mass spectrometry to measure testosterone (T), androstenedione (A4), 11ß-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), cortisol, and cortisone in morning sera obtained from adults in outpatient setting. We performed double immunofluorescence of 3ß-hydroxysteroid dehydrogenase type 2 and cytochrome b5 in adrenal tissue from 19 men, age 23-78 years. RESULTS: We included 590 patients (319 men), aged 18 to 97 years, and 84% white. 11KT and 11KA4 were stable across ages in women, but they declined in men (0.21 and 0.06 ng/dL/year, respectively; P < 0.05). 11OHA4 and 11OHT increased modestly with age in women (0.6 and 0.09 ng/dL/year, respectively; P < 0.01), and both remained stable across ages in men. As body mass index (BMI) increased, 11KA4 decreased in women, and 11KT increased in men, both suggesting higher 17ß-hydroxysteroid dehydrogenase activity in obese individuals. A4 and T declined with age and A4 with BMI in both sexes; T declined with BMI in men. Adrenal androgenic enzyme expressions in aging men were similar to those observed in women. CONCLUSIONS: In contrast with traditional androgens, the production of 11OHA4 and 11OHT is sustained with aging in both sexes. The bioactive androgen 11KT declines in aging men but not in women.

Effect of dehydroepiandrosterone (DHEA) on Akt and protein kinase C zeta (PKCζ) phosphorylation in different tissues of C57BL6, insulin receptor substrate (IRS)1(-/-), and IRS2(-/-) male mice fed a high-fat diet.

We have previously reported that dehydroepiandrosterone (DHEA) suppresses the activity and mRNA expression of the hepatic gluconeogenic enzyme glucose-6-phosphatase (G6Pase), and hepatic glucose production in db/db mice. Tyrosine phosphorylation levels of Insulin receptor substrate (IRS)1 and IRS2 reportedly differ between the liver and muscle tissue and the effect of DHEA on insulin signaling has not been elucidated. Therefore, we examined DHEA's effect on the liver and muscle tissue of IRS1(-/-) and IRS2(-/-) mice. Eight-week-old male C57BL6, IRS1(-/-), and IRS2(-/-) mice were fed a high-fat diet (HFD), or an HFD containing 0.2% DHEA for 4 weeks. In a separate experiment, 8-week-old male C57BL6 mice were fed an HFD or an HFD containing 0.2% androstenedione for 4 weeks. In an insulin tolerance test, DHEA administration decreased the initial plasma glucose levels in the C57BL6, IRS1(-/-), and IRS2(-/-) mice but did not decrease the ratios to the basal blood glucose level. Although DHEA administration increased Akt phosphorylation in the liver of the C57BL6, IRS1(-/-), and IRS2(-/-) mice, androstenedione administration did not increase Akt phosphorylation in the liver of C57BL6 mice. DHEA administration did not increase Akt and PKCζ phosphorylation in the muscle tissue of C57BL6, IRS1(-/-), or IRS2(-/-) mice. However, androstenedione administration increased Akt and PKCζ phosphorylation in the muscle tissue of C57BL6 mice. These findings suggest that the effect of DHEA on insulin action in the liver is self-mediated by DHEA or DHEA sulfate (DHEA-S) in the presence of IRS1, IRS2, or both.

Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production.

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

Effects of photo stimulation and nonstimulation of golden hamsters (Mesocricetus auratus) from birth to early puberty on testes structure and function.

We tested whether puberty in golden hamsters is photoperiodically controlled. Hamsters were raised under 14:10 hours Light:Dark (14L) and 1:23 hours Light:Dark (1L) respectively, from birth to 28 days and tested for various parameters. Body weight, Leydig cell (LC) size and testicular testosterone secretion were greater and plasma thyroxin (T4), testicular androstenedione secretion and LC number were lower (P<0.05) in 1L than 14L hamsters. Volumes of testicular components were similar in the two groups. 3beta-hydroxy steroid dehydrogenase immunohistochemistry demonstrated LC progenitors and newly formed adult LC (ALC) in 14L hamsters, which were absent in 1L hamsters; they contained only fetal LC (FLC). Latter findings suggest the presence and absence of postnatally-differerentiated LC in 14L and 1L hamsters, respectively. Androgen results agreed with these findings, because FLC primarily secrete testosterone, and androstenedione is a major androgen secreted by the newly formed ALC. Reduced T4 in 1L hamsters is attributed to the inhibition of thyroid function by the increased duration of melatonin secretion due to non-photostimulatory conditions. The arrest in LC differentiation in 1L hamsters is attributed to low T4 levels. Although the testis size is unaltered under non-photostimulatory conditions, postnatal LC differentiation is inhibited in golden hamsters, and therefore, it is logical to suggest that their puberty is photoperiodically controlled.

