Conformational dynamics of androgen receptors bound to agonists and antagonists.

The androgen receptor (AR) is critical in the progression of prostate cancer (PCa). Small molecule antagonists that bind to the ligand binding domain (LBD) of the AR have been successful in treating PCa. However, the structural basis by which the AR antagonists manifest their therapeutic efficacy remains unclear, due to the lack of detailed structural information of the AR bound to the antagonists. We have performed accelerated molecular dynamics (aMD) simulations of LBDs bound to a set of ligands including a natural substrate (dihydrotestosterone), an agonist (RU59063) and three antagonists (bicalutamide, enzalutamide and apalutamide) as well as in the absence of ligand (apo). We show that the binding of AR antagonists at the substrate binding pocket alter the dynamic fluctuations of H12, thereby disrupting the structural integrity of the agonistic conformation of AR. Two antagonists, enzalutamide and apalutamide, induce considerable structural changes to the agonist conformation of LBD, when bound close to H12 of AR LBD. When the antagonists bind to the pocket with different orientations having close contact with H11, no significant conformational changes were observed, suggesting the AR remains in the functionally activated (agonistic) state. The simulations on a drug resistance mutant F876L bound to enzalutamide demonstrated that the mutation stabilizes the agonistic conformation of AR LBD, which compromises the efficacy of the antagonists. Principal component analysis (PCA) of the structural fluctuations shows that the binding of enzalutamide and apalutamide induce conformational fluctuations in the AR, which are markedly different from those caused by the agonist as well as another antagonist, bicalutamide. These fluctuations could only be observed with the use of aMD.

Sex and age influence gonadal steroid hormone receptor distributions relative to estrogen receptor ß-containing neurons in the mouse hypothalamic paraventricular nucleus.

Within the hypothalamic paraventricular nucleus (PVN), estrogen receptor (ER) ß and other gonadal hormone receptors play a role in central cardiovascular processes. However, the influence of sex and age on the cellular and subcellular relationships of ERß with ERα, G-protein ER (GPER1), as well as progestin and androgen receptors (PR and AR) in the PVN is uncertain. In young (2- to 3-month-old) females and males, ERß-enhanced green fluorescent protein (EGFP) containing neurons were approximately four times greater than ERα-labeled and PR-labeled nuclei in the PVN. In subdivisions of the PVN, young females, compared to males, had: (1) more ERß-EGFP neurons in neuroendocrine rostral regions; (2) fewer ERα-labeled nuclei in neuroendocrine and autonomic projecting medial subregions; and (3) more ERα-labeled nuclei in an autonomic projecting caudal region. In contrast, young males, compared to females, had approximately 20 times more AR-labeled nuclei, which often colocalized with ERß-EGFP in neuroendocrine (approximately 70%) and autonomic (approximately 50%) projecting subregions. Ultrastructurally, in soma and dendrites, PVN ERß-EGFP colocalized primarily with extranuclear AR (approximately 85% soma) and GPER1 (approximately 70% soma). Aged (12- to 24-month-old) males had more ERß-EGFP neurons in a rostral neuroendocrine subregion compared to aged females and females with accelerated ovarian failure (AOF) and in a caudal autonomic subregion compared to post-AOF females. Late-aged (18- to 24-month-old) females compared to early-aged (12- to 14-month-old) females and AOF females had fewer AR-labeled nuclei in neuroendrocrine and autonomic projecting subregions. These findings indicate that gonadal steroids may directly and indirectly influence PVN neurons via nuclear and extranuclear gonadal hormone receptors in a sex-specific manner.

[Androgen receptor polymorphism in relation to medical conditions characterized by hyper/hypoandrogenism].

An androgen receptor (AR) is a transcription factor consisting of four functional regions. The transactivation region contains a highly polymorphic area characterized by a variable number of CAG trinucleotide repeats encoding a polyglutamine tract. Several in vitro studies demonstrated a negative linear relation between the lengths of CAG repeats and relative AR transactivations. Numerous clinical studies then sought associations between the described polymorphism and clinical parameters of various medical conditions characterized by hyper/hypoandrogenism. In this article, we describe some of those interesting associations. We believe such links should be investigated in any medical condition involving androgens as a key element in its pathogenesis.

Androgen and estrogen (alpha) receptor localization on periaqueductal gray neurons projecting to the rostral ventromedial medulla in the male and female rat.

