Myocytic androgen receptor controls the strength but not the mass of limb muscles.

The anabolic effects of androgens on skeletal muscles are thought to be mediated predominantly through the androgen receptor (AR), a member of the ligand-dependent nuclear receptor superfamily. However, despite numerous studies performed in men and in rodents, these effects remain poorly understood. To characterize androgen signaling in skeletal muscles, we generated mice in which the AR is selectively ablated in myofibers. We show that myocytic AR controls androgen-induced insulin-like growth factor IEa (IGF-IEa) expression in the highly androgen-sensitive perineal muscles and that it mediates androgen-stimulated postnatal hypertrophy of these muscles. In contrast, androgen-dependent postnatal hypertrophy of limb muscle fibers is independent of myocytic AR. Thus, androgens control perineal and limb muscle mass in male mice through myocytic AR-dependent and -independent pathways, respectively. Importantly, we also show that AR deficiency in limb myocytes impairs myofibrillar organization of sarcomeres and decreases muscle strength, thus demonstrating that myocytic AR controls key pathways required for maximum force production. These distinct androgen signaling pathways in perineal and limb muscles may allow the design of screens to identify selective androgen modulators of muscle strength.

Androgen receptor coordinates muscle metabolic and contractile functions.

BACKGROUND: Androgens are anabolic steroid hormones that exert their function by binding to the androgen receptor (AR). We have previously established that AR deficiency in limb muscles impairs sarcomere myofibrillar organization and decreases muscle strength in male mice. However, despite numerous studies performed in men and rodents, the signalling pathways controlled by androgens via their receptor in skeletal muscles remain poorly understood. METHODS: Male ARskm-/y (n = 7-12) and female ARskm-/- mice (n = 9), in which AR is selectively ablated in myofibres of musculoskeletal tissue, and male AR(i)skm-/y , in which AR is selectively ablated in post-mitotic skeletal muscle myofibres (n = 6), were generated. Longitudinal monitoring of body weight, blood glucose, insulin, lipids and lipoproteins was performed, alongside metabolomic analyses. Glucose metabolism was evaluated in C2C12 cells treated with 5α-dihydrotestosterone (DHT) and the anti-androgen flutamide (n = 6). Histological analyses on macroscopic and ultrastructural levels of longitudinal and transversal muscle sections were conducted. The transcriptome of gastrocnemius muscles from control and ARskm-/y mice was analysed at the age of 9 weeks (P < 0.05, 2138 differentially expressed genes) and validated by RT-qPCR analysis. The AR (4691 peaks with false discovery rate [FDR] < 0.1) and H3K4me2 (47 225 peaks with FDR < 0.05) cistromes in limb muscles were determined in 11-week-old wild-type mice. RESULTS: We show that disrupting the androgen/AR axis impairs in vivo glycolytic activity and fastens the development of type 2 diabetes in male, but not in female mice. In agreement, treatment with DHT increases glycolysis in C2C12 myotubes by 30%, whereas flutamide has an opposite effect. Fatty acids are less efficiently metabolized in skeletal muscles of ARskm-/y mice and accumulate in cytoplasm, despite increased transcript levels of genes encoding key enzymes of beta-oxidation and mitochondrial content. Impaired glucose and fatty acid metabolism in AR-deficient muscle fibres is associated with 30% increased lysine and branched-chain amino acid catabolism, decreased polyamine biosynthesis and disrupted glutamate transamination. This metabolic switch generates ammonia (2-fold increase) and oxidative stress (30% increased H2 O2 levels), which impacts mitochondrial functions and causes necrosis in <1% fibres. We unravel that AR directly activates the transcription of genes involved in glycolysis, oxidative metabolism and muscle contraction. CONCLUSIONS: Our study provides important insights into diseases caused by impaired AR function in musculoskeletal system and delivers a deeper understanding of skeletal muscle pathophysiological dynamics that is instrumental to develop effective treatment for muscle disorders.

Transcriptional control of adrenal steroidogenesis: novel connection between Janus kinase (JAK) 2 protein and protein kinase A (PKA) through stabilization of cAMP response element-binding protein (CREB) transcription factor.

In the adrenal gland, adrenocorticotropin (ACTH) acting through the cAMP protein kinase (PKA) transduction pathway is the main regulator of genes involved in glucocorticoid synthesis. The prolactin (PRL) receptor is expressed in the adrenal cortex of most mammals, but experimental proof that PRL ensures direct control on glucocorticoid synthesis in rodents remains elusive. To unravel the physiological importance of PRL in adrenocortical functions, we measured steroidogenic capacity of Prlr-deficient mice (Prlr(-/-)) and explored the influence of JAK/STAT signaling, the major PRL transduction pathway, on the steroidogenic activity of adrenocortical cell cultures. We demonstrate that lack of Prlr does not affect basal (nor stress-induced) corticosterone levels in mice. PRL triggers JAK2/STAT5-dependent transcription in adrenal cells, but this does not influence corticosterone release. In contrast, pharmacological or siRNA-mediated inhibition of JAK2 reveals its essential role in both basal and ACTH/cAMP-induced steroidogenesis. We demonstrate that nuclear JAK2 regulates the amount of active transcription factor CREB (cAMP response element-binding protein) through tyrosine phosphorylation and prevention of proteasomal degradation, which in turn leads to transcriptional activation of the rate-limiting steroidogenic Star gene. Hence, we describe a novel link between PKA and JAK2 by which nuclear JAK2 signaling controls adrenal steroidogenesis by increasing the stability of CREB.

