Inhibition of the androgen receptor by antisense oligonucleotides regulates the biological activity of androgens in SZ95 sebocytes.

One novel strategy for the blockade of the androgen receptor could be the selective inhibition of androgen receptor by antisense oligonucleotides or small interfering RNA molecules. Here we describe the down regulation of the androgen receptor in cultured human SZ95 sebocytes with antisense oligonucleotides modified with phosphorothioates and 2'- O-methylribosyl residues. The ability of antisense oligonucleotides to cross the cellular membrane was enhanced by establishing a transient transfection system based on cationic lipid vesicles. Both antisense oligonucleotide types administered caused assumedly translational arrest. Dose-dependent inhibition of androgen receptor protein expression was observed after SZ95 sebocyte transfection with modified phosphorothioate oligonucleotides and modified 2'- O-methylribonucleotides which were directed against the translational start of the androgen receptor mRNA. The strongest transient inhibition of androgen receptor expression was detected after 14 hours with 1.0 muM antisense 2'- O-methylribonucleotides (88+/-1.3%, p<0.001). With longer recovery times than 24 hours, androgen receptor protein expression returned to the native control levels. Inhibition of the expression of androgen receptor by antisense oligonucleotides, reduced the enhanced proliferation of SZ95 sebocytes challenged by testosterone and 5alpha-dihydrotestosterone. This administration opens new therapeutic possibilities in androgen-associated skin diseases, since we could also show androgen inhibition with these antisense oligonucleotides in a reconstituted human epidermis model (Horm Metab Res 2007; 39:157-165).

[The androgen receptor: molecular pathology]. / Récepteur des androgènes: pathologie moléculaire.

Androgens play a crucial role in the development, maintenance and regulation of male phenotype and reproductive physiology through the androgen receptor, a transcription factor. Testosterone or dihydrotestosterone binding induces a trans-conformation of the androgen receptor and allows its translocation into the nucleus, where it recognizes specific DNA sequences. Recent developments in molecular genetics, as well as structural analysis of the androgen receptor, allow a better understanding of the structure/function relationship of this nuclear receptor. Molecular analyses of androgen insensitivity syndrome, as well as hormone-resistant prostate cancer, Kennedy's disease and isolated male infertility, have been proved useful as privileged models for this purpose. In the absence of identified AR receptor mutations in androgen insensitivity syndromes, abnormalities of transcriptional cofactor should be considered. Finally, identification of androgen-dependent genes will be helpful for evaluating the degree of the molecular defect of androgen action within target cells.

Disorders of androgen action.

Disorders of androgen action are the main cause of male pseudohermaphroditism and include 5alphaR deficiency and androgen receptor defects. 5alphaR deficiency is characterized by female genitalia with some degree of masculinization, clitoromegaly, and severely bifid scrotum corresponding to the so-called pseudovaginal perineoscrotal hypospadias. At the onset of puberty, increased muscle mass, development of pubic hair, and phallic growth are associated with the acquisition of male gender identity. Normal or increased levels of testosterone and an elevated testosterone-to-dihydrotestosterone ratio after human chorionic gonadotropin stimulation testing suggest 5alphareductase deficiency, and the diagnosis can be ascertained by identifying the mutation in the 5alphaR-2 gene. Whatever the patient's age at diagnosis, psychological evaluation with 5alphaRD is vital. Androgen receptor defects encompass two clinical expressions: the complete and partial androgen insensitivity syndromes. Complete androgen insensitivity syndrome should be suspected at birth in the presence of inguinal hernia in a girl without genital ambiguity. At puberty, the sign of alert is primary amenorrhea with normal female phenotype and harmonious mammary development but no pubic hair growth. Partial androgen insensitivity syndrome covers a wide spectrum of undervirilized phenotypes ranging from clitoromegaly at birth to infertile men. In all cases, complementary investigations should include plasma testosterone and luteinizing hormone as well as androgen-binding capacity in cultured genital skin fibroblasts. Diagnosis is confirmed by identification of the androgen receptor gene mutation. Although patients with complete androgen insensitivity syndrome are raised as females, patients with partial androgen insensitivity syndrome should be managed according to age at diagnosis, response to treatment with exogenous androgens, and the presence of an androgen gene mutation. Gonadectomy in complete androgen insensitivity syndrome should be performed before puberty, and androgen substitution may improve the development of external genitalia in some patients with partial androgen insensitivity syndrome. Psychological follow-up is necessary.

