Identification of androgen response elements in the insulin-like growth factor I upstream promoter

Testosterone stimulates the expression of IGF-I in cells and tissues that include prostate, muscle and muscle satellite cells, and the uterus. Here, the molecular mechanisms of this effect of testosterone were explored. Testosterone increased IGF-I mRNA levels in HepG2 and LNCaP cells and stimulated the activity of reporter genes controlled by 1.6 kb of the upstream promoter of the human IGF-I gene. An androgen-responsive region that was located between -1320 and -1420 bases upstream of the first codon was identified by truncation studies. The androgen-responsive region was found to contain two sequences resembling known androgen receptor (AR)-binding sites from the Pem1 gene. Reporter genes incorporating these sequences were strongly stimulated by androgens. Each of the androgen-responsive elements (AREs) bound recombinant AR-DNA-binding domain in gel-shift experiments; binding was greatly enhanced by sequences flanking the apparent AR-binding half-sites. Testosterone induced recruitment of AR to sequences of genomic DNA containing these AREs. The two AREs were activated 5-fold more by AR than glucocorticoid receptor. Collectively, these findings indicate the presence of two AREs within the IGF-I upstream promoter that act in cis to activate IGF-I expression. These AREs seem likely to contribute to the up-regulation of the IGF-I gene in prostate tissues, HepG2 cells, and potentially other tissues.     

Sequence-specific binding of androgen-receptor complexes to prostatic binding protein genes

Prostatic binding protein is a complex glycoprotein comprising three components, C1, C2 and C3, organized into two different heterodimers (C1-C3 and C2-C3). The rat ventral prostate genes encoding all three constituent polypeptides are expressed under androgenic control. Analysis of genomic fragments containing the genes and flanking sequences revealed in each case one androgen receptor-binding region upstream of or within the promoter and another in the first intron. The effect of androgens on the expression of these genes may, therefore, be mediated by these direct receptor-DNA interactions. The genomic fragments which contain androgen receptor-binding regions all contain nucleotide sequences reminiscent of glucocorticoid response elements (GRE). Mutations in these sequences in restriction fragments and in synthetic oligonucleotides significantly decreased their affinity for androgen-receptor complexes and their introduction into nonspecific sequences conferred affinity for androgen-receptor complexes. Based on these data, a consensus sequence for putative androgen response elements (ARE) is proposed. However, despite the specific recognition of these sequences by the androgen receptor in vitro, only the C3(1) intronic fragment could confer significant androgen responsiveness on a heterologous promoter. While this could be due to the fact that the GRE-like sequences present in the other fragments are not strong AREs, alternative hypotheses are being investigated currently. Not least of these is that the similar localization of the binding sites in each gene might underlie a more complex androgen regulation mechanism.     

Prolonged exposure to androgens suppresses follicle-stimulating hormone-induced aromatase activity in rat Sertoli cell cultures

We studied the effects of androgens on basal and FSH-stimulated aromatase activity in Sertoli cell-enriched monolayers. Daily exposure to androgens from the first day of culture results in a time- and dose-dependent decrease in inducible aromatase activity. The inhibition is observed whether FSH, L-isoproterenol or (Bu)2cAMP is used as inducer of the aromatase activity. Basal activity is not affected by preincubation with androgens and the inhibitory effect is not observed after short-term exposure (24 h) to these hormones. The ability of different androgens to decrease inducible aromatase activity does not depend on their ability to serve as a substrate for the aromatase but parallels their ability to stimulate the production of androgen-binding protein. Moreover, the effect of testosterone is neutralized by the antiandrogen cyproterone acetate, suggesting that it is mediated by the androgen receptor. These data suggest that testicular androgens may be responsible for the decrease in FSH-inducible aromatase activity observed in intact rats between days 10 and 30 of life. Similarly, the removal of these androgens during the preparation of Sertoli cell cultures may explain the spontaneous increase in inducible aromatase activity observed when these cultures are maintained in the absence of androgens.     

