Androgen Receptor Expression Plasmid Research Articles

Assessment of the androgen receptor (AR) activity score versus AR expression in muscle invasive bladder cancer (MIBC).

463 Background: Previous studies have demonstrated that a considerable proportion of BCs express androgen receptor (AR), and that AR might drive tumor growth. The RNA-based AR activity score is a novel analytic tool that may better quantify the functional role of AR. Methods: Samples were obtained from patients (pts) with MIBC who had cystectomy or transurethral resection of bladder tumor (TURBT). AR expression was quantified by pathologist scoring of immunohistochemistry (IHC). The AR activity score was quantified using RNA isolates from benign and cancerous sections of slides adjacent to those used for IHC. RT-qPCR of a panel of AR-regulated genes was performed with comparison of transcript levels to those in LNCaP prostate cancer cells. Clinical results were validated in the RT-112 cell line. Results: A total of 37 pts (81.8% male) were analyzed with a median of age of 67. The majority were diagnosed as cT3 (54.5%) and 62.2% underwent neoadjuvant chemotherapy (86.9% with cisplatin/gemcitabine). IHC demonstrated a range of AR positivity from 0.0% to 90.0%, with most samples (46.0%) having no staining. In contrast, the AR activity score was between 2.1% and 178.9% (relative to LNCaP cells) with a median activity of 32.2% in cancerous tissues and 44.0% in benign regions. AR expression and score showed no direct correlation. A strong association was seen between AR activity score in cancerous tissue and DFS (P = 0.005). Interestingly, AR score in benign tissue was inversely correlated with DFS (P = 0.04). Pts who received neoadjuvant chemotherapy were noted to less likely develop metastases (P = 0.05). A more marginal correlation was noted between AR activity score and pathologic downstaging. Treatment of cells transfected with an AR expression plasmid along with an AR-responsive luciferase reporter demonstrated a dose-dependent effect of cisplatin in this assay. Conclusions: IHC and AR activity scores represent independent measures in this MIBC cohort. Our study suggests that pts with higher AR activity may be more susceptible to chemotherapy, and may have prolonged DFS. Additional studies in larger cohorts are warranted.

The potent synthetic androgens, dimethandrolone (7α,11β-dimethyl-19-nortestosterone) and 11β-methyl-19-nortestosterone, do not require 5α-reduction to exert their maximal androgenic effects

Dimethandrolone (DMA: 7α,11β-dimethyl-19-nortestosterone) and 11β-methyl-19-nortestosterone (MNT) are potent androgens in development for hormonal therapy in men. As 5α-reduced androgens, such as 5α-dihydrotestosterone (DHT), may raise the risk of benign prostate hyperplasia, accelerate the development of prostate carcinoma, and increase male pattern baldness and acne, we investigated the role of 5α-reduction in the androgenic activity of DMA and MNT. The authentic 5α-reduced metabolites, 5α-dihydroDMA (5α-DHDMA) and 5α-dihydroMNT (5α-DHMNT), were prepared by chemical synthesis and compared in vitro and in vivo to the parent compounds . Both 5α-reduced androgens bound with high affinity to the rat androgen receptor (AR) and were potent inducers of transactivation of 3XHRE-LUC in CV-1 cells cotransfected with a human AR expression plasmid. To examine in vivo androgenic (stimulation of ventral prostate [VP] and seminal vesicle [SV] weights) and anabolic (stimulation of levator ani [LA] muscle weights) activity, 22-day-old castrate male rats were treated sc for 7 days with various doses of DMA, 5α-DHDMA, or testosterone (T) or MNT, 5α-DHMNT, or T and necropsied on day 8. 5α-DHDMA was at least threefold more potent than T in stimulating growth of the VP but only 30–40% as potent as DMA. 5α-DHMNT was four- to eightfold more potent than T, whereas MNT was approximately equipotent to T. To assess the possible role of 5α-reduction in VP and SV growth, castrate immature rats were treated with maximally effective doses of T, DHT, DMA, MNT, or the related 19-norandrogen, 7α-methyl-19-nortestosterone (MENT), or vehicle, with or without dutasteride (DUT), an inhibitor of 5α-reductases types 1 and 2. In rats treated with T + DUT, serum T was significantly higher ( P < 0.05) than in rats treated with T alone, and serum DHT was decreased ( P < 0.001) to levels observed in castrate vehicle-treated rats. DUT significantly reduced both VP and SV weights in T-treated rats, whereas there was no significant effect of DUT on weights of these accessory sex glands in rats treated with DMA, MNT, DHT, or MENT. These results indicate that inhibition of 5α-reductase activity in vivo does not affect the androgenic potency of DMA, MNT, or MENT.

