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Endocrinology Vol. 147, No. 12 5806-5816
Copyright © 2006 by The Endocrine Society

Transcript Profiling of the Androgen Signal in Normal Prostate, Benign Prostatic Hyperplasia, and Prostate Cancer

David R. Bauman, Stephan Steckelbroeck, Donna M. Peehl and Trevor M. Penning

Department of Pharmacology (D.R.B., S.S., T.M.P.), Center of Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084; and Department of Urology (D.M.P.), Stanford University School of Medicine, Stanford, California 94305

Address all correspondence and requests for reprints to: Trevor M. Penning, Department of Pharmacology, University of Pennsylvania School of Medicine, 130C John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084. E-mail: penning{at}pharm.med.upenn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human prostate adenocarcinoma (CaP) and benign prostatic hyperplasia (BPH) have epithelial and stromal cell origins, respectively. To determine whether the androgen signal is processed differently in these cell types the expression of transcripts for enzymes that control ligand access to the androgen receptor (AR) were measured. Transcripts for type 2 5{alpha}-reductase, ketosteroid reductases [aldo-keto reductase (AKR)1C1-AKR1C4], the major oxidative 3{alpha}-hydroxysteroid dehydrogenase (HSD) retinol dehydrogenase (RODH)-like 3{alpha}-HSD (RL-HSD) and nuclear receptors [AR, estrogen receptor (ER){alpha}, and ERß] were determined in whole human prostate and in cultures of primary epithelial cells (PEC) and primary stromal cells (PSC) from normal prostate, CaP and BPH by real-time RT-PCR. Normal PEC (n = 14) had higher levels of AKR1C1 (10-fold, P < 0.001), AKR1C2 (115-fold, P < 0.001) and AKR1C3 (6-fold, P < 0.001) than normal PSC (n = 15), suggesting that reductive androgen metabolism occurs. By contrast, normal PSC had higher levels of AR (8-fold, P < 0.001) and RL-HSD (21-fold, P < 0.001) than normal PEC, suggesting that 3{alpha}-androstanediol is converted to 5{alpha}-dihydrotestosterone to activate AR. In CaP PEC (n = 14), no significant changes in transcript levels vs. normal PEC were observed. In BPH PSC (n = 21) transcripts for AR (2-fold, P < 0.001), AKR1C1 (4-fold, P < 0.001), AKR1C2 (10-fold P < 0.001), AKR1C3 (4-fold, P < 0.001) and RL-HSD (3-fold, P < 0.003) were elevated to increase androgen response. Differences in the AR:ERß transcript ratios (eight in normal PEC vs. 280 in normal PSC) were maintained in PEC and PSC in diseased prostate. These data suggest that CaP may be more responsive to an ERß agonist and BPH may be more responsive to androgen ablation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN PROSTATE is essential for normal male reproduction because it excretes the prostatic fluid necessary for the maintenance of sperm (1, 2). The growth and maintenance of the mature gland is regulated by the potent androgen 5{alpha}-dihydrotestosterone (DHT) (3, 4). Androgen action is regulated in the prostate at the prereceptor level by enzymes responsible for the formation and elimination of DHT, e.g. type 2 5{alpha}-reductase and the hydroxysteroid dehydrogenases (HSDs). Dysregulation of these enzymes and or nuclear receptors may lead to the development of androgen-dependent diseases such as prostate adenocarcinoma (CaP) and benign prostatic hyperplasia (BPH).

In the normal adult prostate, DHT is formed from the irreversible reduction of circulating gonadal 17ß-hydroxy-androst-4-ene-3-one (testosterone) by type 2 5{alpha}-reductase (3, 4) (Fig. 1Go). By contrast in aging males, DHT production depends upon adrenal androgens, e.g. dehydroepiandrosterone, which is converted by 3ß-HSD/ketosteroid isomerase to the inactive androgen {Delta}4-androstene-3,17-dione. This steroid is then reduced to testosterone by type 2 3{alpha}-HSD/type 5 17ß-HSD also known as aldo-keto reductase (AKR) 1C3 (AKR1C3) (5, 6, 7, 8, 9). The subsequent reduction of testosterone by type 2 5{alpha}-reductase completes the formation of DHT, which then trans-activates the androgen receptor (AR). Intraprostatic levels of DHT are regulated by reductive and oxidative HSDs. AKR1C2 (type 3 3{alpha}-HSD and a 3-ketosteroid reductase) eliminates DHT by reducing it to the inactive androgen 5{alpha}-androstane-3{alpha},17ß-diol (3{alpha}-diol) (9, 10, 11, 12). By contrast, AKR1C1 (20{alpha}-HSD and a 3-ketosteroid reductase) converts DHT to 5{alpha}-androstane-3ß,17ß-diol (3ß-diol) (12) a proapoptotic ligand for estrogen receptor (ER) ß (13, 14, 15). ERß-knockout (KO) mice have increased hyperplastic intraepithelial neoplasia (PIN) compared with wild-type mice, indicating that activation of ERß is an important growth constraint in the gland (13, 16). Thus, the ratio of AKR1C1:AKR1C2 may be an important determinant of whether a proapoptotic signal or an inactive androgen is generated in prostate cell types. A recent comparison of five candidate oxidative 3{alpha}-HSDs also revealed that RODH-like 3{alpha}-HSD (RL-HSD) was most responsible for the oxidation of 3{alpha}-diol back to DHT (17). The back conversion of 3{alpha}-diol to DHT is thought to be an important growth signal across species (18, 19). Androgen responsiveness will thus depend on expression of steroid transforming enzymes and nuclear receptors within discrete portions of the gland.


