Androgen Target Tissues Research Articles

The alternate pathway of androgen metabolism and window of sensitivity.

Since the discovery in 1968 that dihydrotestosterone (DHT) is a major mediator of androgen action, a convincing body of evidence has accumulated to indicate that the major pathway of DHT formation is the 5α-reduction of circulating testosterone in androgen target tissues. However, we now know that DHT can also be formed in peripheral tissues by the oxidation of 5α-androstane-3α,17β-diol (adiol). This pathway is responsible for the formation of the male phenotype. We discuss the serendipitous discovery in the tammar wallaby of an alternate pathway by which adiol is formed in the testes, secreted into plasma and converted in peripheral tissues to DHT. This alternate pathway is responsible for virilisation of the urogenital system in this species and is present in the testes at the onset of male puberty of all mammals studied so far. This is the first clear-cut function for steroid 5α-reductase 1 in males. Unexpectedly, the discovery of this pathway in this Australian marsupial has had a major impact in understanding the pathophysiology of aberrant virilisation in female newborns. Overactivity of the alternate pathway appears to explain virilisation in congenital adrenal hyperplasia CAH, in X-linked 46,XY disorders of sex development. It also appears to be important in polycystic ovarian syndrome (PCOS) since PCOS ovaries have enhanced the expression of genes and proteins of the alternate pathway. It is now clear that normal male development in marsupials, rodents and humans requires the action of both the classic and the alternate (backdoor) pathways.

Classic and Novel Sex Hormone Binding Globulin Effects on the Cardiovascular System in Men.

In men, 70% of circulating testosterone binds with high affinity to plasma sex hormone binding globulin (SHBG), which determines its bioavailability in their target cells. In recent years, a growing body of evidence has shown that circulating SHBG not only is a passive carrier for steroid hormones but also actively regulates testosterone signaling through putative plasma membrane receptors and by local expression of androgen-binding proteins apparently to reach local elevated testosterone concentrations in specific androgen target tissues. Circulating SHBG levels are influenced by metabolic and hormonal factors, and they are reduced in obesity and insulin resistance, suggesting that SHBG may have a broader clinical utility in assessing the risk for cardiovascular diseases. Importantly, plasma SHBG levels are strongly correlated with testosterone concentrations, and in men, low testosterone levels are associated with an adverse cardiometabolic profile. Although obesity and insulin resistance are associated with an increased incidence of cardiovascular disease, whether they lead to abnormal expression of circulating SHBG or its interaction with androgen signaling remains to be elucidated. SHBG is produced mainly in the liver, but it can also be expressed in several tissues including the brain, fat tissue, and myocardium. Expression of SHBG is controlled by peroxisome proliferator-activated receptor γ (PPARγ) and AMP-activated protein kinase (AMPK). AMPK/PPAR interaction is critical to regulate hepatocyte nuclear factor-4 (HNF4), a prerequisite for SHBG upregulation. In cardiomyocytes, testosterone activates AMPK and PPARs. Therefore, the description of local expression of cardiac SHBG and its circulating levels may shed new light to explain physiological and adverse cardiometabolic roles of androgens in different tissues. According to emerging clinical evidence, here, we will discuss the potential mechanisms with cardioprotective effects and SHBG levels to be used as an early metabolic and cardiovascular biomarker in men.

Spatial distribution of isobaric androgens in target tissues using chemical derivatization and MALDI-2 on a trapped ion mobility quadrupole time-of-flight instrument.