Detailed functional studies on androgen receptor mild mutations demonstrate their association with male infertility

Mutations in the androgen receptor (AR) gene can cause the androgen insensitivity syndrome (AIS). For complete and severe partial AIS, well-characterized in vitro functional assays can be used for genotype-phenotype correlation; however, for mild forms of AIS, as associated with male infertility, experimental evidence is scarce or lacking. In particular, optimal in vitro functional tests informative about the genotype-phenotype relation have not been described. The objective of this study was to investigate the association among genotype and phenotype for AR mutations found in infertile males by conventional functional assays and additional in-depth studies performed with several gene reporters. To this aim, we selected four AR missense mutations associated with isolated male infertility (L547F and two novel mutations A474V and S650G) or partial AIS (Y571H). After introduction of the specific mutations in AR expression plasmid, we performed classical in vitro studies (Western immunoblotting, electrophoretic mobility shift assay, hormone-response curves) and transactivation assays with different reporter constructs (MMTV, Sc-ARU-TK, TAT-GRE- 2X, Slp-ARU-TK and PEM). Our results showed that standard functional tests provide sufficient information only for severe AR mutations, whereas for AR mutations found in mild AIS patients with male infertility, only an extensive analysis with different in vitro systems, and in particular with PEM promoter, can give information on the functionality of the AR and therefore on the pathogenicity of the mutations and on genotype-phenotype correlation.

The A645D Mutation in the Hinge Region of the Human Androgen Receptor (AR) Gene Modulates AR Activity, Depending on the Context of the Polymorphic Glutamine and Glycine Repeats

Sufficient androgen receptor (AR) activity is crucial for normal male sexual differentiation. Here we report on two unrelated 46, XY patients suffering from undervirilization and genital malformations. Both patients had a short polyglycine (polyG) repeat of 10 residues and a relatively long polyglutamine (polyQ) repeat of 28 and 30 residues within the transactivation domain of the AR. In addition, they also harbor a rare A645D substitution. We made a set of AR expression plasmid constructs with varying polyQ and polyG tract sizes in context with or without the A645D substitution and analyzed their in vitro transactivation capacity in transfected CHO cells. We found that a short polyG repeat downmodulated AR activity to approximately 60-65% of the wild-type receptor. This effect was aggravated by A645D in context of a long polyQ repeat to less than 50% activity. In contrast, in the context of a short polyQ and a short polyG repeat, the A645D mutation rescues AR activity to almost wild-type levels, demonstrating a contradictory effect of this mutation, depending on the size of the polymorphic repeats. A combination of a short polyG repeat with a long polyQ repeat and an A645D substitution might contribute to the development of virilization disorders and explain the observed phenotypes of our patients as a form of androgen insensitivity. The whole recreation of AR sequence variations including individual polymorphic repeat sizes could unravel possible interference of mutations and variations on AR activity by in vitro transfection.

Dehydroepiandrosterone and its metabolites: Differential effects on androgen receptor trafficking and transcriptional activity

