Androgens are known to modulate many cellular processes such as cell growth and survival by binding to the androgen receptor (AR) and activating the transcription of target genes. Recent data suggested that AR can also mediate non-transcriptional actions outside the nucleus in addition to its ligand-inducible transcription factor function. Here, we describe a transcription-independent activation of the phosphatidylinositol 3-OH kinase (PI3-K) signaling pathway by androgens. Using non-transformed androgen-sensitive epithelial cells, we show that androgens enhance the PI3-K activity by promoting accumulation of phosphoinositide-3-P phospholipids in vitro. This activation is found in conjunction with an increased time-dependent phosphorylation of the downstream kinase AKT/protein kinase B on both Ser 473 and Thr 308 residues. Hormone-stimulated phosphorylation of AKT requires AR since incubation with the anti-androgen bicalutamide completely abolishes the androgen-stimulated AKT phosphorylation. Accordingly, we show that androgens increase AKT phosphorylation level in prostatic carcinoma PC3 cells only once they have been transfected with AR. Downstream, androgens enhance phosphorylation of transcription factor FKHR (Forkhead in rhabdomyosarcoma)-L1 and proapoptotic Bad protein and promote cell survival as they can counteract an apoptotic process. We also report that non-genomic effects of androgens are based on direct interaction between AR and the p85␣ regulatory subunit of class I(A) PI3-K. Together, these novel findings point out an important and physiologically relevant link between androgens and the PI3-K/AKT signaling pathway in governing cell survival.

Cholesterol is the obligate precursor to adrenal steroids but is cytotoxic at high concentrations. Here, we show the role of the liver X receptors (LXRα and LXRβ) in preventing accumulation of free cholesterol in mouse adrenal glands by controlling expression of genes involved in all aspects of cholesterol utilization, including the steroidogenic acute regulatory protein, StAR, a novel LXR target. Under chronic dietary stress, adrenal glands from Lxrαβ −/− mice accumulated free cholesterol. In contrast, wild-type animals maintained cholesterol homeostasis through basal expression of genes involved in cholesterol efflux and storage (ABC transporter A1 [ABCA1], apoE, SREBP-1c) while preventing steroidogenic gene (StAR) expression. Upon treatment with an LXR agonist that mimics activation by oxysterols, expression of these target genes was increased. Basally, Lxrαβ -/-mice exhibited a marked decrease in ABCA1 and a derepression of StAR expression, causing a net decrease in cholesterol efflux and an increase in steroidogenesis. These changes occurred under conditions that prevented the acute stress response and resulted in a phenotype more specific to the loss of LXRα, including hypercorticosteronemia, cholesterol ester accumulation, and adrenomegaly. These results imply LXRα provides a safety valve to limit free cholesterol levels as a basal protective mechanism in the adrenal gland, where cholesterol is under constant flux. IntroductionThe adrenal cortex is responsible for synthesizing glucocorticoid hormones that are essential for survival under stress. This endocrine pathway is acutely regulated by the hypothalamicpituitary-adrenal axis in response to stress through the release of ACTH from the anterior pituitary. ACTH signals the adrenal gland to increase the expression of a cascade of enzymes required for the conversion of cholesterol into biologically active glucocorticoids. The initial and rate-limiting step in this cascade is mediated by the steroidogenic acute regulatory protein (StAR) that transfers cholesterol from the outer to the inner mitochondrial membrane (1, 2). Inside the mitochondria, cytochrome P450 11A1 (CYP11A1) cleaves the cholesterol side chain to form pregnenolone (3), which can be further converted by a series of enzymes (e.g., type I 3β-hydroxysteroid dehydrogenase/D 5 -D 4 -isomerase) to all steroid hormones produced by the adrenal cortex. Because the stress response is intended to be of limited duration, tight regulation of this system is maintained by the negative feedback of circulating glucocorticoids on the hypothalamus and pituitary that decreases ACTH secretion and ultimately turn off glucocorticoid production (4, 5).