Prostate cancer is initially treated via androgen deprivation therapy (ADT), a highly successful treatment in the initial pursuit of tumour regression, but commonly restricted by the eventual emergence of a more lethal ‘castrate resistant’ (CRPC) form of the disease. Intracrine pathways that utilize dehydroepiandrosterone (DHEA) or other circulatory precursor steroids are thought to generate relevant levels of growth-stimulating androgens such as testosterone (T) and dihydrotestosterone (DHT). Decoding this tissue-specific metabolic pathway is key for the development of novel therapeutic treatments. Mass spectrometry imaging (MSI) is an analytical technique that allows the visualization of the distribution of numerous classes of biomolecules within tissue sections. The analysis of androgens by liquid chromatography mass spectrometry (LC/MS)-based methods however presents a challenge due to their generally poor ionization efficiency and low physiological endogenous levels. In MSI, on-tissue chemical derivatization (OTCD) has enabled the limits of steroids to be imaged within tissues to be pushed by overcoming poor ionization performance. However, isobaric interference of key androgen derivatives such as T and DHEA can severely hamper studying the intracrinology in several diseases. Here, we have evaluated the use of laser induced post-ionization (MALDI-2) combined with trapped ion mobility separation (TIMS) and orthogonal time-of-flight (QTOF) MS for the visualization of isobaric derivatized androgens in murine tumour xenograft at about 50 μm spatial resolution. With this combination, isobaric T and DHEA were separated in tissue sections and the signals of derivatized steroids enhanced by about 20 times. The combination of TIMS and MALDI-2 thus shows unique potential to study tissue intracrinology within target tissues. This could offer the opportunity for many novel insights into tissue-specific androgen biology.

Resistance Training Ameliorates Finasteride-Induced Disturbance in Protein Homeostasis in Skeletal Muscle of Rats

PURPOSE Finasteride is a competitive inhibitor of 5-alpha reductase type 2 enzyme that is commonly used to treat benign prostatic hyperplasia and male pattern hair loss. It is not clear about the synergistic effects of finasteride and resistance training on protein homeostasis in androgen-target tissue such as skeletal muscle. Thus, the purpose of this study was to investigate the effects of 8-week finasteride and resistance exercise training on protein synthesis and autophagic degradation in rat skeletal muscle. METHODS Forty-eight male wild-type Sprague Dawley rats (8weeks old) were randomly assigned into 4 groups: (1) sedentary control, (2) finasteride, (3) exercise, and (4) exercise plus finasteride. Rats of the finasteride group received finasteride, dissolved in corn oil (10 mg/kg) by oral gavage. Rats of the exercise group climbed a 1-m ladder inclined at 85° every 3 days for 8 weeks as a means of resistance exercise. They climbed the ladder 8 times with the load of 50%, 75%, 90%, and 100% of its body weight. Rates of the exercise plus finasteride group were given both interventions. We assessed autophagy flux, protein synthesis (mTOR signaling and translation rate using the SUnSET assay), and muscular functions before and after 8-weeks of intervention, and performed one-way ANOVA to determine pre-post differences in each group. RESULTS The finasteride treatment significantly reduced protein synthesis while activating autophagic degradation in skeletal muscle of rats. The exercise training increased both protein synthesis and autophagic degradation. The combined treatments decreased both protein synthesis and autophagy flux in the skeletal muscle (p<0.05). CONCLUSIONS Eight weeks of finasteride treatment appears to disturb the protein homeostasis, which is viewed as a balance of protein synthesis and degradation through the reduction in protein synthesis and activation in autophagic degradation simultaneously in skeletal muscle. Resistance exercise appears to alleviate the disruption of protein homeostasis induced by finasteride treatment. 색인어: í”¼ë‚˜ìŠ¤í Œë¼ì´ë“œ, 자가포식, 자가포식 ìœ ë™, 단백질 항상성, 골격근 Keywords: Finasteride, Autophagy, Autophagic flux, Protein homeostasis, Skeletal muscle

SUN-213 Hsd17b3 Knockout Mice Serve As A Model To Define Mechanisms Behind The Altered Endocrine Phenotype Of Patients With Hsd17b3 Mutations

