Androgen Receptor Biology

Review Article

Androgen Receptor Structure, Function and Biology: From Bench to Bedside *Rachel A Davey and Mathis Grossmann

Department of Medicine, Austin Health, University of Melbourne, Heidelberg, Victoria, 3084, Australia. For correspondence: Associate Professor Rachel Davey, [email protected]

Abstract The actions of androgens such as testosterone and dihydrotestosterone are mediated via the androgen receptor (AR), a liganddependent nuclear transcription factor and member of the steroid hormone nuclear receptor family. Given its widespread expression in many cells and tissues, the AR has a diverse range of biological actions including important roles in the development and maintenance of the reproductive, musculoskeletal, cardiovascular, immune, neural and haemopoietic systems. AR signalling may also be involved in the development of tumours in the prostate, bladder, liver, kidney and lung. Androgens can exert their actions via the AR in a DNA binding-dependent manner to regulate target gene transcription, or in a non-DNA binding-dependent manner to initiate rapid, cellular events such as the phosphorylation of 2nd messenger signalling cascades. More recently, ligandindependent actions of the AR have also been identified. Given the large volume of studies relating to androgens and the AR, this review is not intended as an extensive review of all studies investigating the AR, but rather as an overview of the structure, function, signalling pathways and biology of the AR as well as its important role in clinical medicine, with emphasis on recent developments in this field.

Introduction Androgens (testosterone and dihydrotestosterone (DHT)) are the male sex hormones required for development of the male reproductive system and secondary sexual characteristics.1 Testosterone can be converted to its more biologically active form, DHT, by 5α reductase, and to oestradiol by aromatase. Testosterone and DHT mediate their actions via the AR, a ligand-dependent nuclear transcription factor.2 Other members of the steroid hormone nuclear receptor family include the oestrogen receptor (ER), progesterone receptor (PR), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). The AR, located on the X chromosome, is expressed in a diverse range of tissues and as such androgens have been documented to have significant biological actions in bone, muscle, prostate, adipose tissue and the reproductive, cardiovascular, immune, neural and haemopoietic systems.3 The AR binds androgens with strong affinity in the low nanomolar range4 with DHT being more biologically active than testosterone, binding to the AR with a 2-fold higher affinity and a decreased dissociation rate of 5-fold compared to testosterone.5

Androgen Receptor Structure The AR comprises three main functional domains: the N-terminal transcriptional regulation domain, the DNA binding domain (DBD) and the ligand binding domain (Figure 1).6 The N-terminal domain of the AR is the most variable, whilst the DBD is the most highly conserved region between the different members of the steroid hormone nuclear receptor family. The DBDs of all steroid hormone nuclear receptors consist of two zinc fingers that recognise specific DNA consensus sequences.7 These zinc fingers facilitate direct DNA binding of the AR to the promoter and enhancer regions of AR-regulated genes, thereby allowing the activation functions of the N-terminal and ligand binding domains to stimulate or repress the transcription of these genes. Given the highly conserved nature of the DBD amongst the steroid hormone nuclear receptor family, it has been shown that binding of selective androgen response elements (AREs) allow the specific activation of the AR. The probasin gene is one such example, where the ARE in its promoter is specifically recognised by the AR, but not the GR.8 The DBD is linked to the ligand binding domain by a hinge region. The ligand binding domain also has a similar structure between the nuclear receptors and mediates the interaction between Clin Biochem Rev 37 (1) 2016 3

Davey RA, Grossmann M

Figure 1. Functional domains of the androgen receptor (AR): N-terminal domain, DNA binding domain (DBD), Ligand binding domain. (H – hinge region, AF-1 – transcriptional activating function 1, AF-2 – transcriptional activating function 2, NLS – nuclear localisation signal, NES – nuclear export signal) the AR and heat shock and chaperone proteins, whilst also interacting with the N-terminus of the AR to stabilise bound androgens.7

There are two distinct mechanisms of ligand-dependent AR action, either dependent or independent of DNA binding (Figure 2).

