Serotonin regulates prostate growth through androgen receptor modulation

Abstract

Aging and testosterone almost inexorably cause benign prostatic hyperplasia (BPH) in Human males. However, etiology of BPH is largely unknown. Serotonin (5-HT) is produced by neuroendocrine prostatic cells and presents in high concentration in normal prostatic transition zone, but its function in prostate physiology is unknown. Previous evidence demonstrated that neuroendocrine cells and 5-HT are decreased in BPH compared to normal prostate. Here, we show that 5-HT is a strong negative regulator of prostate growth. In vitro, 5-HT inhibits rat prostate branching through down-regulation of androgen receptor (AR). This 5-HT's inhibitory mechanism is also present in human cells of normal prostate and BPH, namely in cell lines expressing AR when treated with testosterone. In both models, 5-HT's inhibitory mechanism was replicated by specific agonists of 5-Htr1a and 5-Htr1b. Since peripheral 5-HT production is specifically regulated by tryptophan hydroxylase 1(Tph1), we showed that Tph1 knockout mice present higher prostate mass and up-regulation of AR when compared to wild-type, whereas 5-HT treatment restored the prostate weight and AR levels. As 5-HT is decreased in BPH, we present here evidence that links 5-HT depletion to BPH etiology through modulation of AR. Serotoninergic prostate pathway should be explored as a new therapeutic target for BPH.

Figure 1

5-HT, 5-Htr1a specific agonist and 5-Htr1b specific agonist inhibit prostate branching morphogenesis. (a) Photographs of representative VPs at D₀ and at D₄ of culture treated with different 5-HT concentrations. (b) Morphometric analysis of the effect of 5-HT on VPs area and (c) number of peripheral buds (n ≥ 12 VPs per group). (d) Immunofluorescence analysis of 5-Htr1a and (e) 5-Htr1b expression in the rat prostate. (f) Photographs of representative VPs at D0 and at D4 of culture treated with different 8-OH-DPAT concentrations. (g) Morphometric analysis of the effect of 8-OH-DPAT on VPs area and (h) number of peripheral buds (n ≥ 12 VPs per group). (i) Photographs of representative VPs at D0 and at D4 of culture treated with different anpirtoline concentrations. (j) Morphometric analysis of the effect of Anpirtoline on VPs area and (k) number of peripheral buds (n ≥ 12 VPs per group). Error bars indicate s.e.m. ***p < 0.001; two-way ANOVA and Bonferroni post hoc test. VPs, ventral prostate explants; D₀, day 0; D₄, day 4; 5-HT, serotonin.

Figure 2

5-HT, 5-Htr1a specific agonist and 5-Htr1b specific agonist down-regulates AR expression in rat ventral prostate. (a) Western blot analysis of AR expression in prostate explants treated with increasing doses of 5-HT, specific 5-Htr1a agonist, 8-OH-DPAT, and specific 5-Htr1b agonist, anpirtoline. (b) Quantification of AR protein in VPs treated with different concentrations of 5-HT in medium conditions without or with testosterone supplementation, n ≥ 3 (each sample contained a pool of 4 VPs). (c) Quantification of AR protein in VPs treated with different concentrations of 8-OH-DPAT in medium conditions without or with testosterone supplementation, n ≥ 3 (each sample contained a pool of 4 VPs). (d) Quantification of AR protein in VPs treated with different concentrations of Anpirtoline in medium conditions without or with testosterone supplementation, n ≥ 3 (each sample contained a pool of 4 VPs). Error bars indicate s.e.m. n.s. non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA and Bonferroni post hoc test. VPs, ventral prostate explants; AR, androgen receptor; AU, arbitrary units; 5-HT, serotonin.

Figure 3

5-HT, 5-Htr1a specific agonist and 5-Htr1b specific agonist inhibits cell viability in BPH-1 and WPMY-1 human prostatic cells without any effect in PNT1A cells. (a,b,c) Effect of 5-HT on cell viability analyzed by MTS assay in BPH-1, PNT1A and WPMY-1 cells. (d) Immunofluorescence analysis of 5-Htr1a and 5-Htr1b expression in BPH-1, PNT1A and WPMY-1 cells. (e,f,g) Effect of 5-Htr1a specific agonist, 8-OH-DPAT, and (h,i,j) 5-Htr1b specific agonist, Anpirtoline, on cell viability analyzed by MTS assay in BPH-1, PNT1A and WPMY-1 cells. The data are expressed relative to control condition (0 µM 5-HT without testosterone supplementation) and were reproduced in at least three independent experiments. Error bars indicate s.e.m. n.s. non-significant; 5-HT, serotonin; *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA and Bonferroni post hoc test.