Hydroxysteroid (17β) dehydrogenase type 3 (HSD17B3) is known to be the primary HSD17B enzyme converting androstenedione (A) to testosterone (T) in human testes, and thus, to be important for sexual maturation in boys and for reproductive health later in life. In particular, inherited HSD17B3 deficiency is a known cause of 46,XY disorder of sex development in humans, where affected males are born with outwardly female genitalia, but are virilized during puberty. In the present study, Hsd17b3 knockout mice (HSD17B3KO) were established and characterized. HSD17B3KO males were born apparently phenotypically normal, but presented signs of hypogonadism at adulthood, including delayed puberty, subfertility, a shortened anogenital distance and lowered weight of several androgen-sensitive tissues. The loss of HSD17B3 also affected the androgen levels both in the testes and in circulation. Compared with the wild-type mice, androstenedione concentration was increased 10-fold in the testes and 100-fold in the serum in adult HSD17B3KO animals, accompanied with 3-fold increase in serum testosterone. The serum testosterone/androstenedione ratio in the adult HSD17B3KO mice reduced 10-fold. We also observed that circulating LH was increased 40-fold in adult KO animals, with a 200-fold increase in testicular Lhcgr expression. In addition, several genes related to steroid and cholesterol biosynthesis were upregulated in the testes, those including Star, Cyp11a1, Cyp17a1, Cyp51, Nsdhl and Hsd17b7. Our results demonstrate that, similar to humans, HSD17B3 is essential for proper androgen homeostasis in adult mice. However, like in humans, other, still unknown, enzymes can partly compensate the HSD17B3 deficiency, resulting in high amounts of serum T and DHT at adulthood. The persisting hypogonadal phenotype in the presence of high LH levels and high serum T suggest that the lack of HSD17B3 results in a defect in the pituitary-gonadal feedback system, and reduced androgen sensitivity of the extragonadal androgen target tissues. The very similar endocrine disruption present in the adult mice and men with HSD17B3 deficiency provides us the possibility to use the HSD17B3KO mice as a model to further understand the mechanisms behind the altered endocrine phenotype of the patients carrying inactivating mutations at HSD17B3 gene, and to identify potential secondary complications caused by the disease later in the life.

Regulation of androgen action during establishment of pregnancy.

During the establishment of pregnancy, the ovarian-derived hormones progesterone and oestradiol regulate remodelling of the endometrium to promote an environment that is able to support and maintain a successful pregnancy. Decidualisation is characterised by differentiation of endometrial stromal cells that secrete growth factors and cytokines that regulate vascular remodelling and immune cell influx. This differentiation process is critical for reproduction, and inadequate decidualisation is implicated in the aetiology of pregnancy disorders such as foetal growth restriction and preeclampsia. In contrast to progesterone and oestradiol, the role of androgens in regulating endometrial function is poorly understood. Androgen receptors are expressed in the endometrium, and androgens are reported to regulate both the transcriptome and the secretome of endometrial stromal cells. In androgen-target tissues, circulating precursors are activated to mediate local effects, and recent studies report that steroid concentrations detected in endometrial tissue are distinct to those detected in the peripheral circulation. New evidence suggests that decidualisation results in dynamic changes in the expression of androgen biosynthetic enzymes, highlighting a role for pre-receptor regulation of androgen action during the establishment of pregnancy. These results suggest that such enzymes could be future therapeutic targets for the treatment of infertility associated with endometrial dysfunction. In conclusion, these data support the hypothesis that androgens play a beneficial role in regulating the establishment and maintenance of pregnancy. Future studies should be focussed on investigating the safety and efficacy of androgen supplementation with the potential for utilisation of novel therapeutics, such as selective androgen receptor modulators, to improve reproductive outcomes in women.

Preparation and evaluation of flutamide mucoadhesive microparticles loaded poloxamer suppositories

Flutamide (FLT) is a potent non-steroidal anti-androgen drug primarily used in palliative treatment of prostate cancer that inhibits androgen uptake and/or nuclear binding of androgen in target tissues. The objective of the present study was to prepare FLT mucoadhesive microparticles. FLT mucoadhesive microparticles were developed by ionic gelation technique using hydrophilic polymers such as sodium alginate and Carbopol 971P and optimised by one factorial design of response surface methodology. The optimised microparticles were characterised by zeta potential study, in vitro release study, scanning electron microscopy and X-ray diffraction study. The stability studies performed on the optimized batch at 40 °C/75 % RH for 90 days indicated no significant change in the physicochemical properties. Optimised batch of microparticles was further developed as suppository system using different proportions of Poloxamer 407:Poloxamer 188 as suppository base by fusion method.