Within the AR are a number of signal sequences. Two transcriptional activation functions have been identified: the ligand-independent AF-1, located in the N-terminal domain which is required for maximal activity of the AR,9 and the ligand-dependent AF-2, located in the ligand binding domain which is important for forming the coregulator binding site as well as mediating direct interactions between the N-terminal and ligand binding domains (N/C interactions).10,11 Key differences in the contribution of specific conserved residues in the AF-2 core domain between the AR and other steroid hormone nuclear receptors have been identified, which likely account for the observed differences between the AF-2 regions of the AR and other steroid hormone nuclear receptors with respect to their structure and function as well as the coregulatory proteins they interact with.10 A nuclear localisation signal (NLS), responsible for import of the receptor into the nucleus, and a nuclear export signal (NES), responsible for exporting the AR to the cytoplasm upon ligand withdrawal, are located between the DBD and hinge region and in the ligand binding domain respectively.4

DNA Binding-Dependent Actions of the AR The DNA binding-dependent actions of the AR are also commonly referred to in the literature as ‘genomic’, ‘classical’ or ‘canonical’ AR signalling. In the absence of ligand, the AR is cytoplasmic, associated with heat-shock and other chaperone proteins. Androgens bind to the AR, resulting in a conformational change, dissociation of chaperone proteins and exposure of the NLS. The androgen/AR complex translocates to the nucleus where it dimerises and binds to AREs within classical target genes to modulate gene transcription.12 The transcriptional activity of the androgen-bound AR is modulated by specific proteins known as coregulators. Coregulators bind to the activated AR in a ligand-dependent manner to either enhance (coactivator) or repress (corepressor) its ability to transactivate the target gene through chromatin remodelling and histone modifications, as well as being involved in the recruitment of the basal transcriptional machinery.13 Known coregulators of the AR have been extensively reviewed.7,14,15

Figure 2. Mechanisms of ligand-dependent androgen receptor (AR) action: (1) DNA binding-dependent (DBD) and (2) nonDNA binding (DBD)-dependent. (AP-1 – activator protein 1) Reproduced from Rana et al. (ref. 3) with permission.

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Androgen Receptor Biology

Non-DNA Binding-Dependent Actions of the AR The DNA binding independent actions of the AR are also commonly referred to in the literature as ‘non-genomic’, ‘non-classical’ or ‘non-canonical’ AR signalling. The androgen/AR complex can also signal through nonDNA binding-dependent pathways.16 Activation of 2nd messenger pathways including ERK, Akt and MAPK have been identified in a number of cell lines.16-19 These effects occur within seconds to minutes of androgen treatment and are therefore too rapid to have arisen via the DBD actions of the AR to regulate the transcription and translation of target genes. Indirect gene transrepression can also occur, by the AR binding and sequestering transcription factors such as activator protein-1 (AP-1) that are normally required to upregulate target gene expression (e.g. Ngfr20 and Mmp-1321), in the absence of the AR binding to DNA. Evidence exists to suggest that at least some of the non-DNA binding-dependent actions of androgens are mediated via the activation of membrane-bound protein receptors to initiate intracellular signalling pathways22 which can occur even in the presence of low levels of androgens.23 The identification and characterisation of cell surface receptors that can mediate the rapid non-DNA binding-dependent actions of oestrogen and progestins have been documented in a wide range of tissues and cell types,24,25 however, to date, membrane-bound AR receptors have not been studied as extensively. Recently, the iron-regulated transporter-like protein 9 (ZIP9) has been identified to mediate the androgen-induced apoptosis of ovarian follicle cells,26 prostate and breast cancer cells27 and the G-protein coupled receptor GPRC6A has been shown to mediate the non-DNA binding-dependent action of testosterone to phosphorylate ERK in bone marrow stromal and prostate cancer cells in vitro.28 Of interest, it has recently been reported that the bone-derived hormone, osteocalcin, binds to the GPRC6A in Leydig cells of the testes to stimulate testosterone production.29 The non-DNA binding-dependent actions of the AR have been documented in a wide variety of cell types in vitro. In osteoblasts and osteocytes, the ligand binding domain of the AR rapidly phosphorylates Src and ERK to inhibit apoptosis, thereby increasing their life span.18 DHT treatment of osteoblast-like cells increases cell proliferation associated with rapid activation of the Akt signalling pathway.17 These actions can be blocked by siRNA inhibition of AR expression, thus demonstrating non-DNA binding-dependent AR actions in osteoblasts.17 Activation of non-DNA binding-dependent pathways has also been observed in rat myotubes treated with androgens, with an increase in inositol triphosphate (IP3) mass and intracellular calcium seconds after testosterone addition.30