Figure 1
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  		FIG. 1. Androgen metabolism and hormone signaling in human prostate. DHEA, Dehydroepiandrosterone; KSI, ketosteroid isomerase.

 
Knowledge of these pathways has determined approaches to hormone ablation of prostate disease. For example, the type 2 5{alpha}-reductase mechanism-based inactivator, finasteride (20) is used clinically for BPH and reduces intraprostatic levels of DHT by approximately 80% (21, 22, 23) and reduces prostatic volume by greater than 30%. In contrast, prostate cancer chemoprevention trials with finasteride have shown that this agent will reduce the incidence of tumors, but those that form are more aggressive and have higher Gleason grade (24). Because BPH is a disease that typically originates in the stromal cells and CaP typically originates in the epithelial cells, we hypothesized that differences in clinical outcome may be related to differences in how the androgen signal is processed in the two cell types.

We report transcript profiling of proteins associated with androgen signaling in whole human prostate and in primary epithelial cells (PEC) and in primary stromal cells (PSC) from normal and diseased prostate by real-time RT-PCR. mRNA expression was determined for steroid hormone receptors (AR, ER{alpha}, ERß) and relevant steroid transforming enzymes e.g. type 2 5{alpha}-reductase, the 3-, 17-, and 20-ketosteroid reductases [AKR1C1 (20-ketosteroid reductase), AKR1C2 (3-ketosteroid reductase), AKR1C3 (17-ketosteroid reductase), AKR1C4 (3-ketosteroid reductase)], the major oxidative 3{alpha}-HSD (RL-HSD), other oxidative 3{alpha}-HSDs [11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase/type 10 17ß-HSD (ERAB), novel type of human microsomal 3{alpha}-HSD (NT 3{alpha}-HSD) and retinol dehydrogenase 4 (RODH 4)], and cytochrome P450 7B1 (P4507B1). The mRNA expression data indicated that many transcripts displayed a cell type-specific expression. In BPH PSC, there was an increase in the mRNA transcripts for AR, AKR1C1, AKR1C2, AKR1C3, and RL-HSD vs. normal PSC. In CaP PEC, no statistically significant changes were observed vs. normal PEC. Changes in the expression of nuclear receptors were profound and revealed significant differences in the AR:ERß ratio. This ratio was uniformly higher in normal PSC (AR:ERß ratio 280) than in normal PEC (AR:ERß ratio 8), and the ratio increased 3-fold in BPH PSC (AR:ERß ratio 820). Our data suggest that estrogen signaling is an important growth constraint in PEC because the AR:ERß ratio is suppressed 30-fold compared with PSC. Our data support the clinical findings that androgen ablative therapy is an effective treatment for BPH but not for late-stage CaP, where a selective ER modulator (SERM) may be indicated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cultured PEC and PSC
The cultured PEC and PSC were obtained and maintained as previously reported (25, 26). These protocols yielded 14 normal, 14 CaP, and six BPH primary epithelial cell cultures and 15 normal, 16 CaP, and 21 BPH primary stromal cell cultures. Thus, a total of 34 PEC cell lines and 53 PSC cell lines from well-characterized patients were examined under the same culture conditions. CaP PEC and PSC were retrieved from patients with a Gleason Grade 3 + 3 to 4 + 4. Cultured PEC had a phenotype similar to basal and/or transit amplifying cells (27), and cultured PSC were fibroblastic and/or smooth muscle cells (26). Thus, androgen signaling of differentiated secretory epithelial cells was not addressed in this study. All cells were obtained from biopsy samples using an Institutional Review Board-approved protocol at Stanford University.