Analogs and Antagonists of Male Sex Hormones

The steroid testosterone is the major circulating sex hormone in males and is the prototype for the androgens, the anabolic agents, and androgen antagonists. Endogenous androgens are biosynthesized from cholesterol; the majority of the circulating androgens are produced in the testes under the stimulation of luteinizing hormone (LH). The reduction of testosterone to dihydrotestosterone is necessary for androgenic actions of testosterone in many androgen target tissues such as the prostate; the oxidation of testosterone by the enzyme aromatase produces estradiol. The androgenic actions of testosterone and dihydrotestosterone are due to their binding to the androgen receptor, followed by nuclear localization, dimerization of the receptor complex, and binding to specific DNA sequences. This binding of the homodimer to the androgen response element leads to gene expression, stimulation, or repression of new mRNA synthesis, and subsequent protein biosynthesis. The synthetic androgens and anabolics were prepared to impart oral activity to the androgen molecule, to separate the androgenic effects of testosterone from its anabolic effects, and to improve on its biological activities. Novel nonsteroidal androgens, termed selective androgen receptor modulators, were developed to impart agonist activity in selective tissues. Drug preparations are used for the treatment of various androgen-deficient diseases and for the therapy of diseases characterized by muscle wasting and protein catabolism. Androgen antagonists include antiandrogens, which block interactions of androgens with the androgen receptor, and inhibitors of androgen biosynthesis and metabolism. Such compounds have therapeutic potential in the treatment of acne, virilization in women, hyperplasia and neoplasia of the prostate, and baldness. Keywords: androgens; testosterone; dihydrotestosterone; anabolics; antiandrogens; selective androgen receptor modulators; 5α-reductase inhibitors

Dorsomedial hypothalamic lesions counteract decreases in locomotor activity in male Syrian hamsters transferred from long to short day lengths.

The dorsomedial nucleus (DMN) of the hypothalamus has been implicated in seasonal control of reproduction. Syrian hamsters with DMN lesions, unlike control hamsters, do not undergo testicular regression after transfer from a long day length (14 h of light per day; LD) to a short day length (8 h of light per day; SD). SDs also markedly reduce hamster locomotor activity (LMA). To assess whether the DMN is a component of the neural circuitry that mediates seasonal variation in LMA, neurologically intact males (controls) and hamsters that had sustained lesions of the DMN (DMNx) were housed in an LD or SD photoperiod for 26 weeks. DMNx that prevented testicular regression counteracted decreases in LMA during 8 to10 weeks of SD treatment; steroid-independent effects of SDs did not override high levels of LMA in DMNx males. As in previous studies, testosterone (T) restoration increased LMA in LD but not SD castrated control males. In the present study, T also failed to increase LMA in SD-DMNx hamsters. The DMN is not necessary to maintain decreased responsiveness of locomotor activity systems to T in SDs, which presumably is mediated by other central nervous system androgen target tissues. Finally, DMNx did not interfere with the spontaneous increase in LMA exhibited by photorefractory hamsters after 26 weeks of SD treatment. We propose that DMN is an essential part of the substrate that mediates seasonal decreases in LMA as day length decreases but is not required to sustain decreased SD responsiveness to T or for development of refractoriness to SDs.

The impact of exogenic testosterone and nortestosterone-decanoate toxicological evaluation using a rat model.

