CREB-binding protein in androgen receptor-mediated signaling

Abstract

CREB-binding protein (CBP) is a transcriptional coregulator that interacts with different DNA binding proteins and components of the general transcription machinery. CBP enhanced androgen receptor (AR)-dependent transcription under transient transfection conditions in CV-1 cells. The ligand binding domain (LBD) and residues 38-296 of the N-terminal region of AR are not required because the activity of a receptor mutant devoid of these domains was augmented by coexpressed CBP. There is physical interaction between AR and CBP in vivo, as judged by coimmunoprecipitation experiments from cell extracts. Consistent with the role of CBP as a coactivator for AR, the 12S E1A adenoviral protein that inactivates CBP function strongly inhibited AR-dependent transactivation. Exogenous CBP was also capable of overcoming the inhibitory effect of AR on AP-1 activity and diminished the mutual transcriptional repression between AR and NF-kappaB (RelA). Collectively, these data imply that transcriptional interference between AR and AP-1 or NF-kappaB is mediated, at least in part, through competition for intracellular CBP and that this coactivator serves as an integrator between androgen-mediated and other signaling pathways.

Figure 1

CBP stimulates AR-mediated transactivation without altering the amount of receptor protein. (A) CV-1 cells (0.5 × 10⁶ cells per 60-mm dish) were transfected with pARE₄tk-LUC (1.5 μg), AR expression vector (pSG5-rAR, 0.3 μg), and CBP expression plasmid (pSG5-CBP, 3 μg). The total amount of DNA was kept constant by adding empty pSG5 DNA when appropriate. Eighteen hours after transfection, the cells received fresh medium with vehicle or 25 nM testosterone (T) as indicated. After a 30-h culture, the cells were harvested and LUC activity was determined. Reporter gene activities are expressed relative to that achieved with pSG5-rAR in the presence of testosterone, and data are the mean ± SEM of three experiments given as percentages. (B) COS-1 cells were transfected by electroporation with pSG5-rAR with or without pSG5-CBP. Control cells received empty pSG5 DNA. After transfection, the cells were cultured for 30 h in the presence or absence of 25 nM testosterone, after which soluble cell extracts were prepared and subjected for immunoblotting with ARp3 antibody (30).

Figure 2

AR interacts with CBP in transfected COS-1 cells. COS-1 cells were transfected by electroporation with pcDNA 3.1-FLAG-rAR and pSG5-CBP as indicated. After a 30-h culture in the presence or absence of 25 nM testosterone, whole cell extracts were prepared and subjected to immunoprecipitation with mouse monoclonal anti-FLAG antibody. Immunoprecipitated proteins were resolved by SDS/PAGE and subjected to immunoblotting with rabbit anti-CBP antibody. A portion of the cell extract (5%) was subjected to immunoblotting without prior immunoprecipitation with anti-FLAG antibody (lane 4).

Figure 3

Regions of AR involved in the interaction with CBP. CV-1 cells were transfected with 5 μg of pARE₄tk-LUC, 1 μg of pSG5-rARΔ641–902 (A) or 1 μg of pSG5-rARΔ38–296/Δ641–902 (B), and 10 μg pSG5-CBP vectors as indicated. Reporter gene activities are expressed relative to that achieved with pSG5-rAR mutant alone, and data are the mean ± SEM of three experiments given as percentages.

Figure 4

AR-dependent transactivation is repressed by 12S E1A and the repression relieved by coexpressed CBP. (A) CV-1 cells (1.5 × 10⁶ cells per 10-cm dish) were transfected with pARE₂-E1b-CAT (5 μg), AR expression vector (pSG5-rAR, 1 μg), and the indicated amounts of 12S E1A expression vector. Eighteen hours after transfection, the cells received fresh medium containing 25 nM testosterone (T) and were harvested 30 h later to assay CAT activity. Reporter gene activities are expressed relative to that of pSG5-rAR plus testosterone. Data are the mean ± SEM of two experiments. (B) CV-1 cells were transfected with 5 μg of pARE₂-E1b-CAT, 1 μg of pSG5-rAR, 0.1 μg of pRSV-E1A 12S, and the indicated amount of pSG5-CBP (in μg). For the relative CAT activity, the value from the pSG5-rAR plus testosterone result was set as 100. Data are the mean ± SEM of two experiments.

Figure 5

Coexpression of CBP abrogates AR-mediated repression of AP-1 activity. (A) CV-1 cells were transfected with 5 μg of p75–1050CAT reporter, 1 μg of pSG5-rAR, and 10 μg of pSG5-CBP. Total amount of DNA was kept constant by adding empty pSG5 DNA when necessary. The cells were cultured in the presence of 25 nM testosterone (T) for 30 h. For the relative CAT activity, the value of p75–1050CAT alone was set as 100. Data are the mean ± SEM of four experiments. (B) CV-1 cells were transfected with 1 μg of pSG5-rARΔ641–902 instead of pSG5-rAR in experiments otherwise identical with those in A. Data are the mean ± SEM for two experiments.

Figure 6

Coexpression of CBP does not relieve AP-1 repression brought about by GR but increases GR-mediated transcriptional activation. (A) CV-1 cells were transfected with 5 μg of p75–1050CAT reporter, 1 μg of pSG5-GR, and 10 μg of pSG5-CBP. The total amount of DNA was kept constant by adding pSG5 DNA when appropriate. The cells were cultured in the presence of 25 nM dexamethasone (DEX) for 30 h. For the relative CAT activity, the value of p75–1050CAT alone was set as 100. Data are the mean ± SEM values of three experiments. (B) CV-1 cells were transfected with 5 μg of pARE₄-tk-LUC, 1 μg of pSG5-GR, 10 μg of pSG5-CBP, and 0.5 μg of pRSV-E1A 12S as indicated. The cells were treated and the reporter gene activity determined. For the relative LUC value, results of the pSG5-GR plus 25 nM dexamethasone experiment were set at 100. Data are the mean ± SEM of three experiments.

Figure 7

CBP rescues RelA-mediated repression of AR function. CV-1 cells were transfected with pARE₄tk-LUC reporter (5 μg) and indicated amounts (in μg) of pCMV-hAR, pCMV-RelA, and pSG5-CBP vectors in the absence or presence of 25 nM testosterone (T) as indicated. The cells were treated and harvested as described in Fig. 1. LUC activities are expressed relative to that of human AR plus testosterone, and data are the mean ± SEM of two experiments.

Figure 8

CBP stimulates RelA-dependent transactivation and counteracts AR-mediated repression of RelA function. (A) CV-1 cells were transfected with pκB₆tk-LUC reporter (5 μg), pCMV-hAR (1 μg), pCMV-RelA (1 μg), and pSG5-CBP (10 μg) expression vectors. For the relative LUC activity, RelA alone was set at 100. Testosterone (25 nM) was present in all cultures. Data are the mean ± SEM of four experiments are shown. (B) CV-1 cells were transfected with expression vectors for pCMV-RelA (1 μg), pSG5-rARΔ38–296/Δ641–902 (1 μg), and pSG5-CBP (10 μg) and with pκB₆tk-LUC reporter. Data are the mean ± SEM of two experiments.