Dehydroepiandrosterone (DHEA) is a multi-functional steroid that has been implicated in a broad range of biological effects in humans and rodents. Recent studies demonstrated that DHEA acts genomically through the androgen receptor (AR) in addition to its well-known effects on cell surface receptors. However, the relative contribution of DHEA and its major metabolites, including DHEA-Sulfate (DHEA-S), 7α-OH-DHEA, 7β-OH-DHEA, 7-oxo-DHEA, androstenedione (Adione), and androstenediol (Adiol), in the production of genomic effects remains controversial, in part because the metabolism of DHEA varies in different cells and tissues. In the current study, the ability of DHEA and its metabolites to promote AR intracellular trafficking and regulate AR-mediated reporter gene expression, which are characteristic effects of androgens, was determined. Intracellular trafficking of AR-GFP protein was assessed in COS-7 cells while AR transcriptional activity was tested in CV-1 cells transiently co-transfected with AR expression plasmid and an MMTV-ARE-CAT reporter. The results demonstrated that DHEA, the 3β-HSD metabolite Adione, and the 17β-HSD metabolite Adiol, were androgenic. Each promoted AR-GFP intracellular trafficking, the formation of nuclear clusters, and AR-dependent transcriptional activity in a dose-dependent manner. In contrast, DHEA-S, 7α-OH-DHEA, 7β-OH-DHEA, and 7-oxo-DHEA were ineffective and exhibited minimal androgenic activity, even at relatively high concentrations (10 −6 M). These results provide the first systematic comparison of the (i) androgenic activity of DHEA and its sulfated and hydroxylated metabolites, (ii) relative androgenicity of DHEA itself vs. the established androgens Adione and Adiol, and (iii) ability of DHEA and its major metabolites to promote AR-GFP intracellular trafficking. In addition to partitioning DHEA and its metabolites into compounds with (DHEA, Adione, Adiol) and without (DHEA-S, 7α-OH-DHEA, 7β-OH-DHEA, and 7-oxo-DHEA) androgenic activity, the findings improve our understanding of the intracellular processes mediating the genomic effects of DHEA through AR.

Mutations in the Amino-Terminal Domain of the Human Androgen Receptor may be Associated with Partial Androgen Insensitivity and Impaired Transactivation in vitro

The majority of genetic variations in the androgen receptor (AR) gene are point mutations leading to impairment of the DNA- or hormone-binding domains. The N-terminus encoded by the first exon of the AR-gene usually harbors disruptive mutations associated with complete androgen insensitivity syndrome (CAIS) while missense mutations related with partial androgen insensitivity syndrome (PAIS) are seemingly rare. We present a 46,XY male with scrotal hypospadias in whom we detected a S432 F point mutation within the N-terminus. Transient transfections of an AR expression plasmid carrying the S432 F mutation using Chinese Hamster Ovary (CHO) cells revealed a significant partial reduction in transactivation of the co-transfected androgen responsive (ARE) (2)TATA luciferase reporter gene thus confirming PAIS. In two further 46, XY patients with slight to moderate virilization defects, we detected an S411 N mutation, and a 9 base pair deletion leading to the loss of amino acids 409 to 411 (L-A-S), respectively. These mutations did not compromise AR-function under the chosen experimental settings. The S432 F-patient supports particular significance of the AR-N-terminus for mild forms of AIS while the functional role of the two further mutations remains unclear. The N-terminus is a species-specific AR-domain possibly also involved in contributing to target tissue selectivity of AR-actions via mediating co-regulator interactions. Therefore, mild molecular defects of the AR-N-terminus may not necessarily inhibit general transactivation properties using currently established reporter gene models.

Effect of a short CAG (glutamine) repeat on human androgen receptor function.

The human androgen receptor (AR) gene contains an uninterrupted CAG repeat that is polymorphic in length in the general population (range, 11-31 CAG's; median, 21). The CAG repeat encodes a glutamine repeat in the N-terminal transactivation domain of the AR protein. We previously reported that a 17-CAG AR gene was much more common in a cohort of men with prostate cancer (8.5%) than in the general European American population (1.3%). This suggested that a 17-CAG repeat may have pathophysiological consequences. The goal of the present study was to directly test the hypothesis that a 17-CAG repeat might uniquely affect androgen action in human prostate cancer cells. DU145 cells, lacking endogenous AR, were transiently transfected with an AR expression plasmid (with a CAG repeat ranging in length from 14 to 25) and an androgen-responsive reporter plasmid (PSA-luciferase). We found a significant effect of CAG repeat length on AR protein levels per unit amount of DNA transfected (one-way ANOVA, P = 0.02), indicating the need to express transactivation data per unit amount of AR protein. CAG17 AR had 40% more transactivation activity per unit amount of AR protein than CAG21 AR (P < 0.01). Thus, an AR with a 17-CAG repeat may mediate more efficacious growth stimulation of androgen-dependent prostate epithelial cells, and thereby increase the risk that prostate cancer cells develop more efficiently into a clinically significant cancer.