Non-DNA binding-dependent AR signalling pathways have also been identified in cardiomyocytes in vitro,31 and a recent study showed that DHT treatment of isolated rat left atria causes a non-DNA binding-dependent cardiotonic response associated with increased ornithine decarboxylase activity.32 In prostate cancer cells in vitro, Akt phosphorylation of the AR results in suppression of AR target genes, such as p21, which decreases ligand-dependent AR mediated apoptosis.33 For the most part, investigation into the non-DNA bindingdependent actions of the AR have been limited to in vitro studies,34 with the physiological relevance of these actions remaining largely unknown.35 Some studies, however, have documented non-DNA binding-dependent effects of the AR including rapid coronary vasodilation36 and oocyte maturation19,37 in vivo. In addition, non-DNA bindingdependent actions of androgens mediated via a membrane AR (mAR) to promote apoptosis and reduce the size of tumours derived from the prostate cancer cell line LnCap have also been described in vivo.38 There is now significant interest in non-DNA binding-dependent actions of the ER and GR,39 which are functionally very similar to the AR. While the physiological significance of the non-DNA binding-dependent actions of the AR is not yet fully defined, it has been proposed that they may oppose the DNA binding-dependent actions, and serve as a brake to fine-tune androgen action in target tissues. Similar opposing actions of the DNA binding-dependent and non-DNA binding-dependent pathways have been observed for the ER, with DNA binding-dependent activation of the ERα stimulating AP-1 activity,40 but activation of mitogenactivated protein kinases (MAPK) by a non-DNA bindingdependent ERα function suppressing AP-1 activity.41 One of the major difficulties of studying the non-DNA bindingdependent actions of the AR in vivo is the lack of an appropriate animal model that can distinguish between DNA bindingdependent and non-DNA binding-dependent receptor actions. For this purpose we generated a unique AR knockout (ARKO) mouse model with an in-frame deletion of the 2nd zinc finger of the DNA binding domain (DBD-ARKO). Key differences between our DBD-ARKO and other AR-null mouse models are summarised in Table 1. The mutant AR that is expressed in these mice binds ligand but lacks DNA binding-dependent AR signalling while retaining non-DNA binding-dependent signalling evidenced by phosphorylation of the 2nd messenger ERK, both in vitro and in vivo.42 To investigate the potential role of the non-DNA binding-dependent actions of the AR on bone growth and remodelling, gonadally-intact DBD-ARKO males were treated with DHT which cannot be aromatised to oestradiol. While the main action of androgen in cortical bone is considered to be stimulation of bone growth by increasing periosteal bone apposition, most likely via the dominant

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Table 1. Summary of differences between AR-null and DNA binding-dependent androgen receptor knock out (ARKO) mouse models. Null ARKOs

DBD-ARKO

Exon/s of the AR targeted

Exon 1101,102 Exon 2103,104

Exon 3105

Mutation/s

Frame shift deletions of either the N-terminal region or 1st zinc finger of the DNA binding domain

In-frame deletion of 2nd zinc finger of the DNA binding domain

Mutant AR protein

No

Yes Binds ligand, lacks DNA bindingdependent AR signalling, retains nonDNA binding-dependent signalling

