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Endocrinology Vol. 148, No. 5 2264-2272
Copyright © 2007 by The Endocrine Society

Disruption of Prostate Epithelial Androgen Receptor Impedes Prostate Lobe-Specific Growth and Function

Ulla Simanainen, Charles M. Allan, Patrick Lim, Stephen McPherson, Mark Jimenez, Jeffrey D. Zajac, Rachel A. Davey and David J. Handelsman

ANZAC Research Institute (U.S., C.M.A., P.L., M.J., D.J.H.), University of Sydney, Sydney, New South Wales 2139, Australia; Monash Institute of Medical Research (S.M.), Monash University, Clayton, Victoria 3168, Australia; and Department of Medicine (J.D.Z., R.A.D.), Austin Health, University of Melbourne, Melbourne, Victoria 3084, Australia

Address all correspondence and requests for reprints to: Prof. David J. Handelsman, ANZAC Research Institute, Sydney, New South Wales 2139, Australia. E-mail: djh{at}anzac.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostate development and maturation requires stromal-epithelial interactions and androgen action via the androgen receptor (AR) within these compartments. However, the specific roles of epithelial and stromal AR in postnatal prostate differentiation are unclear. We used Cre-LoxP technology to determine the prostate phenotype in mice with epithelial-selective genetic inactivation of the AR leaving the stromal AR functionally intact. We find that prostate development abolished in mice globally lacking a functional AR can be rescued by restricting the AR knockout to the postnatal prostate epithelium. We show that, at 8 wk of age, prostate epithelial AR knockout (PEARKO) mice exhibit prostate development with normal branching morphogenesis but lobe-specific decrease in prostate weight and hindered structural and functional differentiation of the mature prostate epithelium. No change was observed in PEARKO testis weight or serum testosterone compared with littermate controls. The most striking change was increased proliferation and abnormal lesions of epithelial cells predominantly in the anterior lobe of PEARKO mice. These findings highlight the vital role of stromal AR in postnatal prostate growth and structural differentiation and emphasize the requirement of epithelial AR in maintaining functional differentiation and restraining proliferation of epithelial cells in a lobe-specific manner. This unique PEARKO mouse provides a new paradigm with which to define the molecular mechanisms of the androgen signaling in mature prostate lobes in vivo and provides insight into the identification of better targets for treatment of prostate cancer and hyperplasia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROSTATE IS a classically androgen-dependent organ during its three epochs of growth and development. During fetal organogenesis, through pubertal development when the prostate completes structural and functional maturation, and in later life when prostate cells remain androgen sensitive during evolution of prostate disease (1, 2, 3), androgens stimulate proliferation and restrain apoptosis of prostate cells. Prostate diseases during later life are among the major causes of death, disability, and health costs in developed countries. Over 10% of men will develop prostate cancer, which is currently the second most frequent fatal cancer of men (4). Additionally, most men living out their full life expectancy will develop symptoms of prostate hyperplasia, with approximately 25% requiring surgery for lower urinary tract symptoms constituting a major health care cost. Hence, androgen-directed prevention and treatment of prostate diseases has major health, economic, and social impact.

Genetic males lacking a functional androgen receptor (AR) have no prostate (5), whereas only a vestigial organ develops in men with congenital type II 5{alpha} reductase deficiency (6) or after prepubertal orchidectomy (7). Yet, the cellular location of androgen action critical to such impeded prostate growth remains unclear. Tissue recombination experiments proved that androgens induce mouse prostate development and epithelial proliferation indirectly via paracrine signals originating from activation of mesenchymal AR (2). Using recombined urogenital sinus tissue (mesenchyme, epithelium) anlagen with either normal or inactive AR implanted in vivo into an ectopic location, these classical experiments indicated that prostatic glandular differentiation occurred if, and only if, mesenchymal AR was active, whereas epithelial AR activity was not required (2). The ectopic development of rudimentary prostate tissues in these short-term experiments was unable to encompass full prostate development. In mature prostate, epithelial AR expression occurs later in postnatal ontogeny (8, 9) with AR protein expressed mainly in luminal epithelial cells and, to a lesser extent, in epithelial basal cells as well as stromal cells (8, 9, 10, 11, 12). Decisive experimental proof of whether androgen effects in mature prostate are exerted directly on the epithelium and/or indirectly via stromal influence remains difficult to obtain due to the lack of suitable in vivo models. Hence, the precise role of stromal and epithelial AR for proliferation as well as structural and functional differentiation of the mature prostate has remained unclear (13, 14).

