Altered differentiation and proliferation of prostate epithelium in mice lacking the androgen receptor cofactor p44/WDR77

Abstract

Although it has been observed that various cofactors modulate activity of the androgen receptor (AR), the specific relationship between AR cofactors and prostate development and functions has not been well studied. To determine whether AR cofactor p44/WDR77 is important in prostate growth and development, we examined prostate architecture in p44/WDR77-null mice and wild-type (WT) littermates. Prostate glands from p44/WDR77-deficient animals were not only smaller than those from WT mice but also had fewer branches and terminal duct tips and were deficient in production of secretory proteins. The p44/WDR77-null prostate tissue was less differentiated and hyperproliferative relative to WT littermates. In addition, the altered expression of androgen-regulated genes was observed in the p44/WDR77-null prostate. Thus, these results suggest that the AR cofactor p44/WDR77 plays important roles in prostate growth and differentiation by modulating AR-target gene expression.

Figure 1

Generation of the Cre-mediated prostate-specific deletion of the p44/WDR77 gene. A, Diagram of the strategy to generate a prostate-specific p44/WDR77-deficient mouse. Wild allele of the mouse p44/WDR77 gene, the target vector with PGK-neo cassettes and loxP sites, and the predicted MT allele resulting from homologous and Cre-mediated recombination are presented. B, Southern blot analysis of offspring obtained by a heterozygous cross. The WT allele (8.7 kb) and MT allele (4.9 kb) are indicated by arrows. C, Gene typing by PCR. Genomic DNA was isolated from mouse tails and submitted for PCR analysis to detect the presence of the MT loci (the loxP site) (using primers 1 and 2) (top panel). Genomic DNA was isolated from mouse prostates and submitted for PCR analysis to detect the presence of the Cre gene (middle panel, lanes 2 and 4) or the Cre-mediated deletion of the p44/WDR77 gene (using primers 1 and 3) (bottom panel, lane 4). D, Northern blot analysis of p44/WDR77 and β-actin mRNAs in the prostate gland. The mRNA was isolated from the whole prostate glands of p44/WDR77^loxP/loxP (n = 5; lane 1) or p44/WDR77^loxP/loxP;Cre (n = 5; lane 2) mice, fractionated by electrophoresis, and transferred to a Hybond N⁺ membrane. The membrane was hybridized with a p44/WDR77 probe (top panel) or β-actin probe (bottom panel). P1, P2, P3, Primers 1, 2, and 3; TK, thymidine kinase.

Figure 2

Conditional deletion of p44/WDR77 in the prostate luminal epithelium. Immunohistochemical staining of AP, DLP, and VP of the WT (A, C–E, and I; n = 3) and MT (B, F–H, and J; n = 3) mice with anti-p44/WDR77 (A–H) or anti-AR (I and J) antibody and the alkaline phosphophase-labeled (A, B, I, and J) or Alexa 594-labeled (C–H) antirabbit IgG antibody. Panels D and G and panels E and H show the nucleus staining and the merge of the nuclear staining with the p44/WDR77 immunostaining, respectively. Insets in F–H show the immunostaining without the first antibody as a negative control. The signals were detected with a streptavidin-biotin peroxidase system (A, B, I, and J) or observed under a fluorescent microscope (C–H). K, Shows Western blot analysis of AR in protein extracts (20 μg/per sample) prepared from WT (lane 1) or MT (lane 2) whole prostate gland.

Figure 3

Altered development and hyperplasia of the MT mouse prostate. H&E staining of the AP and DLP of WT (panel A; n = 12) and MT (panel B; n = 12) mice at the age of 2 months. Some epithelial enfoldings and hyperplastic lesions are indicated by white and black arrows, respectively. VG, Vesicular gland; UB, urinary bladder.

Figure 4

The p44/WDR77 deletion decreased branches and terminal ductal tips of the prostate gland. A and B Branches and terminal ductal tips of the epithelial cords in AP, DLP, and VP (n = 5) were counted, and data are shown. C, Homozygous p44/WDR77 deletion leads to the small size of prostate. Organs were dissected from WT (n = 5) and MT (n = 10) mice and weighed. Data (mean ± sem) are expressed. The unpaired t test was performed, and the P values are shown.

Figure 5

Loss of p44/WDR77 expression decreased protein secretions in the mouse prostate. A, H&E-stained sections show a basal-like cuboidal epithelium in the MT prostate vs. the tall columnar secretory epithelium in the WT prostate. B, Analysis of secretory proteins from intact (lanes 2–4 and lanes 9–12) or castrated (5,6,7,13,14,15,16) WT (lanes 2, 5, 9, 10, 13, and 14) and MT (lanes 3, 4, 6, 7, 11, 12, 15, and 16) mouse prostate glands. Protein secretions were resolved on a 10–20% SDS-polyacrylamide gradient gel. An equal volume (5 μl) was loaded onto each lane. The migration of molecular weight markers (Bio-Rad) is indicated in lanes 1 and 8.

Figure 6

The p44/WDR77 deletion altered expression of cytokeratines in the MT prostate. Anti-K5 (A and B), anti-K18 (C and D), and anti-p63 (E and F) stainings of prostate of WT (n = 3) and MT (n = 3) mice at the age of 2 months. The alkaline phosphophase-labeled (A, B, E, and F) or the Alexa 594-labeled (C and D) antirabbit IgG antibody was used. DAPI staining of the nucleus was merged with K18 immunostaining in C and D. The black and white arrows indicate the basal and epithelial cells, respectively. G, Shows Western blot analysis of p63 in protein extracts (20 μg/per sample) prepared from WT (lane 1) or MT (lane 2) whole prostate gland.

Figure 7

Loss of p44/WDR77 increased proliferation and apoptosis of prostate epithelial cells. A, In vivo proliferation was assessed using the BrdU incorporation assay. Sections of WT (left) and MT (right) prostate were immunostained with anti-BrdU antibody. The BrdU-positive cells are brown (indicated by black arrows). Insets show regions under high power. B, Percentage of BrdU-positive cells in VP, DLP, and VP of WT and MT mice. C, Apoptosis was assessed using the TUNEL assay with sections of WT and MT prostates. Percentage of apoptotic cells in WT and MT luminal epithelial cells are presented. Data (mean ± sem) from five WT and five MT prostates are expressed. The unpaired t test was performed; *** and ** indicate significant (P < 0.001 and P < 0.05) differences between WT and MT mice.

Figure 8

Loss of p44/WDR77 expression affects androgen-dependent gene expression. Real-time PCR analysis of gene expression in prostates of 7-d-castrated mice (WT, n = 3; MT, n = 3) treated with testosterone propionate for the indicated number of hours. The equation fold change = relative quantification of genes in the presence/absence of androgen. Data (mean ± sem) are expressed. Inset shows Western blot analysis of Maspin in protein extracts (20 μg/per sample) prepared from WT (lane 1) or MT (lane 2) whole prostate gland. The unpaired t test was performed; *** and ** indicate significant (P < 0.0001 and P < 0.001) differences between WT and MT mice.