Androgen Regulation of 5a-Reductase Isoenzymes Figure 4. Regulation of 5a-reductase mRNA level by DHT is AR dependent. A, The AR protein level of each prostate cell line was analyzed by Western blotting. BPH-1-GFP cells were used as a negative control for AR expression. B, The AR protein level was analyzed by Western blotting for LNCaP cells (left) with three control and two AR siRNA treatments. The AR mRNA level was analyzed by qRT-PCR for PWR-1E cells (right), also with three control and two AR siRNA treatments. C, LNCaP and PWR-1E cells were treated with control siRNAs and AR siRNAs and then treated with 2 nM DHT. We measured the mRNA levels of SRD5A1, SRD5A2, and SRD5A3 by using qRT-PCR and normalized the values to b-actin. The changes in mRNA levels with DHT treatment are shown relative to the levels in cells treated with vehicle only. doi:10.1371/journal.pone.0028840.g004 To determine whether AR can directly interact with the nARE of SRD5A3, we carried out EMSA with three oligo probes, each of 40 bp, to cover the 2191/291-bp region of the SRD5A3 promoter (Fig. 6C). Only probe 1 showed a mobility shift (Fig. 6D). To confirm that this mobility shift is specific to AR, we added anti-AR antibodies to the reactions; probe 1 showed a further mobility shift (Fig. 6D). Although unlabeled wild-type probe 1 was able to compete with labeled probe 1 for AR binding in EMSA, it lost that ability when we made mutations in the probe 1 sequence (Figure S5). These results demonstrated that probe 1 contains the nARE and that AR directly binds to this region in vitro. For SRD5A1 and SRD5A2, we also conducted promoter analysis and ChIP, but we did not detect an ARE region or direct AR binding to their proximal promoter regions. The regulational mechanism for their expression is still under investigation. SRD5A3 mRNA increases in the AR-negative SCPC xenograft model On observing the androgen-negative regulation of SRD5A3 in the tested cell lines, we were interested in investigating whether this AR-dependent regulation also occurs in vivo. Therefore, we examined the mRNA level of SRD5A3 in different xenograft models, including MDA PCa 183, an AR-expressing androgen- dependent prostate cancer xenograft, and three AR-negative androgen-independent SCPC xenografts, MDA PCa 144, MDA PCa 146, and MDA PCa 155. On qRT-PCR, we observed a remarkably higher mRNA level of PSA in the MDA PCa 183 xenograft than in the others (Fig. 7, top), which is consistent with the AR status of these cell lines. In contrast, the mRNA level of SRD5A3 in the AR-positive xenograft MDA PCa 183 was much lower than that in the AR-negative SCPC xenografts, except for MDA PCa 155 (Fig. 7, bottom), suggesting a loss of AR-negative regulation in the MDA PCa 144 and MDA PCa 146 xenografts. This result is consistent with the mechanism underlying the androgen-negative regulation of SRD5A3 expression that we found in the prostate cancer cell lines. Discussion The results of this study show that androgens regulate the expression of 5a-reductase isoenzymes in a cell type–specific manner. They also show that androgens regulate the transcription of 5a-reductase isoenzymes and that such regulation is mediated by the AR. Furthermore, to our knowledge, this is the first publication of evidence that SRD5A3 has at least one nARE and that AR directly binds to this nARE region, demonstrating that SRD5A3 is under transcription inhibition by AR in prostate cancer cells. Altogether, our results elucidate a mechanism by which androgens regulate the expression level of 5a-reductase isoen- zymes; this may have clinical implications, by revealing a mechanistic basis for the response and/or resistance to 5a- reductase inhibitors. In this study, the mRNA level of 5a-reductase did not fully correlate with previously reported immunostaining results in PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28840