Transactivation of glucocorticoid-inducible rat aryl sulfotransferase (SULT1A1) gene transcription.

The purpose of the current study was to establish the role of the glucocorticoid receptor (GR) and androgen receptor (AR) transcription factors in the transactivation of rat aryl sulfotransferase (SULT1A1) gene transcription and to identify the functional hormone-responsive element(s) in the SULT1A1 gene. A cis-acting inverted repeat with three intervening bases (IR3) was identified in the 5'-flanking of the SULT1A1 gene that mediates the transactivation of SULT1A1 gene transcription by both the GR and AR. CV-1 cells were cotransfected with SULT1A1-luciferase reporter plasmids and either wild-type or mutant GR or AR expression constructs. In cotransfectants expressing the wild-type GR, treatment with triamcinolone acetonide produced an approximately 4- to 6-fold induction of luciferase activity in IR3-containing SULT1A1 reporter plasmids. IR3-containing SULT1A1 reporter constructs were also activated by treatment with the synthetic androgen R1881 in cells cotransfected with wild-type but not mutant AR. In primary cultured rat hepatocytes, androgen-inducible expression of IR3-containing SULT1A1 reporter plasmids required cotransfection with AR expression plasmid. Targeted disruption of the SULT1A1 IR3 by mutation of a conserved GT sequence in the 3' half-site of the element ablated GR and AR responsiveness. These results indicate that a proximal IR3 element in the 5'-flanking region of the rat SULT1A1 gene is sufficient for the transactivation of SULT1A1 gene transcription by the GR and AR, and that relative to the GR, functional AR activity is reduced in primary cultured rat hepatocytes.

Phosphorylation/Dephosphorylation of Androgen Receptor as a Determinant of Androgen Agonistic or Antagonistic Activity

Protein phosphorylation/dephosphorylation is an important posttranslational modification that plays a critical role in signal transduction. The androgen receptor (AR) is under such control. We demonstrate that androgen receptor phosphorylation determines whether or not AR ligands perform as agonists or antagonists in LNCaP cells. Androgen receptor ligands (such as dihydrotestosterone and β-estradiol) stimulate receptor expression and phosphorylation and, as a result, they act as agonists or partial agonists. In contrast, agents such as bicalutamide and estramustine inhibit the receptor phosphorylation and act as antagonists. This model is supported by gene expression and transactivation assays. Significant increases in levels of both mRNA and protein of prostate-specific antigen (PSA), a natural AR target gene, occur following the treatment of LNCaP cells with DHT, β-estradiol, or hydroxyflutamide. In contrast, exposure of LNCaP cells to bicalutamide or estramustine results in a sharp decrease of PSA expression. Agonistic or antagonistic effect of these compounds on PSA expression parallels the level of phosphorylated, but not dephosphorylated androgen receptors. These agonistic or antagonistic effects are also observed in HeLa cells transfected with wild-type AR expression plasmid (pAR0) and AR-driven luciferase expression plasmid GRE-tk-LUC in the presence of different groups of AR blockers. Our data indicate that the functional status of androgen receptors is strongly correlated with the phosphorylation status of the receptors, and that the phosphorylated androgen receptor is the form of the receptor transcriptionally active in regulation. Thus the androgen receptor phosphorylation/dephosphorylation may serve as a new molecular target for screening androgen antagonists for the treatment of prostate cancer.

Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme.

