Function of androgen receptor in gene regulations

Most of the androgen actions are considered to be mediated by the androgen receptor (AR) of the target genes. The AR is composed of a fairly large molecule because of the long A/B domains of its N-terminal. However, the independent roles of the AR as well as those of the estrogen receptors largely remained unknown mainly due to the lack of the AR knockout (ARKO) mice line. We have succeeded in generating the ARKO mouse by means of a conditional targeting using the Cre/loxP system. The ARKO males grew healthily although they showed a typical feature of the testicular feminization mutation (Tfm) and the hormonal assay revealed significantly lower serum androgen and higher LH levels in comparison with those of the wild type (WT) males. The serum estrogen levels were, however, comparable between both the ARKO and the WT. Another hallmark of the ARKO males was a state of high bone turnover osteopenia, in which the acceleration in the bone resorption clearly exceeded the bone formation. Male-typical behaviors were disrupted in male ARKO mice. Aiming at a quick differentiation of an androgen-dependent polyQ disease such as Kennedy's disease, the authors also developed the Drosophila fly-eye model in which the wild type and the polyQ-expanded human AR (hAR) was induced in the eyes of Drosophila. When androgen was administered to the flies induced with the polyQ-expanded hAR, their optical nerves were devastated.     

The reduced trabecular bone mass of adult ARKO male mice results from the decreased osteogenic differentiation of bone marrow stroma cells

Male mice with androgen receptor knock-out (ARKO) show significant bone loss at a young age. However, the lasting effect of AR inactivation on bone in aging male mice remains unclear. We designed this study to evaluate the effect of AR on bone quality in aging male mice and to find the possible causes of AR inactivation contributing to the bone loss. The mice were grouped according to their ages and AR status and their trabecular bones were examined by micro-CT analysis at 6, 12, 18, and 30 weeks old. We found that bone mass consistently decreased and the bone microarchitectures continuously deteriorated in male ARKO mice at designated time points. To determine the cause of the bone loss in ARKO mice, we further examined the role of AR in bone cell fate decision and differentiation and we conducted experiments on bone marrow stromal cells (BMSC) obtained from wild type (WT) and AR knockout (KO) mice. We found that ARKO mice had higher numbers of colony formation unit-fibroblast (CFU-F), and CD44 and CD34 positive cells in bone marrow than WT mice. Our Q-RT-PCR results showed lower expression of genes linked to osteogenesis in BMSCs isolated from ARKO mice. In conclusion, AR nullification disrupted bone microarchitecture and caused trabecular bone mass loss in male ARKO mice. And the fate of BMSCs was impacted by the loss of AR. Therefore, these findings suggest that AR may accelerate the use of progenitor cells and direct them into osteogenic differentiation to affect bone metabolism.     

Androgen receptor gene knockout male mice exhibit impaired cardiac growth and exacerbation of angiotensin II-induced cardiac fibrosis

Androgen has anabolic effects on cardiac myocytes and has been shown to enhance left ventricular enlargement and function. However, the physiological and patho-physiological roles of androgen in cardiac growth and cardiac stress-induced remodeling remains unclear. We aimed to clarify whether the androgen-nuclear androgen receptor (AR) system contributes to the cardiac growth and angiotensin II (Ang II)-stimulated cardiac remodeling by using systemic AR-null male mice. AR knock-out (ARKO) male mice, at 25 weeks of age, and age-matched wild-type (WT) male mice were treated with or without Ang II stimulation (2.0 mg/kg/day) for 2 weeks. ARKO mice with or without Ang II stimulation showed a significant reduction in the heart-to-body weight ratio compared with those of WT mice. In addition, echocardiographic analysis demonstrated impairments of both the concentric hypertrophic response and left ventricular function in Ang II-stimulated ARKO mice. Western blot analysis of the myocardium revealed that activation of extracellular signal-regulated kinases (ERK) 1/2 and ERK5 by Ang II stimulation were lower in ARKO mice than those of WT mice. Ang II stimulation caused more prominent cardiac fibrosis in ARKO mice than in WT mice with enhanced expression of types I and III collagen and transforming growth factor-beta1 genes and with increased Smad2 activation. These results suggest that, in male mice, the androgen-AR system participates in normal cardiac growth and modulates cardiac adaptive hypertrophy and fibrosis during the process of cardiac remodeling under hypertrophic stress.     