Real-time RT-PCR
Real-time RT-PCR determined relative transcript levels for different proteins involved in androgen signaling. Total RNA pooled from 32 human Caucasian male prostates was purchased from BD Bioscience (Palo Alto, CA) and 1 µg of total RNA was reverse-transcribed using GeneAmp RNA PCR Kit (Applied Biosystems, Foster City, CA). Total RNA from cultured PEC and PSC was isolated as previously reported (28, 29), and 1 µg of RNA was reverse-transcribed using GeneAmp RNA PCR Kit. Real-time PCR was performed using a DNA Engine Opticon2 Continuous Fluorescence Detector (MJ Research Inc., Waltham, MA).

The real-time PCR method and primers used in this study were previously reported for the oxidative 3{alpha}-HSDs (RL-HSD, ERAB, RODH 5, NT 3{alpha}-HSD, and RODH 4) (17), type 2 5{alpha}-reductase (30), P4507B1 and PBDG (31), AR (32), ER{alpha} and ERß (33), and GAPDH (28). Real-time PCR primers for the highly related ketosteroid reductases of the aldo-keto reductase superfamily (AKR1C1-AKR1C4) were designed based on those reported earlier (29), and are listed in Table 1Go. The specificity of these primers was established by demonstrating that each primer pair was only able to amplify the target sequence of interest over 40 cycles even in the presence of 25,000 fg of cDNA of the remaining isoforms using the conditions described below (Fig. 2Go). This important series of controls established primer specificity where each AKR1C isoform shares less than 86% amino acid sequence identity.


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  		TABLE 1. Sequence and localization of oligonucleotide primers for real-time RT-PCR of AKR1C isoforms

 

Figure 2
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  		FIG. 2. Isoform-specificity of the real-time PCR primers for the quantitation of AKR1C1–1C4. Amplification curves of 25,000 fg of (bullet) AKR1C1, ({circ}) AKR1C2, ({blacksquare}) AKR1C3, and ({square}) AKR1C4 authentic full-length standard cDNA templates and of ({triangledown}) a nontemplate control that were obtained by plotting the cycle number vs. the respective DNA-binding dye fluorescent signal determined at the end of each extension step. PCRs were conducted in the presence of the oligonucleotide primer pair designed specifically to bind to AKR1C1 (A), AKR1C2 (B), AKR1C3 (C), and AKR1C4 (D) (see Materials and Methods).

 
Primer specificity for all transcripts was also determined by separating the PCR products on a 3% agarose gel and subjecting them to dideoxysequencing to ensure that only the amplification of the desired gene was achieved. At the end of the PCR, melting curves were performed for those reactions that used SYBR Green (QIAGEN Inc., Valencia, CA) to ensure the specific amplification of the desired product. The RT-PCR method for each transcript was linear (r ≥ 0.995) over a dynamic range (109) as determined by plotting the log10 fluorescence intensity vs. the number of cycles. The conditions for the real-time PCR using SYBR Green were as follows: 95 C for 15 min followed by 40 cycles of 94 C for 15 sec, X C (annealing temperature) for 30 sec and 72 C (extension temperature) for 30 sec (whereby X = 56 C for AKR1C4 (with 4% dimethylsulfoxide); 57 C for AKR1C1; 58 C for AR, P4507B1, GAPDH, and PBGD; 60 C for RL-HSD, NT 3{alpha}-HSD, RODH 4, ER{alpha} and ERß; 61 C for AKR1C2 and AKR1C3; and 63 C for ERAB and RODH 5). Type 2 5{alpha}-reductase primers used were previously reported (30) except that the TaqMan probe (Applied Biosystems) used was 5'-6FAM dCTC ACT TTG TTT CCT TGG GCT GCG AG TAMRA-3'. The cycles for the TaqMan real-time PCR using TaqMan Universal Mix (Roche Diagnostics, Indianapolis, IN) were as follows: 50 C for 3 min then 95 C for 10 min followed by 40 cycles of 94 C for 15 sec, 60 C for 60 sec. Full-length standards (2,500,000 to 0.025 fg) were generated for AKR1C1, AKR1C2, AKR1C3, AKR1C4, ERAB, RL-HSD, RODH 5, NT 3{alpha}-HSD and RODH 4 from their appropriate cDNA plasmids (pcDNA3-AKR1C1, pcDNA3-AKR1C2, pcDNA3-AKR1C3, pcDNA3-AKR1C4, pcDNA2-ERAB, pcDNA3-RL-HSD, pcDNA3-RODH 5, pcDNA3-NT 3{alpha}-HSD and pcDNA3-RODH 4). Total mRNA isolated from liver (BD Bioscience, Palo Alto, CA) was reverse-transcribed to cDNA using GeneAmp RNA PCR Kit and subsequently PCR product standards (2,500,000 to 0.025 fg) were generated for P4507B1, AR, ER{alpha}, ERß, type 2 5{alpha}-reductase, GAPDH, and PBGD by isolating the desired PCR product by gel purification. Correction factors for each transcript GADPH (3.30), PBGD (7.48), AR (11.79), type 2 5{alpha}-reductase (7.08), ER{alpha} (16.53), ERß (11.12), and P4507B1 (10.01) were used to compensate for the differences in molecular weight between full-length cDNA and PCR product standards.