Figure 4

5-HT, 5-Htr1a specific agonist and 5-Htr1b specific agonist inhibits cell proliferation in BPH-1 and WPMY-1 human prostatic cells without any effect in PNT1A cells. (a,b,c) Effect of 5-HT, (d,e,f) 5-Htr1a specific agonist, 8-OH-DPAT, and (g,h,i) 5-Htr1b specific agonist, Anpirtoline, on cell proliferation of BPH-1, PNT1A and WPMY-1 cells as quantified by Ki-67 positive cells/total cell number ratio. The data are expressed relative to control condition (0 µM 5-HT without testosterone supplementation) and were reproduced in at least three independent experiments. Error bars indicate s.e.m. n.s. non-significant; 5-HT, serotonin; *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA and Bonferroni post hoc test.

Figure 5

5-HT, 5-Htr1a specific agonist and 5-Htr1b specific agonist down-regulates AR expression in human prostatic cells. (a) Western blot analysis of AR expression in the three cell lines after 5-HT, 8-OH-DPAT and anpirtoline treatment. (b) Quantification of AR in BPH-1 and (c) WPMY-1 cells after 5-HT treatment in medium conditions without or with Testosterone supplementation. (d) Quantification of AR protein levels in BPH-1 and (e) WPMY-1 cells after 8-OH-DPAT treatment in medium conditions without or with Testosterone supplementation. (f) Quantification of AR protein levels in BPH-1 and (g) WPMY-1 cells after Anpirtoline treatment in medium conditions without or with Testosterone supplementation. The data are expressed relative to control condition (0 µM 5-HT without testosterone supplementation) and were reproduced in at least three independent experiments. Full, uncropped gel images are shown. Error bars indicate s.e.m. n.s. non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; two-way ANOVA and Bonferroni post hoc test. AR, androgen receptor; 5-HT, serotonin.

Figure 6

Genetic deletion of Tph1 increases prostate gland mass. (a) Representative photographs of prostates from 20 week-old wild-type and Tph1 ^−/− mice (Top). Images from H&E staining of wild-type and Tph1 ^−/− prostates (n = 5 per genotype) (Bottom). (b) Prostate-to-body weight ratio of Tph1 ^−/− mice compared to wild-type at different ages (n = 6–7 for wild-type and Tph1 ^−/− mice, for each time point). (c) Representative photographs of prostates and (d) prostate-to-body weight ratio from 20 week-old wild-type, Tph1 ^−/− treated with saline and Tph1 ^−/− treated with 5-HT (n = 7 WT; n = 6 Tph1 ^−/− +Saline; n = 7 Tph1 ^−/− + 5-HT). (e) qRT-PCR for AR expression in dorsolateral lobe of 20 week-old wild-type, Tph1 ^−/− treated with saline and Tph1 ^−/− treated with 5-HT (n = 4 wild-type; n = 4 Tph1 ^−/− +Saline; n = 3 Tph1 ^−/− + 5-HT) (Top). Immunofluorescence analysis of AR expression in dorsolateral prostate of WT and Tph1 ^−/− mice (200x) (Bottom). (f) Seminal vesicle-to-body weight ratio of Tph1 ^−/− mice compared to wild-type mice at different ages (n = 6–7 for wild-type and Tph1 ^−/−; mice, for each time point). (g) Seminal vesicle-to-body weight ratio from 20 week-old wild-type, Tph1 ^−/− treated with saline and Tph1 ^−/− treated with 5-HT (n = 7 WT; n = 6 Tph1 ^−/− + Saline; n = 7 Tph1 ^−/− + 5-HT). (h) prostate-to-body weight ratio and (i) seminal vesical-to-body weight ratio in wild-type mice treated with Saline compared to wild-type mice treated with 5-HT during 10 consecutive days (n = 10 for each group). n.s. non-significant; AR, androgen receptor; 5-HT, serotonin; *p < 0.05; **p < 0.01; ***p < 0.001; (B,F) two-way and (D,G) one-way ANOVA and Bonferroni post hoc test. (H,I) Student t test.

Figure 7

Neuroendocrine hypothesis for etiopathogenesis of benign prostatic hyperplasia. (a) In young human male, prostate transition zone is enriched with 5-HT producing neuroendocrine cells. Serotonin is secreted to the epithelium-stroma interface and through activation of 5-Htr1a and 5-Htr1b, both in epithelium and stroma, the expression of AR is decreased. Yet, more testosterone is delivery to prostate, down-regulated AR limits benign prostate growth. (b) In aged human male, transition zone loses 5-HT producing neuroendocrine cells causing a depletion in local 5-HT. As a consequence 5-Htr1a and 5-Htr1b release their inhibition over AR expression. Although with aging the delivery of testosterone to the prostate is decreased the up-regulation of AR induces the development of BPH. 5-HT, serotonin; AR, androgen receptor; DHT, Dihydrotestosterone; NE, neuroendocrine; T, testosterone.