In mice, prostate growth from the urogenital sinus starts during fetal life and continues until sexual maturity with epithelial branching and canalization completed after birth (1). After the mature size and structure of the prostate is achieved, the cellular turnover in prostate is minimal, and cannot be further induced by exogenous androgens (1). It is assumed that, in benign prostatic hyperplasia and prostate cancer, mature cells reacquire growth responsiveness to androgens, which induce abnormal proliferation of epithelial cells (1). Therefore androgen-dependent developmental mechanisms involved in normal prostate development may also have a role in initiation of prostate diseases. A better understanding of the molecular and cellular mechanism of androgen-regulated prostate growth and branching morphogenesis is vital to developing effective prevention and improving treatment for prostate diseases.

We used a Cre-LoxP system to create mice with selective AR inactivation in the prostatic epithelium. Mice with exon 3 of the AR gene (encoding the second DNA binding zinc finger) flanked by loxP sites (ARfEx3) (15) were crossed with transgenic mice expressing probasin promoter-driven Cre recombinase in prostate epithelial cells [Tg(Pbsn-cre)] (16) to generate prostate epithelial AR knockout mice (PEARKO). Whereas genetic male mice with global excision of AR exon 3 (AR{Delta}Ex3), denoted ARKO, displayed a classical androgen-insensitive testicular feminized mouse (Tfm) phenotype with no prostate development (15), PEARKO mice developed a small prostate with normal ductal branching but reduced and hypofunctional epithelium with lobe-specific differences. We demonstrate that significant stromal influence on epithelial proliferation persists beyond fetal life into maturity, whereas the epithelial AR is required for functional maturation of luminal epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Probasin Cre [Tg(Pbsn-cre); FVB/N background] mice were a kind gift from Dr. Fen Wang of the Center for Cancer Biology and Nutrition (Houston, TX) (16). In Tg(Pbsn-cre) mice, the Cre is controlled by the prostate epithelial cell-specific probasin gene promoter. Mice bearing loxP flanked AR exon 3 (ARfEx3; C57BL/6J background) and global ARKO males were produced as previously described (15). Prostate epithelia-specific exon 3 deletion of AR [Tg(Pbsn-cre x ARfEx3)], denoted PEARKO, were produced by crossing female mice heterozygous for ARfEx3 with homozygous Tg(Pbsn-cre) males. F1 generation from these crosses was used to get experimental PEARKO and littermate control males. All animal procedures were approved by the Animal Welfare Committee of the Sydney South West (formerly Central Sydney) Area Health Service within relevant National Health and Medical Research Council guidelines for animal experimentation.

Genotyping
Genomic DNA from mouse tails was isolated and amplified by PCR. The primer pair sequences used for genotyping mice were as follows: Cre forward 5'-CTGACCGTACACCAAAATTTGCCTG-3' and reverse 5'-GATAATCGCGAACATCTTCAGGTTC-3'; floxed AR-Neo forward 5'-TAGATCTCTCGTGGGATCATTG-3' and reverse 5'-GGGAGACACAGGATAGGAAATT-3, wild-type (wt) AR forward 5'-CTTCTCTCAGGGAAACAGAAGT-3' and reverse 5'-GGGAGACACAGGAT-AGGAAATT-3'.

Sample collection
At 8 wk of age, male mice were killed by cardiac exsanguination under ketamine/xylazine anesthesia. Serum was stored frozen at –20 C. Individual prostate lobes were dissected free of periprostatic fat and connective tissue and weighed separately. Lobes were either snap frozen with liquid nitrogen and stored in –80 C for further RNA extraction, fixed in Bouins solution for 4 h at room temperature for histology, or microdissected after collagenase treatment. One lobe of the ventral, dorsolateral, and anterior prostate was incubated in 0.5% collagenase I in Mg2+- and Ca2+-free Hanks’ balanced salt solution for 15 min at room temperature. Stroma was separated from epithelia under a dissecting microscope by gentle manipulation with 27-gauge needles. The numbers of ductal tips were determined by examining digital microphotographs of microdissected, whole-mount specimens of the isolated epithelium as described previously (17).