Pituitary Androgen Receptor Signalling Regulates Prolactin but Not Gonadotrophins in the Male Mouse

Production of the androgen testosterone is controlled by a negative feedback loop within the hypothalamic-pituitary-gonadal (HPG) axis. Stimulation of testicular Leydig cells by pituitary luteinising hormone (LH) is under the control of hypothalamic gonadotrophin releasing hormone (GnRH), while suppression of LH secretion by the pituitary is controlled by circulating testosterone. Exactly how androgens exert their feedback control of gonadotrophin secretion (and whether this is at the level of the pituitary), as well as the role of AR in other pituitary cell types remains unclear. To investigate these questions, we exploited a transgenic mouse line (Foxg1^Cre/+; AR^fl/y) which lacks androgen receptor in the pituitary gland. Both circulating testosterone and gonadotrophins are unchanged in adulthood, demonstrating that AR signalling is dispensable in the male mouse pituitary for testosterone-dependent regulation of LH secretion. In contrast, Foxg1^Cre/+; AR^fl/y males have a significant increase in circulating prolactin, suggesting that, rather than controlling gonadotrophins, AR-signalling in the pituitary acts to suppress aberrant prolactin production in males.     

Late onset of obesity in male androgen receptor-deficient (AR KO) mice

An androgen receptor (AR) null mutant mice line was generated by means of a Cre-lox P system. The male (AR(L-/Y)) (KO) mice exhibited typical features of testicular feminization mutant (Tfm) disease in external reproductive organs with growth retardation. The growth curve of the male AR KO mice was similar to that of the wild-type female littermates until the 10th week of age, but thereafter a drastic increase in the growth was observed with development of obesity. Clear increase in the wet weights of white adipose tissues, but not of brown adipose tissue, was found in the 30-week-old male AR KO mice. However, no significant alteration in serum lipid parameters and food intake was observed. Thus, the present results suggest that AR may serve as a negative regulator of adipose development in adult males.     

Brain corticosteroid receptor gene expression and neuroendocrine dynamics during aging

The present study examined the stress responsiveness of the hypothalamic-pituitary-adrenal axis in relation to the properties of corticosteroid receptors in the brain and pituitary in old (30 months) and young (3 months) male Brown Norway rats. The data demonstrate that circulating ACTH rather than the corticosteroid plasma level was elevated under basal conditions and following stress. Furthermore, a reduction of mineralocorticoid receptor (MR) number in the hippocampus and of glucocorticoid receptor (GR) number in the hypothalamus and the pituitary correspond to increased neuroendocrine responsiveness and negative feedback following stress. The changes in receptor binding do not parallel the changes in the amount of MR and GR mRNA measured with in situ hybridization. This suggests that the processing rather than the receptor gene expression is affected in senescence.     

Mechanisms of brain glucocorticoid resistance in stress-induced psychopathologies

Exposure to stress activates the hypothalamic–pituitary–adrenal axis and leads to increased levels of glucocorticoid (GC) hormones. Prolonged elevation of GC levels causes neuronal dysfunction, decreases the density of synapses, and impairs neuronal plasticity. Decreased sensitivity to glucocorticoids (glucocorticoid resistance) that develops as a result of chronic stress is one of the characteristic features of stress-induced psychopathologies. In this article, we reviewed the published data on proposed molecular mechanisms that contribute to the development of glucocorticoid resistance in brain, including changes in the expression of the glucocorticoid receptor (GR) gene, biosynthesis of GR isoforms, and GR posttranslational modifications. We also present data on alterations in the expression of the FKBP5 gene encoding the main component of cell ultra-short negative feedback loop of GC signaling regulation. Recent discoveries on stressand GRinduced changes in epigenetic modification patterns as well as normalizing action of antidepressants are discussed. GR and FKBP5 gene polymorphisms associated with stress-induced psychopathologies are described, and their role in glucocorticoid resistance is discussed.     

Sex,stress, and epigenetics: regulation of behavior in animal models of mood disorders

Stress-related psychiatric disorders, such as unipolar depression and post-traumatic stress disorder (PTSD), occur more frequently in women than in men. Emerging research suggests that sex differences in receptors for the stress hormones, corticotropin releasing factor (CRF) and glucocorticoids, contribute to this disparity. For example, sex differences in CRF receptor binding in the amygdala of rats may predispose females to greater anxiety following stressful events. Additionally, sex differences in CRF receptor signaling and trafficking in the locus coeruleus arousal center combine to make females more sensitive to low levels of CRF, and less adaptable to high levels. These receptor differences in females could lead to hyperarousal, a dysregulated state associated with symptoms of depression and PTSD. Similar to the sex differences observed in CRF receptors, sex differences in glucocorticoid receptor (GR) function also appear to make females more susceptible to dysregulation after a stressful event. Following hypothalamic pituitary adrenal axis activation, GRs are critical to the negative feedback process that inhibits additional glucocorticoid release. Compared to males, female rats have fewer GRs and impaired GR translocation following chronic adolescent stress, effects linked to slower glucocorticoid negative feedback. Thus, under conditions of chronic stress, attenuated negative feedback in females would result in hypercortisolemia, an endocrine state thought to cause depression. Together, these studies suggest that sex differences in stress-related receptors shift females more easily into a dysregulated state of stress reactivity, linked to the development of mood and anxiety disorders. The implications of these receptor sex differences for the development of novel pharmacotherapies are also discussed.     