DNA binding-dependent AR pathway,43,44 DHT treatment in DBD-ARKO males inhibited periosteal bone growth.42 These opposing actions of the non-DNA binding-dependent actions of the AR to the DNA binding-dependent actions of the AR on cortical bone, therefore, may reflect a physiological means by which the bone can respond and adapt to changes in mechanical load during growth and development. Ligand-Independent Actions of the AR Evidence also suggests that steroid hormone receptors can act in a ligand-independent manner.45,46 The GR regulates a suite of genes in the absence of ligand that is different to ligand-bound GR target genes, with only a small overlap between these groups, which may contribute to tissue-specific responsiveness.47 Ligand-independent ER actions have been demonstrated in vivo, with EGF- or dopaminergic-mediated activation of ER modulating mouse behaviours.48,49 Ligandindependent activation of the AR by a number of different growth factors has been demonstrated, via phosphorylation of the AR or following interaction with co-activators.50,51 One such pathway identified is IL-6, the circulating levels of which are commonly elevated in patients with metastatic prostate cancer. IL-6 upregulates AR activity in a ligand-independent manner via the protein kinase A (PKA), protein kinase C (PKC) and MAPK pathways, and as such has important clinical implications for prostate cancer patients with low androgen levels as a result of androgen deprivation therapy.52 Ligand-independent AR activation is one mechanism through which prostate cancers develop hormone resistance.53-56 For example, in prostate cancer, the upregulated expression of the Rho guanosine triphosphate guanine nuclear exchange factor, vav3, leads to enhanced activation of the AR by epidermal growth factor (EGF) and insulin-like growth factor (IGF) in

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the absence of androgens, leading to the subsequent activation of AR nuclear localisation and MAPK signalling.56 Similar to the GR, it has been shown in a prostate cancer cell line that ligand-independent AR regulates a distinct group of target genes compared with ligand-bound AR.57 Ligand-independent actions of the AR have also been identified in the myoblast C2C12 cell line, where IGF-I stimulates phosphorylation, nuclear localisation and DNA binding activity of the AR and upregulation of the expression of a number of known AR target genes via the MAPK pathway.58,59 It remains to be determined, however, whether ligand-independent AR pathways are limited to prostate cancer pathophysiology or play a role in normal physiology. The Androgen Receptor in Clinical Medicine Androgen Deficiency Given that the AR is expressed in multiple reproductive tissues, it is not surprising that androgens have important effects on multiple organ systems. These play critical roles in the regulation of many male, and female sexual, somatic and behavioural functions critical to lifelong health. Male androgen deficiency is usually a consequence of reduced testosterone production due to organic hypothalamic-pituitary-gonadal axis pathology leading to reduced AR activation. In general, setting aside the rare condition of androgen resistance, this condition reflects a deficiency of the ligand rather than ARrelated pathology. Because of its clinical importance, this condition is discussed briefly. It should be noted that some of the clinical features attributed to male androgen deficiency may instead be caused by concomitant oestradiol deficiency leading to reduced ER signalling.60 Androgen deficiency is a multi-system syndrome, diagnosed clinically based on symptoms and signs, and confirmed biochemically by

