Interaction Of Androgen Receptor Research Articles

Proteasomes Enhance Androgen Receptor Action

Proteasome inhibitors can elicit apoptosis in different cancer cells types. Lin et al. have explored the molecular details of this effect in prostate cancer cell lines where signals mediated by the steroid hormone androgen play a key role in cancer progression. The study reports that proteasome activity is required for androgen receptor (AR) activity. A link between AR activity and the ubiquitin-proteasome pathway had been suspected, as certain AR coregulators contain ubiquitin-protein ligase activity. Treatment of prostate cancer cells with a proteasome inhibitor suppressed AR transactivation and expression of target genes. A block in AR nuclear translocation and AR interaction with certain coregulators suggests that proteasomes may guide AR to its gene targets, as well as mediate interaction with its coregulators. The proteasome inhibitor did not affect the activity of the glucocorticoid receptor nor did it have a general toxic effect on cells. Overexpression of a proteasome core complex increased AR activity. The authors suggest that the ubiquitin-proteasome pathway may play a nonproteolytic role in modulating AR trafficking and recruitment of transcription factors. H. K. Lin, S. Altuwaljri, W. J. Lin, P.-Y. Kan, L. L. Collins, C. Chang, Proteasome activity is required for androgen receptor transcriptional activity via regulation of androgen receptor nuclear translocation and interaction with coregulators in prostate cancer cells. J. Biol. Chem. 277 , 36570-36476 (2002). [Abstract] [Full Text]

Proteasome Activity Is Required for Androgen Receptor Transcriptional Activity via Regulation of Androgen Receptor Nuclear Translocation and Interaction with Coregulators in Prostate Cancer Cells

Upon binding to androgen, the androgen receptor (AR) can translocate into the nucleus and bind to androgen response element(s) to modulate its target genes. Here we have shown that MG132, a 26 S proteasome inhibitor, suppressed AR transactivation in an androgen-dependent manner in prostate cancer LNCaP and PC-3 cells. In contrast, MG132 showed no suppressive effect on glucocorticoid receptor transactivation. Additionally, transfection of PSMA7, a proteasome subunit, enhanced AR transactivation in a dose-dependent manner. The suppression of AR transactivation by MG132 may then result in the suppression of prostate-specific antigen, a well known marker used to monitor the progress of prostate cancer. Further mechanistic studies indicated that MG132 may suppress AR transactivation via inhibition of AR nuclear translocation and/or inhibition of interactions between AR and its coregulators, such as ARA70 or TIF2. Together, our data suggest that the proteasome system plays important roles in the regulation of AR activity in prostate cancer cells and may provide a unique target site for the development of therapeutic drugs to block androgen/AR-mediated prostate tumor growth.

β-Catenin's Nuclear Receptor Shuttle

It is unclear how the cytoplasmic protein β-catenin moves into the nucleus to regulate transcription. It lacks a nuclear localization signal, is too large to translocate in a passive manner, and moves independently of the nuclear transport pathways involving importin and Ran. Three studies suggest that transport may occur through association with shuttling nuclear receptors. Yang et al. , Mulholland et al. , and Pawlowski et al. report a direct interaction between β-catenin and the androgen receptor (AR). When activated by ligands such as testosterone, AR moves into the nucleus to stimulate transcription of target genes. AR agonists promoted the translocation and nuclear localization of AR and β-catenin with similar kinetics. Other hormone-bound nuclear receptors, including those for progesterone, estrogen, and glucocorticoid, did not act as shuttles. Moreover, AR antagonists did not induce this effect, indicating that translocation of AR alone is not sufficient to carry β-catenin into the nucleus. The COOH-terminal DNA and ligand-binding domain of the AR and the Armadillo repeats of β-catenin were identified as the associating regions. The Wnt signaling pathway, which controls the balance of nuclear and cytosolic pools of β-catenin in some cells, was not activated by AR in the neuronal cell line examined by Pawlowski et al. Mulholland demonstrated that the translocation did not depend on adenomatous polyposis coli, another potential nuclear shuttle for β-catenin. Yang et al. showed that overexpression of the cell-surface protein E-cadherin in a prostate cancer cell line sequestered β-catenin and reduced AR-mediated transcription. The consequence of AR-β-catenin is uncertain as the reports show either suppression or augmentation of AR-mediated transcription in various reporter assays. Steroid receptors appear to offer another convenient ride for β-catenin's trip to the nucleus. J. E. Pawloski, J. R. Ertel, M. P. Allen, M. Xu, C. Butler, E. M. Wilson, M. E. Wierman, Liganded androgen receptor interaction with β-catenin. J. Biol. Chem. 277 , 20702-20710 (2002). [Abstract] [Full Text] F. Yang, X. Li, M. Sharmu, C. Y. Sasaki, D. L. Longo, B. Lim, Z. Sun, Linking β-catenin to androgen-signaling pathway. J. Biol. Chem. 277 , 11336-11344 (2002). [Abstract] [Full Text] D. J. Mulholland, H. Cheng, K. Reid, P. S. Rennie, C. C. Nelson, The androgen receptor can promote β-catenin nuclear translocation independently of adenomatous polyposis coli. J. Biol. Chem. 277 , 17933-17943 (2002). [Abstract] [Full Text]

