Figure 5. The SRD5A3 promoter contains a functional nARE. A, Deletion analysis of SRD5A3 promoter to narrow down the location of the nARE-containing region. LNCaP cells were transfected with luciferase constructs containing a deletion series of SRD5A3 promoter and then treated with ethanol (vehicle only; 2DHT) or 100 nM DHT (+DHT) for 24 hours. Cells were then harvested, and their lysates were used for the luciferase assay. B, SRD5A3 has an nARE. LNCaP cells were transfected with pGL3–4ARE–E4 constructs with and without insertion of the SRD5A3 promoter (2191/272 bp) sequence in both orientations Androgen Regulation of 5a-Reductase Isoenzymes or with insertion of an SRD5A1 promoter fragment (21601/21501 bp). Cells were treated with ethanol (vehicle only) or 100 nM DHT for 24 hours and then harvested for the luciferase assay. C, Mutations in the nARE abolished its suppressive effect. LNCaP cells were transfected with pGL3–4ARE–E4 constructs with and without insertion of SRD5A3 promoter (2191/272 bp) or with insertion of the mutated SRD5A3 promoter (2191/272 bp) and then subjected to DHT treatment and the luciferase assay. doi:10.1371/journal.pone.0028840.g005 human tissues [8], but it is possible that the mRNA level during different stages of prostate cancer development and progression does not fully correlate with the protein levels as measured by immunostaining. Luo and colleagues [7] detected no significant difference in the SRD5A1 mRNA level between tumor samples and BPH or normal samples, although Thomas et al. [8] found greater SRD5A1 immunostaining in tumor samples than they found in BPH samples. It is also possible that the mRNA level of 5a-reductase in this study was modified because we used immortalized cell lines. Additionally, it remains controversial how the 5a-reductase level changes with prostate cancer development and progression, as shown in a recent study by Wako et al. [42]: using semiquantitative immunohistochemical analysis, they found no significant change in SRD5A1 and SRD5A2 levels between localized prostate cancer samples and normal prostatic tissues. This study evaluated multiple cell lines that may represent the processes of prostate cancer initiation and progression. Although these are immortalized cell lines, the results still shed light on how androgen may modulate the mRNA level of 5a-reductase during different stages of prostate cancer pathogenesis and progression. In PWR-1E cells, for example, androgens did not have a significant effect on 5a-reductase expression. However, in BPH-1-AR cells, which represent the transition between normal and cancerous prostate cells,testosterone could up-regulate the mRNA levels of all three 5a-reductase isoenzymes, which would result in high concentrations of DHT in these cells and further increase the expression of these isoenzymes. Such a positive-feedback loop is most likely to lead to constant activation of the AR, which at least partially explains the epidemiologic observation that a high concentration of testosterone level correlates with a high incidence of prostate tumor [43]. In our experiments, the LAPC-4, LNCaP, and C4-2B4 prostate cancer cell lines responded differently to androgen treatment. They also have different androgen sensitivity and AR status, which is an important implication that blocking DHT production with either 5a-reductase inhibitor may not be the gold-standard prevention strategy in every man because the androgen sensitivity, AR status and other factors of each particular prostate cancer may influence expression of 5a-reductase isoenzymes in reponses to blocking of DHT production. Several genes, including PSA, TGF-b1, and maspin, reportedly contain an nARE in their cis-regulatory elements [41,44,45]. The nARE in the PSA gene overlaps with the NF-kB binding site, and AR competes with NF-kB binding, resulting in the negative regulation of PSA [44]. A 250-bp nARE region has also been mapped to the promoter of TGF-b1, although no direct binding of AR to this region has been detected [41]. Further, the AR binds to the nARE of the maspin promoter [45]. However, the nARE in the promoter of SRD5A3 has no notable similarity to that in PSA, TGF-b1, or maspin. Therefore, we believe that we have found a novel nARE and have demonstrated the direct binding of the AR to this region. The SRD5A3 mRNA level was not consistently significantly affected by androgens in PWR-1E cells, was up-regulated in BPH- 1-AR cells, but was down-regulated in LNCaP cells. These results PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28840