The periaqueductal gray (PAG) is involved in many gonadal steroid-sensitive behaviors, including responsiveness to pain. The PAG projects to the rostral ventromedial medulla (RVM), comprising the primary circuit driving pain inhibition. Morphine administered systemically or directly into the PAG produces greater analgesia in male compared to female rats, while manipulation of gonadal hormones alters morphine potency in both sexes. It is unknown if these alterations are due to steroidal actions on PAG neurons projecting to the RVM. The expression of androgen (AR) and estrogen (ERalpha) receptors in the PAG of female rats and within this descending inhibitory pathway in both sexes is unknown. The present study used immunohistochemical techniques (1) to map the distribution of AR and ERalpha across the rostrocaudal axis of the PAG; and (2) to determine whether AR and/or ERalpha were colocalized on PAG neurons projecting to the RVM in male and female rats. AR and ERalpha immunoreactive neurons (AR-IR, ERalpha-IR) were densely distributed within the caudal PAG of male rats, with the majority localized in the lateral/ventrolateral PAG. Females had significantly fewer AR-IR neurons, while the quantity of ERalpha was comparable between the sexes. In both sexes, approximately 25-50% of AR-IR neurons and 20-50% of ERalpha-IR neurons were retrogradely labeled. This study provides direct evidence of the expression of steroid receptors in the PAG and the descending pathway driving pain inhibition in both male and female rats and may provide a mechanism whereby gonadal steroids modulate pain and morphine potency.

Classical androgen receptors in non-classical sites in the brain.

Androgen receptors are expressed in many different neuronal populations in the central nervous system where they often act as transcription factors in the cell nucleus. However, recent studies have detected androgen receptor immunoreactivity in neuronal and glial processes of the adult rat neocortex, hippocampal formation, and amygdala as well as in the telencephalon of eastern fence and green anole lizards. This review discusses previously published findings on extranuclear androgen receptors, as well as new experimental results that begin to establish a possible functional role for androgen receptors in axons within cortical regions. Electron microscopic studies have revealed that androgen receptor immunoreactive processes in the rat brain correspond to axons, dendrites and glial processes. New results show that lesions of the dorsal CA1 region by local administration of ibotenic acid reduce the density of androgen receptor immunoreactive axons in the cerebral cortex and the amygdala, suggesting that these axons may originate in the hippocampus. Androgen receptor immunoreactivity in axons is also decreased by the intracerebroventricular administration of colchicine, suggesting that androgen receptor protein is transported from the perikaryon to the axons by fast axonal transport. Androgen receptors in axons located in the cerebral cortex and amygdala and originating in the hippocampus may play an important role in the rapid behavioral effects of androgens.

The role of cofactors in sex steroid action.

Sex steroid signalling determines female and male sexual development and maintains the female and male phenotype in adults. Steroids carry out their function by activation of their cognate intracellular receptor, which is a ligand-dependent transcription factor. Steroid receptors function by binding to specific structural elements in the regulatory regions of target genes and by recruitment of cofactors by protein-protein interaction. Cofactors might display enzymatic activities that modify histones and other proteins. Cofactors also include proteins that modulate the chromatin structure and protein complexes that function as bridging factors between the multi-protein complexes. This review focuses mainly on the function of the androgen receptor and its cofactors and their role in androgen insensitivity syndrome.

Natural disordered sequences in the amino terminal domain of nuclear receptors: lessons from the androgen and glucocorticoid receptors.

Steroid hormones are a diverse class of structurally related molecules, derived from cholesterol, that include androgens, estrogens, progesterone and corticosteroids. They represent an important group of physiologically active signalling molecules that bind intracellular receptor proteins and regulate genes involved in developmental, reproductive and metabolic processes. The receptor proteins share structurally and functionally related ligand binding and DNA-binding domains, but possess distinct N-terminal domains (NTD) of unique length and amino acids sequence. The NTD contains sequences important for gene regulation, exhibit structure plasticity and are likely to contribute to the specificity of the steroid hormone/receptor response.

[Androgen insensitivity syndrome and co-activator disease].

Androgen insensitivity syndrome(AIS) is a clinical continuum of virilization disorders in XY males. Almost AIS is due to mutations in androgen receptor(AR) gene. Over 300 mutations in the AR gene have been reported. In addition, we have reported the case of complete AIS who has no mutations in AR gene, caused by co-activator abnormality. The recent advances in live-cell imaging techniques have developed a novel concept in AIS.