Human prostatic cell line PNT1A, a useful tool for studying androgen receptor transcriptional activity and its differential subnuclear localization in the presence of androgens and antiandrogens.

The human immortalized prostatic cell line PNT1A has been proved to be a good model for analysis of cellular processes such as the prostatic epithelium proliferation in response to androgens and growth factors. Here we used this cell line for studying the transcriptional activity and trafficking of the androgen receptor (AR) by analyzing several actions of antiandrogens. Transient transfection experiments with PNT1A cells were performed with wild type human AR and an androgen-responsive gene reporter. We demonstrated that the transcription of reporter gene could be triggered by natural androgens (testosterone and dihydrotestosterone) in PNT1A cells as well as in the prostatic carcinoma cell line DU-145. With competitive experiments in the two cell lines, we observed no difference between the antagonistic capacity of cyproterone acetate (CPA) and hydroxyflutamide at 10(-7) M. At this concentration, bicalutamide antagonist activity was lower. In parallel, we compared the subcellular localization of the modified green fluorescent protein (EGFP)-AR in COS-7, PNT1A and DU-145 cell lines under fluorescence microscopy: we found different distributions between nucleus and cytoplasm, depending on the cell line and the culture medium. Androgen induced cluster formation within the nucleus of the PNT1A and DU-145 cells. However, the cytonuclear trafficking of androgen bound EGFP-AR in the same living cell and nuclear foci were easier to examine in the PNT1A cells. The antiandrogen capacity of bicalutamide was manifested by a slower androgen-dependent nuclear transfer of EGFP-AR and a homogeneous nuclear localization. A delayed advent of nuclear clusters was observed in presence of CPA. We conclude that the PNT1A cell line is a better model than the DU-145 cell line to analyze the trafficking of AR and the association of AR on the nuclear matrix, as well as to observe the action of antiandrogens on these critical steps in prostate cells.

Disorders linked to insufficient androgen action in male children.

Virilization of the external genitalia in the male fetus requires testosterone and dihydrotestosterone (DHT), which is formed from testosterone by the action of the enzyme, 5alpha-reductase type 2 (5alphaR-2). Mediation of the effects of both testosterone and DHT requires a functional androgen receptor (AR) located in the cytoplasmic compartment of target cells. DHT (or testosterone) binding induces a conformational change which facilitates AR nuclear transport, phosphorylation and dimerization, ultimately regulating of the rate of transcription of androgen-dependent genes. Any event which impairs DHT formation (mutation within the 5alphaR-2 gene or 5alphaR-2 inhibitors) or normal function of the AR (mutation in the AR gene, antiandrogens) may result in insufficient androgen action in the male fetus and in subsequent undervirilization in the newborn. Hypospadias may be due to a defect in androgen action due to mutation of the 5alphaR-2 or of the AR gene. Mutation of unidentified genes is likely to underlie this displacement of the urethral meatus from the tip to the ventral side of the phallus. An aetiological role for environmental chemical products has been postulated, since ethnic as well as geographical differences in the incidence of hypospadias have been noted. Increasing evidence has been gathered indicating that widely used industrial and agricultural chemicals have deleterious effects on normal male sexual differentiation. Cryptorchidism and micropenis may represent an intersex phenotype, even if they are isolated. Aetiological factors include 5alphaR-2 gene mutation, AR gene mutation or environmental hormonal disruptors. In conclusion, several phenotypes have been attributed to insufficient androgen action during fetal life. Whereas mutations in the 5alphaR-2 gene and AR gene are natural, attention should be focused on environmental endocrine disruptors that are able to mimic steroid 5alpha-reductase deficiency or partial androgen insensitivity syndrome.

Environmental xenoestrogens, antiandrogens and disorders of male sexual differentiation.