All samples were analyzed in triplicate and amounts of the target cDNA (femtograms) were divided by the total cDNA in each reaction (nanograms). The resulting values were subsequently normalized to the relative amount of PBGD and GAPDH amplified (low and high abundance housekeeping genes, respectively) and similar patterns of expression were seen by normalization to either gene. The relative amount of PBGD and GAPDH is defined as the individual fg value determined for each sample for that housekeeping gene divided by the sample set average for that same housekeeping gene. Data normalized to PBGD are presented. This procedure compensated for interindividual variation in housekeeping gene expression.

Statistical analysis of transcripts in normal vs. diseased (CaP and BPH) PEC and PSC
The normal and diseased groups were compared using the Mann-Whitney rank sum test by the statistical analysis program Sigma Stat (Port Richmond, CA). This test was selected as the groups were of unequal sizes and assumes that the data followed non-Gaussian distributions. The statistical analysis was performed to compare PEC [normal n = 14 and diseased (CaP n = 14, BPH n = 6)] as well as for PSC [normal n = 15 and diseased (CaP n = 16, BPH n = 21)]. In comparing transcript levels in normal PEC vs. normal PSC fourteen comparisons were made so that the Bonferroni correction becomes P < 0.05/14 or P < 0.00357 to reject the null hypothesis. In comparing normal PSC vs. CaP PSC, normal PSC vs. BPH PSC, and CaP PSC vs. BPH PSC, the same 14 comparisons were made across the paired groups and so the Boneferroni correction was unchanged. The procedure was repeated for the comparison of normal PEC vs. CaP PEC, normal PEC vs. BPH PEC, and CaP PEC vs. BPH PEC. In comparing receptor levels, three receptors AR, ER{alpha}, and ERß were compared in three related cells (normal PEC, CaP PEC, and BPH PEC), making nine comparisons so the Boneferroni correction applied was P = 0.05/9 or P < 0.0055 to reject the null hypothesis. The procedure was replicated for normal PSC, CaP PSC, and BPH PSC.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Relative expression of mRNA transcripts that regulate androgen signaling in whole human prostate
The relative expression of mRNA transcripts that regulate the androgen signal in whole human prostate by real-time RT-PCR were determined. Transcripts included those for nuclear hormone receptors (AR, ER{alpha}, and ERß) and those for steroid transforming enzymes: type 2 5{alpha}-reductase, the ketosteroid reductases (AKR1C1-AKR1C4), the oxidative 3{alpha}-HSDs (RL-HSD, ERAB, RODH 4, RODH 5, and NT 3{alpha}-HSD) and P4507B1. AKR1C1, AKR1C2, and AKR1C3 can regulate the androgen signal by increasing the amount of 3ß-diol (proapoptotic ligand for ER), 3{alpha}-diol (inactive androgen) and testosterone formed, respectively (5, 8, 9, 11, 12). Oxidative 3{alpha}-HSDs can oxidize 3{alpha}-diol back to DHT; of these, RL-HSD was recently identified as the major oxidative 3{alpha}-HSD in normal human prostate (17). P4507B1 can also regulate the hormonal signal by converting 3ß-diol to 5{alpha}-androstane-3ß,7{alpha},17ß-triol, thus decreasing the availability of this proapoptotic ligand for ERß (16, 34). Transcripts were measured in total RNA isolated and pooled from 32 normal whole human prostates. The mRNA expression levels were normalized to the high-copy housekeeping gene GAPDH and the low-copy housekeeping gene PBGD, and similar patterns were observed.