Androgen Receptor Biology

documenting abnormally low serum testosterone levels.61 Organic androgen deficiency, also known as classical hypogonadism, has to be distinguished from the ageassociated decline in testosterone levels occurring in some men. While this decline can be associated with non-specific symptoms resembling androgen deficiency, whether this association is causal is uncertain. Testosterone levels decline with age by about 0.5–2% per year in community-dwelling men,62 however this decline in testosterone is accelerated by age-related accumulation of comorbidities, especially obesity.63 This suggests that, in older men, low testosterone is a robust marker of poor health rather than a causal factor. Therefore clinical management of the older man with modest testosterone reductions should focus on treating (and preventing) comorbidities with lifestyle measures and appropriate pharmacotherapy (such as antiglycaemic agents for diabetes). If successful, such measures including weight loss do not only have multiple health benefits but can even lead to normalisation of reduced testosterone levels.64 In contrast, there is no evidence that testosterone therapy in older men without classical hypogonadism is effective or safe, because adequately-powered randomised controlled clinical trials (RCTs) designed and powered to examine clinically important health outcomes are not yet available. Role of the AR in Prostate Cancer Prostate cancer is the most common solid organ cancer in men and a major cause of cancer death. Since Huggins and Hodges first demonstrated the responsiveness of prostate cancer to androgen deprivation, it has been clear that prostate cancer is dependent on AR activation for growth and survival.65 Androgen deprivation therapy (ADT) is the mainstay of therapy in advanced prostate cancer, and also improves prognosis in appropriately-selected men with highrisk localised prostate cancer.66 Conventional ADT involves deprivation of the AR of testosterone produced by the testicles, achieved either by surgical or medical orchidectomy. This is sometimes complemented by the addition of an AR antagonist to achieve so-called complete androgen blockade, although the incremental benefit of this combined approach using firstgeneration AR antagonists is marginal.67 ADT is not curative, and after a median of 1–2 years of ADT, clinical progression occurs.68 While this has traditionally been thought to represent androgen-independent prostate cancer, this is now known to be generally incorrect. Instead, surprisingly, in most cases ADT-resistant prostate cancer remains androgen-dependent, and this stage is now termed castrate-resistant prostate cancer (CRPC).68 Despite very low circulating levels of serum testosterone, AR signalling is maintained by multiple mechanisms including activating AR mutations or truncations, AR amplification or overexpression resulting in increased protein expression, changes in AR cofactor balance and

extragonadal androgen production, including in the tumour tissue itself.69 Clinical proof-of-principle that CRPC remains androgen-driven has been provided by recent RCTs showing survival benefit with innovative, more effective androgen blockade. Abiraterone, an androgen biosynthesis inhibitor, prevents not only testicular but also adrenal and intratumoural androgen synthesis resulting in a survival benefit in CRPC.70 Similar evidence exists for enzalutamide, a second- generation AR antagonist with higher affinity for the AR.71 However, therapy-induced resistance to these newer agents is occurring, and additional strategies to suppress AR activation are being explored in mechanistic studies and in clinical trials. Indeed the prostate cancer treatment landscape is changing rapidly, with several options to the treatment of CRPC now available, but the optimal choice, timing sequencing and combinations of these agents awaits evidence from ongoing clinical trials. Not surprisingly, there is evidence that advanced prostate cancer can become truly androgen-insensitive, via activation of signalling pathways that bypass AR dependence including the use of alterative steroid receptor pathways such as the GR.72 While comparatively less toxic than chemotherapy, ADT has a number of important side effects.73 These side effects are a consequence of the induced severe sex steroid deficiency, and, consistent with the widespread expression of sex steroid receptors, affect multiple somatic and psychosocial domains. Sexual dysfunction and fatigue are almost universal, the latter contributed to by anaemia, hot flushes, loss of muscle mass and, perhaps, by sex steroid deprivation of the central nervous system. Cardiometabolic and musculoskeletal complications of ADT are of particular concern because of their impact on morbidity and mortality. These include sarcopaenic obesity leading to insulin resistance and diabetes, and possibly increased risk of cardiovascular events. In addition, there is accelerated loss of bone mass leading to increased fragility fractures.73 Given that most men with prostate cancer do not die from the disease, but rather from conditions similar to those in the general male population such as cardiovascular disease, and given the high prevalence of cardiometabolic and skeletal morbidity in men with prostate cancer already prior to commencing ADT, even modest adverse effects of ADT on these conditions may substantially diminish the riskbenefit ratio of ADT.74 Therefore the use of ADT has to be individualised. It should only be used in situations where there is proven benefit, and comorbidities should be managed proactively.75 Accumulating data suggests that intermittent ADT, by allowing for testosterone recovery, improves the tolerability of ADT without compromising clinical antitumour outcomes in appropriately-selected patients.76

