Helen E. MacLean

Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice

To identify mechanisms of anabolic androgen action in muscle, we generated male and female genomic androgen receptor (AR) knockout (ARKO) mice, and characterized muscle mass, contractile function, and gene expression. Muscle mass is decreased in ARKO males, but normal in ARKO females. The levator ani muscle, which fails to develop in normal females, is also absent in ARKO males. Force production is decreased from fast‐twitch ARKO male muscle, and slow‐twitch muscle has increased fatigue resistance. Microarray analysis shows up‐regulation of genes encoding slow‐twitch muscle contractile proteins. Realtime PCR confirms that expression of genes encoding polyamine biosynthetic enzymes, ornithine decarboxylase (Odc1), and S‐adenosylmethionine decarboxylase (Amd1), is reduced in ARKO muscle, suggesting androgens act through regulation of polyamine biosynthesis. Altered expression of regulators of myoblast progression from proliferation to terminal differentiation suggests androgens also promote muscle growth by maintaining myoblasts in the proliferate state and delaying differentiation (increased Cdkn1c and Igf2, decreased Itg1bp3). A similar pattern of gene expression is observed in orchidectomized male mice, during androgen withdrawal‐dependent muscle atrophy. In conclusion, androgens are not required for peak muscle mass in females. In males, androgens act through the AR to regulate multiple gene pathways that control muscle mass, strength, and fatigue resistance.—MacLean, H. E., Maria Chiu, W. S., Notini, A. J., Axell, A.‐M., Davey, R. A., McManus, J. F., Ma, C., Plant, D. R., Lynch, G. S., Zajac, J. D. Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice. FASEB J. 22, 2676–2689 (2008)

Localization of functional domains in the androgen receptor

Functional domains of the androgen receptor (AR) have been localized through a combination of studies on naturally occurring AR gene mutations, in vitro mutagenesis studies and comparison with the structure of other members of the steroid/nuclear receptor superfamily. Two activation domains exist within the amino-terminal domain, and a ligand-dependent activation domain is present in the ligand binding domain. The poly(Gln) stretch within the amino-terminal domain may inhibit the transactivation function of the receptor. Different ligands or binding to different promoters may recruit the use of different activation domains, which may provide promoter-specific effects of receptor action. Co-activator proteins that modulate or enhance AR action have been identified, many of which interact with the ligand binding domain of the AR. Tissue-specific expression of such co-activators, and promoter-specific protein interactions, may also help control the specificity of androgen action. Target Ser residues for phosphorylation have been identified, which may be the site of action for cross-talk from protein kinase signalling pathways. However, the role of phosphorylation in AR function in general is still unclear. It is now clear that interactions occur between receptor domains, modulating functions including ligand dissociation, dimerization and transactivation. By studying the functional domains of the AR, and how they control receptor function in response to different activation signals, we are beginning to understand the mechanisms controlling the specificity of receptor action.

Calcitonin Receptor Plays a Physiological Role to Protect Against Hypercalcemia in Mice

It is well established that calcitonin is a potent inhibitor of bone resorption; however, a physiological role for calcitonin acting through its cognate receptor, the calcitonin receptor (CTR), has not been identified. Data from previous genetically modified animal models have recognized a possible role for calcitonin and the CTR in controlling bone formation; however, interpretation of these data are complicated, in part because of their mixed genetic background. Therefore, to elucidate the physiological role of the CTR in calcium and bone metabolism, we generated a viable global CTR knockout (KO) mouse model using the Cre/loxP system, in which the CTR is globally deleted by >94% but <100%. Global CTRKOs displayed normal serum ultrafiltrable calcium levels and a mild increase in bone formation in males, showing that the CTR plays a modest physiological role in the regulation of bone and calcium homeostasis in the basal state in mice. Furthermore, the peak in serum total calcium after calcitriol [1,25(OH)2D3]‐induced hypercalcemia was substantially greater in global CTRKOs compared with controls. These data provide strong evidence for a biological role of the CTR in regulating calcium homeostasis in states of calcium stress.