Over the past 20 years, the documented increase in the disorders of male sexual differentiation, such as hypospadias, cryptorchidism, and micropenis, has led to the suspicion that environmental chemicals are detrimental to normal male genital development in utero. Male sexual differentiation is critically dependent on the normal action of androgens, and unbalanced androgen/estrogen ratios can disturb it. Environmental xenoestrogens (such as herbicides, pesticides, PCBs, plasticizers, and polystyrenes) that mimic estrogens or environmental antiandrogens (such as polyaromatic hydrocarbons, linuron, vinclozolin, and pp'DDE) that disturb endocrine balance, cause demasculinizing effects in the male foetus. These environmental chemicals are often referred to as endocrine disruptors: they are thought to mimic endogenous estrogens by entering the cell, binding to the receptor and activating transcription, they may also antagonize normal androgen action. We have established numerous cell lines to assess the estrogenicity and antiandrogenicity of compounds found in the environment and to identify new products present in wastewater effluents that are able to disrupt endocrine functions. Several cell lines responding to estrogens have been obtained in our group, including cells with different enzymatic equipment and cells expressing chimeric receptor or natural estrogen receptors alpha and beta. These cell lines have proved to be useful for assessing the biological activity of pesticides, fungicides, and chemicals found in plastic or discarded in the environment. In order to generate a powerful tool for the investigation of androgen action and the rapid screening of potential antagonists, we developed a new stable prostatic cell line. The PALM cell line is an original cellular model to characterize the response of hAR, and it provides an easy and rapid bioluminescent test to identify new antagonists. We also developed a model based on a fusion protein between the androgen receptor (AR) and the green fluorescent protein (GFP) to study the intracellular dynamics of AR. The GFP-AR model was applied to define the ability of several xenoestrogens and antiandrogens to inhibit the nuclear transfer of AR. The ubiquitous presence of endocrine disruptors in the environment and the increased incidence of neonatal genital malformation support the hypothesis that disturbed male sexual differentiation may in some cases be caused by increased exposure to environmental xenoestrogens and/or antiandrogens.

Antimineralocorticoid 11beta-substituted spirolactones exhibit androgen receptor agonistic activity: a structure function study.

In humans, spironolactone and mespirenone are well known antimineralocorticoids without C-11beta substituents. These compounds display antagonist properties by acting through the human androgen receptor (hAR). In contrast, we demonstrate here that synthetic mineralocorticoid antagonists bearing hydrophobic C-11beta substituents and C-17gamma-lactone are potent hAR agonists in vitro. The three-dimensional construction of both the ligand binding domain (LBD) of the hAR and the human mineralocorticoid receptor (hMR), based on the crystal structure of the LBD of the human progesterone receptor, revealed not only that the interactions with the steroidal A- and D-rings seemed to be crucial for stabilization of active hMR or hAR conformation, but that other steroidal substitutions could influence the agonist versus antagonist activity of ligands. The docking of synthetic compounds bearing C-11beta hydrophobic substituents within the ligand binding pocket of hAR demonstrated that precise positions of the steroid, such as C-11 and C-17, are in close contact with some residues on the receptor, C-11 with Gly 708 and C-17 with Asn705 and Thr877. These contacts are crucial for the stabilization of the active receptor conformation. Mutation of Asn705 by alanine altered the 11beta-substituted spirolactone-mediated trans-activation function of hAR, suggesting an anchoring of the C-17-lactone carbonyl group (C-22) with this residue. The stabilizing effect of the H12 helix in its active conformation is also induced by hydrophobic contacts between the Gly708 and C-11beta substituents, as recently observed with the A773G-hMR mutant in the presence of similar drugs. The study of the role of these substituents suggests efficient new directions for the drug design of selective androgen agonists.

A stable prostatic bioluminescent cell line to investigate androgen and antiandrogen effects.

