Two androgen response elements in the androgen receptor coding region are required for cell-specific up-regulation of receptor messenger RNA

In most cells and tissues containing androgen receptors (ARs), androgen regulates the levels of AR messenger RNA (mRNA). As the AR concentration is correlated with androgen responsiveness, this autoregulation of AR mRNA may affect cellular sensitivity to androgens. Androgens decrease levels of AR mRNA in many cell lines and tissues; however, in some tissues and possibly also at certain developmental stages, AR mRNA is up-regulated by androgens. Sequences within the 5'-flanking region and AR promoter do not appear to be sufficient for androgen regulation of AR mRNA. We have previously shown that both down- and up-regulation of AR mRNA by androgen can be reproduced in cell lines expressing a transfected human AR complementary DNA (cDNA). Sequences within the AR cDNA confer this autoregulation in transfected cells, suggesting that sequences within the transcribed region of the AR gene are sufficient for autoregulation. In this study we have determined the mechanism of androgenic up-regulation of AR mRNA encoded by the human AR cDNA in the prostate cancer cell line, PC3, and have identified the cis-acting sequences of the AR cDNA that are required. The observations that actinomycin D blocked androgenic up-regulation of AR mRNA but cycloheximide had no effect are consistent with a model in which AR is directly involved in transcriptional up-regulation of AR cDNA expression. Nuclear run-on assays showed that androgen treatment resulted in increased transcription of the AR cDNA. Furthermore, a 350-bp AR cDNA fragment inserted 5' of a thymidine kinase promoter-chloramphenicol acetyltransferase gene conferred androgen induction of chloramphenicol acetyltransferase activity in PC3 cells. This 350-bp fragment, which is located in the AR coding region, contains two putative androgen response elements (AREs) separated by 182 bp. The 5'-most ARE (ARE- 1, 5'-TGTCCT-3') resembles a half-site of the palindromic consensus hormone response element, recognized by several steroid receptors, including AR, and the 3'-sequence (ARE-2, 5'-AGTACTCC-3') is identical to a portion of an androgen-responsive region found in the rat probasin gene promoter. Analysis of either ARE-1 or ARE-2 mutants revealed that these elements function synergistically. AR protein binds to the 350-bp fragment, as demonstrated by electrophoretic mobility shift assays using a glutathione-S-transferase-AR fusion protein containing the DNA- and steroid-binding domains of AR. These results indicate that the AR coding region contains an androgen-responsive region that is involved in cell line-specific up-regulation of AR mRNA.