Increased prostate cell proliferation and loss of cell differentiation in mice lacking prostate epithelial androgen receptor

Abstract

Developmental studies of the prostate have established that ductal morphogenesis, epithelial cytodifferentiation, and proliferation/apoptosis are regulated by androgens acting through stromal androgen receptor (AR). Here, we found mice lacking epithelial AR within the mature prostate (pes-ARKO) developed prostate tissue that was less differentiated and hyperproliferative relative to WT littermates. Epithelial AR protein was significantly decreased in 6-week-old mice and was nearly absent by >/=24 weeks of age. Circulating levels of testosterone, external genitalia, or fertility were not altered in pes-ARKO mice. A significant (P < 0.05) increase in bromo-deoxyuridine-positive epithelia was observed in ventral and dorsal-lateral prostates of pes-ARKO mice at 24 weeks of age. Less differentiation was observed as indicated by decreased epithelial height and glandular infolding through 24 weeks of age, differentiation markers probasin, PSP-94, and Nkx3.1 were sig nificantly decreased, and epithelial sloughing and luminal cell apoptosis increased from 6 to 32 weeks of age in pes-ARKO mice. Gain of function occurred by crossing pes-ARKO to the T857A transgenic mice containing constitutively activated AR. In T857A-pes-ARKO mice prostates were of normal size, contained glandular infoldings, and maintained high secretory epithelium, and the appropriate prostatic epithelial proliferation was restored. Collectively, these results suggest that prostatic epithelial AR plays an important role in the homeostasis of the prostate gland. These data support the hypothesis that epithelial AR controls prostate growth by suppressing epithelial proliferation in the mature gland.

Fig. 1.

Characterization of pes-ARKO mice. (a) Genotypes of WT (Left) and pes-ARKO mice (Right, KO). The presence of transgenes cre (110 bp) and floxAR (540 bp) were observed only within pes-ARKO mice. Only WT mice have WT AR gene. IL-2 RNA is present in both mice and serves as PCR genotyping control. (b) Evaluation of AR and floxed AR using RT-PCR was performed by using primers that span exons 1–3 of the AR gene. This result shows one band for WT AR (305 bp) in WT mice. In pes-ARKO mice, which lack exon 2, both WT band (WT AR is present in stroma) and the truncated transcript band (153 bp; deletion of exon 2) appear within DLP and VP, but only the WT band appears in other tissues. (c and d) Evaluation of external (c) and internal (d) organs demonstrated no differences between strains, except for the larger VP size in pes-ARKO mice. (e) Assessment of protein expression of AR (Left) and probasin (Right) in VPs of WT vs. pes-ARKO mice demonstrate a decrease over time in pes-ARKO mice VPs but not in WT mice VPs. (f) To assess fertility, pups per litter from WT female × WT males (red bar) or × pes-ARKO (blue bar) males were compared and found to be not significantly different (Left). Serum testosterone levels (Right) were similar in WT (red bar) and pes-ARKO (blue bar) male mice at weeks 12 and 24, and thus the changes in prostatic phenotypes are not likely to be due to changes in circulating androgens. SV, seminal vesicle; Kid, kidney; U, ureter; AP, anterior prostate; Pr, all lobes of prostate; T, testis; Pe, glans penis. *, P < 0.05; ***, P < 0.001.

Fig. 2.

Histomorphological changes in the VP of pes-ARKO mice. (a) H&E staining of VPs of WT mice shows glandular infoldings (arrowheads) and tall secretory epithelium through week 32. In pes-ARKO (KO) littermates, we saw these features only at weeks 3 and 6. In week-9 pes-ARKO mice, some VP ducts lose glandular infoldings (*) and have short, poorly differentiated epithelial cells compared with WT littermates. The change in epithelia is evident in ≈50% of ducts within the VP of week 14 pes-ARKO mice. At week 24 (and older), in pes-ARKO mice nearly all glandular infolding and high secretory cells are lost, and the enlarged ducts have many sloughed epithelial cells, fragmented nuclei, and immune cells. By week 32, pes-ARKO mice lack glandular infolding and have squat epithelial cells. (Magnification: ×100; Inset, ×400.) (b–e) At week 14, in pes-ARKO mice, the VP continues to lose glandular infolding. Layers of sloughed epithelial cells are abundant in the prostate lumen. (b) In week 14 pes-ARKO mice, some glandular infoldings (arrow) are present. (c–e) In pes-ARKO mice, infoldings (arrow) become smaller and shorter (c), and infoldings (arrow) constrict (red arrow) at their base (d). Ultimately (e), putative glandular infoldings (arrow) are lost, and sloughed luminal cells appear within the lumen.

Fig. 3.

Loss of epithelial AR leads to loss of androgen-regulated protein and gene expression and increased proliferation. (a) Androgen-regulated gene transcription decreases as epithelial ARs are lost. Quantitative PCR was done on VPs from WT (red bar) and pes-ARKO (blue bar) mice. Androgen-regulated genes probasin, PSP94, and Nkx3.1 are all down-regulated in pes-ARKO prostates compared with WT prostates. (Magnification: ×100; Inset, ×400.) (b) VPs were collected from WT (red bar) and pes-ARKO (blue bar) mice during different stages and analyzed for proliferation. Proliferation, as determined by BrdU-positive nuclei primarily occurs in epithelial cells at all stages evaluated. (c) Epithelial cell proliferation is significantly (P < 0.01) higher in pes-ARKO than in WT littermates. Prepuberty, 2–3 weeks; puberty, 4–6 weeks; postpuberty, 7–8 weeks; early adult, 9–22 weeks; late adult, 24–32 weeks. *, P < 0.05.

Fig. 4.

Expression of T857A mutant AR transgene in pes-ARKO mice reverts VP phenotype to WT VPs. To determine whether reexpression of AR(T857A) could rescue the pes-ARKO phenotype, we created compound transgenic mice, and their prostates were evaluated. (a) H&E staining of 32 week-old VPs from WT, pes-ARKO, and pes-ARKO/T857A mice. Note that epithelium in pes-ARKO/T857A mice are very similar in morphology, cell height, architecture, and glandular infolding to WT mice. (Magnification: ×100; Inset, ×400.) (b) pes-ARKO/T857A mice have normal AR-regulated gene transcription levels and proliferation rates. Quantitative PCR for probasin (orange bars) expression in VPs at 32 weeks. In pes-ARKO/T857A mice probasin expression is significantly increased compared with pes-ARKO mice but not different compared with WT. Quantitative PCR for PSP-94 (green bars) expression in VPs at week 32. In pes-ARKO/T857A mice PSP-94 expression is significantly increased compared with pes-ARKO mice but not different compared with WT. BrdU-labeling index (blue bars) in week 32 VPs. In pes-ARKO/T857A mice epithelial cell proliferation is significantly decreased compared with pes-ARKO mice but not different compared with WT. *, P < 0.05.