We developed a new stable prostatic cell line expressing the human androgen receptor (AR) and the AR-responsive reporter gene to generate a powerful tool for investigating androgen action and for rapid screening of agonists and antagonists. The AR-deficient PC-3 cells were stably transfected with pSG(5)-puro-hAR and pMMTV-neo-Luc. After selection with puromycin and neomycin, one highly inducible clone was isolated and named PALM, for PC-3-Androgen receptor-Luciferase-MMTV. The expression of hAR was confirmed by western blot and steroid-binding assays on the whole cells. The transcriptional activity of the clone was measured after incubation of cells with increasing concentrations of synthetic R1881 or natural androgens (DHT and testosterone). The three agonists had the same maximal activity at 0.1 microM and the fold induction was equal to 20. The agonist and antagonist activities of the steroidal antiandrogens (cyproterone acetate and RU2956) and the non-steroidal antiandrogens (nilutamide, bicalutamide, inocoterone and hydroxyflutamide) measured with the PALM cells were in good correlation with the results obtained with transiently transfected cells. The selectivity in steroid transactivation was demonstrated with estradiol, progesterone, cortisol, dexamethasone and aldosterone. Spironolactone and RU486 showed partial agonist and antagonist activities, whereas R5020 presented only a partial antagonist activity. We here demonstrate that this stable transfectant provides an accurate tool for studying wild-type human AR activation and its regulation by androgens and antiandrogens in a human prostatic epithelial cell, which is routinely available and remains androgen-responsive in vitro.

[Androgen-independent prostate carcinoma and androgen-receptor: recent progress in molecular genetics]. / Cancer de la prostate hormono-indépendant et récepteurs des androgènes: les avancées en génétique moléculaire.

Prostate cancer is an androgen-dependent tumor which presents an androgen-independent regrowth after clinical regression in response to antiandrogen treatment. Four hypotheses have been developed to understand how androgen signal transduction pathway mediate androgen-independent tumor progression: over expression of the wild-type androgen-receptor gene, androgen-receptor gene mutation, excessive recruitment of transcriptional co-activator ARA-70 and a cross-talk between the androgen-receptor and the growth factor receptor pathways. In this work, C. Sawyers's group elegantly demonstrates, in LAPC-4 androgen-independent prostate cancer sublines, that forced hyperexpression of HER-2/Neu receptor tyrosine kinase allowed androgen-independent growth, that HER-2/Neu activated the androgen-receptor pathway in the absence of androgens and synergized with low levels of androgen to superactivate the pathway. These important data could have therapeutic implications for the management of androgen-independent prostate cancer.

Conformational change in the human glucocorticoid receptor induced by ligand binding is altered by mutation of isoleucine 747 by a threonine.

Limited proteolysis experiments were performed to study conformation changes induced by ligand binding on in vitro produced wild-type and I747T mutant glucocorticoid receptors. Dexamethasone-induced conformational changes were characterized by two resistant proteolysis fragments of 30 and 27 kDa. Although dexamethasone binding affinity was only slightly altered by the I747T substitution (Roux, S., Térouanne, B., Balaguer, P., Loffreda-Jausons, N., Pons, M., Chambon, P., Gronemeyer, H., and Nicolas, J.-C. (1996) Mol. Endocrinol. 10, 1214-1226), higher dexamethasone concentrations were required to obtain the same proteolysis pattern. This difference was less marked when proteolysis experiments were conducted at 0 degrees C, indicating that a step of the conformational change after ligand binding was affected by the mutation. In contrast, RU486 binding to the wild-type receptor induced a different conformational change that was not affected by the mutation. Analysis of proteolysis fragments obtained in the presence of dexamethasone or RU486 indicated that the RU486-induced conformational change affected the C-terminal part of the ligand binding domain differently. These data suggest that the ligand-induced conformational change occurs via a multistep process. In the first step, characterized by compaction of the ligand binding domain, the mutation has no effect. The second step, which stabilizes the activated conformation and does not occur at 4 degrees C, seems to be a key element in the activation process that can be altered by the mutation. This step could involve modification of the helix H12 position, explaining why the conformation induced by RU486 is not affected by the mutation.

Transcriptional interferences between normal or mutant androgen receptors and the activator protein 1--dissection of the androgen receptor functional domains.