Androgen receptor (AR) plays a key role in cell growth both in the normal prostate and in prostate cancer. Androgen ablation and prolonged antiandrogen therapy can give rise to AR-dependent prostate tumors, which nonetheless can grow in the androgen-deprived milieu. Here we describe the ribozyme approach to selectively degrading the AR mRNA and thereby inhibiting AR function. A trans-acting hammerhead ribozyme was designed to cleave the rat AR mRNA at the position +1827/ 1828, a region predicted to be minimally involved in generating stable secondary structures. Using AR mRNA fragments as substrates, it was established that this ribozyme can specifically cleave the RNA target in a sequence-specific manner. Kinetic experiments determined a Km for the substrate of 77 nM and a kcat/Km value of 1.8 x 10(7) M(-1) x min(-1), suggesting a catalytic efficiency similar to that of protein enzymes such as the relatively nonspecific ribonuclease A and a sequence-specific endonuclease EcoRI. Transient cotransfections of prostate-derived PC3 cells with three plasmids, an AR-inducible chloramphenicol acetyltransferase (CAT) reporter, an AR expression vector, and a ribozyme expression vector, showed that the ribozyme was capable of reducing the functional activity of AR. At an equimolar ratio of the AR expression plasmid to ribozyme expression plasmid, androgen-inducible CAT activity was inhibited 70%. Similar extents of inhibition were also observed at the cellular mRNA level using ribonuclease protection assays, indicating that the ribozyme functioned as an AR mRNA cleaving enzyme in cellulo. Immunocytochemical examination revealed a decline of AR immunoreactivity in ribozyme-transfected cells. In addition, no morphologically detectable cellular abnormalities were associated with ribozyme expression, indicating the absence of deleterious side effects. These results offer a new avenue for the control of AR function and cell growth, especially in the case of androgen-resistant, but AR-dependent, prostate cancer cells.

Tissue-specific and Androgen-repressible Regulation of the Rat Dehydroepiandrosterone Sulfotransferase Gene Promoter

Dehydroepiandrosterone sulfotransferase (Std) catalyzes sulfonation of androgenic steroids and certain aromatic procarcinogens. In rats, this enzyme is selectively expressed in the liver, and its expression is strongly repressed by androgens. DNase I footprinting and electrophoretic mobility shift analyses revealed two hepatocyte nuclear factor-1 (HNF1), three CCAAT/enhancer-binding protein (C/EBP), and one consensus palindromic thyroid hormone response elements within the first 215 base pairs (bp) of the promoter sequence of rat Std. This promoter is normally inactive in fibroblast-derived NIH 3T3 cells. However, overexpression of HNF1 and C/EBP resulted in synergistic activation of the Std promoter in this cell type, indicating essential roles of these two trans-regulators in liver-selective expression of the rat Std gene. On the other hand, point mutations at any one of five cis elements proximal to the -215 bp region markedly reduced reporter gene expression, suggesting that all of these sites are important for overall promoter function. Androgenic repression of the Std gene in rat liver can be recapitulated in androgen receptor (AR)-negative HepG2 hepatoma cells after cotransfection with an AR expression plasmid. Functional assay of a nested set of 5'-deleted promoters mapped the negative androgen response region between positions -235 and -310. Antibody supershift and oligonucleotide competition identified three OCT-1 and two C/EBP elements between bp -231 and -292. An additional OCT-1 site was found to overlap with a C/EBP element at the -262/-252 position. Mutational inactivation of any one of five cis elements within the -231/-292 region abolished negative androgen response. However, none of these cis elements showed DNase I protection by recombinant AR in footprinting assay, suggesting the absence of a direct AR-DNA interaction. Thus, these studies on rat Std promoter function indicate that (i) HNF1 and C/EBP are responsible for liver specificity of the rat Std gene; (ii) androgenic repression of the gene requires the presence of all of the OCT-1 and C/EBP elements between positions -231 and -292; and (iii) AR may exert its negative regulatory effect indirectly through transcriptional interference of OCT-1 and C/EBP rather than through a direct DNA-AR interaction.

Repression of cAMP-induced expression of the mouse P450 17 alpha-hydroxylase/C17-20 lyase gene (Cyp17) by androgens.