Blood pressure in middle-aged women: are androgens involved? A population-based study of Swedish women: the Women's Health in the Lund Area study.

OBJECTIVE: To assess the prevalence of hypertension and use of antihypertensive drug therapy in relation to menopausal status and to delineate perceived associations between androgens and blood pressure in perimenopausal women. METHODS: A population-based sample of women aged 50-59 (n = 6893). Women were divided into three groups according to their hormonal status: premenopausal, postmenopausal without hormone therapy, and postmenopausal with hormone therapy. RESULT: In the premenopausal, postmenopausal without hormone therapy, and postmenopausal with hormone therapy groups, the prevalence of high blood pressure (>/= 140 mmHg systolic or >/= 90 mmHg diastolic) was 43.9, 49.9 and 45.8%, respectively. In women with normal blood pressure, adjusting for age, body mass index and smoking, there were negative associations between serum testosterone and systolic blood pressure in the total sample (P < 0.01) and the postmenopausal without hormone therapy group (P < 0.05). In women using antihypertensive drug therapy with a blood pressure of at least 140/90 mmHg, positive associations were found between serum testosterone and systolic blood pressure in the total series (P < 0.05) and in the postmenopausal without hormone therapy group (P < 0.05). CONCLUSION: Abnormal blood pressure is common in middle-aged women regardless of hormonal status. Our findings suggest that testosterone could have a dual influence on blood pressure in perimenopausal women.

Preeclampsia and maternal breast cancer risk by offspring gender: do elevated androgen concentrations play a role?

Among older mothers, preeclampsia in the first pregnancy was associated with a reduction in maternal breast cancer risk that was significantly more pronounced in women bearing male than female infants. Androgen concentrations in male, preeclamptic pregnancies were consistent with the hypothesis that elevated pregnancy androgens might mediate this apparent modifying effect of fetal gender.

Role of steroid hormones and prolactin in canine mammary cancer.

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.

Sexually mature male goldfish release large quantities of androstenedione into the water where it functions as a pheromone.

Previous studies have demonstrated that ovulatory female goldfish release a variety of sex steroids into the water where they function as a pheromonal blend dominated by C21 steroids that stimulates male hormone release, sperm production and behavior. This study investigated whether male goldfish might also release sex steroids with pheromonal activity. It found that spermiated male goldfish release substantial quantities of androstenedione (AD; about 50 ng/h) together with smaller (10-20 ng/h) quantities of several other related C19 steroids but only very small quantities (<5 ng/h) of C21 steroids. Further, when sexually aroused by females and/or their pheromones, males released even greater quantities of AD (up to 1 microg/h) while C21 steroid release rate changed little. This created a ratio of C19 to C21 steroids of about 50:1 that was dramatically different from that emitted by females (1:7). The male olfactory system was also found to be extremely sensitive to AD, detecting it to near picomolar concentrations. Together with previous studies that have shown water-borne AD to increase male aggressive behavior while suppressing responsiveness to female pheromones, this study establishes AD as a male pheromone in the goldfish. Because ovulating females also release AD but in the presence of C21 steroids, recognition of the male-derived steroid pheromone is presumably mixture dependent.

Ovarian steroids modulate neuroendocrine dysfunction in polycystic ovary syndrome.

OBJECTIVE: Neuroendocrine dysfunction in polycystic ovary syndrome (PCOS) was addressed by studying the steroid hormone changes in women with PCOS with either high or normal LH levels leading to inferences regarding the primacy of elevated LH in the pathophysiology of PCOS. METHODS: A cross-sectional study was designed in an academic clinical facility involving 234 women with PCOS. Patients were divided into two groups based on an LH/FSH ratio < or >1 and hormonal and metabolic studies were performed in both groups. Factors were determined by binomial logistic regression that predicted group membership of these women. RESULTS: Higher follicular phase estradiol (E2) and androstenedione (A4) levels as well as greater insulin sensitivity were the only factors that predicted the presence of neuroendocrine dysfunction with elevated A4 being necessary for neuroendocrine dysfunction. CONCLUSIONS: It was concluded that uncoupling of hypothalamic E2 inhibition by elevated ovarian A4 associated with E2 related sensitization of pituitary LH leads to neuroendocrine dysfunction in PCOS.