[The effect of hyperprolactinemia on morphology and function of androgen receptor expressing cells in rat testis, epididymis and prostate]. / Wplyw polekowej hiperprolaktynemii na morfologie i funkcje komórek z receptorem androgenowym w jadrze, najadrzu i prostacie szczura.

UNLABELLED: The effects of hyperprolactinemia on the testis, epididymis and prostate are not fully understood. The aim of this study was to determine the influence of hyperprolactinemia induced with metoclopramide (MCP) on expression of the androgen receptor (AR) in the testis, epididymis and the lateral and dorsal lobes of the prostate in rats. Male, sexually mature, inbred Wistar rats were divided into two groups of ten rats each. Hyperprolactynemia was induced in the study group with metoclopramide intraperitoneally at 2.2 mg/kg b. w. for 14 days. Rats of the control group were given saline. Prolactin (PRL) levels in serum were measured with an enzyme immunoassay. Serum levels of testosterone (T) were measured with a radioimmunoassay. The testis, epididymis and lateral and dorsal lobes of the prostate were obtained for light and electron microscopy. The immunohistochemical reaction to AR was assessed by optical density measurements with a computer image analyzer. Ultrastructural studies of androgen receptor expressing cells were carried out with transmission and scanning electron microscopes. RESULTS: PRL concentrations in MCP rats were increased by more than twice, while T concentrations were reduced by half. The intensity of the immunohistochemical reaction to AR in the testis, epididymis and lateral and dorsal lobes of the prostate differed significantly in the study and control groups. Hyperprolactinemia produced structural changes in cells expressing the androgen receptor in the testis, epididymis and the lateral and dorsal lobes of prostate.

Localization of androgen and estrogen receptors in rat and primate tissues.

There is now evidence that estrogens and androgens are exerting their effects in different tissues throughout the body. In order to determine the sites of action of these steroids, studies have been performed to identify at the cellular level the localization of androgen receptor (AR) and the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, specially in the rat, monkey and human. In the prostate, AR was observed in the secretory and stromal cells. In the testis, Sertoli, Leydig and myoid cells were labelled. In the epididymis and seminal vesicles, both epithelial and stromal cells contained AR. In the ovary, AR was detected in granulosa and interstitial cells. In the uterus, epithelial, stromal and muscle cells were all immunopositive for AR. In the central nervous system, AR-containing neurons were found to be widely distributed throughout the brain. In the mammary gland, epithelial cells in acini and ducts and stromal cells were demonstrated to express AR. In the skin, AR was detected in keratinocytes, sebaceous and sweat glands, and hair follicles. In addition, AR was also found in anterior pituitary, thyroid, adrenal cortex, liver, kidney tubules, urinary bladder, cardiac and striated muscle, and bone. The ER subtypes are in general differentially expressed. While ERalpha has been predominantly found in anterior pituitary, uterus, vagina, testis, liver and kidney, ERbeta is predominant in thyroid, ovary, prostate, skin, bladder, lungs, gastro-intestinal tract, cartilage and bone. In tissues which contain both receptor subtypes, such as ovary, testis and various regions of the brain, a cell-specific localization for each ER subtype has been generally observed. Altogether, the recent results on the cellular localization of sex steroid receptors will certainly contribute to a better understanding of the specific role of these steroids in different target organs.

Immunohistochemical findings on androgen receptor in mouse androgen-dependent tumor (Shionogi Carcinoma 115) and its independent sublines.

To examine the androgen receptor in androgen-dependent and -independent tumor immunohistochemically, an indirect immunofluorescence study with an antibody to human androgen receptor was performed. Shionogi Carcinoma 115 (SC 115) cells are an androgen-dependent mouse tumor, but the growth is sustained without androgen when fetal bovine serum is added to serum-free medium. Cells obtained from successive culture (A (-)X cells; X is generations after removal of androgen) were androgen-independent but showed binding to androgen. SC 115 cells, A (-) cells and CS 2 cells which are the other androgen-independent cells derived from SC 115, were used in the study. The androgen receptor (AR) in SC115 cells was stained as small-sized oval granules localized in the nucleus, and the number of the granules was 10-20 per cell. Removal of testosterone for one day as well as one week did not change the size of the AR, but some of the AR in A (-) 10 cells and in generations thereafter appeared to be large. Other small ones were similar to that in SC 115 cells. The nuclear location of the AR did not change in A (-) cells. The ratio of cells containing large AR to the total number of cells increased with each generation after the removal of testosterone from the culture. The addition of testosterone to the culture changed the AR in A (-) 40 cells to small ones, but did not influence the form of the AR in A (-) 60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and regulation of estrogen, progestin and androgen receptors in the seminal vesicle of the rhesus monkey.