Endometrium in PCOS: Implantation and predisposition to endocrine CA

Polycystic ovarian syndrome (PCOS) is a common endocrinopathy characterized by oligo/anovulatiaon and elevated circulating androgens or evidence of hyperandrogenism after all known potential causes have been excluded. In addition, insulin resistance and accompanying hyperinsulinemia commonly occur in women with PCOS. There is increasing evidence that the endocrinologic and metabolic abnormalities in PCOS may have complex effects on the endometrium, contributing to the infertility and endometrial disorders observed in women with this syndrome. Androgen receptors and steroid receptor co-activators are over-expressed in the endometrium of women with PCOS. Also, biomarkers of endometrial receptivity to embryonic implantation-such as alpha(v)beta3-integrin and glycodelin-are decreased, and epithelial expression of estrogen receptor alpha (ERalpha) abnormally persists in the window of implantation in endometrium in women with PCOS. In addition to being responsive to the steroid hormones estradiol, progesterone, and androgens, the endometrium is also a target for insulin, the receptor for which is cyclically regulated in normo-ovulatory women. In vitro, insulin inhibits the normal process of endometrial stromal differentiation (decidualization). In addition, insulin-like growth factors (IGFs) and their binding proteins are regulated in and act on endometrial cellular constituents, and hyperinsulinemia down-regulates hepatic IGFBP-1, resulting in elevated free IGF-I in the circulation. Thus, elevated estrogen (without the opposing effects of progesterone in the absence of ovulation), hyperinsulinemia, elevated free IGF-I and androgens, and obesity all likely contribute to endometrial dysfunction, infertility, increased miscarriage rate, endometrial hyperplasia, and endometrial cancer common in women with PCOS. The potential mechanisms underlying these disorders, specifically in women with PCOS, are complex and await additional transdisciplinary research for their complete elucidation.     

Sex hormone adjuvant therapy in rheumatoid arthritis

RA is an autoimmune rheumatic disorder resulting from the combination of several predisposing factors, including the relation between epitopes of possible triggering agents and histocompatibility epitopes, the status of the stress response system, and the sex hormone status. Estrogens are implicated as enhancers of humoral immunity, and androgens and progesterone are natural immune suppressors. Sex hormone concentrations have been evaluated in RA patients before glucocorticoid therapy and have frequently been found to be altered, especially in premenopausal women and male patients. In particular, low levels of gonadal and adrenal androgens (testosterone and DHT, DHEA and DHEAS) and a reduced androgen:estrogen ratio have been detected in body fluids (i.e., blood, synovial fluid, smears, saliva) of male and female RA patients. These observations support a possible pathogenic role for the decreased levels of the immune-suppressive androgens. Exposure to environmental estrogens (estrogenic xenobiotics), genetic polymorphisms of genes coding for hormone metabolic enzymes or receptors, and gonadal disturbances related to stress system activation (hypothalamic-pituitary-adrenocortical axis) and physiologic hormonal perturbations such as during aging, the menstrual cycle, pregnancy, the postpartum period, and menopause may interfere with the androgen:estrogen ratio. Sex hormones might exert their immune-modulating effects, at least in RA synovitis, because synovial macrophages, monocytes, and lymphocytes possess functional androgen and estrogen receptors and may metabolize gonadal hormones. The molecular basis for sex hormone adjuvant therapy in RA is thus experimentally substantiated. By considering the well-demonstrated immune-suppressive activities exerted by androgens, male hormones and their derivatives seem to be the most promising therapeutic approach. Recent studies have shown positive effects of androgen replacement therapy at least in male RA patients, particularly as adjuvant treatment. Interestingly, the increase in serum androgen metabolism induced by RA treatment with CSA should be regarded as a possible marker of androgen-mediated immune-suppressive activities exerted by CSA, at least in RA and at the level of sensitive target cells and tissues (i.e., synovial macrophages). The absence of altered serum levels of estrogens in RA patients and the reported immune-enhancing properties exerted by female hormones have represented a poor stimulus to test estrogen replacement therapy in RA. The different results obtained with OC use seem to depend on dose-related effects and the different type of response to estrogens in relation to the cytokine balance between Th1 cells (cellular immunity, i.e., RA) and Th2 cells (humoral immunity, i.e., SLE). The androgen replacement obtained directly (i.e., testosterone, DHT, DHEAS) or indirectly (i.e., antiestrogens) may represent a valuable concomitant or adjuvant treatment to be associated with other disease-modifying antirheumatic drugs (i.e., MTX, CSA) in the management of RA.     

Androgens and male sexual behavior from mice to men.