Increased adiposity in DNA binding-dependent androgen receptor knockout male mice associated with decreased voluntary activity and not insulin resistance

In men, as testosterone levels decrease, fat mass increases and muscle mass decreases. Increased fat mass in men, in particular central obesity, is a major risk factor for type 2 diabetes, cardiovascular disease, and all-cause mortality. Testosterone treatment has been shown to decrease fat mass and increase fat-free mass. We hypothesize that androgens act directly via the DNA binding-dependent actions of the androgen receptor (AR) to regulate genes controlling fat mass and metabolism. The aim of this study was to determine the effect of a global DNA binding-dependent (DBD) AR knockout (DBD-ARKO) on the metabolic phenotype in male mice by measuring body mass, fat mass, food intake, voluntary physical activity, resting energy expenditure, substrate oxidation rates, serum glucose, insulin, lipid, and hormone levels, and metabolic gene expression levels and second messenger protein levels. DBD-ARKO males have increased adiposity despite a decreased total body mass compared with wild-type (WT) males. DBD-ARKO males showed reduced voluntary activity, decreased food intake, increased serum leptin and adiponectin levels, an altered lipid metabolism gene profile, and increased phosphorylated CREB levels compared with WT males. This study demonstrates that androgens acting via the DNA binding-dependent actions of the AR regulate fat mass and metabolism in males and that the increased adiposity in DBD-ARKO male mice is associated with decreased voluntary activity, hyperleptinemia and hyperadiponectinemia and not with insulin resistance, increased food intake, or decreased resting energy expenditure.

Defects of androgen receptor function: from sex reversal to motor neurone disease

The androgen receptor (AR) is a ligand-dependent DNA transcription factor that binds androgens which cause masculinisation of the developing male fetus. Classical abnormalities of receptor function result in the syndrome of androgen resistance, with resultant failure of normal male differentiation. In more recent years, however, mutations in the AR gene have been described in a number of diverse clinical conditions, from male infertility to prostate and breast cancer through to a form of motor neurone disease (Kennedys disease). This review discusses the various AR gene mutations found in androgen insensitivity syndrome (AIS) and the other conditions described above, and relates how different mutations, or disruption of different functional domains, contributes to the various phenotypes. Mutations that cause complete AIS usually disrupt the DNA or steroid binding ability of the receptor. In partial AIS, mutations generally decrease receptor affinity for ligand, affect thermostability of the protein, or affect the ability of the receptor to activate transcription of responsive genes. Isolated mutations occur in the steroid binding domain of the receptor in prostate cancer, and many cancers have an identical mutation. Similarly, in the two cases of male breast cancer in which AR gene mutations have been described, the mutations in the DNA binding domain of the receptor are alike. In Kennedys disease a trinucleotide repeat expansion occurs in exon A of the AR gene, which appears to affect ability of the receptor to bind ligand and activate transcription, although the mechanism of neuronal degeneration remains unknown.

Genetically modified animal models as tools for studying bone and mineral metabolism

Genetic modification of mice is a powerful tool for the study of bone development and metabolism. This review discusses the advantages and disadvantages of various approaches used in bone‐related research and the contributions these studies have made to bone biology.

A floxed allele of the androgen receptor gene causes hyperandrogenization in male mice

We previously generated a conditional floxed mouse line to study androgen action, in which exon 3 of the androgen receptor (AR) gene is flanked by loxP sites, with the neomycin resistance gene present in intron 3. Deletion of exon 3 in global AR knockout mice causes androgen insensitivity syndrome, characterized by genotypic males lacking normal masculinization. We now report that male mice carrying the floxed allele (AR(lox)) have the reverse phenotype, termed hyperandrogenization. AR(lox) mice have increased mass of androgen-dependent tissues, including kidney, (P < 0.001), seminal vesicle (P < 0.001), levator ani muscle (P = 0.001), and heart (P < 0.05). Serum testosterone is not significantly different. Testis mass is normal, histology shows normal spermatogenesis, and AR(lox) males are fertile. AR(lox) males also have normal AR mRNA levels in kidney, brain, levator ani, liver, and testis. This study reaffirms the need to investigate the potential phenotypic effects of floxed alleles in the absence of cre in tissue-specific knockout studies. In addition, this androgen hypersensitivity model may be useful to further investigate the effects of subtle perturbations of androgen action in a range of androgen-responsive systems in the male.

