COUP-TF II inhibits the N/C terminal interaction of AR.

<p>(A) Mammalian two-hybrid assay. PPC-1 cells were transfected with 5XGAL4-Luc3 together with or without VP-AR1-660, GAL-AR624-919, and COUP-TF II expression plasmids. Cells were treated with or without 10 nM DHT for 24 h. At least three independent experiments were combined and values represent the mean±SEM. ***, P<0.001. (B) GST pull-down competition assay. Immobilized GST-AR LBD proteins were incubated with [³⁵S] methionine-labeled AR AF1DBDh proteins produced by <i>in vitro</i> translation. For competition analysis, 5 and 10-fold excess of <i>in vitro</i> translated COUP-TF II proteins was added together with radiolabeled AR AF1DBDh proteins. Data are representative of three independent experiments. AF1DBDh: AF1+DBD+hinge region.</p

COUP-TF II-induced AR repression involves neither the mislocalization of AR nor recruitment of HDACs.

<p>(A) Effects of COUP-TF II on the subcellular localization of AR. PPC-1 cells were transfected with RFP-fused AR and GFP-fused COUP-TF II expression plasmids. Twenty-four hours after transfection, the cells were treated with 10 nM DHT or vehicle for 4 h. Fluorescence was analyzed with a laser scanning confocal microscope. The cellular nuclei were stained with DAPI (blue). Data are representative of three independent experiments. (B) HDAC activity is not involved in the COUP-TF II-mediated repression of AR transactivation. PPC-1 cells were transfected as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049026#pone-0049026-g002" target="_blank">Figure 2A</a>. The cells were treated with or without 100 nM TSA or 5 mM NaBut in the presence or absence of 10 nM DHT 24 h prior to harvesting. At least three independent experiments were combined and values represent the mean±SEM. ns, not significant; **, P<0.01; ***, P<0.001.</p

COUP-TF II represses the transactivation of AR.

<p>(A) Dose-dependent inhibition of AR transactivation by COUP-TFs. PPC-1 cells were cotransfected with 350 ng of pPBARE×7-tk-Luc reporter and 50 ng of AR expression plasmid along with increasing concentration (100, 250, and 500 ng) of COUP-TF I or COUP-TF II. Cells were treated with or without 3 nM DHT for 24 h. (B) COUP-TF II-mediated repression of AR transactivation on natural AR-target promoters. PPC-1 cells were transfected as in “A”, with PSA-luc or MMTV-luc reporter. (C) COUP-TF II-mediated repression of endogenous AR transactivation. LNCaP cells were transfected as in “A”, without the AR expression plasmid. (D) Repression of androgen-induced PSA mRNA expression by COUP-TF II. LNCaP cells were infected with AdGFP or AdCOUP-TF II. After 24 h of recovery, the cells were treated with 10 nM DHT, and cultured for another 24 h prior to harvesting. Quantitative RT-PCR analysis was conducted using specific primers for PSA and β-actin. The relative PSA mRNA expression was normalized by β-actin expression. At least three independent experiments were combined and values represent the mean±SEM (A–D). **, P<0.01; ***, P<0.001. (E) Repression of androgen-induced PSA protein expression by COUP-TF II. LNCaP cells were infected and processed as in “D”, and cultured for another 48 h prior to harvesting. Western blot analysis of total proteins was conducted using anti-PSA, anti-AR (sc-815), anti-HA, and anti-α-tubulin. Data are representative of three independent experiments (left). The relative PSA protein expression was quantified by normalizing with tubulin expression (rignt).</p

COUP-TF II inhibits AR recruitment to the PSA promoter and competes with AR coactivators to modulate AR transactivation.