Oxysterol nuclear receptors liver X receptor (LXR)alpha and LXRbeta are known to regulate lipid homeostasis in cells exposed to high amounts of cholesterol and/or fatty acids. In order to elucidate the specific and redundant roles of the LXRs in the testis, we explored the reproductive phenotypes of mice deficient of LXRalpha, LXRbeta, and both, of which only the lxralpha;beta-/- mice are infertile by 5 months of age. We demonstrate that LXRalpha-deficient mice had lower levels of testicular testosterone that correlated with a higher apoptotic rate of the germ cells. LXRbeta-deficient mice showed increased lipid accumulation in the Sertoli cells and a lower proliferation rate of the germ cells. In lxralpha;beta-/- mice, fatty acid metabolism was affected through a decrease of srebp1c and increase in scd1 mRNA expression. The retinoid acid signaling pathway was also altered in lxralpha;beta-/- mice, with a higher accumulation of all-trans retinoid receptor alpha, all-trans retinoid receptor beta, and retinoic aldehyde dehydrogenase-2 mRNA. Combination of these alterations might explain the deleterious phenotype of infertility observed only in lxralpha;beta-/- mice, even though lipid homeostasis seemed to be first altered. Wild-type mice treated with a specific LXR agonist showed an increase of testosterone production involving both LXR isoforms. Altogether, these data identify new roles of each LXR, collaborating to maintain both integrity and functions of the testis.

The androgen receptor (AR) is a ligand-responsive transcription factor known to play a central role in the pathogenesis of prostate cancer. However, the regulation of AR gene expression in the normal and pathological prostate remains poorly understood. This study focuses on the effect of the phosphoinositide 3-kinase (PI 3-kinase)/Akt axis on AR expression in vas deferens epithelial cells (VDEC), a suitable model to study androgen regulation of gene expression, and LNCaP cells (derived from a metastasis at the left supraclavicular lymph node from a 50-year-old patient with a confirmed diagnosis of metastatic prostate carcinoma). Taken together, our data show for the first time that the PI 3-kinase/Akt pathway is required for basal and dihydrotestosterone-induced AR protein expression in both VDEC and LNCaP. Inhibition of the PI 3-kinase/Akt pathway reduced AR expression and the decline in AR protein level correlated with a decrease in AR mRNA in VDEC but not in LNCaP. Since PI 3-kinase/Akt axis is active in prostate cancer, cross-talk between PI 3-kinase/Akt and AR signalling pathways may have implications for endocrine therapy.

Male mice were raised in cohabitation with females from birth to 90 days. Testosterone was measured every 10 days in plasma and testes. Sex difference in body weight was related to the pre-pubertal increase of testosterone levels in males. The weight of the seminal vesicle was positively correlated with circulating testosterone levels between 1 and 40 days but not between 50 and 90 days Testosterone concentrations in the plasma and testes were high at birth: 630 pg/ml and 58.0 \ m=+-\ 17.7 ng/100 mg; they subsequently decreased during the first days of life and remained low until day 20: 240 \m=+-\ 110 pg/ml and 0.1 \ m=+-\0.03 ng/100 mg. The testosterone levels then increased rapidly between days 20 and 30 and especially between 30 and 40 reaching their maxima: 5770 \ m=+-\ 1720 pg/ml and 123.7 \ m=+-\ 18.3 ng/100 mg testis. This increase was transitory and testosterone levels fell after day 40. By 90 days, the testosterone levels, 440 \ m=+-\ 65 pg/ml and 43.2 \ m=+-\ 5.5 ng/100 mg testis, were comparable to those measured at birth. Plasma testosterone and age were positively correlated between 1 and 40 days, and negatively between 50 and 90 days. The first fertile matings occurred at age 40 days.In spite of a large body of research, the physiological mechanisms responsible for sexual maturation remain poorly understood (Odell 8c Swerdloff 1976). One important factor for their understanding involves a systematic study of the secretion of sex hormones from birth to puberty. The present report deals with the variations of plasma and testicular testosterone in mice between 1 and 90 days of age and the correlations between the levels of this hormone and the development of seminal vesicle, spermatogenesis and the appearance of fertility. MATERIALS AND METHODSMice of the Swiss strain (CD-I, Charles River) were raised in the laboratory under identical conditions of temperature (20 ± 1°C), lighting (daylight) and nutrition (com¬ plete pelleted chow and water given freely). Immediately after delivery the litters were reduced to 6 pups, 3 males and 3 females. After weaning on the 20th day, the 6 pups stayed in cohabitation until sacrifice. Litters which appeared in the cages were immediately removed. 15 males were sacrificed every 10 days from birth to 90 days. Blood was obtained by cardiac puncture without anticoagulant under light ether anaes¬ thesia. Testosterone was measured in the right testis of each male. Testosterone was determined on pooled samples of plasma (15 animals) on the one day and ten day old mice, in individual samples for the other stages. Testosterone was also measured in females, in pooled samples of plasma in the one day (100 animals), 10 -20 -30 and 40 (15 animals each) day-old animals.Testosterone was measured by the radioimmunoassay method previously described (Veyssiere et al. 1976). The antibody was prepared in rabbits against testosterone 3-0-carboxy-methyl-oxime bovine albumine. The major cross reacting steroid, 5a-dihydrotestosterone (74°/o) was separated by chromatography on a celite colum...