The real-time RT-PCR data indicated that the mRNA transcripts of proteins that regulate the androgen signal were expressed in normal human prostate (Fig. 3AGo). The mRNA transcripts for type 2 5{alpha}-reductase and the AR were more abundant than those observed for other genes (by ~50-fold). AKR1C1 and ER{alpha} were the next most abundantly expressed transcripts, followed by P4507B1, ERAB, RL-HSD, AKR1C2, ERß, and AKR1C3. Finally, very low levels of RODH 5, NT 3{alpha}-HSD, and RODH 4 were detected. The transcript for AKR1C4 was not detected in normal prostate and confirms that it shows liver-specific expression (9). AKR1C1 was 13-fold more highly expressed than AKR1C2, suggesting that conversion of DHT to 3ß-diol is favored unless this ratio changes in diseased prostate. The data also indicated that the AR was much more highly expressed than ER{alpha} (10-fold) and ERß (66-fold), whereas ER{alpha} was approximately 6-fold more highly expressed than ERß. These results gave estimates of the transcript levels for steroid transforming enzymes involved in androgen signaling and the nuclear receptors that process these signals in whole normal human prostate. However, they do not provide information on expression levels by cell-type or by disease status.


Figure 3
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  		FIG. 3. Relative expression of transcripts involved in androgen signaling in whole prostate (A), prostate epithelial and prostate stromal cells (B), and the ratio of cDNA expression in prostate epithelial and stromal cells (C), normalized to PBGD as determined by real-time PCR. One microgram of total RNA from pooled whole human prostate (n = 32), was reverse-transcribed to cDNA from the prostate and 12.5 ng of cDNA was added to each real-time PCR experiment (A). One microgram of total RNA from normal PEC (n = 14) and normal PSC (n = 15) were reverse-transcribed to cDNA and 50 ng of each cDNA was added to each real-time PCR experiment (B). Each real-time experiment was performed in triplicate with the mean shown. Data are normalized to the housekeeping gene PBGD and is expressed as femtograms of each transcript per nanogram of total cDNA. C, Ratio of the expression of transcripts involved in androgen signaling in normal PEC vs. normal PSC. Positive values represent higher expression in normal PEC, whereas negative values represent higher expression in normal PSC. *, Statistically different expression levels between PEC and PSC with p values (P < 0.001) for significance.

 
Transcripts of proteins that regulate androgen signaling show a cell type-specific expression: comparison of normal prostate PEC and PSC
The expression of mRNA transcripts for proteins involved in androgen signaling was determined in normal prostate PEC and PSC cultures. The results indicated a cell type-specific expression of the transcripts (Fig. 3BGo and Table 2Go). In normal PEC, the ketosteroid reductases (AKR1C1-AKR1C3) were most highly expressed. In the same cells, ERAB was the next highest expressed followed by NT 3{alpha}-HSD, AR, type 2 5{alpha}-reductase, and P4507B1. Also RODH 5, ER{alpha}, and ERß were detected in normal PEC, but at low levels. In normal PSC, AKR1C1 was the highest expressed followed by AR, ERAB, RL-HSD, AKR1C3, and AKR1C2 and to a lesser extent RODH 5 and type 2 5{alpha}-reductase. AKR1C4 was not detected in either normal PEC or normal PSC consistent with liver-specific expression (9).


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  		TABLE 2. Transcript levels in primary cultures of prostate epithelial and stromal cells

 
The ratio of mRNA transcripts in normal PEC vs. normal PSC indicated that many transcripts associated with androgen signaling had a cell type-specific preference (Fig. 3CGo). The following transcripts were expressed at higher amounts in PEC: NT 3{alpha}-HSD (~3000-fold, P < 0.001), AKR1C2 (~115-fold, P < 0.001), P4507B1 (~75-fold, P < 0.001), RODH 4 (~20-fold, P < 0.001), AKR1C1 (~10-fold, P < 0.001), AKR1C3 (~6-fold, P < 0.001), ERAB (~4-fold, P < 0.001) and ERß (~4-fold, P < 0.001) over those levels seen in PSC using median values (Fig. 3CGo and Table 2Go). In contrast, there were transcripts that showed a preferential expression in normal PSC over that seen in normal PEC and these included: RL-HSD (~20-fold, P < 0.001) and AR (~8-fold, P < 0.001) using median values. Type 2 5{alpha}-reductase, RODH 5 and ER{alpha} were expressed almost equally in both normal PEC and PSC. Although slight increases were noted for type 2 5{alpha}-reductase in normal PEC (~2.5-fold, P = 0.121) and a slight increases were noted for RODH 5 (~1.5-fold, P = 0.198) and ER{alpha} (~1.5-fold, P = 0.121) in normal PSC they did not reach significance. The detection of type 2 5{alpha}-reductase in epithelial cells and stromal cells is consistent with the findings of others (35).

Changes in the expression of mRNA transcripts in normal and diseased prostate PEC and PSC
The expression levels of mRNA transcripts for proteins involved in androgen signaling were compared in normal and diseased prostate PEC and PSC using real-time RT-PCR (Table 2Go and Figs. 4Go and 5Go). Because the values were derived from unmatched samples and non-Gaussian distributions were observed, we used the Mann-Whitney rank sums test to determine whether changes in the expression of transcripts were statistically significant between median values and the Boneferroni correction was applied.