We have used monoclonal antibodies against the estrogen (E), progestin (P) and androgen (A) receptors (R) to study receptor localization and regulation in the seminal vesicles of rhesus monkeys under different hormonal conditions. The antibodies caused substantial shifts of the appropriately labeled receptors on sucrose gradients. ER levels were lower in intact males than in immature, castrate, and estrogen-treated castrates. With immunocytochemistry, ER were detectable only in stromal and smooth muscle cells, not the epithelium. The number of ER-positive stromal cells was significantly lower in intact males than in immature, castrate, and estrogen-treated castrates, and low in a DHT-treated castrate animal. Androgen receptors were localized in epithelial as well as stromal and smooth muscle cells, and the number of AR-positive stromal cells was highest in intact adults and lowest in castrated and immature animals. Estrogen treatment at the time of castration induced PR in the ER-positive stromal cells, prevented a decline in the number of AR-positive stromal cells, and caused stromal hypertrophy. In summary, in the seminal vesicle, as in the prostate, ER is restricted to the fibromuscular stroma, is suppressed by androgens, and can mediate induction of PR on estrogen treatment. Androgen receptors are present in epithelial as well as stromal and smooth muscle cells, but variations in hormonal state appear to affect regulation of AR more in the stroma than the epithelium.

Androgen modulation of prostate cancer cell androgen receptor content is cell line specific.

Using methods for cell lysis and fractionation which yield essentially quantitative recovery of rat prostate cancer cell cytosolic and nuclear androgen receptors, we examined androgen modulation of androgen receptor content of clonally derived prostate cancer cell lines. We showed that testosterone elicited a concentration-dependent 2.3-fold increase in T5 cell androgen receptor content which was maximum after 48 h and was maintained through at least 72 h of culture. Testosterone caused only a 1.4-fold elevation in D2 cell androgen receptor content which was maximum between 6 and 12 h of culture and was maintained through at least 72 h culture. In contrast, testosterone did not cause a change in C3 cell androgen receptor content. Cycloheximide inhibition showed that both the testosterone-mediated increase in and maintenance of basal prostate cancer cell androgen receptor content required protein synthesis. Because testosterone and the nonmetabolizable androgen R1881 were essentially equipotent as effectors of the increase in T5 cell androgen receptor content, findings using testosterone appear to represent maximum effects. RU 23908 antagonized both R1881 and testosterone promoted elevations of prostate cancer cell androgen receptor content. Effectiveness of RU 23908 was comparable to the relative binding affinity of R1881, testosterone and RU 23908 for androgen receptors. This implies that at least part of the androgen-promoted increase in prostate cancer cell androgen receptor content is mediated through the action of androgen receptors and suggests that androgen receptors may act as both cis and trans regulatory elements. The mechanisms which determine basal or androgen-modulated prostate cancer cell androgen receptor content remain to be elucidated.

Intersex mice composed of androgen insensitive Tfm and wild-type cells analysed by 3H dihydrotestosterone autoradiography.

The X-linked testicular feminization mutation (Tfm) in the mouse is characterized by an androgen receptor defect. Due to random X-chromosome inactivation, XTfm/X+ heterozygotes are mosaics with respect to Tfm. They are composed of androgen receptor deficient XTfm cells and normal X+ wild-type cells. If Tfm heterozygotes are converted to XX males by the sex reversal factor (Sxr) the mosaicism is expressed. Therefore in sex reversed Tfm heterozygotes (XTfm/X+-Sxr) intersexual sex organs develop. In five intersexes with small male accessory glands and hypospadia and one heavily feminized intersex with vagina and caudally dislocated deferent ducts the mosaic is visualized by 3H-DHT-autoradiography. In the epididymis differentiated wild-type cells show nuclear labeling, whereas undifferentiated Tfm cells are unlabeled. Unlabeled Tfm cells are also encountered in the vesicular glands of the heavily feminized animal, demonstrating that Tfm cells can participate in the formation of male sex glands. The urethral glands of the mosaic animals are composed of unlabeled Tfm lobules exhibiting the female phenotype of the glands, and of labeled wild-type lobules exhibiting the male phenotype. Formation of a vagina and deviation of the deferent ducts is correlated with lack of androgen binding sites in the connective tissue.