Epithelial cell isolation
Epithelial cells were isolated as described by (18) with some modifications. Individual prostate lobes were separated in ice-cold DMEM/F12 medium and incubated in 1% trypsin (Sigma, St Louis, MO) for 60 min at 4 C. The stroma was manually separated from epithelium with 27-gauge needles and stored for mRNA analysis. Epithelium from four separate animals was combined (repeated three times) and further incubated in DMEM/F12 medium containing collagenase III (300 U/ml; Worthington, Templestowe, Australia), hyaluronidase (1 mg/ml; Sigma), and DNase (0.04%; Sigma) at 37 C for 60 min. The cell suspension was filtered with a 70-µm Falcon cell strainer, and a single-cell suspension was incubated overnight in DMEM/F12 medium containing 10% fetal calf serum at 37 C to let the stromal cells attach to the plate. Supernatant containing floating epithelial cells was collected, and cells were washed twice with ice-cold PBS and stored in –80 C for mRNA separation.

X-gal staining
Functional recombinase activity of transgenic Cre in male reproductive organs was analyzed using Tg(Pbsn-cre) mice crossed with R26R (ROSA) reporter mice (19), in which a universally expressed floxed lacZ gene is inactive until reconstituted after excision of loxP-flanked stop sequences by Cre recombinase expression. Expression of functional Cre recombinase by lacZ activity in tissues was detected by whole mount ß-galactosidase staining of dissected male reproductive organs. Briefly, organs were prefixed in 2% paraformaldehyde for 1 h at room temperature, then subjected to 5-bromo-galactopyranoside (X-gal; 1 mg/ml) staining for 4 h at 37 C, followed by rinsing with PBS and postfixation overnight in 4% paraformaldehyde at 4 C.

RNA extraction and RT-PCR
Total RNA was extracted using RNeasy Micro Kit or RNeasy Mini Kit (Qiagen, Doncaster, Australia) with DNase treatment (Ambion, Austin, TX) and cDNA synthesized with Omniscript reverse transcriptase (Qiagen) from 200 or 500 ng of total RNA, respectively, using oligo dT (Invitrogen Australia Pty, Mount Waverley, Victoria, Australia). Final RT reactions were diluted 1:5 for storage at –20 C.

Primer sequences for detecting normal and exon-3-excised AR were 5'-GGACAGTACCAGGGACCAT-3' for forward and 5'-CCAAGTTTCTTCAGCTTACGA-3' for reverse primers and located in exon 2 and 4 of AR cDNA, respectively. The size of the amplicon is 288 and 171 bp for wt AR and exon-3-excised AR, respectively. Stromal contamination of separated epithelial cell suspensions were analyzed by expression of smooth muscle {alpha}-actin using primer sequences as previously described (20).

Real-time RT-PCR
Quantitative real-time RT-PCR analyses were performed on cDNA using QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 2000 System (Corbett Research, Mortlake, Australia). Standard curves were generated using purified PCR products from the same primers designed for quantitative PCR, purified using QIAquick PCR Purification Kit (Qiagen). Quantitative PCR for standard series were carried out in triplicate, and dilutions used for each gene were 10–5–10–10. Negative controls included in each run contained all the components of the reaction mixture but water replacing the template. Primer sequences, product size, and annealing temperatures were as previously described (20, 21). Primer sequences for Cre were 5'-GCCTGCATTACCGGTCGAT-3' for forward and 5'-CAGGTTCTGCGGGAAACCA-3' for reverse primers. Relative expression of Cre recombinase mRNA was analyzed from the same cDNA used for all the mRNA analyses in the paper. Expression of exclusively wt AR in separated stromal and epithelial cells was quantified by real-time RT-PCR using forward primer 5'-GGACAGTACCAGGGACCAT-3' and exon-3-specific reverse primer 5'-CAGAGTCATCCCTGCTTCAT-3'.

Molecular analysis of cellular differentiation
Functional activity of specific prostate lobes was measured by real-time PCR quantitation of gene expression for androgen-dependent secreted products from each lobe using renin-1 (22) as marker for anterior, probasin (23) for dorsolateral, and MP25 (24) for ventral lobes. Results were standardized using both cyclophilin as a housekeeping control expressed by all prostate cell types (25) as well as cytokeratin 8 (CK8) as a marker for differentiated luminal epithelial cells (26).