Exogenous androgenic hormones or drugs that affect endogenous androgen levels are now used in a variety of human applications [cf. Bardin et al. (1991)]. Therefore, an increasing number of well-controlled physiological, clinical, and psychological studies have examined the effects of androgens on muscle, prostate, lipids, and bone, as well as the cardiovascular, immune, and nervous systems. The increasing number of androgens and delivery systems that are currently available or under development should also increase the prevalence of androgen therapy (Table 1). As androgen use increases, a crucial area of research is the effect of androgens on sexual behavior, which encompasses sexual desire, motivation, performance, and satisfaction. There is a large body of animal data documenting hormonal regulation of male sexual behavior and the neural sites of action of these hormones. Clinical data on the effects of androgens on human male sexuality come from correlative studies of endogenous testosterone levels and sexual function, treatment of men who are testosterone deficient, and men using hormonal contraceptives.     

New perspectives on Mars and Venus: unravelling the role of androgens in gender differences in cardiovascular biology and disease

There are substantial gender differences in the pattern, severity and clinical outcomes of coronary heart disease independent of environmental risk factor exposure. As a consequence, there has been considerable interest in the potential role of sex hormones in atherogenesis, particularly the potential protective effects of oestrogen. However, the failure of the recent clinical randomised trials to show a cardioprotective effect for oestrogen coupled with a growing interest in androgen replacement therapy in elderly men has refocused interest on the role of androgens in cardiovascular biology and disease. Over the last decade, compelling evidence has emerged that sex differences in vascular biology are not only determined by gender-related differences in sex steroid levels but also by gender-specific tissue and cellular characteristics which mediate sex-specific responses to a variety of stimuli. In the vasculature, androgens often act in a gender-specific manner, with differential effects in male and female cells. This gender-dependent regulation may have important implications for understanding the basis of the gender gap in atherosclerosis and may eventually lead to the development of sex-specific treatments for cardiovascular disease. This review will summarise the current data for the role of androgens in gender differences in coronary heart disease and cardiovascular biology.     

Testosterone and 5 alpha-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene.

Testosterone and its 5 alpha-reduced derivative 5 alpha-dihydrotestosterone exert different actions in the male during embryogenesis and in postnatal life. Nevertheless the two hormones bind to the same intracellular androgen receptor, and genetic and endocrinological studies in the Tfm mouse suggest that the actions of both hormones are mediated by this single receptor. Previous studies indicate that dihydrotestosterone binds more tightly to the androgen receptor but that the Bmax of binding of the two hormones is the same. To determine whether these differences in binding parameters could explain the mechanism by which the two hormones exert different physiological actions via the same receptor, we introduced a plasmid encoding the androgen receptor cDNA and a reporter plasmid encoding MMTV-CAT into Chinese hamster ovary cells. These cells do not express endogenous androgen receptor and do not convert testosterone to dihydrotestosterone. Therefore, it was possible to examine the relation between the concentration of each of the steroids and reporter gene expression. Both hormones enhanced CAT activity, but dihydrotestosterone was approximately 10 times as potent (half maximal of 0.018 nM) as testosterone (half maximal of 0.2 nM); the maximal activity achieved was the same for the two androgens. These findings are nearly identical to the apparent Kd values for the interaction of the two hormones with the androgen receptor. Although testosterone and dihydrotestosterone may influence the expression of other genes differently, these findings are compatible with a model system in which the differential effects can be explained as a consequence of different binding affinities to the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Minireview: sex differentiation.

Mammalian sex differentiation is a hormone-dependent process in the male following the determination of a testis from the indifferent gonad through a cascade of genetic events. Female sex differentiation is not dependent on ovarian hormones, yet there is evidence that members of the Wnt family of developmental signaling molecules play a role in Müllerian duct development and in suppressing Leydig cell differentiation in the ovary. The testis induces male sex differentiation (including testis descent) through a time-dependent production of optimal concentrations of anti-Müllerian hormone, insulin-like factor(s) and androgens. Observations in several human syndromes of disordered fetal sex development corroborate findings in murine embryo studies, although there are exceptions in some gene knockout models. The ubiquitously expressed AR interacts in a ligand-dependent manner with coregulators to control the expression of androgen-responsive genes. Preliminary studies suggest the possibility of hormone resistance syndromes associated with coregulator dysfunction. Polymorphic variants in genes controlling androgen synthesis and action may modulate androgenic effects on sex differentiation.     