The impact of exogenic testosterone (T): 1.5 and 3.0 mg/kg.bw) and 19-nortestosterone 17-decanoate (ND): 1.5 and 7.5 mg/kg.bw) in castrated male rats was evaluated based on: (a) weight increase of the androgen target tissues, respecting the Hershberger methodology; (b) the 17α and β-testosterone, 17 α and β-estradiol and 17 α and β-nortestosterone levels using the GC-MS/MS technique; and (c) observation of the serum free thyroxine levels (T4). Results revealed that T and ND significantly increased the weight of androgen target tissues as follows: ND was more influential on seminal vesicles, levator ani-bulbocavernosus muscle (LABC) and Cowper's glands and T (at a dose of 3.0 mg/kg.bw) influenced the weight of the ventral prostate and glans penis. Serum samples analyzed for steroid hormone levels showed the presence of 17β-testosterone, 17β-estradiol and 17β-nor-testosterone, in castrated male rats injected with testosterone and nortestosterone, but no significant differences were found between thyroid responses and thyroid hormone levels. The results of this research proved the disrupting activity of T and ND when administered in high doses and the useful application of the Hershberger bioassay in the case of ND.

The Dark Side of 5α-Reductase Inhibitors' Therapy: Sexual Dysfunction, High Gleason Grade Prostate Cancer and Depression

With aging, abnormal benign growth of the prostate results in benign prostate hyperplasia (BPH) with concomitant lower urinary tract symptoms (LUTS). Because the prostate is an androgen target tissue, and transforms testosterone into 5α-dihydrotestosterone (5α-DHT), a potent androgen, via 5α-reductase (5α-R) activity, inhibiting this key metabolic reaction was identified as a target for drug development to treat symptoms of BPH. Two drugs, namely finasteride and dutasteride were developed as specific 5α-reductase inhibitors (5α-RIs) and were approved by the U.S. Food and Drug Administration for the treatment of BPH symptoms. These agents have proven useful in the reducing urinary retention and minimizing surgical intervention in patients with BPH symptoms and considerable literature exists describing the benefits of these agents. In this review we highlight the adverse side effects of 5α-RIs on sexual function, high grade prostate cancer incidence, central nervous system function and on depression. 5α-Rs isoforms (types 1-3) are widely distributed in many tissues including the central nervous system and inhibition of these enzymes results in blockade of synthesis of several key hormones and neuro-active steroids leading to a host of adverse effects, including loss of or reduced libido, erectile dysfunction, orgasmic dysfunction, increased high Gleason grade prostate cancer, observed heart failure and cardiovascular events in clinical trials, and depression. Considerable evidence exists from preclinical and clinical studies, which point to significant and serious adverse effects of 5α-RIs, finasteride and dutasteride, on sexual health, vascular health, psychological health and the overall quality of life. Physicians need to be aware of such potential adverse effects and communicate such information to their patients prior to commencing 5α-RIs therapy.

The sexually dimorphic adipose fin is an androgen target tissue in the brown trout (Salmo trutta fario)

An investigation has been described on the relationship of body length, age and sex with adipose fin length and the number of androgen receptor (AR)-containing cells in the adipose fin as a secondary sexual characteristic for brown trout (Salmo trutta fario). Firstly, body and adipose fin lengths of 2- to 5-year-old brown trout were measured. Thereafter, these fish were killed by decapitation, then their sexes were determined, and adipose fins were excised. The cellular bases of AR binding activities in the adipose fins were analyzed with an antibody against human/rat AR peptide. Immunocytochemistry and western blotting techniques were performed with this antibody. Analysis of morphological measurements indicated that body length and age had a linear relationship with adipose fin length. The coefficients of determination for the body length and age were 0.92 and 0.85 in the male fish and 0.76 and 0.73 in the female fish against the adipose fin length, respectively. At 2 years of age, cells in the adipose fin did not exhibit AR immunoreactivity. However, AR-immunopositive cells were abundant in the adipose fin of 3- to 5-year-old fish. Moreover, the number of AR-immunopositive cells was significantly (P < 0.05) high in males and increased with age. These observations indicate that the adipose fin in the brown trout is a probable target for androgen action and that tissue function or development may to some extent be androgen dependent. In addition, it is likely that such an effect will be mediated by specific androgen receptors.