Histology and stereology
Serial 5-µm sections were cut from fixed, paraffin-embedded prostate lobes. Every 10th section was stained with hematoxylin and eosin. CASTGRID version 1.10 (Olympus Corp., Albertslund, Denmark) software generated counting frames and a point grid. Sections were mapped manually for tissue boundaries, and sampling was conducted at uniform random intervals along x- and y-axes. At least 100 counts per tissue compartment (stroma, lumen, and epithelia) were obtained. Point counts were combined to get a reference volume for each lobe, and relative volumes for each tissue compartment were determined. Absolute volume estimates were obtained by multiplying the relative volume of each compartment by the weight of the organ.

Immunohistochemistry
Immunohistochemistry for AR, neuroendocrine (chromogranin A), and basal epithelial cells (high-molecular-weight cytokeratin, CKHMW) was performed on 5 µm thick dewaxed paraffin sections. Antibodies used were rabbit anti-AR (N-20; 1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA) for AR, rabbit anti-chromogranin A (Zymed, San Francisco, CA; 1:200; Invitrogen Australia Pty) and anti-CKHMW (Dako, Carpentaria, CA; 1:100). For AR and chromogranin A, microwave-induced antigen retrieval was done with 0.01 M citrate buffer, pH 6. Signal was visualized with Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) and followed by color development with 3,3'-diaminobenzidine tetrahydrochloride chromogenic substrate (Dako). Sections were counterstained with 0.5% toludine blue. CKHMW immunohistochemistry was performed as described in Ref. 27 . Cell proliferation was determined using proliferating cell nuclear antigen (PCNA) kit (Zymed) according to manufacturer’s instructions. For anterior prostate epithelial proliferating cell index, CASTGRID V1.10 (Olympus Corp.) software was used to generate counting frames after manually mapping tissue boundaries. Each section of anterior prostate contained both distal and a proximal end of lobe, and sampling was conducted at uniform random intervals along x- and y-axes. At least 500 cells were counted.

Testosterone assay
Serum testosterone levels were measured as previously described (28).

Statistics
Statistical analysis was performed using one-way ANOVA with the least significant difference method as a post hoc test using SPSS software (SPSS, Chicago, IL). In case of nonhomogenous variances (Levene’s test, P < 0.01), the nonparametric Kruskal-Wallis ANOVA was used followed by the Mann-Whitney U test. P values less than 0.05 were considered statistically significant. Data are expressed as mean and SEM unless otherwise specified.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transgenic Cre expression
To identify the temporal onset of Cre expression in male reproductive organs, Pbsn-cre mice were crossed with transgenic ROSA mice (19). Three days after birth, weak Cre expression was detected in vas deferens and epididymis, but no staining was observed in whole mounts of prostate lobes (data not shown). At 1 wk of age, Cre expression was much stronger in the epididymis and vas deferens and staining of epithelial cord in anterior and ventral prostate was detectable (Fig. 1Go, A and B). At 2 wk of age, Cre expression was evident in epithelial branches of all prostate lobes and epididymis. Low level of Cre expression was also detected in the seminal vesicles at 2 wk of age (Fig. 1Go, C and D). Nontransgenic ROSA mice revealed no staining (data not shown).


Figure 1
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  		FIG. 1. LacZ expression after Cre recombination detected by whole-mount X-Gal staining of 1 (A and B)- and 2-wk-old (C and D) male reproductive organs. Arrows (A and B) show staining in prostatic epithelial cords. Ventral prostate (VP), dorsolateral prostate (DLP), anterior prostate (AP), seminal vesicles (SV), bladder (Bl), testis (T), epididymis (E), and vas deferens (VD).

 
Molecular analysis of AR expression
Truncated exon-3-excised AR (171-bp PCR product) was strongly expressed in the isolated prostatic epithelial cells from anterior, dorsolateral, and ventral prostate lobes (Fig. 2AGo). Conversely, prostatic stroma contained intact wt AR (288-bp PCR product) with only traces of exon 3 excised (171-bp PCR product) barely visible (Fig. 2AGo). Littermate control epithelial and stromal cells expressed only wt AR (data not shown). Stromal cell contamination in epithelial cell preparation was demonstrated by detectable levels of smooth muscle {alpha}-actin cDNA (Fig. 2AGo).


Figure 2
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  		FIG. 2. A, Molecular analysis (RT-PCR) of AR and smooth muscle {alpha}-actin marker (ACTA) in manually separated PEARKO ventral (VP), dorsolateral (DLP), and anterior prostate (AP) epithelial cells and stroma. Cyclophilin (Cy) used as a loading control. PCR product for wt AR is 288 and 171 bp for exon-3-excised AR{Delta}Ex3. B, Relative mRNA abundance of Cre for anterior, dorsolateral, and ventral lobe in PEARKO. mRNA abundance Cre was determined by real-time RT-PCR and normalized to epithelial cell marker CK8. Values are mean ± SEM and n = 6.

