Figure 1. The expression pattern of 5a-reductase in prostate cell lines varies. Graphs depict the relative mRNA levels of the SRD5A1, SRD5A2, and SRD5A3 isoenzymes in PWR-1E, BPH-1-AR, LAPC-4, LNCaP, and C4-2B4 cells as determined on qRT-PCR analysis. doi:10.1371/journal.pone.0028840.g001 which are derived from a xenograft bone metastasis of human prostate cancer. DHT also down-regulated the mRNA level of SRD5A3 in VCaP cells (Figure S1), indicating that androgen- negative regulation of SRD5A3 is not specific to LNCaP or LNCaP-derived cell lines. Testosterone and R1881 had effects similar to those of DHT on the expression of 5a-reductase in all the cell lines tested (Figures S2 and S3). Our data thus demonstrate that androgens regulate the mRNA level of 5a-reductase isoenzymes in a cell type–specific manner. Androgen Regulation of 5a-Reductase Isoenzymes Regulation of 5a-reductase mRNA level occurs through transcription Androgens could regulate 5a-reductase expression by control- ling 5a-reductase transcription or by affecting mRNA stability. To understand the mechanism underlying the regulation of 5a- reductase by androgens, we treated BPH-1-AR cells with actinomycin D, a transcription inhibitor. As shown by the results of qRT-PCR analysis in Figure 3, DHT induced increased expression of all three 5a-reductase isoenzymes relative to that in the ethanol (vehicle) control in BPH-1-AR cells. However, the mRNA level of 5a-reductase did not change with DHT treatment when the cells were also treated with actinomycin D at 1 mg/mL and 5 mg/mL concentrations (Fig. 3A). These results indicate that the up-regulation of 5a-reductase expression by androgens is sensitive to actinomycin D treatment, suggesting that androgens regulate the transcription of 5a-reductase. Similarly, when LNCaP cells were treated with actinomycin D, testosterone did not significantly increase SRD5A1 or decrease SRD5A2 and SRD5A3 mRNA levels (Fig. 3B). As a negative control, we also treated PWR-1E cells with actinomycin D, followed by DHT treatment (Fig. 3C). In the case of the DMSO- only control, DHT did not affect the mRNA level of SRD5A1, SRD5A2, or SRD5A3. With actinomycin D treatment, DHT did not significantly alter the mRNA level of these genes, either. Regulation of 5a-reductase mRNA level by androgens is AR dependent By using Western blotting, we verified the expression of AR in each of the studied cell lines by comparison with that of the AR- negative BPH-1-GFP cells (Fig. 4A). To determine whether AR is required to mediate regulation of 5a-reductase expression by androgens, we performed Western blotting and qRT-PCR analysis after treating LNCaP and PWR- 1E cells with AR siRNAs and then with 2 nM DHT. Western blotting and qRT-PCR validated the effective knockdown of AR expression by the AR siRNAs (Fig. 4B). qRT-PCR showed that the control siRNAs did not significantly alter the effect of DHT on 5a-reductase levels in LNCaP cells (Fig. 4C, top). DHT treatment alone resulted in increased SRD5A1 mRNA but decreased SRD5A2 and SRD5A3 mRNA (Fig. 4C, top). In contrast, the 5a-reductase levels in LNCaP cells treated with AR siRNAs did not change in response to treatment with DHT (Fig. 4C, top). When we treated LAPC-4 cells in a similar way, SRD5A1 mRNA similarly increased with DHT or testosterone treatment alone, but not when cells were also treated with AR siRNA (Figure S4). When we treated PWR-1E cells in the same way, DHT did not affect the mRNA level of 5a-reductase in PWR-1E cells, no matter whether they were treated with control siRNAs or AR siRNAs (Fig. 4C, bottom). Taken together, these results indicate that the regulation of 5a- reductase by androgens is AR dependent. SRD5A3 promoter contains an nARE We showed that androgen regulates the transcription of 5a- reductase and that AR is necessary to mediate this transcriptional regulation. AR directly binds to AREs and regulates the transcription of the AR-targeted genes. Thus, we investigated whether AR can directly regulate the transcription of 5a- reductase. We cloned a promoter region of SRD5A3 (21027 to +155 bp) into the pGL2-basic vector. In LNCaP cells, this construct drives luciferase expression and, when they are treated with 100 nM DHT, a 75% reduction of luciferase activity results (Fig. 5A). This is similar to the response of endogenous SRD5A3 to PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28840