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Androgen Insensitivity Syndrome Androgen insensitivity syndrome (AIS) is a disorder of sexual differentiation due to inactivating AR mutations conferring resistance to circulating testosterone, with more than 400 different AR mutations reported.77 The clinical phenotype largely depends on the residual degree to which the mutated AR can be transactivated. Mutations rendering the AR completely unresponsive cause complete AIS (CAIS), whereas mutations with residual AR function lead to partial AIS (PAIS). Individuals with CAIS have XY sex reversal and present with a female phenotype and normal female gender identity. At puberty, normal breast development and pubertal growth occurs because these XY women have adequate levels of oestradiol derived from aromatisation of testosterone which circulates in the high male reference interval. Female internal genitalia are absent, and hence CAIS commonly presents as primary amenorrhea in an adolescent female. Sexual hair is androgen-dependent and therefore reduced. Clinical management is complex and involves a multidisciplinary approach including psychologists, endocrinologists, urologists and gynaecologists. Because of malignancy risk, gonads, usually located in the abdomen or inguinal canal, are commonly removed, requiring subsequent oestrogen replacement to maintain feminisation. The phenotype of PAIS varies depending on the degree of residual AR function, ranging from male-appearing genitalia to severe undermasculinisation resembling female genitalia.77 Management of PAIS, including gender assignment, is very complex. AR CAG Repeat Number and Disease While the DNA and ligand binding domains of the AR are highly conserved during evolution, the transactivation domain is more polymorphic and genetic changes therein may affect AR-mediated gene regulation of AR-responsive genes. Indeed, the variability of AR size is in part due to microsatellite trinucleotide repeats located in the transactivation domain within the first exon, a CAG polymorphism encoding polyglutamine regions. The normal range of the CAG repeat is 11–31 triplets in length, and the transactivational activity of the AR is inversely associated with the number of CAG repeats.78 Whether variations of the CAG repeat lengths within the normal range are associated with clinical changes in tissue androgenisation is doubtful. Most likely subtle decreases in transactivational activity due to longer CAG repeat chains are counterbalanced by subtle upregulation of the activity of the hypothalamic-pituitary-gonadal axis. Similarly, there is no conclusive evidence that AR CAG repeat polymorphisms modulate the responsiveness to exogenous testosterone replacement in hypogonadism, although larger studies are required to fully characterise the impact of modest variability in CAG repeat length. It is possible that the impact of CAG variability within the normal range may be greater in men 8 Clin Biochem Rev 37 (1) 2016

with gonadal axis pathology. For example, a cross-sectional study in men with Klinefelter’s syndrome reported that a higher CAG repeat number is associated with smaller testis volume and more severe gynaecomastia.79 In contrast to variations within the normal range, more marked expansion of the CAG repeat is associated with Kennedy’s disease, also known as bulbospinal muscular atrophy (BSMA). This is a rare, recessive adult-onset neurodegenerative disorder.80 Due to the X-linked transmission (the AR gene is located on the X chromosome), BSMA exclusively affects males. BSMA is transmitted by female carriers who are usually asymptomatic or have a mild clinical phenotype. BSMA presents with slowly progressive muscle weakness and wasting involving facial, bulbar and limb muscles.81 Although the pathogenesis is not fully understood, BSMA is caused by pathological CAG tandem-repeat expansion in the order of 40–62 CAG repeats. Most likely, this leads to a toxic gain of function of the elongated AR within the expanded polyglutamine tract in the AR protein leading to AR misfolding and nuclear aggregation within motor neurons and possibly other cell types, causing toxicity to these cells.80 Longer AR repeat chains correlate with earlier disease onset. Although traditionally viewed as a primary motor neuron disease, preclinical studies have suggested more recently that muscle cells may be an important target of mutant AR toxicity.82 In addition to this toxic gain of function, the mutant receptor is less responsive to circulating androgens, leading to androgen resistance. Therefore features of androgen deficiency are a clinical component of BMSA and include gynaecomastia, sexual dysfunction and reduced fertility. Interestingly, intracellular aggregation of the mutant AR requires the presence of androgens. Therefore, androgen deprivation treatment is a potential therapeutic option, although controlled studies are lacking due to the rarity of this condition. One drawback of androgen deprivation is the resultant exacerbation of androgen deficiency-associated symptoms. Conversely, testosterone treatment to alleviate androgen deficiency symptoms in men with Kennedy’s disease carries the risk of accelerating the progression of muscle weakness via enhanced mutant AR aggregation. Other treatment approaches involving inhibition of histone acetylation or acceleration of AR degradation remain experimental.81 Should the preclinical studies identifying muscle as a site of direct mutant AR toxicity82 be confirmed in humans, this may allow targeting the periphery rather than the central nervous system (CNS) for treatment. This not only has practical advantages for delivery of therapeutic agents, but may avoid side effects thought to be primarily due to AR inhibition in the CNS such as reduced libido.83 AR Genetically Modified Animal Models Due to the widespread expression of the AR, its biological