 
Real-time RT-PCR was used to quantify wt AR and smooth muscle {alpha}-actin cDNA (as marker of stromal cell contamination) in PEARKO epithelial cells. Wt AR represented 4% (range 1.5–8.8%) of AR expression comparable with the stromal cell contamination estimated as 5% (3.7–8.4%) according to {alpha}-actin expression with no significant differences between the lobes (three independent epithelial separations for each lobe).

Cre mRNA expression relative to epithelial cell marker CK8 was similar between the lobes (Fig. 2BGo).

Phenotype
At 8 wk of age, PEARKO and global ARKO genetic males were healthy with normal appearance and physical activity. PEARKO and global ARKO males had normal body weight when compared with their respective littermate controls (data not shown). Testis weight as well as serum testosterone levels were normal in PEARKO males compared with littermates expressing wt AR but they were reduced by 95% (P < 0.001) and 75% (P < 0.05) in global ARKO males, respectively (Fig. 3AGo).


Figure 3
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  		FIG. 3. A, Effect of global AR deficiency in ARKO males compared with selective AR deficiency in PEARKO males on testis and epididymis weight and serum testosterone concentrations. B, Gross pelvic anatomy of dissected littermate control (CTR), PEARKO, and ARKO males. Values are mean ± SEM; animal number in each group is shown in respective bars. *, Significantly different (at least P < 0.05) from controls. ND, Not detectable.

 
Global ARKO had the classical androgen-insensitive (Tfm-like) phenotype with absent epididymis (Fig. 3Go, A and B), prostate, and seminal vesicles (Fig. 3BGo) as well as immature, incompletely descended testes, no scrotum, and short ano-genital distance.

Conversely, PEARKO males developed prostates with macroscopically normal appearance, including development of lobar structures (Fig. 3BGo). However all prostate lobes, as well as seminal vesicles, were smaller than controls (Fig. 4AGo) with weights of anterior, dorsolateral, and ventral lobes reduced by 64, 53, and 20%, respectively, and seminal vesicles by 45% compared with littermate controls.


Figure 4
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  		FIG. 4. Effect of epithelial AR deficiency in PEARKO males on male sex accessory organ weights (A) and histology (B, x10 magnification) at 8 wk of age. Values are mean ± SEM; animal number in each group is shown in respective bars. *, Significantly different (at least P < 0.05) from controls (CTR). Ventral prostate (VP), dorsolateral prostate (DLP), anterior prostate (AP), seminal vesicles (SV), stroma (S), and lumen (L).

 
Microscopy and histochemistry
Histological examination of PEARKO (compared with littermate control) prostate revealed marked reduction in stroma and limited eosinophilic secretion in prostatic lumen, with the epithelium appearing relatively more prominent (Fig. 4BGo). Abnormal clustering of epithelial cells was evident particularly in the anterior lobe (Fig. 5Go, A and B) and, to a lesser extent, in dorsolateral and ventral prostate. Histological examination of PEARKO seminal vesicles revealed abnormal multilayered epithelium, whereas the eosinophilic intraluminal secretions were still present (Fig. 4BGo).


Figure 5
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  		FIG. 5. Abnormal cell clustering (arrow) mainly observed in anterior prostate of PEARKO males [A, x20; B, higher magnification of area shown by square in A (x40)]. AR immunohistochemistry for anterior prostate (C, negative control staining substituting primary antibody with isotype control, x20; D, positive AR staining for littermate control, x20; E, AR staining for PEARKO, x20). F, Hematoxylin and eosin stained adjacent section to area shown by square in E to highlight cell clusters, x40.

 
AR immunohistochemistry, using an antibody that detects both wt AR and in-frame mutated AR{Delta}Ex3, confirmed AR expression also in the PEARKO prostate epithelial cells. In littermate control, prostate AR was detectable mainly in the nucleus, whereas, in PEARKO mice, prostate nuclear staining was more frequently cytoplasmic as shown for anterior prostate in Fig. 5Go, C–E. Cytoplasmic AR staining appeared characteristic of the clusters of epithelial cells (Fig. 5Go, E and F), but was also detected outside these foci.

