DNA methyl transferase 1 reduces expression of SRD5A2 in the aging adult prostate

Abstract

5-α Reductase type 2 (SRD5A2) is a critical enzyme for prostatic development and growth. Inhibition of SRD5A2 by finasteride is used commonly for the management of urinary obstruction caused by benign prostatic hyperplasia. Contrary to common belief, we have found that expression of SRD5A2 is variable and absent in one third of benign adult prostates. In human samples, absent SRD5A2 expression is associated with hypermethylation of the SRD5A2 promoter, and in vitro SRD5A2 promoter activity is suppressed by methylation. We show that methylation of SRD5A2 is regulated by DNA methyltransferase 1, and inflammatory mediators such as tumor necrosis factor α, NF-κB, and IL-6 regulate DNA methyltransferase 1 expression and thereby affect SRD5A2 promoter methylation and gene expression. Furthermore, we show that increasing age in mice and humans is associated with increased methylation of the SRD5A2 promoter and concomitantly decreased protein expression. Artificial induction of inflammation in prostate primary epithelial cells leads to hypermethylation of the SRD5A2 promoter and silencing of SRD5A2, whereas inhibition with tumor necrosis factor α inhibitor reactivates SRD5A2 expression. Therefore, expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases.

Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Methylation status of SRD5A2 promoter correlates with SRD5A2 protein expression in benign prostatic hyperplasia samples. A: Representative figures for immunohistochemical analysis of benign prostatic hyperplasia samples. The immunohistochemistry was defined as follows: <10% and >10% were defined as negative and positive, respectively. B: Representative figures for SRD5A2 protein expression by immunoblot and methylation status of SRD5A2 promoter by methylation pull-down assay. Original magnification, ×400 (A). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WB, Western blot.

Figure 2

Methylation of the SRD5A2 promoter is regulated by DNMT1. A: A representative figure for DNMT1 protein expression by immunoblot. B: DNMT1 activity assay. C: Benign prostatic hyperplasia (BPH)-1 cells were treated with 5-AZC or DNMT1 siRNA for 48 hours, and then cells were harvested for immunoblot analysis. D: BPH-1 cells were transfected with DNMT1, DNMT3a, or DNMT3b siRNA, and then cells were harvested for immunoblot analysis. E:SRD5A2 promoter-luciferase constructs were methylated in vitro by M.Sssi. Then, the methylated plasmids and mock-treated controls were transfected into BPH-1 cells. After 48 hours of incubation, cells were harvested and luciferase was quantified. F:DNMT1 cDNA and mock plasmids were transfected into BPE cells. After 48 hours of incubation, cells were harvested for immunoblot and methylation pull-down assay. G:DNMT1 cDNA and mock plasmids were transfected into BPE cells for 48 hours and then nuclear extracts were subjected to chromatin immunoprecipitation assay using a DNMT1 antibody or IgG. All experiments were repeated independently at least three times with similar results. 5-AZC, aza-2′-deoxycytidine; BPE, benign prostate epithelial; DMSO, dimethyl sulfoxide; M-CpG, methylation-CpG; SAM, S-Adenosyl methionine.

Figure 3

Tumor necrosis factor alpha (TNF-α) contributes to silencing of SRD5A2 by regulating DNMT1 activity. A: Levels of TNF-α (blue bars) and IL-6 (red bars) in benign prostatic hyperplasia (BPH) samples were measured by the MILLIPLEX MAP Human Cytokine/Chemokine assay (Millipore). B: Representative figures for protein expression of TNF-α and TNFR1 in BPH samples by immunoblot. C: BPH-1 cells were treated with a different dosage of TNF-α cytokines for 48 hours, and then cells were harvested for immunoblot analysis. D: BPH-1 cells were treated with TNFR siRNA for 48 hours, and then cells were harvested for immunoblot analysis and DNMT1 activity assay. The data represent means of average determinants ± SEM. All experiments were repeated independently at least three times with similar results. ∗∗P < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