In primary cultures of mouse Leydig cells, testosterone represses the cAMP-induced de novo synthesis of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) protein and the accumulation of P450c17 mRNA, via an androgen receptor (AR)-mediated mechanism. To examine the mechanism by which androgens repress the cAMP-induced expression of the mouse Cyp17 gene, constructs containing 5'-flanking sequences of the mouse Cyp17 linked to the chloramphenicol acetyltransferase (CAT) reporter gene were cotransfected into MA-10 tumor Leydig cells with a mouse AR expression plasmid. In the presence of dihydrotestosterone, the cAMP-induced expression of a reporter construct containing -1021 bp of Cyp17 promoter sequences was repressed. In contrast, no repression by dihydrotestosterone was observed when the -1021 bp Cyp17-CAT construct was cotransfected with a human AR expression plasmid missing the second zinc finger of the DNA-binding domain, indicating that DNA binding is involved in AR-mediated repression of Cyp17 expression. Analysis of deletions -346 bp of 5'-flanking region of the mouse Cyp17 promoter are sufficient to confer androgen repression of the cAMP-induced expression of Cyp17. Deoxyribonuclease I footprinting analysis indicated that the AR interacts with sequences between -330. and -278 bp of the Cyp17 promoter. This region overlaps with the previously identified cAMP-responsive region located between -346 and -245 bp of the Cyp17 promoter. These results suggest that AR-mediated repression involves binding of the AR to sequences in the cAMP-responsive region of the Cyp17 promoter, possibly interfering with the binding of the protein(s) that mediate cAMP induction of Cyp17.

Regulation of interleukin-6, osteoclastogenesis, and bone mass by androgens. The role of the androgen receptor.

Interleukin-6 is an essential mediator of the bone loss caused by loss of estrogens. Because loss of androgens also causes bone loss, we have examined whether the IL-6 gene is regulated by androgens, and whether IL-6 plays a role in the bone loss caused by androgen deficiency. Both testosterone and dihydrotestosterone inhibited IL-6 production by murine bone marrow-derived stromal cells. In addition, testosterone, dihydrotestosterone, and adrenal androgens inhibited the expression of a chloramphenicol acetyl transferase reporter plasmid driven by the human IL-6 promoter in HeLa cells cotransfected with an androgen receptor expression plasmid; however, these steroids were ineffective when the cells were cotransfected with an estrogen receptor expression plasmid. In accordance with the in vitro findings, orchidectomy in mice caused an increase in the replication of osteoclast progenitors in the bone marrow which could be prevented by androgen replacement or administration of an IL-6 neutralizing antibody. Moreover, bone histomorphometric analysis of trabecular bone revealed that, in contrast to IL-6 sufficient mice which exhibited increased osteoclast numbers and bone loss following orchidectomy, IL-6 deficient mice (generated by targeted gene disruption) did not. This evidence demonstrates that male sex steroids, acting through the androgen-specific receptor, inhibit the expression of the IL-6 gene; and that IL-6 mediates the upregulation of osteoclastogenesis and therefore the bone loss caused by androgen deficiency, as it does in estrogen deficiency.

Stimulation of androgen-regulated transactivation by modulators of protein phosphorylation.

The effect of modulators of protein phosphorylation on the transcriptional activity of the androgen receptor (AR) was studied under transient expression conditions. Activators of protein kinase-A [8-bromo-cAMP (8-Br-cAMP)] and protein kinase-C (phorbol 12-myristate 13-acetate) or an inhibitor of protein phosphatase-1 and -2A (okadaic acid) influenced minimally pMMTV-chloramphenicol acetyl-transferase (CAT) activity in CV-1 cells cotransfected with an AR expression plasmid in the absence of androgen. In the presence of testosterone, however, all compounds enhanced AR-mediated transactivation by 2- to 4-fold. A nonsteroidal antiandrogen, Casodex, behaved as a pure antagonist; it blunted the action of testosterone and was not rendered agonistic by activators of protein kinase-A. A reporter plasmid containing two androgen response elements (AREs) in front of the thymidine kinase promoter (pARE2tk-CAT) was also used to examine promoter specificity. It was activated by 8-Br-cAMP, forskolin, or okadaic acid even without AR or androgen. However, when forskolin or okadaic acid was used together with androgen and AR, the resulting AR-dependent transactivation of pARE2tk-CAT was more than additive. Intact DNA- and ligand-binding domains, but not the N-terminal amino acid residues 40-147, of the receptor were mandatory for the synergism between protein kinase-A activators and androgen. Immunoreactive AR content in transfected COS-1 cells was not influenced by exposure to 8-Br-cAMP. Similar results were obtained by ligand binding assays. Quantitative or qualitative differences were not observed in DNA-binding characteristics between receptors extracted from cells treated with testosterone with or without protein kinase-A activator. Collectively, the synergistic stimulation of AR-dependent transactivation by androgen and protein kinase activators is not due to changes in cellular AR content or affinity of the receptor for the cognate DNA element; rather, this phenomenon seems to result from altered interaction of ligand-activated AR with other proteins in the transcription machinery.