Using PCNA to identify proliferating cells, PEARKO anterior prostate had increased proportion of proliferating epithelial cells compared with littermate controls (Fig. 6Go, A–D). Abnormal cell clusters in PEARKO were generally negative for PCNA staining, although occasional epithelial cell clusters had markedly increased PCNA staining (Fig. 6CGo).


Figure 6
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  		FIG. 6. PCNA immunostaining was performed to detect proliferative cells in anterior prostate. A, Control (CTR), x40; B, PEARKO, x40 showing increased epithelial PCNA staining; C, PEARKO, x40 showing occasional increase in PCNA staining inside the abnormal cell clusters. D, Percentage of epithelial cells staining for PCNA in anterior prostate. Values are mean ± SEM and n = 3. *, Significantly different (at least P < 0.05) from littermate control. CKHMW immunostaining for basal cells. E, Littermate control, x40 showing discontinuous layer of basal epithelial cells. F, PEARKO, x40 showing intense basal epithelial staining surrounding the abnormal cell clusters (arrow).

 
Basal epithelial cells, identified immunohistochemically by CKHMW, formed a discontinuous layer in the control anterior prostate (Fig. 6EGo), whereas, in PEARKO epithelium, there was an increased density of basal epithelial cells (Fig. 6FGo). In addition, abnormal epithelial cell clusters were surrounded by basal epithelial cells (Fig. 6FGo) and were negative to chromogranin A staining (data not shown).

Branching morphogenesis
All prostate lobes in PEARKO males exhibited normal epithelial branching (Fig. 7AGo), and the numbers of ductal tips were not different from those in control tissues (data not shown).


Figure 7
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  		FIG. 7. Representative pictures of microdissected epithelial branching in littermate control and PEARKO ventral (VP), dorsolateral (DLP), and anterior (AP) prostate (A) and stereological analysis of absolute and relative volumes of AP, DLP, and VP from control and PEARKO animals at 8 wk of age (B). Note relative volumes of stroma, lumen, and epithelia add to 100% for each genotype. Values are mean ± SEM and n = 4. *, Significantly different (at least P < 0.05) from littermate control.

 
Stereological analysis of prostate
In accordance with reduced total weight of the prostate lobes in PEARKO males, in the dorsolateral and anterior prostate lobes, the absolute volume of each cellular compartment (stroma, lumen, and epithelium) was significantly reduced compared with littermate controls. However, in ventral prostates with least reduction in absolute weight, the changes were less apparent with stromal volume unchanged, whereas luminal volume showed nonsignificant reduction although absolute epithelial volume was significantly reduced (Fig. 7BGo).

Volumetric proportion of stroma, epithelia, and lumen (expressed as percentage of total volume) were similar for PEARKO and control males for the ventral prostate. However, for anterior and dorsolateral prostate lobes, the epithelium was increased from 38 to 54% (P < 0.05) and from 32 to 48% (P < 0.05), respectively, whereas lumen was decreased from 29 to 18% (P < 0.05) and 27 to 20%, respectively, in PEARKO compared with littermate control males (Fig. 7BGo).

Functional differentiation of epithelial cells
Lobe-specific androgen-dependent gene expression in prostate of PEARKO males was reduced in each isolated lobe with the most prominent effects in dorsolateral and anterior lobe epithelium (Fig. 8Go). Relative expression of luminal epithelial CK8 to cyclophilin was similar in the prostates of PEARKO and littermate control mice (Fig. 8Go).


Figure 8
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  		FIG. 8. Relative mRNA abundance of lobe-specific androgen-responsive secretory protein markers and luminal epithelial cell marker CK8 in PEARKO and littermate control. mRNA abundance of CK8, Renin-1, probasin, and MP25 were determined by real-time RT-PCR and normalized to cyclophilin. Values are mean ± SEM and n = 8. *, Significantly different (P < 0.05). AP, Anterior prostate; DLP, dorsolateral prostate; VP, ventral prostate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Elucidating the cellular basis of androgen action in the prostate is critical to understanding prostate development, physiology, and pathology. Tissue localization of androgen action has remained unclear and difficult to study in vivo in mature prostate. Ablate-replace animal models to study hormone action suffer the limitation that intimate structural and functional interrelationships of stroma and epithelium remain difficult to disentangle. Embryological studies have previously elucidated the pivotal role of androgens interacting with mesenchymal AR to induce prostate epithelial differentiation (1, 29), but the findings were limited to primordial prostate development at an ectopic reimplantation site so that full prostate development could not be studied. In our study, the Cre-LoxP strategy has allowed prostate epithelia-selective AR deficiency in mature prostate and provides an in vivo model to analyze the role of epithelial AR in maintaining the differentiated, functional prostate. We revealed that prostate development abolished in mice globally lacking a functional AR can be rescued by restricting the AR knockout to the prostate epithelium. PEARKO mice exhibit normal branching morphogenesis but hindered structural and functional cellular differentiation of the prostate epithelium. The effects were nonuniform, with greatest impact on the anterior and dorsolateral compared with ventral lobes.