-Catenin's Nuclear Receptor Shuttle

It is unclear how the cytoplasmic protein β-catenin moves into the nucleus to regulate transcription. It lacks a nuclear localization signal, is too large to translocate in a passive manner, and moves independently of the nuclear transport pathways involving importin and Ran. Three studies suggest that transport may occur through association with shuttling nuclear receptors. Yang et al., Mulholland et al., and Pawlowski et al. report a direct interaction between β-catenin and the androgen receptor (AR). When activated by ligands such as testosterone, AR moves into the nucleus to stimulate transcription of target genes. AR agonists promoted the translocation and nuclear localization of AR and β-catenin with similar kinetics. Other hormone-bound nuclear receptors, including those for progesterone, estrogen, and glucocorticoid, did not act as shuttles. Moreover, AR antagonists did not induce this effect, indicating that translocation of AR alone is not sufficient to carry β-catenin into the nucleus. The COOH-terminal DNA and ligand-binding domain of the AR and the Armadillo repeats of β-catenin were identified as the associating regions. The Wnt signaling pathway, which controls the balance of nuclear and cytosolic pools of β-catenin in some cells, was not activated by AR in the neuronal cell line examined by Pawlowski et al. Mulholland demonstrated that the translocation did not depend on adenomatous polyposis coli, another potential nuclear shuttle for β-catenin. Yang et al. showed that overexpression of the cell-surface protein E-cadherin in a prostate cancer cell line sequestered β-catenin and reduced AR-mediated transcription. The consequence of AR-β-catenin is uncertain as the reports show either suppression or augmentation of AR-mediated transcription in various reporter assays. Steroid receptors appear to offer another convenient ride for β-catenin's trip to the nucleus.J. E. Pawloski, J. R. Ertel, M. P. Allen, M. Xu, C. Butler, E. M. Wilson, M. E. Wierman, Liganded androgen receptor interaction with β-catenin. J. Biol. Chem. 277, 20702-20710 (2002). [Abstract] [Full Text]F. Yang, X. Li, M. Sharmu, C. Y. Sasaki, D. L. Longo, B. Lim, Z. Sun, Linking β-catenin to androgen-signaling pathway. J. Biol. Chem. 277, 11336-11344 (2002). [Abstract] [Full Text]D. J. Mulholland, H. Cheng, K. Reid, P. S. Rennie, C. C. Nelson, The androgen receptor can promote β-catenin nuclear translocation independently of adenomatous polyposis coli. J. Biol. Chem. 277, 17933-17943 (2002). [Abstract] [Full Text]

Liganded Androgen Receptor Interaction with β-Catenin: NUCLEAR CO-LOCALIZATION AND MODULATION OF TRANSCRIPTIONAL ACTIVITY IN NEURONAL CELLS