Figure 4
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  		FIG. 4. Representative scatter box plots of transcripts of enzymes that regulate ligand access to nuclear receptors (A) and for transcripts of nuclear receptors (B) in normal and diseased (CaP and BPH) PEC and PSC using real-time RT-PCR. One microgram of total RNA was reverse-transcribed to cDNA from the PEC and PSC and 50 ng of cDNA was added to each real-time PCR experiment that was performed in triplicate with the mean shown for each sample. Data are normalized to the housekeeping gene PBGD and is expressed as femtograms of each transcript per nanogram of total cDNA. Normal (n = 14), CaP (n = 14), and BPH (n = 6) PEC and normal (n = 15), CaP (n = 16), and BPH (n = 21) PSC were used for the study.

 

Figure 5
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  		FIG. 5. Ratios of transcripts altering ERß signaling. A, Ratio of AKR1C1:AKR1C2 transcripts; B, ratio of AR:ER{alpha} transcripts; C, ratio of AR:ERß transcripts; and D, ratio of ER{alpha}:ERß transcripts in normal and diseased prostates. Normal PEC (n = 14), CaP PEC (n = 14), and BPH PEC (n = 6) and normal PSC (n = 15), CaP PSC (n = 16), and BPH PSC (n = 21) were used for the study. Data taken from Table 2Go.

 
In CaP PEC, no significant changes in transcript levels were evident when compared with normal PEC. The levels of steroid transforming enzymes and steroid receptors were not significantly altered in CaP PEC vs. the already high levels observed in normal PEC. Similarly, in BPH PEC no significant changes were observed in the expression of transcripts for steroid transforming enzymes or steroid receptors over normal PEC. This may not be surprising because BPH originates in stromal cells of the prostate (36).

In CaP PSC, only a significant increase in mRNA expression of RODH 4 (2-fold, P < 0.005) was evident when compared with normal PSC. The remainder of the steroid transforming enzymes and steroid receptors were unaltered at the transcript level. In contrast, in BPH PSC significant increases in mRNA expression for ERAB (2-fold, P < 0.001), AKR1C1 (4-fold, P < 0.001), AKR1C2 (10-fold, P < 0.001), AKR1C3 (4-fold, P < 0.001), AR (2-fold, P < 0.001), RL-HSD (3-fold, P < 0.005) were observed, but the increase in type 2 5{alpha}-reductase expression (2-fold, P = 0.072) and decrease in ER{alpha} expression (2-fold, P = 0.072) were not significant. The increases in transcripts for the oxidative 3{alpha}-HSD (RL-HSD) and AR could lead to increased androgen gene transcription in BPH PSC.

Representative scatter box plots for transcripts in normal and diseased prostate PEC and PSC
We plotted all the transcripts investigated in this study as scatter box plots to visually identify potential trends. Representative scattered box plots for type 2 5{alpha}-reductase, RL-HSD, AKR1C1, AKR1C2, and AKR1C3, as well as for the nuclear hormone receptors (AR, ER{alpha}, and ERß) in normal and diseased (CaP and BPH) PEC and PSC, are shown (Fig. 4Go, A and B, respectively). These data indicate that several of the transcripts are more highly expressed in PEC than PSC. However, transcripts in PEC were often unaltered in disease, whereas transcripts in PSC were altered, which is in agreement with the statistical analysis. In BPH PSC, increased expression in AKR1C3 (P < 0.005) > RL-HSD (P > 0.005) > type 2 5{alpha}-reductase (not significant) suggest that these cells have gained steroid transforming enzymes necessary to produce excess DHT. Furthermore, elevated expression of AR (P < 0.001) was noted and may lead to an increase in androgen signaling in these cells. Elevated mRNA expression of AKR1C1 (P < 0.001) and AKR1C2 (P < 0.001) was observed and could indicate increased capacity to metabolize DHT in the BPH PSC. The scatter box plots also permit the preferential expression of the transcripts for the indicated proteins to be directly compared because the axes are the same for each individual transcript between normal and diseased prostate PEC and PSC.

Altered ratio of AKR1C1:AKR1C2 transcripts in normal and diseased prostate PEC and PSC
The ratio of AKR1C1:AKR1C2 mRNA expression in human prostate will determine whether DHT is converted either to 3ß-diol, a proapoptotic ligand for ERß or to 3{alpha}-diol an inactive androgen. In whole prostate, the ratio of AKR1C1:AKR1C2 transcripts was 13, but was reduced to 1.9 in normal PEC (Fig. 5AGo). This transcript ratio remained unaltered in PEC in CaP and BPH. Thus, it is predicted that PEC will produce less 3ß-diol and are less growth constrained. By contrast, the AKR1C1:AKR1C2 transcript ratio of 13 observed in whole prostate was elevated to 22 in PSC. This indicates that normal PSC generates more 3ß-diol than 3{alpha}-diol. The AKR1C1:AKR1C2 ratio decreased in diseased PSC to 13 in CaP and to eight in BPH and indicates that the 3ß-diol signal will be attenuated in this disease.