Human Cytosolic Hydroxysteroid Dehydrogenases of the Aldo-ketoreductase Superfamily Catalyze Reduction of Conjugated Steroids

Aldo-ketoreductase 1C (AKR1C) enzymes catalyze the NADPH-dependent reduction of ketosteroids to hydroxysteroids. They are Phase I metabolizing enzymes for natural and synthetic steroid hormones. They convert 5alpha-dihydrotestosterone (Dht, potent androgen) to 3alpha/beta-androstanediols (inactive androgens) and the prodrug tibolone (Tib) to estrogenic 3alpha/beta-hydroxytibolones. Herein we demonstrate for the first time that human AKR1C enzymes (AKR1C1-4) are able to reduce conjugated steroids such as Dht-17beta-glucuronide (DhtG), Dht-17beta-sulfate (DhtS), and Tib-17beta-sulfate (TibS). Product identities were characterized by liquid chromatography-mass spectrometry, and kinetic parameters of the reactions were determined. The product profile of the reduction of each steroid conjugate by the individual AKR1C isoform was similar to that of the corresponding free steroid except for the reduction of DhtG catalyzed by AKR1C2, where a complete inversion in stereochemical preference to 3beta-reduction (with DhtG) from 3alpha-reduction (with Dht and DhtS) was observed. The catalytic efficiency of 3-keto reduction was modestly affected by the presence of a 17-sulfate group but severely impaired by the presence of a 17-glucuronide group for AKR1C1-3 isoforms. AKR1C4, however, showed superior catalytic efficiencies versus the other isoforms, and those were unaffected by steroid conjugation. Our findings provide evidence for alternative pathways of steroid metabolism where the phase I reaction (reduction) occurs after the phase II reaction (conjugation). Specifically, it is indicated that Dht is metabolized to its metabolite 3alpha-androstanediol-17-glucuronide via the previously unrecognized "conjugation pathway" involving the sequential reactions of UGT2B17 and AKR1C4 in liver but via the conventional "reduction pathway" involving the sequential reactions of AKR1C2 and UGT2B15/17 in prostate.

Inactivation of androgens by UDP-glucuronosyltransferases in the human prostate

In the human prostate, dihydrotestosterone (DHT) -- the natural androgen having the highest affinity for the androgen receptor -- is not released directly into the systemic circulation from peripheral target tissues but it is rather converted in situ into two metabolites which have a low affinity for the androgen receptor: androsterone (ADT) and androstane-3alpha,17beta-diol (3alpha-DIOL). Several clinical observations indicate that these two androgen metabolites are further inactivated in the prostate by glucuronidation. In the human, the family of UDP-glucuronosyltransferase (UGT) enzymes comprises 18 members in three subfamilies: UGT1A, UGT2A and UGT2B. Identification of the substrates for each member has revealed that three UGT2B enzymes are mainly responsible for DHT, ADT and 3alpha-DIOL glucuronidation: UGT2B7, UGT2B15 and UGT2B17. Tissue distribution and cellular localization of UGT2B transcripts and proteins clearly indicate that only UGT2B15 and UGT2B17 are expressed in the prostate. Using the human prostate carcinoma LNCaP cell line, it was shown that UGT2B expression and activity are negatively regulated by several factors, including androgens. On the other hand, inhibition of UGT2B115/17 expression by small interfering RNA (siRNA) resulted in an induced response to DHT of androgen-receptor target genes such as PSA, KLK4, NKX3.1, TMPRSS2, SLC16A6 and VEGF. It is suggested that the conjugating activity of UGT enzymes in androgen target tissues is a mechanism for modulating the action of steroids and/or protecting the tissues from deleterious high concentrations of androgens.