The zebrafish as an in vivo model system for glucocorticoid resistance

Glucocorticoids regulate a wide range of systems in vertebrate organisms, and their effects are mediated by the glucocorticoid receptor (GR). The responsiveness to glucocorticoids differs largely between individuals. Resistance to glucocorticoids is an important medical problem, since it limits the efficacy of glucocorticoids when they are used to treat immune-related diseases like asthma and rheumatoid arthritis. Glucocorticoid resistance also contributes to the pathogenesis of other diseases, like major depression because of the decreased negative feedback on the hypothalamic pituitary adrenal axis. In this review, we present the zebrafish as an excellent in vivo model system to study glucocorticoid resistance. First, the zebrafish is the only non-primate animal model in which a β-isoform of GR occurs, which is a splice variant with dominant-negative activity. Zebrafish are easily genetically modified, so the expression of GRβ can be varied, creating an in vivo model for GRβ-induced glucocorticoid resistance. Second, by performing a forward-genetic screen using the glucocorticoid-induced decrease in POMC expression in the pituitary gland as a readout, several zebrafish mutants have been obtained which appear to be resistant to glucocorticoid treatment. We present here two types of in vivo models for studying glucocorticoid resistance, that will be used to study the molecular mechanism of glucocorticoid signaling and resistance. Finally these models will be used to screen for small molecules that can alleviate glucocorticoid resistance.     

Regional Difference in Sex Steroid Action on Formation of Morphological Sex Differences in the Anteroventral Periventricular Nucleus and Principal Nucleus of the Bed Nucleus of the Stria Terminalis

Sex steroid action is critical to form sexually dimorphic nuclei, although it is not fully understood. We previously reported that masculinization of the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), which is larger and has more neurons in males than in females, involves aromatized testosterone that acts via estrogen receptor-α (ERα), but not estrogen receptor-β (ERβ). Here, we examined sex steroid action on the formation of the anteroventral periventricular nucleus (AVPV) that is larger and has more neurons in females. Morphometrical analysis of transgenic mice lacking aromatase, ERα, or ERβ genes revealed that the volume and neuron number of the male AVPV were significantly increased by deletion of aromatase and ERα genes, but not the ERβ gene. We further examined the AVPV and BNSTp of androgen receptor knockout (ARKO) mice. The volume and neuron number of the male BNSTp were smaller in ARKO mice than those in wild-type mice, while no significant effect of ARKO was found on the AVPV and female BNSTp. We also examined aromatase, ERα, and AR mRNA levels in the AVPV and BNSTp of wild-type and ARKO mice on embryonic day (ED) 18 and postnatal day (PD) 4. AR mRNA in the BNSTp and AVPV of wild-type mice was not expressed on ED18 and emerged on PD4. In the AVPV, the aromatase mRNA level was higher on ED18, although the ERα mRNA level was higher on PD4 without any effect of AR gene deletion. Aromatase and ERα mRNA levels in the male BNSTp were significantly increased on PD4 by AR gene deletion. These results suggest that estradiol signaling via ERα during the perinatal period and testosterone signaling via AR during the postnatal period are required for masculinization of the BNSTp, whereas the former is sufficient to defeminize the AVPV.     

Glucocorticoid receptor represses the Dex-mediated induction of human androgen response element-linked Luc activity

A human androgen response element (hARE), identified within intron 8 of the human sterol regulatory element-binding protein cleavage-activating protein, interacts with both glucocorticoid receptor (GR) and androgen receptors (AR). The aim of this study was to test the hypothesis that human GR (hGR) might modulate the expression of a hARE-linked reporter gene by dexamethasone (Dex). The hypothesis was tested by: a) co-transfecting HepG2 cells with a hGR and a luciferase (Luc)-reporter gene for performing in vitro investigations and b) by their co-injection into the tail vein of mice for in vivo investigation. In vitro co-transfected cells and the in vivo co-injected mice were then treated with Dex. Our results have led us to concluded that both transfection and injection of the hGR leads to a repression in the Dex-mediated induction of hARE-linked Luc activity both in vitro and in vivo settings. These findings suggest that this assay system allows screening of drug candidates affecting to a signal transduction pathway of the GR and AR and may help in the future discovery and analysis of novel and selection of GR and AR agonists.     

