Discovery of the selective androgen receptor modulator MK-0773 using a rational development strategy based on differential transcriptional requirements for androgenic anabolism versus reproductive physiology.

Selective androgen receptor modulators (SARMs) are androgen receptor (AR) ligands that induce anabolism while having reduced effects in reproductive tissues. In various experimental contexts SARMs fully activate, partially activate, or even antagonize the AR, but how these complex activities translate into tissue selectivity is not known. Here, we probed receptor function using >1000 synthetic AR ligands. These compounds produced a spectrum of activities in each assay ranging from 0 to 100% of maximal response. By testing different classes of compounds in ovariectomized rats, we established that ligands that transactivated a model promoter 40-80% of an agonist, recruited the coactivator GRIP-1 <15%, and stabilized the N-/C-terminal interdomain interaction <7% induced bone formation with reduced effects in the uterus and in sebaceous glands. Using these criteria, multiple SARMs were synthesized including MK-0773, a 4-aza-steroid that exhibited tissue selectivity in humans. Thus, AR activated to moderate levels due to reduced cofactor recruitment, and N-/C-terminal interactions produce a fully anabolic response, whereas more complete receptor activation is required for reproductive effects. This bimodal activation provides a molecular basis for the development of SARMs.

Identification of anabolic selective androgen receptor modulators with reduced activities in reproductive tissues and sebaceous glands.

Androgen replacement therapy is a promising strategy for the treatment of frailty; however, androgens pose risks for unwanted effects including virilization and hypertrophy of reproductive organs. Selective Androgen Receptor Modulators (SARMs) retain the anabolic properties of androgens in bone and muscle while having reduced effects in other tissues. We describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC(50), 38 nm) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5alpha-dihydrotestosterone (DHT), TFM-4AS-1 acts as a gene-selective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all. This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, we identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands.

A regulatory circuit mediating convergence between Nurr1 transcriptional regulation and Wnt signaling.

The orphan nuclear receptor Nurr1 is essential for the development and maintenance of midbrain dopaminergic neurons, the cells that degenerate during Parkinson's disease, by promoting the transcription of genes involved in dopaminergic neurotransmission. Since Nurr1 lacks a classical ligand-binding pocket, it is not clear which factors regulate its activity and how these factors are affected during disease pathogenesis. Since Wnt signaling via beta-catenin promotes the differentiation of Nurr1(+) dopaminergic precursors in vitro, we tested for functional interactions between these systems. We found that beta-catenin and Nurr1 functionally interact at multiple levels. In the absence of beta-catenin, Nurr1 is associated with Lef-1 in corepressor complexes. Beta-catenin binds Nurr1 and disrupts these corepressor complexes, leading to coactivator recruitment and induction of Wnt- and Nurr1-responsive genes. We then identified KCNIP4/calsenilin-like protein as being responsive to concurrent activation by Nurr1 and beta-catenin. Since KCNIP4 interacts with presenilins, the Alzheimer's disease-associated proteins that promote beta-catenin degradation, we tested the possibility that KCNIP4 induction regulates beta-catenin signaling. KCNIP4 induction limited beta-catenin activity in a presenilin-dependent manner, thereby serving as a negative feedback loop; furthermore, Nurr1 inhibition of beta-catenin activity was absent in PS1(-/-) cells or in the presence of small interfering RNAs specific to KCNIP4. These data describe regulatory convergence between Nurr1 and beta-catenin, providing a mechanism by which Nurr1 could be regulated by Wnt signaling.

Androgenic induction of growth and differentiation in the rodent uterus involves the modulation of estrogen-regulated genetic pathways.

The androgen receptor (AR) is expressed in the uterus; however, the role of AR in female reproductive physiology is poorly understood. Here we examined the effects of androgens on uterine growth and gene expression in adult ovariectomized rats. Nonaromatizable AR-selective agonists potently stimulate hypertrophy and induce significant myometrial expansion distinct from that induced by 17beta-estradiol (E2). In the endometrium, androgens only modestly increase epithelial cell height and antagonize the trophic effects of E2. To identify underlying mechanisms, global changes in RNA levels 24 h after stimulation with E2 and 5alpha-dihydrotestosterone (DHT) were compared. A total of 491 genes were differentially expressed after E2 treatment, including key regulators of tissue remodeling, cell signaling, metabolism, and gene expression. Of the 164 transcripts regulated by DHT, 86% were also affected by E2, including trophic genes like IGF-I and epithelial secretory genes such as uterocalin. In estrogen receptor (ER)alpha knockout mice, DHT cannot induce uterine growth, suggesting a key role for ERalpha. However, DHT appears not to activate ERalpha directly because DHT induction of IGF-I is blocked by the AR antagonist bicalutamide, and multiple genes regulated directly by ERalpha were not induced by DHT. The similarity between estrogens and androgens instead could reflect general trophic signaling in reproductive tissues because 93 of the 503 genes regulated in the uterus are similarly affected during prostate growth. Thus androgens regulate the trophic environment and architecture of the rodent uterus via a gene expression program that is overlapping but distinct from the estrogen response.