Altered ratios of steroid receptor (AR:ER{alpha}, AR:ERß and ER{alpha}:ERß) transcripts in normal and diseased prostate PEC and PSC
The levels of steroid receptor transcripts (AR:ER{alpha}, AR:ERß, and ER{alpha}:ERß) were compared in normal and diseased PEC and PSC (Fig. 5Go, B–D). In whole prostate, the ratio of AR:ER{alpha} transcripts was 6 and the ratio of AR:ERß transcripts was 50. In normal PEC, the ratio of AR:ER{alpha} was also 6 (P < 0.05) and the ratio of AR:ERß was 8 (P < 0.005). By contrast ER{alpha} and ERß mRNA transcripts were expressed in equal amounts in normal PEC (P = 0.765).

In CaP PEC, the ratio of AR:ER{alpha} transcripts increased from 6 to 30 (P < 0.005) and the ratio of AR:ERß transcripts increased from 8 to 50 (P < 0.001) and was due to elevated AR expression, whereas ER{alpha} and ERß transcripts remained constant. This would suggest that, in CaP PEC, the androgen signal can be amplified by AR. In BPH PEC, changes in the ratio of AR:ER{alpha}, AR:ERß, and ER{alpha}:ERß did not reach significance.

In normal PSC, the ratios between AR and ER transcripts were much higher than in normal PEC. The AR mRNA transcript was expressed approximately 50-fold higher than ER{alpha} (P < 0.001) and 280-fold higher than ERß (P < 0.001). This is due to a 10-fold increase in the expression of the AR transcript and a 4-fold decrease in the expression of the ERß-transcript in normal PSC when compared with normal PEC. This suggests that androgen signal is more important in normal PSC than in normal PEC, whereas the estrogen signal is more important in normal PEC than in normal PSC. The transcript level for ER{alpha} indicated that it was 5-fold higher than ERß in normal PSC (P < 0.001), suggesting that estrogen signaling may be through ER{alpha}, and is consistent with the finding that {alpha}ERKO mouse prostate cells fail to respond to DES (37, 38).

In CaP PSC, the ratio of AR:ER{alpha} transcripts increased from 50 to greater than 60 (P < 0.001), whereas the ratio of AR:ERß transcripts remained unchanged at about 275, Fig. 5Go, B and C. The ER{alpha}:ERß transcript ratio decreased slightly compared with normal (ratio 4, P < 0.001). In BPH PSC, the ratio of AR:ER{alpha} transcripts increased from 50 to greater than 180 and the ratio of AR:ERß transcripts increased from 280 to 820 (Fig. 5Go, B and C). The ER{alpha}:ERß transcript ratio decreased slightly compared with normal PSC but did not reach significance. This suggests that, in BPH PSC, androgen signaling is increased due to an elevated AR:ERß ratio.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Profiles of transcripts involved in androgen signaling were measured by real-time RT-PCR in whole normal human prostate and in normal and diseased PEC and PSC and indicated that this signal may be processed differently by cell type and disease status.

Total RNA from 32 pooled whole normal human prostates showed that all the transcripts to modulate the androgen signal were present. Type 2 5{alpha}-reductase and AR mRNA were the highest expressed. The mRNA for the major oxidative 3{alpha}-HSD (RL-HSD) was also present. Thus, normal prostate contains steroid transforming enzymes that can activate the AR either by reducing testosterone to DHT (type 2 5{alpha}-reductase) or by oxidizing 3{alpha}-diol back to DHT (RL-HSD) (17). The ketosteroid reductases (AKR1C1-AKR1C3) were also expressed in whole human prostate. AKR1C1 and AKR1C2 metabolize DHT to 3ß-diol or 3{alpha}-diol, respectively, whereas AKR1C3 reduces {Delta}4-androstene-3,17-dione to testosterone (5, 9, 11, 12). AKR1C1 was the dominant AKR1C isoform expressed and may provide a growth constraint in normal prostate, as its 3ß-diol product is a proapoptotic ligand for ERß. Expression profiles of AR, ER{alpha}, and ERß in whole normal prostate indicated that the AR was more than 10- and 66-fold higher than the transcripts for ER{alpha} and ERß, respectively.

