The Amino Terminus of the Human AR Is Target for Corepressor Action and Antihormone Agonism

Abstract

Antiandrogens inhibit the ligand-induced transactivation by the androgen receptor (AR) and have a widespread use in the treatment of prostate cancer but their mode of action is not fully understood. Here we show that the ability of the antiandrogen cyproterone acetate (CPA) to inhibit transactivation by the human AR (hAR) involves the corepressor SMRT (silencing mediator for retinoic acid and thyroid hormone receptor). We detect binding of SMRT to hAR when treating with the antiandrogen CPA, but not with the antihormones casodex or hydroxyflutamide. Interestingly, we find that SMRT binds to the N terminus of the hAR. Thereby, SMRT modulates the activity of hAR in receptor-negative CV1 cells. In addition, we have used receptor point mutants that exhibit normal transactivation potential and unchanged partial agonistic activity when treated with CPA, but lack both SMRT binding and SMRT-mediated inhibition of CPA-bound AR. This indicates that mechanisms involved in hAR-mediated transactivation are distinct from antihormone-induced receptor inactivation. Furthermore, we show that treatment of transfected cells with a cAMP analog or coexpression of the catalytic subunit of PKA, known to activate hAR, inhibits the binding of SMRT to the AR. This suggests that the association of SMRT with hAR is regulated at the level of cross-talk mechanisms and that ligand-independent receptor activation is due to corepressor dissociation. Taken together, we provide novel insights in AR regulation, antihormone action, and functional nuclear receptor-corepressor interaction.

NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth

“…6a, left panel). The deletion of the entire N-terminus is transcriptional inactive, which is in line with previous observations that the major transactivation domain is localized in the AR amino-terminus [22,[34][35][36] and thus NBBS is unable to inhibit. These data suggest that the HBD is required for the NBBS mediated inhibition of AR transactivation.…”

“…The expression vector for the human AR or AR T877A, pSG-hAR or pSG-hAR T877A and deletions are described in reference [22]. The plasmid (DR 4 ) 2 -tk-luc is described in reference [23], for expression of human GR in reference [24] and human TRβ in reference [25].…”

“…Six hours later cells were transfected by using the CaPO 4 method [22]. The DNA mixture for transfections consisted of 1 µg of the appropriated luciferase reporter constructs, 0.2 µg of the appropriated mammalian steroid receptor expression vector and 0.2 µg of the cytomegalovirus (CMV)-driven β-galactosidase expressions vector, as internal control for transfection efficiency.…”

“…After 7 days, cells were seeded into six-well plates at 1.5×10 5 per well. Twenty-four hours later cells were transfected by using the CaPO 4 method [22] with 2 µg reporter plasmid, 0.2 µg receptor expressions vector and 0.2 µg of the cytomegalovirus (CMV)-driven β-galactosidase for normalization. After 14 h media were replaced either with or without the addition of the appropriate hormones together with the indicated compounds.…”

“…6a, right panel). Deletion of the HBD leads to a hormone-independent transactivation of AR [22], which however, cannot be suppressed by NBBS, indicating that the HBD is required for NBBS-mediated antagonism. Deletion of the first 171 amino acids of the AR resulted in reduced transactivation that was inhibited by NBBS (Fig.…”