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

Abstract

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.

In vitro evidence for complex modes of nuclear β-catenin signaling during prostate growth and tumorigenesis

“…TCFs) may heighten expression of a factor(s) capable of the observed activity. As myc has been shown to play an intimate role in expression of the AR gene itself (Grad et al, 1999), its induction by b-catenin/TCF may impact on AR protein output. However, this idea is inconsistent with the observed lack of b-catenin/TCF signaling in LNCaP cells, given their reported ability to support bcatenin-enhanced AR function (Truica et al, 2000).…”

Androgens and the male reproductive tract: an overview of classical roles and current perspectives

“…Interestingly, studies conducted with monkey kidney COS-1 cells (37) and androgen independent cell lines PC3 and DU145 (38), all transiently transfected with human AR cDNA and stimulated with R1881, have indicated the presence of regulatory regions within the AR coding sequence that are important in AR gene homologous regulation. In fact, studies using PC3 cells transiently transfected with human AR cDNA have confirmed the presence of functional AREs in exons 4 and 5 of AR gene (39).…”

Regulation of Caveolin-1 Expression and Secretion by a Protein Kinase Cε Signaling Pathway in Human Prostate Cancer Cells

“…These responses to c-Myc activation inhibit ubiquitin-mediated proteolysis in lymphoma cells and could potentially contribute to AR stabilization in recurrent CWR22 tumors. The AR promoter does not contain a consensus E box for direct c-Myc transactivation (10), and there is no evidence of a change in AR mRNA in CWR-R1 cells. These observations reinforce the idea that there may be a regulatory link between c-Myc activation, changes in proteolysis, and AR stability in human prostate cancer cells.…”

“…Several independent lines of evidence from gene transfer, antisense, and pharmacological experiments are presented in this paper that support the notion that PKC⑀ is capable of sequentially activating MEK, ERK, c-myc, and AR to promote caveolin-1 expression and the proliferation of CWR-R1 cells in culture. Detailed descriptions of the factors governing the activity of the molecular components featured within this PKC⑀ signaling cascade have been proposed and additional intermediary events should be anticipated (9,10,13). There is no clear and direct evidence that explains how caveolin-1 is diverted from the classic exocytotic pathway to reach, and cross, the plasma membrane and then function as an autocrine/paracrine factor in prostate cancer progression.…”

“…In rodent prostate cancer cells, c-Myc has been reported to repress caveolin-1 expression at the transcriptional level (9). At the same time, there is evidence that c-Myc-Max heterodimers interact with a novel exonic region of the androgen receptor (AR) gene to enhance AR autoregulation in the human PC3 prostate cancer cell line (10). The latter finding may be important because testosterone stimulates caveolin-1 transcription in the androgensensitive LNCaP prostate cancer cell line (11).…”