<p>(A) COUP-TF II inhibits the recruitment of AR to PSA enhancer. LNCaP cells were infected with AdCOUP-TF II or AdGFP. After 24 h of infection, cells were treated with 10 nM DHT or vehicle for 6 h, and then harvested for ChIP assays. Anti-AR antibody (PG-21) was used for immunoprecipitation. Immunoprecipitates were analyzed by PCR using a specific primer pair spanning the AR binding site of the PSA enhancer region. A control PCR for non-specific immunoprecipitation was performed using specific primers to the β-actin coding region. (B) AR coactivators relieve the COUP-TF II-mediated repression of AR transactivation. PPC-1 cells were cotransfected with 50 ng of AR, 250 ng of COUP-TF II and 500 ng of AR coactivator expression plasmids. (C) ARA70 relieves COUP-TF II repression of AR transactivation in a dose-dependent manner. PPC-1 cells were transfected as in “B” with increasing concentration (250, 500, and 1000 ng) of ARA70. (D) COUP-TF II represses ARA70-elevated AR transactivation in a dose-dependent manner. PPC-1 cells were transfected with 50 ng of AR, 250 ng of ARA70 and increasing concentration (250, 500, and 1000 ng) of COUP-TF II expression vector. Cells were treated with or without 3 nM DHT for 24 h. At least three independent experiments were combined and values represent the mean±SEM. *, P<0.05;**, P<0.01; ***, P<0.001.</p

COUP-TF II inhibits androgen-dependent proliferation of prostate cancer cells.

<p>(A) COUP-TF II expression in human prostate cancer cell lines. Protein expression levels of COUP-TF II were determined by Western blot analysis of total proteins using anti-COUP-TF II, anti-AR (sc-815), and anti-α-tubulin antibodies. mRNA expression levels of COUP-TF II were determined by RT-PCR of total RNAs. The expression of tubulin and β-actin was used as an internal control. (B) COUP-TF II inhibits the growth of prostate cancer cells. Soft agar colony formation assay was conducted with complete growth medium (left panel) or with medium containing charcoal-stripped serum and supplemented with or without 1 nM DHT (right panel). LNCaP cells were infected with AdGFP or AdCOUP-TF II for 24 h, and were processed for colony formation assay as indicated in “Materials and Methods”. Colonies larger than diameter of 300 mm were scored. Data are representative of three independent experiments. (C) COUP-TF II decreases the rate of DNA synthesis in prostate cancer cells. LNCaP cells were infected with AdGFP or AdCOUP-TF II in medium containing charcoal-stripped serum and supplemented with or without 1 nM DHT. Their DNA synthesis rate was then analyzed by [³H]-thymidine incorporation assay. At least three independent experiments were combined and values represent the mean±SEM. *, P<0.05; **, P<0.01; ***, P<0.001.</p

Smad3 physically interacts with Nur77.

<p>(<b>A</b>) mKG_N-MC-NLS-Smad3 and mKG_C-MC-Nur77 were transfected into HeLa cells for 24 hours. Interaction between Nur77 and NLS-Smad3 yielded fluorescent green signals in the nucleus. The single fusion protein alone (mKG_N-MC-NLS-Smad3 or mKG_C-MC-Nur77) and another pair (mKG_N-MC-NLS-Smad3 and mKG_C-MN-Nur77) gave no fluorescent signal. The scale bars represent 25 µm. (<b>B</b>) [³⁵S] methionine-labeled Smad3 produced by <i>in vitro</i> translation was incubated with the GST-Nur77 fusion protein and its deletion mutants. Coomassie blue staining shows the protein level of the purified GST, GST-Nur77 and GST-Nur77 deletion mutant (bottom). (<b>C</b>) [³⁵S] methionine-labeled Smad3 deletion mutants were incubated with GST-Nur77 fusion protein. The data are representative of three independent experiments.</p

TGF-β1 signaling regulates steroidogenic gene expression, affecting testicular testosterone levels in mice.

<p>(<b>A</b>) Decreased Tgfbr2^fl°^x allele in purified primary Leydig cells isolated from mice harboring the Cyp17iCre transgene. The genomic DNA isolated from primary Leydig cells of Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice was amplified for Tgfbr2 intron region containing the LoxP site. A pair of β-actin primers was used as the control for the amount of genomic DNA. (<b>B</b>) Decreased TGF-β1-mediated repression of steroidogenic gene expression with Tgfbr2 silencing. Purified primary Leydig cells from the testes of 12-week-old Tgfbr2^flox/flox (n = 6) and Tgfbr2^flox/flox Cyp17iCre (n = 6) mice were treated with 300 µM of 8-Br-cAMP and 2 ng/ml of TGF-β1 for 24 hours, and mRNA expression levels were measured using qRT-PCR. β-actin expression was used as a loading control. The data are presented as the mean ± SEM. **, P<0.01; ***, P<0.01. (<b>C</b>) Testicular testosterone levels were measured by RIA in the testes of 5 week-old Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice. (<b>D</b>) Total protein (100 µg) from the testes of 5 week-old Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice was subjected to western blot analysis for protein levels of steroidogenic genes. The relative level of each protein/GAPDH was quantified by densitometric analysis using Image J software. In panels C and D, the data are presented as the mean ± SD (n = 10). **, P<0.01.</p

TGF-β1 signaling interferes with Nur77 binding to DNA.