Inactivation by UDP-glucuronosyltransferase enzymes: The end of androgen signaling

Conjugation by UDP-Glucuronosyltransferase (UGT) is the major pathway of androgen metabolism and elimination in the human. High concentrations of glucuronide conjugates of androsterone (ADT) and androstane-3α,17β-diol (3α-diol) are present in circulation and several studies over the last 30 years have concluded that the serum levels of these metabolites might reflect the androgen metabolism in several tissues, including the liver and androgen target tissues. Three UGT2B enzymes are responsible for the conjugation of DHT and its metabolites ADT and 3α-diol: UGT2B7, B15 and B17. UGT2B7 is expressed in the liver and skin whereas UGT2B15 and B17 were found in the liver, prostate and skin. Very specific antibodies against each UGT2B enzyme have been obtained and used for immunohistochemical studies in the human prostate. It was shown that UGT2B17 is expressed in basal cells whereas UGT2B15 is only localized in luminal cells, where it inactivates DHT. By using LNCaP cells, we have also demonstrated that the expression and activity of UGT2B15 and B17 are modulated by several endogenous prostate factors including androgen. Finally, to study the physiological role of UGT2B enzymes, transgenic mice bearing the human UGT2B15 gene were recently obtained. A decrease in reproductive tissue weight from transgenic animals compared to those from control animals was observed. In conclusion, the conjugation by UGT2B7, B15 and B17, which represents a non-reversible step in androgen metabolism, is an important means by which androgens are regulated locally. It is also postulated that UGT enzymes protect the tissue from deleteriously high concentrations of active androgen.

Cell-Specific Activation of the Human Skeletal α-Actin by Androgens

Although it is evident that androgens increase muscle mass and strength, little is known about the critical molecular targets of androgens in skeletal muscle. In rodents, the skeletal alpha-actin gene is a tissue-specific gene expressed only in the levator ani and other skeletal muscles but not in the prostate or preputial gland, the well-known androgen target tissue. We identified tissue-specific androgen-regulated genes in the skeletal muscle in rats after oral administration of androgens and focused on androgen-dependent up-regulation of the skeletal alpha-actin gene. To investigate the mechanism of action, an in vitro system with various cell lines and a series of deletion mutants of the alpha-actin promoter were used. The human skeletal alpha-actin promoter was activated by androgens in the muscle cell line C2C12 but not in the liver, prostate, or breast cancer cell lines in which exogenous human androgen receptor is expressed. The sequence of the promoter is sufficient for cell-specific androgen response, providing a model for the tissue specificity demonstrated in vivo. Using a series of deletion mutants, the androgen response can be maintained using just the proximal promoter region. The importance of androgen regulation of this small portion of the human skeletal alpha-actin promoter was demonstrated by the correlation between muscle and the alpha-actin promoter activity for an array of selective androgen receptor modulators (SARMs), including an orally active SARM LGD2226. Taken together, the results suggest that the regulation of skeletal alpha-actin by androgens/SARMs may represent an important model system for understanding androgen anabolic action in the muscle.

PRMT2, a member of the protein arginine methyltransferase family, is a coactivator of the androgen receptor

The basal transcriptional activity of nuclear receptors (NRs) is regulated by interactions with additional comodulator proteins (coactivator/corepressor). Here, we describe a new androgen receptor (AR)-associated coactivator, PRMT2, which belongs to the arginine methyltransferase protein family. To search for AR-interacting proteins a fragment of the AR was used in a library screen exploiting the yeast two-hybrid technique and identifying the C-terminal region of PRMT2. We demonstrated that PRMT2 acts as a strong coactivator of the AR, had modest or none influence on transcriptional activation mediated by other NRs. Interestingly, PRMT2 interaction with the estrogen receptor (ER) was strongly dependent on the cellular background, thus, suggesting the involvement of additional, differentially expressed coregulators. We also demonstrated synergistic interaction of PRMT2 with other known nuclear receptor coactivators, such as GRIP1/TIF-2. Potentiation of AR-mediated transactivation by PRMT2 alone and in synergism with GRIP1 was prevented by a competitive inhibitor of methyltransferase activity. The PRMT2 expression profile overlaps with the distribution of AR, with strongest PRMT2 abundance in androgen target tissues. Immunofluorescence experiments showed that the intracellular localization of PRMT2 depends on the presence of the cognate receptor ligand. Under androgen-free conditions, both AR and PRMT2 are confined to the cytoplasm, whereas in the presence of androgens both proteins colocalize and translocate into the nucleus. Treatment with the AR antagonist hydroxyflutamide results in nuclear translocation of the AR, but not the coactivator PRMT2. Thus, it appears that the ligand-dependent AR conformation is essential for the recruitment and nuclear translocation of PMRT2 which acts as AR-coactivator, presumably by arginine methylation.