Transfection with steroid-responsive reporter constructs shows glucocorticoid rather than androgen responsiveness in cultured Sertoli cells

It remains unclear why it has proven so difficult to identify androgen target genes in cultured Sertoli cells. Given the lack of useful endogenous reporter genes, we studied the androgen and glucocorticoid responsiveness of these cells by transfection with three different steroid-responsive reporter constructs. The constructs were driven by the tyrosine aminotransferase steroid-responsive region (TAT-GRE4x-Luc), the mouse mammary tumor virus promoter (MMTV-Luc) and the Pem homeobox gene proximal promoter respectively (Pem-Luc). These constructs can be activated either by both the glucocorticoid receptor (GR) and the androgen receptor (AR) (TAT-GRE4x-Luc and MMTV-Luc) or selectively by the AR (Pem-Luc). Despite high transfection efficiency (30-40%) none of the constructs could be activated by treatment of the Sertoli cells with testosterone, 5alpha-dihydrotestosterone or synthetic androgens. Even pretreatment with follicle-stimulating hormone to raise AR levels (from 31 up to 82fmol/mg protein) did not result in androgen responsiveness. In contrast, treatment with dexamethasone markedly stimulated TAT-GRE4x-Luc and MMTV-Luc activity. GR levels reached a value of 172fmol/mg protein in the cultured cells and both AR and GR displayed homogeneous distribution by immunocytochemical evaluation. Androgen responsiveness was restored and glucocorticoid responsiveness was increased by cotransfection with AR or GR expression constructs. Under cotransfection conditions, 1nM of testosterone (a concentration that is some 100 times lower than that estimated to be present in the testis) was sufficient to stimulate the TAT-GRE4x-Luc maximally. Our data indicate that cultured Sertoli cells respond better to glucocorticoids than to androgens and that one of the factors limiting androgen responsiveness is the availability of AR. Other factors limiting the transactivation capacity of the (endogenous) AR, however, cannot be excluded.     

Developmental changes in neural corticosteroid receptor binding capacity in altricial nestlings

Altricial nestlings typically do not show an adrenocortical response during the early post‐hatch period. This may be a result of an immature hypothalamic‐pituitary‐adrenal axis, or an enhanced control of the axis by negative feedback. To examine whether the dampened adrenocortical response is due to higher receptor densities in hypothalamus and hippocampus, the major sites for negative feedback and tonic inhibition, we explored the ontogenetic changes in glucocorticoid (GR) and mineralocorticoid receptor (MR) binding capacities in the brain of white‐crowned sparrow nestlings. During the 10‐day nestling period, MR binding capacity decreased with age, whereas GR capacity was not affected. In addition, this overall decline in MR levels was driven entirely by a decline in cerebellar MR. No age‐related changes were observed in hippocampal or hypothalamic areas. Our findings suggest that enhanced negative feedback does not play a major role in the attenuated adrenocortical responses seen in white‐crowned sparrow nestlings. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 853–861, 2010     

The Glucocorticoid Receptor and its Expression in the Anterior Pituitary and the Adrenal Cortex: A Source of Variation in Hypothalamic-Pituitary-Adrenal Axis Function; Implications for Pituitary and Adrenal Tumors

ObjectiveTo review the expression of the glucocorticoid receptor (GR) in anterior pituitary and adrenocortical cells and tumors derived from these tissues as well as factors that may influence its expression.MethodsWe present an overview of the relevant literature, with a focus on data generated from our studies.ResultsThe expression of the GR is an essential element of the negative feedback that closes the loop formed by corticotropin-releasing hormone, adrenocorticotropic hormone, and cortisol in the context of the hypothalamicpituitary-adrenal (HPA) axis. Although the GR expression in anterior pituitary cells—and in particular the corticotrophs—was first demonstrated several years ago, it was not known until relatively recently where, by what cells, and in what form the GR is expressed in the adrenal cortex.The variability in the expression of the GR in pituitary and adrenocortical cells may underlie the substantial differences in HPA axis function across individuals, especially when testing for tumors associated with hypercortisolemia. This expression is influenced by a multitude of tissue-specific factors, which may explain why it is so difficult to interpret (or reproduce) studies that are based on GR functional polymorphisms on different cohorts of patients or even different sets of laboratory animals.ConclusionThis review highlights the variability in expression and function of the GR in pituitary and adrenocortical cells as one of the reasons for the appreciable differences in HPA axis function across individuals. Particular attention was paid to interactions that may affect the interpretation of diagnostic testing of the HPA axis in patients with pituitary adenomas (Cushing disease) or adrenocortical tumors (Cushing syndrome). (Endocr Pract. 2011;17:941-948)