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Multiple Androgen Response Elements and a Myc Consensus Site in the Androgen Receptor (AR) Coding Region Are Involved in Androgen-Mediated Up-Regulation of AR Messenger RNA

Department of Molecular and Cellular Pharmacology University of Miami School of Medicine Miami, Florida 33101

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.

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