Androstenedione increases cytochrome P450 aromatase messenger ribonucleic acid transcripts in nonluteinizing bovine granulosa cells.

The objective of this study was to determine if androgens regulate granulosa cell steroidogenesis at physiological doses found in small bovine follicles. Bovine granulosa cells were cultured under serum-free conditions that permit the induction and maintenance of FSH-dependent estradiol secretion. Increasing androstenedione concentrations from 0.1 to 1 or 10 microM significantly increased estradiol accumulation and cytochrome P450 aromatase (P450arom) mRNA abundance. No increase in progesterone accumulation or abundance of mRNA for P450 side-chain cleavage or 3beta-hydroxysteroid dehydrogenase enzymes was observed. The addition of 0.1, 1, or 10 microM progestins or estrogens had no stimulatory effect on P450arom mRNA levels. An analysis of the 5'-untranslated region of P450arom mRNA transcripts indicated that the majority was derived from Cyp19 ovary-specific promoter 2, with some contribution from promoters 1.1 and 1.5. Transcripts from these three promoters were all significantly increased by androstenedione. Testosterone increased promoter 1.1 and 1.5-derived transcripts, but only promoter 2-derived transcripts at the highest dose tested (100 microM). Dihydrotestosterone (DHT) did not affect Cyp19 expression. Collectively, these data show that androgens may exert specific stimulatory effects on P450arom mRNA concentrations in granulosa cells. Interestingly, different androgens had different effects on Cyp19 promoter usage, suggesting differential regulation of aromatase gene expression in the developing follicle.

The effect of circulating androgens on bone mineral density in postmenopausal women.

BACKGROUND: Osteoporosis is a common problem in postmenopausal period. Recent studies have suggested that endogenous and exogenous androgens may influence the bone mineral density in women. There is limited data about the effect of circulating androgens on bone density in postmenopausal women. AIM: The aim of this study was to evaluate the effect of circulating androgens of ovarian and adrenal origin on bone mineral density in postmenopausal women. MATERIALS AND METHODS: This cross-sectional study included 178 postmenopausal women, who had never been treated with hormonal therapy or calciotropic agents. Serum free testosterone, dehydroepiandrosterone sulfate and androstenedione levels and their relationship with bone mass (dual X-ray absorptiometry) were evaluated. RESULTS: Serum free testosterone and DHEAS levels were correlated positively with bone mineral density at lumbar spine and femoral neck (P < 0.001). However, stepwise linear regression analyses revealed a differential effect of androgens on bone density. Serum free testosterone was among the independent predictor of bone density at lumbar spine (trabecular bone), whereas serum DHEAS level was of bone density at femoral neck (cortical bone). CONCLUSION: This study suggests that endogenous androgens are influential on bone density in postmenopausal women. However, regression analyses revealed a differential effect of androgens on different bone types.

Hormonal regulation of androgen receptor messenger ribonucleic acid expression in human tooth pulp.

Tooth pulp contains steroid receptors and therefore is likely to respond to steroids. Steroids and cytokines together can alter steroid receptor content in many tissues; thus, similar mechanisms may exist in tooth pulp. In this study, reverse-transcription/polymerase chain-reaction was used to screen human pulp for the mRNAs encoding receptors for androgen (AR), estrogens (ERbeta), and hepatocyte growth factor (HGF: c-Met). AR mRNA content was greater in male pulp vs. female pulp in all age groups. In both genders, AR mRNA content diminished with age. In pulp cell cultures, androstenedione, estradiol-17beta, and HGF each stimulated AR mRNA accumulation. Testosterone inhibited, whereas 5alpha-dihydrotestosterone did not affect, AR mRNA content. ERbeta was not hormonally altered in pulp cell cultures. By showing steroid- and cytokine-orchestrated regulation of AR mRNA in vitro, it is possible that age- and/or pathogen-dependent changes in available steroids and cytokines can affect any androgen-responsiveness of pulp.

Mechanisms of hypothalamic-pituitary-gonadal disruption in polycystic ovarian syndrome.