<p>(<b>A and B</b>) TGF-β1 inhibits the recruitment of Nur77 to the P450c17 promoter. ChIP assays were performed using purified primary Leydig cells treated with 300 µM of 8-Br-cAMP and 10 ng/ml of TGF-β1 for 2 hours (A) and R2C cells treated with 10 ng/ml of TGF-β1 for the indicated time (B). Anti-Nur77 antibody was used for immunoprecipitation. The immunoprecipitates were analyzed by PCR using a pair of specific primers spanning a region containing the Nur77 binding site of the P450c17 promoter. A negative control PCR for nonspecific immunoprecipitation was performed using primers specific to the GAPDH coding region. (<b>C</b>) The interference with Nur77 binding to NBRE by Smad3. The GST-Nur77 fusion protein was incubated with α-³²P-labeled NBRE oligonucleotide, along with increasing amounts of purified GST-Smad3 (lanes 7 and 8) proteins. A 100-fold excess of cold NBRE oligomer (lane 9) or nonspecific oligomer (ARE, lane 10) was added. Positions of the specific protein-DNA complex and the free probe are indicated. The data are representative of three independent experiments.</p

ALK5 signaling inhibits Nur77 transactivation of steroidogenic gene promoters.

<p>(<b>A and B</b>) MA-10 cells were transiently transfected with the ALK5 WT (wild type), ALK5 mutant (TD; constitutively active form or KR; inactive form), and Nur77 expression plasmids, along with the indicated reporter. The CMVβ expression plasmid was used as a control for transfection efficiency. (<b>C</b>) Whole cell extracts and subcellular fractions of primary Leydig cells, which were treated with 300 µM of 8-Br-cAMP and 2.5 ng/ml of TGF-β1 for 4 hours, were analyzed by western blot analysis with anti-Nur77, anti-pSmad3, anti-α-Tubulin (cytoplasmic marker) and anti-Lamin B (nuclear marker) antibodies. (<b>D</b>) MA-10 cells were transiently transfected with scrambled or Nur77 siRNA, ALK5 (TD) expression plasmid and P450c17 promoter reporter (top). Silenced Nur77 protein levels in HEK293T cells, which were transiently transfected with scrambled or Nur77 siRNA, Flag-Nur77 and CMVβ expression vector for 48 hours, were determined by western blot analysis (bottom). The data are presented as the mean ± SEM of at least three independent experiments. **, P<0.01; ***, P<0.001; ns, not significant.</p

ALK5-activated Smad3 represses Nur77 transactivation of steroidogenic gene promoters.

<p>(<b>A</b>) MA-10 cells were transiently transfected with siRNA, Nur77, ALK5 (TD) and an indicated reporter for 48 hours and were harvested for luciferase assay. The CMVβ expression plasmid was used as a control for transfection efficiency (bottom). The silencing efficiencies of Smad2 and Smad3 siRNA were determined by western blot analysis (top). (<b>B</b>) MA-10 cells were transiently transfected with Nur77, increasing amounts of Smad (60 and 150 ng) expression plasmids and the NBRE reporter construct. (<b>C–E</b>) MA-10 cells were transiently transfected with ALK5 WT, ALK5 mutant (TD or KR), Smad3 and Nur77 expression plasmids, along with the indicated reporter construct. (<b>F</b>) MA-10 cells were transiently transfected with expression plasmids of Nur77, ALK5 (TD), Flag-Smad3 (WT) or a phosphorylation mutant (S3A or S3D), and NurRE-luc reporter construct (top). A similar amount of expressed protein was confirmed by western blot analysis (bottom). The data are presented as the mean ± SEM of at least three independent experiments. *, P<0.5; **, P<0.01; ***, P<0.001; ns, not significant.</p