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Dynamic Expression of 5-alpha Reductase 2 in Aging Prostate is Regulated by DNA Methyltransferase 1
Rongbin Ge, MD, PhD, Zongwei Wang, Ph.D, Chin-lee Wu, MD PhD, Shahin Tabatabaei, MD, Aria F. Olumi, MD.
MGH, Boston, MA, USA.

BACKGROUND:
5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth, is utilized as an inhibitory target by Finasteride for patients with BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter contains a CpG island, we hypothesized that somatic methylation of the promoter may account for absence of SRD5A2 expression and DNA methyltransferases may play a role in silencing SRD5A2 expression.
METHODS:
Western blot and ELISA were applied to evaluate protein expression. Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. In addition, prostate samples from human male patients who were treated by TURP for bladder outlet obstruction secondary to BPH were used. Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). DNMT1-siRNA and 5-AZA-C were used to determine the methylation status of SRD5A2 in benign prostatic cells (BPH-1). To determine the effect of CpG methylation, SRD5A2 promoter-luciferase constructs were methylated in vitro using M.SssI methylase.

RESULTS:
Benign prostatic tissues from wild-type mice of different ages (3 months, 6 months and 12 months, n=6 per group) were harvested. We found that expression of SRD5A2 and DNMT1, 3a, 3b were not significantly changed in mice at the age of 3 and 6 months. However, 33% of mice at age of 12 months (2/6) had low of SRD5A2 with concomitant high DNMT1 protein levels, while expression of DNMT3a and 3b were constant with age. Consistent with reduced expression of SRD5A2 and increased DNMT1, the methylation of SRD5A2 promoter was increased as assayed by MIRA. Moreover, silencing DNMT1 with siRNA led to re-expression of SRD5A2. Similarly, exposure of BPH-1 cells to 5-AZA-C led to re-expression of SRD5A2 and reduced expression of DNMT1, but did not affect DNMT3a or 3b expression. We used SRD5A2 promoter-luciferase constructs that were methylated in vitro using M.SssI methylase. When the methylated SRD5A2 promoter constructs were ectopically expressed in BPH-1 cells, we found that methylation of the SRD5A2 promoter caused reduction of luciferase expression by 80%, suggesting that methylation of the SRD5A2 promoter regulates the expression of SRD5A2. Since age-associated increased IL-6 has been shown to regulate DNMT1, we next evaluated expression of IL-6 in aging prostate tissue. We found that expression of IL-6 and its downstream molecule, phosphorylated-STAT3 correlated with expression of DNMT1. Concurrent with our animal and in-vitro studies, we analyzed 36 benign human prostate samples and found 12/36 (33%) of benign human prostate samples did not express the SRD5A2 protein with concurrent methylation of SRD5A2promoter, suggesting that methylation and expression of SRD5A2 are closed linked (p=0.0019, Fisher’s exact test). This data suggests that expression of SRD5A2 is highly variable in human adult prostate tissue.

CONCLUSIONS:
Expression of SRD5A2 in aging prostates is a dynamic process that is regulated by methylation of SRD5A2 promoter. DNMT1 regulates the methylation and expression of SRD5A2. Dynamic and variable expression of SRD5A2 has implications for management of BPH and chemopreventive strategies for prostate cancer.


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