The promotion and progression of prostate cancer (PCa) are associated with androgen receptor (AR) signalling. AR functions are modulated by a variety of co-factors amongst which we identified the nucleophosmin (NPM/ B23), a member of the histone chaperone family. Here, we show that NPM is overexpressed in PCa compared to normal adjacent tissues. AR and NPM interact in vitro and in vivo, and NPM is critical for androgen-dependent transcriptional activation in LNCaP cells as an anti-NPM siRNA downregulates transcription of a transfected androgen response element (ARE)-containing reporter promoter as well as expression of the endogenous androgen responsive prostate-specific antigen (PSA) gene. By investigating the effect of NPM on AR, we have also observed that NPM enhances AR binding to an ARE in vitro in electrophoretic gel mobility-shift assay experiments. Chromatin immunoprecipitation studies further demonstrated that both AR and NPM associate with AREs of the PSA gene in vivo. Altogether, our data suggest that the molecular histone chaperone NPM could regulate AR functions by promoting assembly of ARcontaining regulatory complexes and that high levels of NPM might alter AR functions in PCa.

Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5-6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD 50 . In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis.

The human aldose reductase, AKR1B1, participates in glucose metabolism and osmoregulation and is supposed to play a protective role against toxic aldehydes derived from lipid peroxidation and steroidogenesis that could affect cell growth/differentiation when accumulated. Adrenal gland is a major site of expression of AKR1B1, and we asked whether changes in its expression could be associated with adrenal disorders. Therefore, we examined AKR1B1 gene expression in human fetal adrenals, adrenocortical cell line, and tumors and compared the results with the expression of steroidogenic genes (StAR and CYP11A) and regulators of adrenal cortex development [steroidogenic factor-1 (SF-1) and dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1)]. Using specific antibodies, Northern blotting, and enzymatic assays, we present evidences that AKR1B1 detectable in 15-wk-old fetal glands is regulated by cAMP in NCI-H295 cells and thus that AKR1B1 is functionally related to the ACTH-responsive murine akr1b7/mvdp gene rather than to its direct ortholog, the mouse aldose reductase akr1b3 gene. Although low DAX1 expression in aldosterone-producing adenomas (n = 5) was confirmed (P < 0.05), no correlation was found between the expression of all other genes and the tumors endocrine activity. In contrast, relative abundance of AKR1B1 mRNA was decreased in adrenocortical carcinomas (n = 5; mean +/- sem, 0.95 +/- 0.2) when compared with adenomas (n = 12; 9.29 +/- 3.05; P < 0.001). Most (seven of eight) adrenocortical carcinomas (19.0 +/- 5.4) had very low relative AKR1B1 protein levels when compared with benign tumors (cortisol-producing adenomas, n = 5, 63.0 +/- 9.8; nonfunctional adenomas, n = 5, 58.0 +/- 10.4; aldosterone-producing adenomas, n = 4, 65.3 +/- 7.7; P < 0.001), Cushing's hyperplasia (n = 5, 54.6 +/- 5.3; P < 0.01), or normal adrenals (n = 4; 37.1 +/- 5.3; P < 0.001). These properties provide the first evidence that expression of cAMP-regulated AKR1B1 is decreased in adrenocortical cancer. This might take part in adrenal tumorigenesis and could be investigated as a marker of malignancy for the diagnosis of adrenal tumors.