Androgen Receptor Biology

and physiological effects following activation are numerous. Significant advances in our understanding of AR actions in specific tissues have been gained through the use of genetically modified animal models where the AR is globally deleted, that is, in every cell and tissue (Global ARKO). Studies in Global ARKO male mice have focussed on bone, brain, the cardiovascular system, glucose metabolism and fat, the immune and haemopoietic systems, muscle and the prostate. A summary of the key findings from these studies in male Global ARKOs is provided in Table 2. Studies have also investigated the phenotype of homozygous female Global ARKO mice but these studies are, for the most

part, limited to the reproductive actions of the AR. The AR has been shown to be important for normal folliculogenesis, mammary gland development, and uterine morphology and development.84-88 We have also shown, using our unique DBD-ARKO mouse model, that the DNA binding-dependent actions of the AR promote cardiac growth, kidney hypertrophy, cortical bone growth and regulate trabecular bone architecture in females.89 A more detailed understanding of the AR within specific tissues and/or cell types has been made through the use of the Cre/loxP system in which deletion of the AR has been targeted to a precise tissue or cell type at defined stages of

Table 2. Overview of key insights gained into androgen receptor (AR) biology in males from Global AR Knockout (ARKO) male mice. Tissue

Phenotype

Reference

Bone

Bones of reduced size, thickness and volume. Increased bone turnover.

Yeh S et al.,104 MacLean HE et al.,89 Kawano H et al.106 and Venken K et al.107

Brain

Absence of male sexual and aggressive behaviours.

Sato T et al.108

Cardiovascular System

Reduced heart size, impaired contraction, exacerbation of angiotensin II-induced cardiac fibrosis.

Ikeda et al.109

Glucose Metabolism and Fat

Increased subcutaneous and visceral fat mass, decreased voluntary activity. Late-onset obesity, hyperinsulinemia, increased serum levels of leptin and adiponectin.

Rana K et al.110

Reduced Neutrophil count. Increased self-renewal of bone marrow mesenchymal stem cells. Accelerated wound healing.

Lai JJ et al.113 Huang CK et al., 2013114

Muscle

Decreased skeletal muscle and perineal muscle mass, decreased strength.

MacLean HE et al.,116 Altuwaijri S et al.117 and Ophoff J et al.118

Prostate

No prostate development.