In PEARKO males, the size of all prostate lobes as well as functional lobe-specific markers of androgen action were uniformly decreased, although to differing extents. Histologically, this reduced function was most prominently represented by a reduction in luminal caliber and reduced or absent luminal secretion in each lobe. Thus, the decrease in prostate weight can be attributed to disrupted functional cytodifferentiation leading to smaller, less functionally active epithelial cells and reduced secretions. Yet a crucial finding is that the effect on lobe weight or functional cytodifferentiation was less severe than expected compared with orchidectomy, which produces a dramatic reduction in prostate size due to loss of epithelial cells (30) primarily by freeing the androgenic restraint on apoptosis (31, 32). This is also supported by the morphometric analyses, which revealed that the stromal to epithelial ratio was reduced in PEARKO dorsolateral and anterior prostate, whereas, after orchidectomy, a significant increase in stromal to epithelial ratio is observed (33). Furthermore, the increased proliferation of epithelial cells in PEARKO mice support the recent suggestion that androgen action in the epithelia suppresses proliferation and maintains terminal differentiation (34), whereas epithelial apoptosis is restrained by androgenic stimulation of prostatic stromal AR independent of epithelial AR activation (35, 36). This strongly suggests that functional stromal AR signaling, still present in our model, is vital in maintaining prostate epithelial structure and also contributes to the cytodifferentiation in the mature prostate. In effect, this finding extends the classical Cunha model of mesenchymal AR-driven embryological prostate development to the mature prostate, where stromal androgen action appears crucial in regulating epithelial proliferation.

An unexpected consequence of epithelial-specific AR inactivation was the increased proliferation and abnormal clustering of epithelial cells in foci throughout the prostatic tissues, most prominently in the anterior lobe of PEARKO mice. Abnormal cell clusters in PEARKO epithelium were surrounded by a high density of basal epithelial cells. In the normal prostate, the basal cell population is not actively proliferating (37) and, therefore, the increased density of proliferating basal cells in our model implies that lack of androgen-dependent signaling from basal or luminal epithelial cells is directly or indirectly (via stromal cells) regulating basal cell proliferation. The inner cells of these cell clusters were not basal epithelial cells based on CKHMW immunostaining, indicating that these cells may reflect abnormal maturation of luminal epithelial cells due to loss of AR-regulated balance in epithelial proliferation and survival.

A key feature of this PEARKO model is that mutated AR{Delta}Ex3 with an intact ligand binding domain but deleted second zinc finger produces complete androgen insensitivity in mice (15) and men (Androgen Receptor Mutations Database, http://www.androgendb.mcgill.ca/), whereas the nongenomic androgen actions may still remain functional. Interestingly, in PEARKO prostates, some cytoplasmic AR localization appeared to be associated with the clustering cells, with reduced nuclear and increased cytoplasmic localization of mutated AR. This is consistent with a previous report showing cytoplasmic localization of a different mutated AR but also with inactivated DNA binding (38). In that study, mutated AR could still activate nongenomic signaling and proliferation of human genital fibroblasts, and such nongenomic mechanisms may remain unimpeded in the PEARKO mouse prostate. Whether or not these aberrant cell clusters are directly related to the cytoplasmic localization of mutated AR remains to be determined.

Epithelial branching was normal in all lobes of the PEARKO prostate, indicating that epithelial AR is not required for completion of branching morphogenesis in the prostate. This is in agreement with findings that 70–80% of ductal tips of a normal mature prostate are already present after first 2 wk of neonatal development when circulating androgen levels are low (39). In our model, functional Cre expression was detectable at 1 wk of age in rudimental epithelial cords of prostate lobes. At 2 wk of age, Cre expression was strongly evident in all prostate lobes and epididymis. In mice, epithelial AR expression is detected around a week after birth and, therefore, in our model, AR inactivation covers most postnatal prostatic development with known epithelial AR expression (40). In our model, transient epithelial AR activity may have occurred neonatally, as the composite probasin promoter used to produce Tg(Pbsn-cre) mice is mostly but not totally androgen dependent (41). However, this is unlikely to explain the completion of epithelial branching as functional AR is not required in fetal epithelium for rudimentary epithelial branching (1). In this study, we show that epithelial AR is not required for the completion of epithelial branching after the first week, indicating the cardinal importance of stromal AR signaling for development as well as maintenance of prostate ductal architecture.