Although the pathogenesis of polycystic ovarian syndrome (PCOS) is still controversial, a series of investigations has demonstrated an array of neuroendocrine abnormalities as a major component of the syndrome. From a neuroendocrine perspective, patients with PCOS exhibit an accelerated frequency and/or higher amplitude of LH pulses, augmentation of LH secretory burst mass, and a more disorderly LH release. Elevated in vitro LH bioactivity and a preponderance of basic LH isoforms, which correlate positively with elevated serum 17-hydroxyprogesterone, androstenedione, and testosterone concentrations, also characterize adolescents with PCOS. Heightened GnRH drive of gonadotropin secretion and a steroid-permissive milieu appear to jointly promote elevated secretion of basic LH isoforms. Positive feedback is implied, because hypersecretion of highly bioactive LH in PCOS probably contributes to inordinate androgen output. However, the precise nature of feedback disruption remains uncertain. Indeed, recent data suggest that PCOS is marked by anomalies of both feedforward and feedback signaling between GnRH/LH and ovarian androgens. From a single hormone perspective, the individual patterns of LH and androstenedione release are consistently more irregular in patients with PCOS. Bihormonal analysis has disclosed concomitant uncoupling of the pairwise synchrony of LH and testosterone, LH and androstenedione, and testosterone and androstenedione secretion. The foregoing ensemble of findings points to deterioration of both orderly uniglandular and coordinate bihormonal output in PCOS. Additional studies are needed to establish the primary pathophysiologic mechanisms underlying this disorder.

The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1.

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.

Do androgens have a direct effect on endometrial function? An in vitro study.

OBJECTIVE: To test the hypothesis that androgens have a direct effect on the function of endometrial epithelial cells. DESIGN: In vitro study. SETTING: Academic research center. PATIENT(S): Endometrial epithelial cells were prepared from biopsy samples obtained from normal fertile women. INTERVENTIONS: Cells were incubated with androstenedione, testosterone, dihydrotestosterone, and DHEA. MAIN OUTCOME MEASURE(S): Secretion of glycodelin A into the culture fluid was used to assess secretory activity. Uptake of (3)H-thymidine and immunostaining for Ki67 was used to assess cell growth. The specific action of the androgens was confirmed by incubation with an antiandrogen, cyproterone acetate. RESULT(S): Androstenedione (10(-6) M and 10(-7) M) caused a dose-dependent decrease in glycodelin A secretion, uptake of (3)H-thymidine, and percentage of positive Ki67 cells in cultured human endometrial epithelial cells. Testosterone, dihydrotestosterone, and DHEA had no effect on glycodelin A secretion or (3)H-thymidine uptake. The direct effect of androgens on endometrial function were confirmed by demonstrating the presence of androgen receptors in cultured endometrial epithelial cells and showing that the direct effects of the androgens were not observed when cyproterone acetate was added to the cultures. CONCLUSION(S): The results suggest that androstenedione can inhibit human endometrial cell growth and secretory activity. Infertility and miscarriage associated with high androgen levels (e.g., that caused by the polycystic ovary syndrome) may be due to an adverse effect of high androgen levels on the endometrium.

Yolk androgens reduce offspring survival.

Females may favour some offspring over others by differential deposition of yolk hormones. In American kestrels (Falco sparverius), we found that yolks of eggs laid late in the sequence of a clutch had more testosterone (T) and androstenedione (A4) than yolks of first-laid eggs. To investigate the effects of these yolk androgens on nestling 'fitness', we injected both T and A4 into the yolks of first-laid eggs and compared their hatching time, nestling growth and nestling survival with those of first-laid eggs in which we injected vehicle as a control. Compared to controls, injection of T and A4 at a dose intended to increase their levels to those of later-laid eggs delayed hatching and reduced nestling growth and survival rates. Yolk androgen treatment of egg 1 had no effect on survival of siblings hatching from subsequently laid eggs. The adverse actions of yolk androgen treatment in the kestrel are in contrast to the favourable actions of yolk T treatment found previously in canaries (Serinus canaria). Additional studies are necessary in order to determine whether the deposition of yolk androgens is an adaptive form of parental favouritism or an adverse by-product of endocrine processes during egg formation. Despite its adaptive significance, such 'transgenerational' effects of steroid hormones may have helped to evolutionarily shape the hormonal mechanisms regulating reproduction.