A yeast two-hybrid assay was employed to identify androgen receptor (AR) protein partners in gonadotropin-releasing hormone neuronal cells. By using an AR deletion construct (AR-(Delta371-485)) as a bait, beta-catenin was identified as an AR-interacting protein from a gonadotropin-releasing hormone neuronal cell library. Immunolocalization of co-transfected AR and FLAG-beta-catenin demonstrated that FLAG-beta-catenin was predominantly cytoplasmic in the absence of androgen. In the presence of 5alpha-dihydrotestosterone, FLAG-beta-catenin completely co-localized to the nucleus with AR. This effect was specific to AR because liganded progesterone, glucocorticoid, or estrogen alpha receptors did not translocate FLAG-beta-catenin to the nucleus. Agonist-bound AR was required because the AR antagonists casodex and hydroxyflutamide failed to translocate beta-catenin. Time course experiments demonstrated that co-translocation occurred with similar kinetics. Nuclear co-localization was independent of the glycogen synthase kinase-3beta, p42/44 ERK mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways because inhibitors of these pathways had no effect. Transcription assays demonstrated that liganded AR repressed beta-catenin/T cell factor-responsive reporter gene activity. Conversely, co-expression of beta-catenin/T cell factor repressed AR stimulation of AR-responsive reporter gene activity. Our data suggest that liganded AR shuttles beta-catenin to the nucleus and that nuclear interaction of AR with beta-catenin may modulate transcriptional activity in androgen target tissues.

Androgen receptor interactions with Oct-1 and Brn-1 are physically and functionally distinct

POU domain proteins interact positively or negatively with steroid hormone receptors, depending on the precise array of these and other factors assembled on target gene promoters. Octamer transcription factor 1 (Oct-1), a ubiquitous POU factor, is implicated in androgen induction of the mouse sex-limited protein ( Slp) gene based on protein–DNA interaction studies. However, direct evidence for a role of Oct-1 in the hormone response has been difficult to obtain. Brain 1 (Brn-1), another POU factor, is more tissue-specific, expressing in brain and also in kidney, which is a major site of Slp synthesis. We compared the interaction of the androgen receptor (AR) with Oct-1 and Brn-1 to reveal the more likely candidate for regulation of Slp. In transfection, addition of either Oct-1 or Brn-1 reduced AR activation, regardless of the presence of an octamer-like sequence in the enhancer, suggesting interference was indirect. However, when the octamer-like element was changed to a consensus octamer site, Brn-1, but not Oct-1, strongly enhanced androgen activation. This correlated with Brn-l's preference for the consensus octamer sequence in DNA binding assays. Direct interaction of AR with glutathione-S-transferase-(GST)–fused Oct-1 was DNA-dependent, while Brn-l–AR association was not. Chimeric Brn-1 and Oct-1 POU domains demonstrated that the DNA-dependent AR interaction relied on the origin of the POU homeodomain. However, in the context of full-length Brn-1 and Oct-1 chimeric proteins, the POU homedomain was not sufficient to confer the distinct behaviors of these factors in vivo, but instead revealed the importance of an N-terminal transactivation domain in Brn-1. These results demonstrate that functional interaction of Oct-1 and Brn-1 with AR is determined by the precise sequence of the octamer binding site, and by differential interaction of the POU factors with AR and other components of the transcriptional machinery.

Tip60 Is a Co-activator Specific for Class I Nuclear Hormone Receptors

The nuclear hormone receptor superfamily is composed of a group of hormone-dependent transcription factors that play prominent roles in homeostatic events in vertebrates. A prerequisite for steroid hormone receptor activity is the binding of co-activator molecules to the activation function-2 domain of the receptor. The LXXLL motif/nuclear receptor box, contained within a number of co-activator molecules, mediates the interaction with nuclear hormone receptors. Tip60 (Tat-interactive protein 60 kDa), previously shown to bind to and enhance androgen receptor (AR)-mediated transactivation, contains a single nuclear receptor box at its extreme C terminus. We demonstrate that unlike members of the p160 co-activator family that interact predominantly with the N terminus of the AR in an LXXLL motif-independent manner, the LXXLL motif of Tip60 is required and is sufficient for AR interaction. Furthermore, by using the mammalian two-hybrid system and transient transfection experiments, we show that Tip60 preferentially interacts with and up-regulates class I nuclear receptors, suggesting that Tip60 is a steroid hormone receptor-specific co-activator. We conclude that Tip60 may specifically regulate a subset of nuclear hormone receptors, giving an indication to how regulated nuclear receptor activation can be achieved.