Our findings show the ventral prostate is the least sensitive to prostate epithelial AR inactivation compared with anterior and dorsolateral lobes. Conversely, this could also be interpreted as the ventral prostate lobe being more reliant upon the persistence of stromal AR activation. As similar levels of Cre expression and the presence of predominantly exon-3-excised AR was verified in each lobe, uneven Cre expression cannot explain the difference in the response of the lobes. Although both epithelial and stromal cell preparations appeared to have almost exclusive expression of mutated and wt AR, respectively, a small degree of expression of the alternative molecular form of AR was detectable in each tissue. This was observed in all prostate lobes at similar levels. In the stroma, the degree of mutated AR expression was too low to quantify. Similarly, the epithelial cell preparation displayed less than 5% of wt AR, a proportion very similar to the degree of contamination as detected by the smooth muscle marker {alpha}-actin. Nevertheless, low levels of incomplete Cre expression could not be fully ruled out, but Cre-mediated excision is likely to be at least 95% complete in the epithelia of PEARKO mice. Altogether, it is concluded that the strong prostatic phenotype observed in our model demonstrates that the loss of most, if not all, epithelial AR function has a major impact on prostate development, maturation, and function.

In addition to prostate lobes, the growth of seminal vesicles and epididymis were also significantly affected in PEARKO males. Reduced epididymal and seminal vesicle weights in mature PEARKO mice is consistent with both rescue from their failure to form in the global ARKO and with strong expression of Cre detected in epididymis at 2 wk of age in this study and at 8 wk of age in seminal vesicles in a previous study with the same Cre line (16) as well as in different transgenic line using probasin promoter (42). However, epididymal Cre expression and evidence of functional activity is at variance with a previous report that interpreted weak epididymal staining as nonspecific (16). Normal testis weight and serum testosterone concentrations in PEARKO males excludes the possibility that the reduction in weight of these androgen-responsive sex accessory glands might be due to reduced circulating androgen levels. The dichotomy of normal testis development with reduced epididymal weight is very unusual and may be worthy of further study to examine the role of androgen action in epididymal function and functional sperm maturation.

AR has a pivotal role in not only prostate growth and development but also in the origins of prostate cancer and hyperplasia. The vast majority of prostate cancers express AR, are androgen dependent for their growth, and initially respond to androgen ablation therapy (34, 43, 44). Furthermore, prostate cancer that recurs after androgen ablation therapy also expresses AR. In a previous study, mutated AR transgene expression that is able to modulate the AR function in the prostate epithelial cells resulted in cancer (34). However, the relationship between AR expression, activation, or function, and the initiation or progression of prostate cancer is not well understood. Therefore, our model will be a valuable tool for identifying roles of AR signaling in the early stages of prostate diseases. We demonstrate that significant stromal influence on epithelial proliferation persists beyond fetal life into maturity. In turn, this reinforces the validity of efforts to identify stromal-originating paracrine signals as potential novel agents to inhibit early stages of androgen-dependent prostate diseases.


   Acknowledgments
 
We thank Dr. Fen Wang for the generous supply of the Probasin Cre [Tg(Pbsn-cre)] mice. We thank Susan Schmidt and Keely McNamara for their valuable help with stereology and cell counting and Daniel Liske for genotyping.


   Footnotes
 
This study was supported by Academy of Finland Postdoctoral Fellowship (107825), Finnish Cultural Foundation of Northern Savo, Endeavor Australia Postdoctoral Fellowship, Cure Cancer Australia, and National Health and Medical Research Council.

Disclosure Statement: The authors state no conflict of interest.

First Published Online February 22, 2007

Abbreviations: AR, Androgen receptor; CK8, cytokeratin 8; CKHMW, high-molecular-weight cytokeratin; PEARKO, prostate epithelial AR knockout; wt, wild type.

Received September 7, 2006.

Accepted for publication February 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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