PGC1alpha expression is controlled in skeletal muscles by PPARbeta, whose ablation results in fiber-type switching, obesity, and type 2 diabetes.

Mice in which peroxisome proliferator-activated receptor beta (PPARbeta) is selectively ablated in skeletal muscle myocytes were generated to elucidate the role played by PPARbeta signaling in these myocytes. These somatic mutant mice exhibited a muscle fiber-type switching toward lower oxidative capacity that preceded the development of obesity and diabetes, thus demonstrating that PPARbeta is instrumental in myocytes to the maintenance of oxidative fibers and that fiber-type switching is likely to be the cause and not the consequence of these metabolic disorders. We also show that PPARbeta stimulates in myocytes the expression of PGC1alpha, a coactivator of various transcription factors, known to play an important role in slow muscle fiber formation. Moreover, as the PGC1alpha promoter contains a PPAR response element, the effect of PPARbeta on the formation and/or maintenance of slow muscle fibers can be ascribed, at least in part, to a stimulation of PGC1alpha expression at the transcriptional level.

Adrenocorticotropin-dependent changes in SF-1/DAX-1 ratio influence steroidogenic genes expression in a novel model of glucocorticoid-producing adrenocortical cell lines derived from targeted tumorigenesis.

We established the first adrenocortical tumor cell lines with complete zona fasciculata (ZF) cell phenotype from tumors induced in transgenic mice by large T-antigen of simian virus 40 under the control of the aldose reductase-like akr1b7 gene promoter. Adrenocortical tumor cell lines produced high amounts of corticosterone and were responsive to ACTH. All genes that are supportive for glucocorticoid synthesis including cyp21a1 and cyp11b1 were expressed, and most of them were transiently up-regulated by ACTH at transcriptional level: stimulation culminated after 3-6 h and returned to basal levels after 24 h. Taking advantage of these cells, we have examined the effect of ACTH on DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on X-chromosome, gene 1) and SF-1 (steroidogenic factor 1), two transcription factors known to respectively repress and activate adrenocortical steroidogenesis by acting on common target genes. According to their antagonistic activities, DAX-1 mRNA and protein levels were transiently down-regulated by ACTH, whereas those of SF-1 were stimulated, with kinetics paralleling those of steroidogenic genes expression, notably of two known SF-1 target genes, star and akr1b7. This suggests an essential role of SF-1/DAX-1 proteins ratio to achieve proper ACTH control of steroidogenic gene expression in cells derived from ZF. This was confirmed in mice adrenals, where repression of dax-1 gene and concomitant up-regulation of sf-1, star, and akr1b7 genes were observed in response to ACTH stimulation. In conclusion, using both unique differentiated cell lines and in vivo approaches, we provide the first evidence that hormonally induced changes in SF-1/DAX-1 ratio are part of the molecular arsenal of ZF cells to fine tune ACTH responsiveness.

The transcriptional coregulators TIF2 and SRC-1 regulate energy homeostasis by modulating mitochondrial respiration in skeletal muscles.

The two p160 transcriptional coregulator family members SRC-1 and TIF2 have important metabolic functions in white and brown adipose tissues as well as in the liver. To analyze TIF2 cell-autonomous functions in skeletal muscles, we generated TIF2((i)skm)⁻(/)⁻ mice in which TIF2 was selectively ablated in skeletal muscle myofibers at adulthood. We found that increased mitochondrial uncoupling in skeletal muscle myocytes protected these mice from decreased muscle oxidative capacities induced by sedentariness, delayed the development of type 2 diabetes, and attenuated high-caloric-diet-induced obesity. Moreover, our results demonstrate that SRC-1 and TIF2 can modulate the expression of the uncoupling protein 3 (UCP3) in an antagonistic manner and that enhanced SRC-1 levels in TIF2-deficient myofibers are critically involved in the metabolic changes of TIF2((i)skm)⁻(/)⁻ mice. Thus, modulation of the expression and/or activity of these coregulators represents an attractive way to prevent or treat metabolic disorders.

The upstream Sal repeat-containing segment of Arabidopsis thaliana ribosomal DNA intergenic region (IGR) enhances the activity of adjacent protein-coding genes.

The sequence containing 'upstream Sal repeats' (USR) from the Arabidopsis thaliana ribosomal DNA intergenic region (IGR) was tested for its influence on the in vivo activity of nearby protein coding genes. On average, the presence of the IGR fragment leads to a four-fold increase in the expression of a reporter gene, beta-glucuronidase, under control of the strong CaMV 35S promoter. With the help of the site-specific cre-lox recombination system, we have also obtained pairs of transgenic lines with or without the USR-containing fragment, both integrated at the same chromosomal position. Results with these transgenic lines, which contain an NPT II (kanamycin resistance) gene under control of the nos promoter as a test gene, confirmed the results obtained with the CaMV 35S-driven GUS gene. Moreover, they show that the IGR sequence can oppose tendencies of gene silencing. We hypothesize that the described effect relates to features of the chromatin structure in the proximity of the upstream Sal repeats.