New roles for estrogens in rheumatoid arthritis

Sex hormones appear to play an important role as modulators of autoimmune disease onset/perpetuation. Steroid hormones are implicated in the immune response, with estrogens as enhancers at least of humoral immunity, and androgens and progesterone (and glucocorticoids) as natural immune suppressors. Serum levels of estrogens have been found to be normal in rheumatoid arthritis (RA) patients. Synovial fluid levels (SF) of proinflammatory estrogens relative to androgens are significantly elevated in both male and female RA patients as compared to controls, which is most probably due to an increase in local aromatase activity. Thus, available steroid pre-hormones are rapidly converted to proinflammatory estrogens in the synovial tissue in the presence of inflammatory cytokines (i.e. TNF alpha, IL-1, IL-6). The increased estrogen concentrations observed in RA SF of both sexes are characterized mainly by the hydroxylated forms, in particular 16 alpha-hydroxyestrone, showing a mitogenic stimulating role. Indeed, recent studies by us indicate that 17-beta estradiol (E2) clearly enhanced the expression of markers of cell growth and proliferation, whereas testosterone (T) induced an increase in markers indicating DNA damage and apoptosis. In particular, our data further shows that the enhancing role of estrogens on the immune/inflammatory response is exerted by activating the NFkB complex. In conclusion, locally increased estrogens may exert activating effects on synovial cell proliferation, including macrophages and fibroblasts, suggesting new roles for estrogens in RA.     

Androgen regulation of prostate morphoregulatory gene expression: Fgf10-dependent and -independent pathways

Androgens are essential and sufficient for prostate gland morphogenesis; however, the downstream gene targets that mediate this action are unclear. To identify androgen-regulated genes involved in prostate development, we used short-term organ culture and examined the effect of testosterone on the expression of several critical prostate morphoregulatory genes. Rat ventral prostates (VP) and lateral prostates (LP) were collected at birth, and contralateral lobes were cultured for 18 h in the presence or absence of 10 nM testosterone with or without OH-flutamide to block residual androgens. Gene expression was quantitated using real-time RT-PCR. Although expression of Fgf10, Nkx3.1, and Ptc was increased in both prostate lobes, other genes were regulated by testosterone in a lobe-specific manner. This included up-regulation of epithelial genes FgfR2iiib, Shh, Hoxb13, and Bmp7 in the VP specifically and down-regulation of mesenchymal genes Wnt5a (VP) and Bmp4 (LP). Thus, in addition to stimulation of homeobox genes and paracrine-acting growth factors, androgens may positively regulate prostatic development through suppression of growth inhibitory genes. Because previous studies revealed a similar gene regulation pattern in response to exogenous Fgf10, experiments were performed to identify androgen-regulated genes mediated through Fgf10 signaling. Short-term VP and LP cultures with FgfR antagonist PD173074 and Mek inhibitor U0126 identified epithelial Shh and Hoxb13 up-regulation by androgens to be Fgf10-dependent. We propose that androgen regulation of prostate development is mediated through positive and negative regulation of multiple morphoregulatory genes acting in combination through complex gene networks. Lobe-specific responses may provide a developmental basis for prostate gland heterogeneity.     

Consistent variation in yolk androgens in the Australian Brush-turkey, a species without sibling competition or parental care

Maternal hormones are an excellent pathway for the mother to influence offspring development, and birds provide exceptional opportunities to study these hormone-mediated maternal effects. Two dominant hypotheses about the function of yolk androgens in avian eggs concern maternal manipulation of sibling competition and post hatching paternal care. In megapodes, however, neither sibling competition nor post hatching parental care exists. Eggs are incubated by external heat sources, and chicks dig themselves out of their underground nest and live independently of their parents and their siblings. In this first study on egg androgens of such a megapode, the Australian Brush-turkey Alectura lathami, we found nevertheless substantial amounts of maternal androgens. Since size of the incubation mound, incubation temperature, egg size and laying date greatly vary in this species, we analysed variation in testosterone (T), androstenedione (A4) and dihydrotestosterone (DHT) in relation to these factors. T concentrations were significantly higher in eggs from bigger mounds and laid at greater depth, which may compensate via anabolic effects for the longer duration and higher energetic requirements of chicks when digging themselves out. T concentrations were higher in smaller eggs, and both yolk A4 and T concentrations increased with laying date, perhaps as a compensatory measure, while DHT concentrations only varied across different mounds. These results indicate that maternal androgens may influence offspring development outside the contexts of sibling competition or parental care.     

Regulation of alpha and thyrotrophin-beta subunit mRNA levels by androgens in the female rat.

