Figure 1.
Differential effects of DHT in LNCaP and C4-2B cells: AR transactivation, and nuclear AR, p65-Nrf1 & Nrf2 levels.
(A). Cells were cotransfected with psPSA-luc and pRL-TK (internal control). The effect of 24 hrs stimulation with either 1 nM or 10 nM DHT on fold changes in luciferase activity (firefly/renilla RLU) are shown (n = 3). DHT-induced AR transactivation is significantly (***; p<0.001) higher in C4-2B cells as compared to LNCaP cells. (B). DHT-induced AR nuclear localization in LNCaP and C4-2B cells. Following 24 hrs of DHT (0, 1 and 10 nM) stimulation (n = 4) western immunoblots show changes in AR nuclear levels. Both C4-2B and LNCaP cells showed similar levels of nuclear AR following DHT-stimulation. (C). p65-Nrf1 and Nrf2 levels following DHT stimulation. Western immunoblots showing nuclear p65-Nrf1 and Nrf2 levels following 24 hr of DHT stimulation (n = 3). Differences in nuclear p65-Nrf1 and Nrf2 were observed in LNCaP and C4-2B cells. Fold changes and ±SEM values represent relative differences in the expression of AR, p65-Nrf1, and Nrf2. In both (B) and (C), data were normalized to TBP levels in each sample.
Figure 2.
Effect of Nrf1 modulation on DHT-induced AR transactivation.
(A) Effect of Nrf1 knockdown on AR transactivation in DHT-stimulated C4-2B cells. Cells were co-transfected with the psPSA-luc vector and with either the Nrf1 siRNA or control siRNA (NC1). Fold changes in luciferase activity (firefly/renilla RLU) following Nrf1 knockdown are shown (n = 3; p<0.01). (B) Nuclear p65-Nrf1 protein levels after siRNA mediated knockdown in C4-2B cells (n = 2). Data were normalized to TBP levels. (C) Effect of p65-Nrf1 overexpression on DHT-induced AR transactivation in LNCaP cells. Cells were co-transfected with the psPSA-luc vector and with either the control vector (pcDNA3.1) or the p65-Nrf1 expression vector (p65-Nrf1-V5-His). Fold change in luciferase activity following Nrf1 overexpression are shown (n = 3; p<0.001). (D) Changes in nuclear V5 protein (tag) in pcDNA3.1 (control) or p65-Nrf1-V5-His transfected LNCaP cells (n = 2). Fold changes represent relative (V5/TBP) differences in Nrf1.
Figure 3.
Effect of Nrf2 on DHT-induced AR nuclear localization and AR transactivation.
The effects of Nrf2 overexpression on DHT-stimulated AR activity were monitored in both LNCaP (A) and C4-2B (B) cells. Cells were transfected with the psPSA-luc reporter plasmid, and with either the Nrf2 expression vector (pCMV-Nrf2) or the control vector (pcDNA3.1) and stimulated with DHT (0–10 nM) for 24 hrs (n = 5). Significant differences in luciferase activity (firefly/renilla RLU) from controls are represented as *; p<0.05, ***; p<0.001 and ****; p<0.0001. In both (A) and (B), panels above the bar graphs represent changes in nuclear Nrf2 levels after transfection with pCMV-Nrf2. In (C) and (D), effects of Nrf2 overexpression on nuclear levels of AR in both untreated and DHT (0, 1, 10 nM) treated LNCaP (C) and C4-2B (D) cells are shown. Data were normalized to nuclear TBP levels. Fold changes and ±SEM values represent differences in nuclear AR levels as compared to untreated cells.
Figure 4.
Nrf1 association with the AR transactivation complex at the ARE.
(A) Co-IP/IB studies of Nrf1 binding to AR in nuclear extracts from DHT-stimulated cells. AR was immunoprecipitated (IP) from nuclear extracts of LNCaP and C4-2B cells, electrophoresed on SDS-PAGE and immunoblotted (IB) with Nrf1 antibodies. Changes in p120-Nrf1 (120 kDa) and p65-Nrf1 (65 kDa) interaction with nuclear AR are shown (n = 3). In the bottom lane, nuclear AR levels in DHT-stimulated cells are shown as a positive control. Data were normalized to nuclear TBP levels (not shown) and fold change and ±SEM values represent relative differences in Nrf1 proteins. (B) AR and Nrf1 interactions at the androgen response element. ChIP assays were carried out using nuclear extracts from DHT treated LNCaP and C4-2B cells. Protected chromatin regions were amplified using ARE specific PCR primers. The ‘input’ represents PCR products amplified directly from the sheared chromatin. Products generated following no antibody incubation was used as a negative control, and those generated following incubation with either the anti-AR or anti-Nrf1 antibody were used to show Nrf1-AR interactions at the ARE sequences. A representative showing the ARE PCR products from each treatment group is shown. In (C) & (D), Nrf1-AR interaction with the labeled ARE was monitored by EMSA and specificity of binding established with competition with either antibodies or oligonucleotides. (C) Nuclear extracts from DHT-treated C4-2B cells were pre-incubated with Nrf1 antibody before addition of the labeled ARE oligo. (D) Competition with excess (50-fold) of unlabeled ARE oligos, unlabeled TCF11, or unlabeled TCF11/MafG (Nrf1 specific) oligos (n = 2). In each experiment, fold changes represent relative differences in protein complex formation at the ARE.
Figure 5.
Role of p120-Nrf1 in Nrf2 mediated repression of AR transactivation.
(A) Effect of DHT on total Nrf2 protein levels in LNCaP and C4-2B cells (n = 3). Data were normalized to GAPDH protein levels in respective samples. Fold changes and ±SEM values represent relative differences in the expression of Nrf2. (B) Effect of Nrf2 on nuclear p65-Nrf1 and p120-Nrf1 levels in C4-2B cells. Cells were transfected with either pcDNA3.1 or pCMV-Nrf2 and stimulated with DHT (0–10 nM) for 24 hrs. Nuclear extracts were evaluated for changes in nuclear p65-Nrf1 and p120-Nrf1 protein levels (n = 2). (C) Effect of DHT treatment on nuclear protein levels of both p65-Nrf1 and p120-Nrf1 in LNCaP and C4-2B cells. In both (B) and (C), densitometric values for each band was normalized to TBP levels and fold changes compared to control (0 nM DHT) are shown (n = 3). (D) Effect of p120-Nrf1 overexpression on DHT-induced AR activity in C4-2B cells. Cells were cotransfected with the psPSA-luc vector and with either pcDNA-3.1 (control) or the p120-Nrf1 expression vector (p120-Nrf1-V5-His) and exposed to DHT (0–10 nM) for 24 hrs and luciferase assays were performed using whole cell extracts. Significant differences in luciferase activity (firefly/renilla RLU) from controls are represented as **; p<0.01 (n = 3). Panels above each bar graph show nuclear V5-His expression in cells transfected with either control or p120-Nrf1 expression vectors (n = 2).
Figure 6.
Mechanism of AR transactivation in CRPC cells: role of Nrf2, p65-Nrf1 and p120-Nrf1.
In CRPC cells (e.g. C4-2B), despite ADT, residual androgens (e.g. DHT) increase nuclear p65-Nrf1 and simultaneously decrease both total Nrf2 and nuclear p120-Nrf1 levels. Nuclear p65-Nrf1 associates with nuclear AR and with the AR transcription complex bound to the ARE sequences in promoter/enhancer regions of genes that regulate prostate tumor growth. Therefore, CRPC cells may utilize the AR coactivator p65-Nrf1 to enhance AR transactivation and facilitate the recurrence of PCa.