Sato T et al.,108 Yeh S et al.104 and Notini AJ et al.105

Immune and Haemopoietic Systems

Lin HY et al.111 and Fan W et al.112

Lai JJ et al.115

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development. For example, we have shown that within bone, the AR acts in osteoblasts at the mineralisation stage of their development to inhibit bone resorption and to coordinate the synthesis of bone matrix with its subsequent mineralisation.90 Much of what is currently known about AR function in the testis has been gained through the use of Cre/loxP technology to target AR deletion in specific cell types residing within the testes, such as the leydig, sertoli and peritubular myloid cells. The studies utilising tissue-specific ARKO mouse models to investigate AR function and physiology are both complex in nature and numerous. Their findings in the prostate,91 bone,3,92 reproductive,93,94 cardiovascular,95 metabolic3 and muscle3,96,97 as well as hormone-related tumours in the liver, bladder, kidney and lung98 have been reviewed extensively. Comparisons of these studies and their principal findings and limitations have been elegantly reviewed recently by De Gendt and Vanderscheuren.96 Of importance, a number of factors need to be taken into consideration when assessing the phenotype of genetically modified mice to ensure accurate interpretation of the data such as genetic background, environmental factors including diet and housing, and the use of appropriate control groups, which is of particular importance when using Cre/loxP technology. These factors, as well a number of other considerations, are discussed in depth in our previous reviews.3,99,100 Conclusion Activation of the AR has diverse biological effects in health and disease. As such, understanding the role of androgen action mediated via both the DNA binding-dependent and non-DNA binding-dependent actions of the AR, in addition to the potential ligand-independent actions of the AR, in normal physiology as well as in pathological conditions is crucial for the future development of targeted therapies for a wide range of AR-related clinical conditions. For example, understanding the structure and function of the ligand binding domain of the AR and its interaction with coregulators is important for the design of new AR antagonists and agonists. Likewise, the development of selective AR modulators (SARMs) that target the specific actions of the AR within a specific cell/tissue type will be pertinent to large groups of patients who cannot be administered testosterone due to potential side-effects, including women, ageing men and prostate cancer patients undergoing ADT. However, it has to be emphasised that, apart from men with classical hypogonadism due to organic hypothalamic-pituitary-gonadal pathology, the efficacy or safety of treatment with testosterone or other AR modulators has not been established in other populations, including in men with age-related decline in testosterone.

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Acknowledgements R Davey was supported by a National Health and Medical Research Council of Australia Project Grant (APP1058189) and M Grossmann was supported by a National Health and Medical Research Council of Australia Career Development Fellowship (# 1024139). Competing Interests: None declared. References 1. MacLean HE, Chu S, Warne GL, Zajac JD. Related individuals with different androgen receptor gene deletions. J Clin Invest 1993;91:1123-8. 2. Chang C, Saltzman A, Yeh S, Young W, Keller E, Lee HJ, et al. Androgen receptor: an overview. Crit Rev Eukaryot Gene Expr 1995;5:97-125. 3. Rana K, Davey RA, Zajac JD. Human androgen deficiency: insights gained from androgen receptor knockout mouse models. Asian J Androl 2014;16:16977. 4. Tan MH, Li J, Xu HE, Melcher K, Yong EL. Androgen receptor: structure, role in prostate cancer and drug discovery. Acta Pharmacol Sin 2015;36:3-23. 5. Grino PB, Griffin JE, Wilson JD. Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone. Endocrinology 1990;126:1165-72. 6. MacLean HE, Warne GL, Zajac JD. Localization of functional domains in the androgen receptor. J Steroid Biochem Mol Biol 1997;62:233-42. 7. Heinlein CA, Chang C. Androgen receptor (AR) coregulators: an overview. Endocr Rev 2002;23:175200. 8. Schoenmakers E, Verrijdt G, Peeters B, Verhoeven G, Rombauts W, Claessens F. Differences in DNA binding characteristics of the androgen and glucocorticoid receptors can determine hormone-specific responses. J Biol Chem 2000;275:12290-7. 9. Callewaert L, Van Tilborgh N, Claessens F. Interplay between two hormone-independent activation domains in the androgen receptor. Cancer Res 2006;66:543-53. 10. Slagsvold T, Kraus I, Bentzen T, Palvimo J, Saatcioglu F. Mutational analysis of the androgen receptor AF-2 (activation function 2) core domain reveals functional and mechanistic differences of conserved residues compared with other nuclear receptors. Mol Endocrinol 2000;14:1603-17. 11. Wilson EM. Analysis of interdomain interactions of the androgen receptor. Methods Mol Biol 2011;776:113-29. 12. Eder IE, Culig Z, Putz T, Nessler-Menardi C, Bartsch G, Klocker H. Molecular biology of the androgen receptor: from molecular understanding to the clinic. Eur Urol

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Androgen Receptor Structure, Function and Biology: From Bench to Bedside.

The actions of androgens such as testosterone and dihydrotestosterone are mediated via the androgen receptor (AR), a ligand-dependent nuclear transcri...
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