Induction of Apoptosis by Protein Inhibitor of Activated Stat1 through c-Jun NH2-terminal Kinase Activation

Members of the protein inhibitor of activated signal transducer and activator of transcription (STAT) family (PIAS family) of proteins act as negative regulators of STATs in cytokine signaling. We report here that PIAS proteins have proapoptotic activity. PIAS1 induced apoptosis in both human 293T cells and human osteosarcoma U2OS cells. PIAS1 is localized in the nucleus as distinct nuclear dots. Ectopic expression of PIAS1 in U2OS cells activated JNK1 (c-Jun NH(2)-terminal kinase). A dominant-negative JNK1, capable of inhibiting PIAS1-induced JNK1 activation, blocked PIAS1-mediated apoptosis. Furthermore, a mutant PIAS1, lacking the first 9 amino acid residues, failed to repress Stat1-mediated gene activation although it retained its ability to activate JNK and to trigger apoptosis. Our results identify a novel function of PIAS1 in the induction of JNK-dependent apoptosis, independent of the previously known inhibitory activity of PIAS1 in STAT-mediated gene activation.

Inhibition of androgen receptor-mediated transcription by amino-terminal enhancer of split.

A yeast two-hybrid assay has identified an androgen-dependent interaction of androgen receptor (AR) with amino-terminal enhancer of split (AES), a member of the highly conserved Groucho/TLE family of corepressors. Full-length AR, as well as the N-terminal fragment of AR, showed direct interactions with AES in in vitro protein-protein interaction assays. AES specifically inhibited AR-mediated transcription in a well-defined cell-free transcription system and interacted specifically with the basal transcription factor (TFIIE) in HeLa nuclear extract. These observations implicate AES as a selective repressor of ligand-dependent AR-mediated transcription that acts by directly interacting with AR and by targeting the basal transcription machinery.

Disrupted amino- and carboxyl-terminal interactions of the androgen receptor are linked to androgen insensitivity.

We have compared the functional consequences of seven single-point mutations in the ligand-binding domain (LBD) of the androgen receptor (AR). The mutations span helices 3 to 11 and are present in patients suffering from androgen insensitivity syndromes (AIS) and other male-specific disorders. The mutants, except M742V, bound to androgen response elements in vivo and in vitro and showed a testosterone-dependent conformational change. With regard to functional activity, the mutant M742V had severely blunted ability to transactivate or exhibit the androgen-dependent amino/carboxyl-terminal (N/C) interaction; mutants F725L, G743V, and F754L showed reduced transactivation potential and attenuated N/C interaction; and mutants V715M, R726L, and M886V had minor functional impairments. The mutants belonging to the first two groups also displayed reduced response to coexpressed GRIP1. In addition, mutations of amino acids M894 and A896 in the putative core activation domain 2 (AF2) in helix 12 confirmed that this helix is important for N/C interactions. Thus, amino acids located between helices 3 and 4 (F725 and R726), in helix 5 (M742, G743, and F754), and in helix 12 (M894 and A896) play critical roles in mediating the N/C interaction of AR. The data also show that disrupted N/C interaction is a potential molecular abnormality in AIS cases in which LBD mutations have not resulted in markedly impaired ability to bind androgen.