We investigated the interferences of the normal or mutated androgen receptor with the activator protein-1 (AP-1) by assessing their effects on transcriptional activity in CV-1 cells. A luciferase reporter gene was constructed downstream from either a promoter for the mouse vas deferens protein, or a trimerized 12-O-tetradecanoyl phorbol-13-acetate-response element site whose transcriptions are activated by androgen and 12-O-tetradecanoyl phorbol-13-acetate, a potent AP-1 activator. The blockade of dephosphorylation by protein phosphatases identifies the protein phosphatases that modulate the AP-1/androgen receptor cross-talk. Using engineered or naturally occurring androgen receptor mutants that are responsible for complete or partial androgen insensitivity syndromes, we defined the subregions involved in the cross-talk of the androgen receptor with the AP-1 factors. First, it appears that the 188 first amino acids of the N-terminal domain of the androgen receptor are necessary to obtain a full transrepression. Second, a functional and intact ligand binding domain is critical for the modulation of androgen/AP-1 pathway interactions. Third, normal DNA binding capacity of the androgen receptor is not required. Two mutants at positions 568 and 581 of the DNA binding domain demonstrate that the transactivation and transrepression functions of the androgen receptor can be dissociated. Collectively, these data indicate that several segments of the androgen receptor are involved in cross-talk with the AP-1 pathway. Mutations within the DNA binding domain of the androgen receptor highly impair these interferences.

Trafficking of androgen receptor mutants fused to green fluorescent protein: a new investigation of partial androgen insensitivity syndrome.

The naturally occurring mutations of the androgen receptor (AR), detected in patients with androgen insensitivity syndrome (AIS), are currently analyzed by in vitro assays. Unfortunately, these assays do not always permit the demonstration of a direct relationship between the in vitro activity of the receptor and the severity of the phenotype (in particular, for mutations detected in patients with partial AIS). We recently studied the trafficking of wild-type AR, fused to the green fluorescent protein (GFP) in living cells. In the present study, we applied this method for the analysis of AR mutants to find out whether it could be a complementary method of investigation of AIS. After construction of the GFP-AR mutant fusion proteins, the androgen-binding characteristics, nuclear transfer capacities, and transcriptional activities were evaluated. The nuclear transfer was quantified in the presence of various concentrations of dihydrotestosterone (DHT). We studied two mutants associated with partial AIS: G743V and R840C. The androgen-binding characteristics of both mutants were affected, in comparison with normal AR. Although the affinities were similar, the dissociation rate of GFP-AR-G743V was twice that of GFP-AR-R840C. In transcriptional assay, both mutants were active only at high concentrations of androgen. The nuclear trafficking of the mutants was evaluated by two parameters: 1) the rate of nuclear transfer; and 2) the maximal amount of receptors imported into the nucleus. At 10(-6) mol/L DHT, the GFP-AR mutants entered into the nucleus in a fashion similar to that of GFP-AR-wt. At 10(-7) mol/L DHT, the rate and maximal degree of nuclear import were both reduced, even more, for GFP-AR-G743V. The difference between mutants was more pronounced at 10(-9) mol/L DHT, because GFP-AR-G743V entered into the nucleus with even slower kinetics. Though the androgen-binding affinity and transcriptional activity assays did not reveal major differences between mutants, the dissociation rate and the trafficking capacity measurements permitted the activity of the mutants to be differentiated. We observed that the nuclear transfer capacities of these mutants are in correlation with the severity of the phenotype. The GFP-AR model provides an opportunity both to observe the dynamics of the hormone/receptor complex in living cells and to study the impact of the ligand-binding domain mutation, as opposed to certain in vitro techniques. Because the nuclear import capacity correlates well with the degree of androgen insensitivity, the GFP-AR is a useful complementary tool to understanding the phenotype/genotype relationship of AR function in patients with AIS.

Glucocorticoid receptors in bronchial epithelial cells in asthma.