Synthesis and Biological Evaluation of [18F]Bicalutamide, 4-[76Br]Bromobicalutamide, and 4-[76Br]Bromo-thiobicalutamide as Non-Steroidal Androgens for Prostate Cancer Imaging

Androgen receptors (AR) are overexpressed in most primary and metastatic prostate cancers. To develop a nonsteroidal AR-mediated imaging agent, we synthesized and radiolabeled several analogs of the potent antiandrogen bicalutamide: [18F]bicalutamide, 4-[76Br]bromobicalutamide, and [76Br]bromo-thiobicalutamide. Two of these analogs, 4-[76Br]bromobicalutamide and [76Br]bromo-thiobicalutamide, were found to have a substantially increased affinity for the androgen receptor (AR) compared to that of bicalutamide. The synthesis of [18F]bicalutamide utilized a pseudocarrier approach to effect addition of a carbanion generated from tracer-level amounts of a radiolabeled precursor to an unlabeled carbonyl precursor. 4-[76Br]Bromobicalutamide and [76Br]bromo-thiobicalutamide were labeled through electrophilic bromination of a tributylstannane precursor. The former could be prepared in high specific activity, and its tissue distribution was tested in vivo. Androgen target tissue uptake was evident in castrated adult male rats; however, in DES-treated, AR-positive, tumor-bearing male mice, tumor uptake was low.

The promoter of the rat 5α-reductase type 1 gene is bidirectional and Sp1-dependent

In many androgen target tissues, testosterone is reduced to the more potent androgen, dihydrotestosterone, by steroid 5α-reductase. Two isoforms of 5α-reductase, type 1 and type 2, have been cloned. They are differentially expressed and regulated. To determine the mechanisms of regulation of 5α-reductase type 1 expression, we have cloned its 5′upstream region and defined its promoter. The proximal 5′upstream region of 5α-reductase type 1 displays all the features of a CpG island and has numerous Sp1 binding sites. By transient transfection assays, we have identified a bidirectional promoter activity in this region; this activity was highest in the negative orientation, in the direction of the methyltransferase Nsun2 (predicted) gene. Promoter activity, in either orientation, was lost in Sp1 deficient cells but was rescued following co-transfection with a Sp1 expression vector. Thus, the 5′upstream region of rat 5α-reductase type 1 contains a bidirectional promoter with an activity that is Sp1-dependent.

Synthesis and biological evaluation of a nonsteroidal bromine-76-labeled androgen receptor ligand 3-[76Br]bromo-hydroxyflutamide

Androgen receptors (ARs) are overexpressed in normal tissues and in most primary and metastatic prostate cancers. In our efforts to develop a nonsteroidal AR-specific imaging agent, we synthesized (+/-)-3-[(76)Br]bromo-hydroxyflutamide ((76)Br-), an analog of hydroxyflutamide, the active metabolite of the AR antagonist ligand flutamide. (76)Br- was synthesized in three steps, starting with commercially available compounds. Labeling of (76)Br- was achieved through the nucleophilic opening of an epoxide intermediate, and a labeled compound was obtained in high specific activity and good radiochemical yield. (+/-)-3-Bromo-hydroxyflutamide has a significantly higher affinity for ARs compared to hydroxyflutamide, its parent compound. The androgen target-tissue uptake of (76)Br- in diethylstilbestrol-treated male rats was examined; however, AR-mediated uptake was minimal due most likely to the rapid metabolic debromination of the radiolabeled ligand. This study is part of our first look at a novel class of nonsteroidal AR antagonists as positron emission tomography (PET) imaging agents, which are alternatives to steroidal AR agonist-based imaging agents. Although (76)Br- has a significant affinity for ARs, it showed limited promise as a PET imaging agent because of its poor target-tissue distribution properties.