NF-κB indirectly impacts SRD5A2 expression by regulating DNMT1 activity. A: BPH-1 cells were transfected with p65 siRNA for 48 hours, and then cells were harvested for immunoblot analysis and DNMT1 activity assay. B: BPH-1 cells were transfected with p65 cDNA plasmids for 48 hours, and then cells were harvested for immunoblot analysis and DNMT1 activity assay. C: Global methylation assay by the MethylFlash Methylated DNA quantification Kit (Epigentek). D: TNFR siRNA was transfected into BPH-1 or BPH-1/p65 cells for 48 hours, and then cells were harvested for immunoblot analysis. E: DNMT1 activity assay. F: p65 siRNA or p65 cDNA plasmids were transfected into BPH-1 cells for 48 hours, and then nuclear extracts were subjected to chromatin immunoprecipitation assay using a DNMT1 antibody or IgG. The data represent the means of average determinants ± SEM. All experiments were repeated independently at least three times with similar results. ∗P < 0.05, ∗∗P < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

NF-κB regulates the methylation status of SRD5A2 indirectly through an IL-6 pathway. A: BPH-1 cells were treated with 100 ng/mL IL-6 for 24 hours and then cells were harvested for immunoblot analysis. PBS-treated samples were used as control. B:DNMT1 activity assay. C: BPH-1 cells were transfected with IL-6 siRNA for 48 hours, and then cells were harvested for immunoblot analysis. D: DNMT1 activity assay. E: BPH-1 cells were transfected with IL-6 siRNA for 48 hours, and then cells were harvested for immunoblot analysis. F: BPH-1 cells were transfected with p65 siRNA for 48 hours, and then cells were harvested for immunoblot analysis. G: BPH-1 cells were transfected with p65 cDNA plasmids for 48 hours, and then cells were harvested for immunoblot analysis. H: BPH-1 cells were transfected with p65 siRNA or a combination of p65 siRNA and IL-6 cDNA plasmids for 48 hours, respectively, and then cells were harvested for immunoblot analysis. I: DNMT1 activity assay. J: p65 siRNA or combination of p65 siRNA and IL-6 cDNA plasmids were transfected into BPH-1 cells for 48 hours, respectively, and then nuclear extracts were subjected to a chromatin immunoprecipitation assay using a DNMT1 antibody or IgG. The data represent the means of average determinants ± SEM. All experiments were repeated independently at least three times with similar results. ∗P < 0.05. PBS, phosphate-buffered saline.

Figure 6

Aging is associated with a reduced expression of SRD5A2. A: Definition of middle- and old-age group. Levels of TNF-α and IL-6 in benign prostatic hyperplasia samples were measured by the MILLIPLEX MAP Human Cytokine/Chemokine assay (Millipore, lower panel). B: The benign prostatic tissues were harvested from C57BL/6 mice of different ages (3 months, 6 months, and 12 months; n = 6). Representative figures for protein expression of DNMT1, DNMT3a, and DNMT3b by immunoblot assay. C: Representative figures for methylation status of the SRD5A2 promoter by a methylation pull-down assay. D:DNMT1 activity assay. E: Global methylation assay by MethylFlash Methylated DNA quantification Kit (Epigentek). F: Levels of TNF-α and IL-6 in murine prostates were measured by the MILLIPLEX MAP Human Cytokine/Chemokine assay. G: Representative figures for protein expression in murine prostates by immunoblot. H: BPEs were treated with 100 ng/mL LPS or a combination of LPS and 1 μg/mL TNF-α inhibitor for 48 hours, and then cells were harvested for immunoblot analysis. The data represent means of the average determinants ± SEM. All experiments were repeated independently at least three times with similar results. ∗P < 0.05, ∗∗P < 0.01. BPE, benign prostate epithelial cell.

Figure 7

Schematic diagrams of cytokine-mediated activation of DNMT1 and silencing of SRD5A2 in benign prostatic tissues. With increasing age, inflammatory mediators, TNF-α, NF-κB, and IL-6 lead to up-regulation of DNMT1 and methylation of the SRD5A2 promoter and suppression of gene expression. NF-κB regulates DNMT1 through an IL-6–dependent pathway.