Androgen Regulation of 5a-Reductase Isoenzymes Figure 3. Androgen regulation of 5a-reductase mRNA level is sensitive to actinomycin D treatment. BPH-1-AR (A) and LNCaP (B), and PWR-1E (C) cells were treated for 30 minutes with DMSO (vehicle-only control) or actinomycin D at 1 mg/mL and 5 mg/mL concentrations. Treatment with either ethanol (vehicle only) or 10 nM DHT or testosterone followed. We quantified the mRNA levels of SRD5A1, SRD5A2, and SRD5A3 by qRT- PCR. doi:10.1371/journal.pone.0028840.g003 androgen treatment in LNCaP cells. Thus, an ARE may reside in this 1-kb promoter region of SRD5A3. To identify the putative ARE, we made a series of deletion constructs in the SRD5A3 promoter region; the constructs retained responsiveness to androgen treatment until the 2191/291 bps were deleted, suggesting that the putative ARE is located in this region (Fig. 5A). To further evaluate whether this region indeed contains an nARE, we inserted the region of 2191/272 bp between the 4ARE and E4 core promoter in the pGL3–4ARE–E4–luc construct [41], transfected LNCaP cells with it, and then treated the cells with DHT. The pGL3–4ARE–E4–luc construct contains four tandem repeats of the ARE of the PSA gene and an E4 core promoter [41]. As shown in Figure 5B, DHT treatment induced about a 24-fold increase in luciferase activity of the pGL3–4ARE– E4–luc construct. When the 2191/272-bp region of the SRD5A3 promoter was inserted between 4ARE and E4, luciferase activity was increased only 3- to 6-fold by DHT treatment, suggesting that this region contains an nARE. When a 101-bp region derived from the SRD5A1 promoter (21601/21501 bp) was similarly inserted between 4ARE and E4 as a control, DHT treatment still induced a 26-fold increase in luciferase activity. Furthermore, mutations in the 2191/272-bp region of SRD5A3 abolished its repressive ability (Fig. 5C). Together, these data suggest that SRD5A3 has a functional nARE in its promoter. AR is recruited to the nARE-containing region of SRD5A3 To investigate whether AR is recruited to the promoter of SRD5A3 in vivo, we performed ChIP using genomic DNA fragments from LNCaP cells (150–900 bp; Fig. 6A) with primers specifically targeting the nARE region of SRD5A3. As shown on PCR, AR was enriched at the nARE region when cells grew in the presence of DHT (Fig. 6B). Normal mouse immunoglobulin G (as a negative control) was also used for immunoprecipitation with LNCaP genomic DNA; the immunoglobulin G did not pull down any AR-associated DNA sequences of the SRD5A3 promoter (Fig. 6B). These results suggest that AR is recruited to the nARE region in the promoter of SRD5A3. PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28840