Disrupted Amino- and Carboxyl-Terminal Interactions of the Androgen Receptor Are Linked to Androgen Insensitivity

Disrupted Amino- and Carboxyl-Terminal Interactions of the Androgen Receptor Are Linked to Androgen Insensitivity

Coregulator Small Nuclear RING Finger Protein (SNURF) Enhances Sp1- and Steroid Receptor-mediated Transcription by Different Mechanisms

The small nuclear RING finger protein SNURF is not only a coactivator in steroid receptor-dependent transcription but also activates transcription from steroid-independent promoters. In this work, we show that SNURF, via the RING finger domain, enhances protein binding to Sp1 elements/GC boxes and interacts and cooperates with Sp1 in transcriptional activation. The activation of androgen receptor (AR) function requires regions other than the RING finger of SNURF, and SNURF does not influence binding of AR to cognate DNA elements. The zinc finger region (ZFR) together with the hinge region of AR are sufficient for contacting SNURF. The nuclear localization signal in the boundary between ZFR and the hinge region participates in the association of AR with SNURF, and a receptor mutant lacking the C-terminal part of the bipartite nuclear localization signal shows attenuated response to coexpressed SNURF. Some AR ZFR point mutations observed in patients with partial androgen insensitivity syndrome or male breast cancer impair the interaction of AR with SNURF and also render AR refractory to the transcription-activating effect of SNURF. Collectively, SNURF modulates the transcriptional activities of androgen receptor and Sp1 via different domains, and it may act as a functional link between steroid- and Sp1-regulated transcription.

Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA.

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.

Multiple Androgen Response Elements and a Myc Consensus Site in the Androgen Receptor (AR) Coding Region Are Involved in Androgen-Mediated Up-Regulation of AR Messenger RNA

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5′-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.

Promotion of agonist activity of antiandrogens by the androgen receptor coactivator, ARA70, in human prostate cancer DU145 cells.

Although hormone therapy with antiandrogens has been widely used for the treatment of prostate cancer, some antiandrogens may act as androgen receptor (AR) agonists that may result in antiandrogen withdrawal syndrome. The molecular mechanism of this agonist response, however, remains unclear. Using mammalian two-hybrid assay, we report that antiandrogens, hydroxyflutamide, bicalutamide (casodex), cyproterone acetate, and RU58841, and other compounds such as genistein and RU486, can promote the interaction between AR and its coactivator, ARA70, in a dose-dependent manner. The chloramphenicol acetyltransferase assay further demonstrates that these antiandrogens and related compounds significantly enhance the AR transcriptional activity by cotransfection of AR and ARA70 in a 1:3 ratio into human prostate cancer DU145 cells. Our results suggest that the agonist activity of antiandrogens might occur with the proper interaction of AR and ARA70 in DU145 cells. These findings may provide a good model to develop better antiandrogens without agonist activity.

Interaction of Androgen Receptors with Androgen Response Element in Intact Cells: ROLES OF AMINO- AND CARBOXYL-TERMINAL REGIONS AND THE LIGAND

Promoter interference assay was employed to examine in intact cells the roles of the functional domains of androgen receptor (AR) and the ligand for specific DNA interactions using a cytomegalovirus-(androgen response element)-chloramphenicol acetyltransferase reporter (pCMV-ARE2-CAT). Native rat and human ARs interfered with pCMV-ARE2-CAT expression in a hormone-dependent fashion. Low steroid-independent interference seemed to occur because of the ligand binding domain (LBD), which was transcriptionally inhibitory also in a heterologous context. AR devoid of LBD (rARDelta641-902) decreased pCMV-ARE2-CAT activity by 50%. The rARDelta46-408 mutant devoid of the NH2-terminal transcription activation region exhibited ligand-dependent promoter interference of a similar magnitude. Ligand and DNA binding-deficient mutants (hARM807R and rARC562G, respectively) did not influence pCMV-ARE2-CAT expression, although hARM807R binds to ARE in vitro. Non-steroidal anti-androgens casodex and hydroxyflutamide antagonized agonist-dependent promoter interference, whereas cyproterone acetate, RU 56187, RU 57073, and RU 59063 were partial agonists/antagonists. Collectively, interaction of ARs with ARE in intact cells does not require the presence of the COOH-terminal or NH2-terminal domain and/or their interaction. In the context of native AR, however, the androgen-induced conformational change in LBD is mandatory for generation of a transcriptionally competent receptor that binds to DNA in intact cells.