Androgen-mediated improvement of body composition and muscle function involves a novel early transcriptional program including IGF1, mechano growth factor, and induction of {beta}-catenin.

Androgens promote anabolism in the musculoskeletal system while generally repressing adiposity, leading to lean body composition. Circulating androgens decline with age, contributing to frailty, osteoporosis, and obesity; however, the mechanisms by which androgens modulate body composition are largely unknown. Here, we demonstrate that aged castrated rats develop increased fat mass, reduced muscle mass and strength, and lower bone mass. Treatment with testosterone or 5alpha-dihydrotestosterone (DHT) reverses the effects on muscle and adipose tissues while only aromatizable testosterone increased bone mass. During the first week, DHT transiently increased soleus muscle nuclear density and induced expression of IGF1 and its splice variant mechano growth factor (MGF) without early regulation of the myogenic factors MyoD, myogenin, monocyte nuclear factor, or myostatin. A genome-wide microarray screen was also performed to identify potential pro-myogenic genes that respond to androgen receptor activation in vivo within 24 h. Of 24 000 genes examined, 70 candidate genes were identified whose functions suggest initiation of remodeling and regeneration, including the type II muscle genes for myosin heavy chain type II and parvalbumin and the chemokine monocyte chemoattractant protein-1. Interestingly, Axin and Axin2, negative regulators of beta-catenin, were repressed, indicating modulation of the beta-catenin pathway. DHT increased total levels of beta-catenin protein, which accumulated in nuclei in vivo. Likewise, treatment of C2C12 myoblasts with both IGF1Ea and MGF C-terminal peptide increased nuclear beta-catenin in vitro. Thus, we propose that androgenic anabolism involves early downregulation of Axin and induction of IGF1, leading to nuclear accumulation of beta-catenin, a pro-myogenic, anti-adipogenic stem cell regulatory factor.

Control of rat tail skin temperature regulation by estrogen receptor-beta selective ligand.

OBJECTIVE: To test the role of ERbeta in the control of estrogen-dependent thermoregulation in rats. METHODS: Test the ability of an ERbeta-selective ligand to suppress the elevation in basal rat tail skin temperature (TST) caused by ovariectomy (OVX). RESULTS: ERbeta-19 is a tetrahydrofluorenone ERbeta-selective ligand that displaces 0.1 nM estradiol from ERbeta with an IC50 of 1.8 nM compared to an IC50 of 141 nM for ERalpha. Like estradiol, it acts as an agonist on ERbeta-mediated transactivation and transrepression with 25- and 60-fold selectivity, respectively, over ERalpha-controlled transcription. Administration of estradiol to estrogen-depleted rats suppresses the ovariectomy-induced elevation of TST. Similar treatment of OVX rats with ERbeta-19 also results in suppression of elevated TST. However, in contrast to estradiol, ERbeta-19 does not suppress body weight, does not increase uterine weight, nor does it stimulate uterocalin biomarker expression which is under the control of ERalpha. Thus, the ERbeta-19 suppression of rat TST is mediated by ERbeta without eliciting the activity of ERalpha. CONCLUSION: Estrogen-sensitive thermoregulation in ovariectomized rats can be controlled by an ERbeta-selective ligand.

Identification of genetic pathways activated by the androgen receptor during the induction of proliferation in the ventral prostate gland.

The androgen receptor (AR), when complexed with 5alpha-dihydrotestosterone (DHT), supports the survival and proliferation of prostate cells, a process critical for normal development, benign prostatic hypertrophy, and tumorigenesis. However, the androgen-responsive genetic pathways that control prostate cell division and differentiation are largely unknown. To identify such pathways, we examined gene expression in the ventral prostate 6 and 24 h after DHT administration to androgen-depleted rats. 234 transcripts were expressed significantly differently from controls (p < 0.05) at both time points and were subjected to extensive data mining. Functional clustering of the data reveals that the majority of these genes can be classified as participating in induction of secretory activity, metabolic activation, and intracellular signaling/signal transduction, indicating that AR rapidly modulates the expression of genes involved in proliferation and differentiation in the prostate. Notably AR represses the expression of several key cell cycle inhibitors, while modulating members of the wnt and notch signaling pathways, multiple growth factors, and peptide hormone signaling systems, and genes involved in MAP kinase and calcium signaling. Analysis of these data also suggested that p53 activity is negatively regulated by AR activation even though p53 RNA was unchanged. Experiments in LNCaP prostate cancer cells reveal that AR inhibits p53 protein accumulation in the nucleus, providing a post-transcriptional mechanism by which androgens control prostate cell growth and survival. In summary these data provide a comprehensive view of the earliest events in AR-mediated prostate cell proliferation in vivo, and suggest that nuclear exclusion of p53 is a critical step in prostate growth.