Identification of a negative regulatory cis-element in the enhancer core region of the prostate-specific antigen promoter: implications for intersection of androgen receptor and nuclear factor-kappaB signalling in prostate cancer cells

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Abstract
The NF-κB (nuclear factor-κB) transcription factors mediate activation of a large number of gene promoters containing diverse κB-site sequences. Here, PSA (prostate-specific antigen) was used as an AR (androgen receptor)-responsive gene to examine the underlying mechanism by which the NF-κB p65 transcription factor down-regulates the transcriptional activity of AR in cells. We observed that activation of NF-κB by TNFα (tumour necrosis factor α) inhibited both basal and androgen-stimulated PSA expression, and that this down-regulation occurred at the promoter level, as confirmed by the super-repressor IκBα (S32A/S36A), a dominant negative inhibitor of NF-κB. Using a linker-scanning mutagenesis approach, we identified a cis-element, designated XBE (X-factor-binding element), in the AREc (androgen response element enhancer core) of the PSA promoter, which negatively regulated several AR-responsive promoters, including that of PSA. When three copies of XBE in tandem were juxtaposed to GRE4 (glucocorticoid response element 4), a 4–6-fold reduction of inducible GRE4 activity was detected in three different cell lines, LNCaP, ARCaP-AR and PC3-AR. Bioinformatics and molecular biochemical studies indicated that XBE is a κB-like element that binds specifically to the NF-κB p65 subunit; consistent with these observations, only NF-κB p65, but not the NF-κB p50 subunit, was capable of inhibiting AR-mediated PSA promoter transactivation in LNCaP cells. In addition, our data also showed that AR binds to XBE, as well as to the κB consensus site, and that the transfection of AR inhibits the κB-responsive promoter in transient co-transfection assays. Collectively, these data indicate that cross-modulation between AR and NF-κB p65 transcription factors may occur by a novel mechanism involving binding to a common cis-DNA element.