Evidence for an Anti-parallel Orientation of the Ligand-activated Human Androgen Receptor Dimer

Domain interactions of the human androgen receptor (AR) dimer were investigated using a protein-protein interaction assay in which the NH2- and carboxyl-terminal regions of human AR were fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and herpes simplex virus VP16 transactivation domain to produce chimeric proteins. Transcriptional activation of a GAL4 luciferase reporter vector up to 100-fold was greater than Fos/Jun leucine zipper binding, indicating stable AR interaction between AR NH2-terminal residues 1-503 and steroid-binding domain residues 624-919 that was specific for and dependent on androgen binding to the steroid-binding domain and was inhibited by anti-androgen binding. Deletion mutagenesis within the NH2-terminal region indicated transactivation domain residues 142-337 were not required for dimerization, whereas deletions near the NH2 terminus (delta 14-150) or NH2-terminal to the DNA-binding domain (delta 339-499) reduced or eliminated the AR interaction, respectively. An NH2-/NH2-terminal interaction was also observed, but no interaction was detected between ligand-free or bound steroid-binding domains. The results indicate that high affinity androgen binding promotes interactions between the NH2-terminal and steroid-binding domains of human AR, raising the possibility of an androgen-induced anti-parallel AR dimer.

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.

In vivo upregulation of adipocyte alpha 2-adrenoceptors by androgens is consequence of direct action on fat cells.

Adipose precursor cells from male hamsters were exposed to testosterone in primary culture. The effect of the sex steroid on alpha 2-adrenoceptor (AR) expression was studied. Testosterone enhanced alpha 2-AR expression during adipose conversion to an identical extent to that determined in vivo without modification of the differentiation state as estimated by glycerol-3-phosphate dehydrogenase activity. The expression of preadipocyte alha 2-ARs from female hamster was also submitted to the testosterone control. The upregulating effect was androgen specific, since only testosterone and dihydrotestosterone were efficient in inducing an increase of alpha 2-AR density, whereas estradiol, dexamethasone, progesterone, and androstenedione were without any action. To conclude, androgens act directly on fat cells by upregulating alpha 2-AR expression. The effects are not mediated by aromatization into estrogens and probably involve androgen receptor interactions.

Immunolocalization of Androgen Receptor in the Small, Preovulatory, and Postovulatory Follicles of Laying Hens

Previous studies have indicated that androgens may act directly on the ovarian follicles to regulate their functions. The aim of this study was to localize androgen receptor (AR) in the small, preovulatory and postovulatory follicles of laying hens by an immunocytochemical method and Western blot analysis. Small follicles embedded in the stroma (SF), small white follicles protruding from the surface of ovary (SWF), the third largest (F3) and largest follicles (F1), and the most recent postovulatory follicle (POF) were obtained from hens approximately 4 or 10 hr before the expected time of ovulation. Frozen sections of these follicles were immunostained by using anti-human AR antibody. Furthermore, the granulosa cells and theca tissue of preovulatory follicles (F1 and F2) approximately 4 hr before the expected time of ovulation were processed for western blot analysis for AR. The majority of granulosa cell of SF showed negligible AR immunoreaction, whereas all of the granulosa cells of SWF, F3, F1, and POF exhibited a strong AR immunoreaction. Thecal interstitial ceils in SWF, F3, and F1 stained positive for AR, and those in POF showed only a weak immunoreaction. The thecal fibroblasts of SWF, F3, and F1 also showed a positive AR immunoreaction, whereas those of POF stained weakly. No significant difference in the AR localization in the ovary was observed between 4 and 10 hr before the expected time of ovulation. Western blot analysis indicated that the granulosa cells and thecal tissue contained AR protein of molecular weight of approximately 120,000. These results suggest that ovarian tissues are target sites of androgens and the interaction of AR with androgens may have a role in regulation of the functions of the follicles.