Increased frequency of long androgen receptor CAG repeats in male breast cancers

We have investigated the possible link between androgen hyposensitivity caused by long androgen receptor (AR) CAG repeats, and breast carcinogenesis, in men. AR gene mutations have been described in men with androgen insensitivity syndrome and breast carcinoma, and some studies have shown long CAG repeats are associated with increased risk of breast cancer in women. DNA was isolated from male breast cancer biopsies, and the AR CAG repeat sized. Forty one male breast cancer samples were studied, including one sample from a man with spinal and bulbar muscular atrophy (SBMA), which is caused by an AR CAG repeat expansion. The man with breast cancer and SBMA had 49 CAG repeats (normal range 6–35), but all other breast cancer samples had repeats within the normal range. The frequency of CAG repeats ≥24 was significantly higher in the breast cancer group (excluding the SBMA subject) than in the normal population (p<0.05), and was more marked in grade I and II tumors (p=0.001). There was no correlation between AR CAG repeat length and age at diagnosis. In conclusion, longer AR CAG repeats are more common in men with breast cancer than in the control male population. Androgen hyposensitivity, caused by long AR CAG repeats, may increase the risk of breast cancer in men.

Intersex disorders : shedding light on male sexual differentiation beyond SRY

 Male sexual differentiation involves a cascade of events initiated by the presence on the Y chromosome of the SRY gene, which causes the indifferent gonad to develop into a testis. Hormonal products of the testis, predominantly testosterone and Mu¨llerian inhibiting substance (MIS), then control the sexual differentiation of the developing foetus. SRY is a transcription factor; however, target genes for its action have yet to be identified, because the DNA recognition sequence for SRY is found in many genes. Therefore the study of intersex disorders is being used to identify other genes active in the pathway of sexual differentiation. Genes identified as being important in the differentiation of the indifferent gonad include WT1 (abnormal in Denys Drash syndrome) and SF‐1. The DSS locus may contain a gene that controls ovarian differentiation, and SOX9 (identified from campomelic dysplasia) is required for testis differentiation. In addition to playing a role in the development of the bipotential gonad, SF‐1 may also activate MIS gene expression in the testis, causing regression of Mu¨llerian structures. Luteinizing hormone and its receptor are required for Leydig cell differentiation, and the testosterone biosynthetic enzymes (P450scc, 3β‐hydroxysteroid dehydrogenase, P45017α and 17β‐hydroxysteroid dehydrogenase) are all necessary for masculinization of external genitalia. 5α‐Reductase is required for the production of dihydrotestosterone, and the androgen receptor mediates the action of both testosterone and dihydrotestosterone. The identification of abnormal genes in other disorders of sexual differentiation is likely to provide further information about the factors required for testicular development and function.

DNA-binding-dependent androgen receptor signaling contributes to gender differences and has physiological actions in males and females.

We used our genomic androgen receptor (AR) knockout (ARKO) mouse model, in which the AR is unable to bind DNA to: 1) document gender differences between males and females; 2) identify the genomic (DNA-binding-dependent) AR-mediated actions in males; 3) determine the contribution of genomic AR-mediated actions to these gender differences; and 4) identify physiological genomic AR-mediated actions in females. At 9 weeks of age, control males had higher body, heart and kidney mass, lower spleen mass, and longer and larger bones compared to control females. Compared to control males, ARKO males had lower body and kidney mass, higher splenic mass, and reductions in cortical and trabecular bone. Deletion of the AR in ARKO males abolished the gender differences in heart and cortical bone. Compared with control females, ARKO females had normal body weight, but 14% lower heart mass and heart weight/body weight ratio. Relative kidney mass was also reduced, and relative spleen mass was increased. ARKO females had a significant reduction in cortical bone growth and changes in trabecular architecture, although with no net change in trabecular bone volume. In conclusion, we have shown that androgens acting via the genomic AR signaling pathway mediate, at least in part, the gender differences in body mass, heart, kidney, spleen, and bone, and play a physiological role in the regulation of cardiac, kidney and splenic size, cortical bone growth, and trabecular bone architecture in females.