The expression of the glucocorticoid receptor (GR) in untreated or in steroid-dependent asthmatic patients is poorly understood. We therefore studied GR mRNA and protein levels in bronchial biopsies obtained from seven untreated asthmatic patients, seven control volunteers, and seven patients with chronic bronchitis. We also studied in bronchial epithelial cells obtained by brushing from 13 untreated asthmatics, 18 steroid-dependent asthmatics, 11 control volunteers, and 12 patients with chronic bronchitis, GR and heat shock protein 90 kD (hsp90) mRNA as well as the immunoreactivity of GR, intercellular adhesion molecule (ICAM-1), and granulocyte macrophage-colony-stimulating factor (GM-CSF). GR mRNA and protein level was similar in all subject groups in both biopsies and bronchial epithelial cells. Hsp90 mRNA level was also similar in all subject groups. ICAM-1 expression was significantly increased in bronchial epithelial cells from untreated asthmatics, but ICAM-1 was not expressed in those from steroid-dependent asthmatic patients. GM-CSF expression was significantly increased in bronchial epithelial cells from untreated and steroid-dependent asthmatic patients. GR expression within the airways is unaltered by oral long-term steroid treatment in asthma, but the expression of some but not all specific markers for asthma is modified by oral steroid.

Quantitation of androgen receptor messenger RNA from genital skin fibroblasts by reverse transcription--competitive polymerase chain reaction.

To gain further information concerning the regulation by androgen of AR mRNA expression in cultured genital skin fibroblasts (GSF), we first developed a quantitative reverse transcription-competitive polymerase chain reaction (RT-PCR). This method used an ethidium bromide stain analysis of the PCR products for the accurate quantitation of low levels of human androgen receptor (hAR) mRNA in GSF. To control for variations due to sample preparation, and to minimize the disparity of the reverse transcriptase efficiency between samples after the RT procedure, we produced an initial PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and then adjusted the amount of cDNA to that of this housekeeping gene. Competitive PCR for hAR was then immediately performed on normalized cDNA with a competitor DNA that exhibited a 13 bp deletion as compared to the 163 bp for the target fragment, and the PCR products were easily separated by 3.5% agarose gel electrophoresis. This quantitation procedure involved no additional steps, such as enzymatic cleavage of the PCR products, nor the use of radioactivity. In GSF from individuals, we found that the normal amount of AR mRNA was 5.6 attomoles/microg RNA, (+/-1.0, s.e.m.) with an intra- and an inter-assay of 8.4 and 14.7%, respectively. We observed a biphasic pattern of AR mRNA expression in normal human GSF in the presence of physiological concentration of androgen. Quantitative RT-PCR of AR mRNA may be useful for studying AR mRNA expression in experimental or clinical conditions.

Trafficking of the androgen receptor in living cells with fused green fluorescent protein-androgen receptor.

The trafficking of the androgen receptor (AR) in transfected cells was studied using a green fluorescent protein (GFP)-AR chimera. The reporter molecule GFP enabled the localization of AR in living cells with a good spatial and temporal resolution. After the construction of GFP-AR and verification of the size of the fusion protein produced, we demonstrated that GFP-AR conserves the functional properties of the AR: GFP-AR had the same androgen-binding affinity as AR, and GFP-AR efficiently transactivated an androgen-responsive gene in response to synthetic androgen at 30 degrees C. The fusion protein was first detected throughout the cytoplasm without hormone, fluorescence becoming nuclear rapidly after androgen incubation. This hormone dependence of AR trafficking was confirmed by the use of the mutant GFP-AR-del4, which lacked the androgen-binding function. The mutant was localized in the cytoplasm in the absence of hormone, but the distribution was not modified by androgen incubation. An ACAS 570 scanning laser cytometer was used to quantify fluorescence in a single living cell, first without and then with hormone. Different hormones and antihormones were tested to determine the dynamics of GFP-AR translocation into the nucleus. All the drugs used were able to induce nuclear translocation, and steady state level was rapidly attained within 1 h. The ratio of receptors in cytoplasmic and nuclear compartments was related to both affinity and concentration of ligand. The data from this follow-up study demonstrated for the first time the intracellular dynamics of the hormone-dependent trafficking of AR in a single living cell.

Mutation of isoleucine 747 by a threonine alters the ligand responsiveness of the human glucocorticoid receptor.