Thyroid and steroid hormones act by similar mechanisms to influence gene expression in the anterior pituitary gland. The genes encoding the common alpha and TSH-beta glycoprotein subunits are known to be regulated by thyroid hormones; we report here the effects of androgen administration on levels of alpha and TSH-beta mRNA in pituitary cytoplasm in the euthyroid and hypothyroid female rat. Dihydrotestosterone (DHT) suppressed both alpha and TSH-beta mRNAs to levels lower than those found in untreated animals; a similar reduction was seen in hypothyroid animals treated with DHT. A biphasic response of TSH-beta mRNA was seen following administration of tri-iodothyronine (T3) to hypothyroid rats, with early stimulation followed by later inhibition; these changes were also evident after administration of T3 to androgen-treated animals, although mRNA levels were again suppressed. The effects of testosterone were similar to those of DHT. In contrast to the changes in mRNA levels, androgen administration did not lead to significant alterations in serum TSH concentrations or pituitary TSH content. These results indicate that, like thyroid hormones, androgens suppress both alpha and TSH-beta subunit mRNA levels in the female rat. Androgens, however, exert differential effects on TSH synthesis and release which contrast with those of thyroid hormones.     

Influence of sex hormones on brain excitability and epilepsy

Epilepsy is one of the most common neurologic problems worldwide. In spite of the many studies carried out, our understanding of generalized epileptogenesis remains far from complete. In recent years many data have clarified the effects of sexual hormones on brain excitability. Female and male sexual hormones may be considered pharmacoactive compounds that alter the seizure threshold, changing the frequency and semeiology of the seizures. In particular, estrogens may increase neuronal excitability while progesterone enhances inhibitions and increases the seizure threshold; on the other hand, androgens can decrease ictal activity in the human brain. This review provides an overview of the current knowledge in this field and highlights some of the prevailing hypotheses about the effects of sexual hormones on neuronal excitability analysing data from both animal and clinical studies.     

No direct mitogenic effect of sex hormones on antlerogenic cells detected in vitro.

Deer pedicles, antecedents of antlers, develop from a specialized periosteum (antlerogenic periosteum) which overlies the lateral crest of the deer frontal bone. The initiation of pedicle growth is triggered by androgen hormones. Thus far, it is not known whether pedicle initiation is caused by direct stimulation of androgen hormones on the antlerogenic periosteum or whether some intermediate mechanisms are necessary. The present study took an in vitro approach to investigate whether sex hormones have direct mitogenic effects on primary cultured antlerogenic periosteal cells (antlerogenic cells). Antlerogenic cells were obtained from two 5-month-old red deer calves. The cells were passaged twice and then treated with testosterone, dihydrotestosterone, and estradiol. The proliferation assays showed that no direct mitogenic effects on the second passage antlerogenic cells could be detected with any of the sex hormone treatments (P > 0.05). Testosterone-binding studies showed that at the second passage, specific testosterone-binding sites were present in the antlerogenic cells. Therefore, we conclude that androgens do not have mitogenic effects on antlerogenic cells in vitro. Our results suggest that pedicle formation may not be the result of direct stimulation of androgen hormones on antlerogenic tissue. Instead, androgen hormones may only allow the process to proceed by increasing the sensitivity of antlerogenic cells to mitogens, e.g., some growth factors.     

Androgens modulate structure and function of the suprachiasmatic nucleus brain clock

Gonadal hormones can modulate circadian rhythms in rodents and humans, and androgen receptors are highly localized within the core region of the mouse suprachiasmatic nucleus (SCN) brain clock. Although androgens are known to modulate neural plasticity in other CNS compartments, the role of androgens and their receptors on plasticity in the SCN is unexplored. In the present study, we ask whether androgens influence the structure and function of the mouse SCN by examining the effects of gonadectomy (GDX) on the structure of the SCN circuit and its responses to light, including induction of clock genes and behavioral phase shifting. We found that after GDX, glial fibrillary acidic protein increased with concomitant decreases in the expression of the synaptic proteins synaptophysin and postsynaptic density 95. We also found that GDX exerts effects on the molecular and behavioral responses to light that are phase dependent. In late night [circadian time (CT)21], GDX increased light-induced mPer1 but not mPer2 expression compared with intact (INT) controls. In contrast, in early night (CT13.5), GDX decreased light induced mPer2 but had no effect on mPer1. At CT13.5, GDX animals also showed larger phase delays than did INT. Treatment of GDX animals with the nonaromatizable androgen dihydrotestosterone restored glial fibrillary acidic protein, postsynaptic density 95, and synaptophysin in the SCN and reinstated the INT pattern of molecular and behavioral responses to light. Together, the results reveal a role for androgens in regulating circuitry in the mouse SCN, with functional consequences for clock gene expression and behavioral responses to photic phase resetting stimuli.