Androgen receptor acetylation sites differentially regulate gene control

Androgen receptor (AR) function is modulated by post-translational modifications such as acetylation, ubiquitylation, sumoylation, and phosphorylation. Concerning acetylation, three lysines residues located in a consensus KxKK motif of the AR hinge domain have been identified. For a better evaluation of the role of this modification, the activity of AR modified at different acetylation sites was determined by comparing the effects on natural and synthetic promoters. We found that mutation of AR acetylation sites affected both potency and efficacy of androgen-dependent response. Remarkably, elimination of all three acetylation sites was still compatible with strong AR activity on the PSA and MMTV promoters, but not on the Pem promoter. This differential effect was seen at various wild-type (wt) to mutant AR receptor ratios and at changing hormone concentrations. Subcellular localization studies showed that both mutated and wt AR efficiently translocated into the cell nucleus. Plasmid immunoprecipitation revealed comparable binding of both receptor forms to the Pem promoter. The differential effects observed for the Pem promoter were partially due to an androgen response element (ARE) named ARE-1 which was only poorly stimulated by the AR acetylation site mutant. Finally, AR mutants impaired in their N/C interaction elicited intact stimulation of the Pem promoter, suggesting that AR acetylation was not influenced by this inter-domain communication. The promoter-selective effects seen for the AR acetylation site mutants strongly suggest this post-translational modification to be important in the fine-tuning of the effects of androgens on different target genes.     

Androgen receptor regulation of the versican gene through an androgen response element in the proximal promoter

Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor (AR)-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and dihydrotestosterone stimulate expression of a versican promoter-driven luciferase reporter vector (versican-Luc). Further, both basal and androgen-stimulated versican-Luc activities were significantly diminished in LNCaP cells, when AR gene expression was knocked down using a short hairpin RNA. Methylation-protection footprinting analysis revealed an AR-protected element between positions +75 and +102 of the proximal versican promoter, which strongly resembled a consensus steroid receptor element. Electrophoretic mobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated versican-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce versican promoter activity and mRNA levels in AR-positive prostate stromal fibroblasts. Interestingly, overexpression of beta-catenin in the presence of androgen augmented versican promoter activity 10- and 30-fold and enhanced versican mRNA levels 2.8-fold in fibroblasts. In conclusion, we demonstrate that AR transactivates versican expression, which may augment tumor-stromal interactions and may contribute to prostate cancer progression.