Mutation of isoleucine 747 to threonine in the C-terminal part of the ligand-binding domain (LBD) of the human glucocorticoid receptor (GR) alters the ligand specificity for transactivation. Natural glucocorticoids such as cortisol or corticosterone were completely inactive with the mutant 1747T, whereas synthetic steroids like dexamethasone efficiently stimulated GR 1747T-mediated transactivation. However, the corresponding ligand dose-response curve for dexamethasone-induced transactivation was shifted to higher concentrations when compared with that obtained with the wild type GR. Neither this shift nor the inability of cortisol to activate the 1747T mutant was due to an altered in vitro ligand-binding affinity. In the canonical three-dimensional structure of nuclear receptor LBDs, isoleucine 747 is in the direct vicinity of residues that contribute to the ligand-binding pocket. Moreover, it is located in the C-terminal LBD region, which harbors the conserved core of the activation function AF-2 and undergoes a ligand-induce transconformation, required to generate the surface interacting with putative transcriptional intermediary factors/coactivators of AF-2. The phenotype of 1747T mutant is discussed in view of the possible consequences of the mutation on the various events which, according to the model, lead to a transcriptionally competent AF-2.

A novel substitution (Leu707Arg) in exon 4 of the androgen receptor gene causes complete androgen resistance.

A wide spectrum of androgen receptor (AR) gene mutations has been reported in complete androgen insensitivity syndromes. The molecular basis of androgen resistance was investigated in a female newborn with complete testicular feminization. Sequencing identified a point mutation in exon 4 responsible for a leucine (CTG) to arginine (CGG) replacement at codon 707. This novel mutation is located in the amino-terminal part of the ligand-binding domain of the AR. To determine the functional properties of the mutated AR and to establish the correlation with the clinical phenotype of androgen resistance, the mutation was reproduced in AR wild-type complementary DNA, and the plasmid was transfected into AR-free mammalian cells. In vitro studies showed that the mutant AR was functionally defective as an androgen-binding molecule. Electrophoretic mobility shift assay revealed that the binding of mutated AR to DNA was reduced. Finally, the mutant was unable to induce the transcriptional activation of androgen-responsive reporter gene. This amino acid defect in the primary sequence probably involves the rupture of hydropathicity in a region that is conserved among members of the steroid receptor subfamily. Our data substantiate the major contribution of leucine 707 to normal AR function and demonstrate that its substitution by an arginine caused the complete androgen insensitivity in this patient. Our findings also contribute to the elaboration of the structure-function map of the AR based on naturally occurring mutations.

Glucocorticoids induced down-regulation of glucocorticoid receptor mRNA expression in asthma.

Although their precise mechanism of action remains to be elucidated, glucocorticoids represent the most effective therapy in the treatment of asthma. Interactions between the glucocorticoid receptor and the AP-1 complex have been shown to regulate the transcription of some genes, including glucocorticoid receptor itself. The aim of the present study was to compare the expression of mRNA for glucocorticoid receptor in human blood monocytes obtained from seven unstable untreated asthmatic patients who were subsequently treated with high doses of parenteral corticosteroid (methyl prednisolone 120 mg/day) for 10 days. mRNA expression was identified after RNA extraction using RNAzol and analysed after reverse transcriptase, by polymerase chain reaction using a semiquantitative competitive hybridization assay. All asthmatic patients showed an improvement in their FEV1 values after corticosteroid treatment (per cent of predicted value 68.28 +/- 4.93 versus 95.57 +/- 6.41, P < 0.02), and a significant decrease for glucocorticoid receptor mRNA expression (P < 0.02) was observed in their monocytes. This is the first report of an ex vivo down-regulation for the glucocorticoid receptor mRNA expression, following corticosteroid treatment.

Nonradioactive quantification of glucocorticoid receptor expression during differentiation of human monocytic cells (U937).

We describe a method for relative quantification of specific mRNA using a nonradioactive assay based on DNA strand competition between identical sequences of biotin- and fluorescein-labeled amplicon (probe) and unlabeled amplicon (target) during hybridization. As the target quantity increased, that of the double-labeled probe decreased in accordance with the mass action law. This technique was successfully applied to evaluate differences in glucocorticoid receptor expression in U937 cells before and after the addition of potent differentiation inducers: 12-O-tetradecanoylphorbol 13-acetate (TPA) and a combination of all-trans retinoic acid (RA) and 1,25-dihydroxyvitamin D2 (VD). We observed that TPA treatment was associated with an increase in specific binding of [3H]dexamethasone and up-regulation of GR mRNA while no enhanced GR expression was perceived with RA/VD treatment.