The Promoter of the Prostate-Specific Antigen Gene Contains a Functional Androgen Responsive Element

Expression of prostate-specific antigen (PA) mRNA was tested at various time periods after incubation of the human prostate tumor cell line LNCaP with the synthetic androgen R1881. Androgen-stimulated expression was observed within 6 h after addition of R1881 to the cells. Run-on experiments with nuclei isolated from LNCaP cells showed that expression of the PA gene could be regulated by R1881 on the level of transcription. DNase I footprints of the promoter region of the PA gene (-320 to +12) with nuclear protein extracts from LNCaP cells showed at least four protected regions. The protected areas include the TATA-box, a GC-box sequence, and a sequence AGAACAgcaAGTGCT at position -170 to -156, which closely resembles the reverse complement of the consensus sequence GGTACAnnnTGTTCT for binding of the glucocorticoid receptor and the progesterone receptor. Fragments of the PA promoter region were cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression plasmid into COS cells in a transient expression assay. CAT activity of COS cells grown in the presence of 1 nM R1881 was compared to untreated controls. A 110-fold induction of CAT activity was found if a -1600 to +12 PA promoter fragment was used in the construct. By further deletion mapping of the PA promoter a minimal region (-320 to -155) was identified as being essential for androgen-regulated gene expression. Mutation of the sequence AGAACAgcaAGTGCT (at -170 to -156) to AAAAAAgcaAGTGCT almost completely abolished androgen inducibility of the reporter gene constructs. One or more copies of the sequence AGAACAgcaAGTGCT cloned in front of a thymidine kinase promoter-CAT reporter gene confers androgen regulation to the reporter gene. These findings provide strong evidence for transcription regulation of the PA gene by androgens via the sequence AGAACAgcaAGTGCT. Interestingly, in addition to the AGAACAgcaAGTGCT element, an upstream region (-539 to -320) is needed for optimal androgen inducibility of the PA promoter.

Ontogenesis and sexual steroids in Salamandra salamandra

Dehydroepiandrosterone (DHEA) is a multi-functional steroid that has been implicated in a broad range of biological effects in humans and rodents. Recent studies demonstrated that DHEA acts genomically through the androgen receptor (AR) in addition to its well-known effects on cell surface receptors. However, the relative contribution of DHEA and its major metabolites, including DHEA-Sulfate (DHEA-S), 7α-OH-DHEA, 7β-OH-DHEA, 7-oxo-DHEA, androstenedione (Adione), and androstenediol (Adiol), in the production of genomic effects remains controversial, in part because the metabolism of DHEA varies in different cells and tissues. In the current study, the ability of DHEA and its metabolites to promote AR intracellular trafficking and regulate AR-mediated reporter gene expression, which are characteristic effects of androgens, was determined. Intracellular trafficking of AR-GFP protein was assessed in COS-7 cells while AR transcriptional activity was tested in CV-1 cells transiently co-transfected with AR expression plasmid and an MMTV-ARE-CAT reporter. The results demonstrated that DHEA, the 3β-HSD metabolite Adione, and the 17β-HSD metabolite Adiol, were androgenic. Each promoted AR-GFP intracellular trafficking, the formation of nuclear clusters, and AR-dependent transcriptional activity in a dose-dependent manner. In contrast, DHEA-S, 7α-OH-DHEA, 7β-OH-DHEA, and 7-oxo-DHEA were ineffective and exhibited minimal androgenic activity, even at relatively high concentrations (10−6 M). These results provide the first systematic comparison of the (i) androgenic activity of DHEA and its sulfated and hydroxylated metabolites, (ii) relative androgenicity of DHEA itself vs. the established androgens Adione and Adiol, and (iii) ability of DHEA and its major metabolites to promote AR-GFP intracellular trafficking. In addition to partitioning DHEA and its metabolites into compounds with (DHEA, Adione, Adiol) and without (DHEA-S, 7α-OH-DHEA, 7β-OH-DHEA, and 7-oxo-DHEA) androgenic activity, the findings improve our understanding of the intracellular processes mediating the genomic effects of DHEA through AR.