Transcript levels in PEC and PSC may be altered by culture conditions, but these cells are superior to immortalized transformed cell lines (27). In this study, PEC and PSC from normal, CaP and BPH patients are compared from multiple individuals in which the cell culture conditions were identical providing confidence in the trends observed. In normal prostate PEC, AKR1C transcripts were the highest expressed followed by ERAB, type 2 5{alpha}-reductase, AR, and P4507B1. The expression of AR was eight times lower in PEC than in normal PSC and is consistent with the immunohistochemical localization of the AR, which shows a stromal cell preference (39, 40). Additionally, ER{alpha} showed equal expression in PEC and PSC, but ERß showed a marked increase in expression in PEC over PSC. These data are consistent with the differential cellular localization of ER{alpha} and ERß in prostate reported by others (40). This suggests the following model of androgen signaling in normal PEC and its relationship to stromal cells in an aging male (Fig. 6AGo). {Delta}4-Androstene-3,17-dione is reduced by AKR1C3 to yield testosterone, which is subsequently reduced by type 2 5{alpha}-reductase to yield DHT. DHT can then preferentially activate the stromal cell AR. The stromal cell may then influence epithelial cell growth in an androgen-dependent paracrine manner. Additional fates exist for DHT in normal PEC are that it can be converted to 3ß-diol by AKR1C1 to activate ERß or it can be converted to the inactive androgen 3{alpha}-diol by AKR1C2. Furthermore, P4507B1 could eliminate 3ß-diol by metabolizing it to 5{alpha}-androstane-3ß,7{alpha},17ß-triol that is excreted.


Figure 6
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  		FIG. 6. Summary of androgen signaling in normal PEC and PSC. A, Androgen signaling in normal PEC with the differences in expression indicated for CaP PEC and BPH PEC as determined by real-time RT-PCR. The ratio between AR and ERß is indicated in parentheses. B, Androgen signaling in normal PSC with the differences in expression indicated for CaP PSC and BPH PSC as determined by real-time RT-PCR. The ratio between AR and ERß is indicated in parentheses. AD, Androstenedione; T, testosterone; triol, 5{alpha}-androstane-3ß,7{alpha},17ß-diol.

 
Estrogen signaling by 3ß-diol may have a more important regulatory growth function in PEC than PSC due to the ratio of AR:ERß (8 in PEC compared with 280 in PSC). This is substantiated by both the ERß-KO mouse and the P4507B1-KO mouse (13, 16). ERß-KO mice had increased hyperplastic foci in the epithelial cells of the prostate, which were identified as low-grade PIN compared with wild-type mice (13, 16). The hyperplasia was reduced in the wild-type mice with 3ß-diol, but was unaltered in the ERß-KO mice. Also, P4507B1-KO mice displayed hypo-proliferative prostates compared with wild-type mice (16). P4507B1 regulates the local concentration of 3ß-diol, a ligand for the ERß (13, 14, 41, 42). These results indicate the importance of both the estrogen signal and the proapoptotic ligand 3ß-diol in regulating the normal growth of prostatic epithelial cells.

In normal prostate PSC, the AKR1C isozymes were highly expressed, albeit 10-fold less than in normal PEC. Type 2 5{alpha}-reductase, AR and RL-HSD were also present. In these cells, the expression of AKR1C3 was still 6-fold greater than type 2 5{alpha}-reductase. However, the ratio of AR to ER dramatically favored androgen signaling because the AR was expressed more than 50-fold higher than ER{alpha} and 280-fold higher than ERß. This leads to the following model of androgen signaling in normal prostate PSC in aging males (Fig. 6BGo). {Delta}4-Androstene-3,17-dione is reduced by AKR1C3 to yield testosterone, which is subsequently reduced by type 2 5{alpha}-reductase to yield DHT. An additional source of DHT is available via RL-HSD, which oxidizes 3{alpha}-diol back to DHT. DHT can be reduced either to 3ß-diol or to 3{alpha}-diol by AKR1C1 and AKR1C2, respectively. The cells are highly responsive to androgens due to the high expression of AR and the highly favorable AR:ERß ratio. Activation of stromal cell AR would increase androgen-dependent paracrine signaling to the epithelial cells.

CaP originates in the epithelial cells of the prostate but the originating cell (stem cell, basal or transitory amplifying, or secretory cell) is unknown; thus, our conclusions using CaP PEC must be tempered (27). In CaP PEC few changes over normal PEC were apparent. The largest changes were in the ratio of AR:ER{alpha} which increased from 6- to 30-fold and the ratio of AR:ERß, which changed from 8- to 50-fold vs. normal PEC These changes support an adaptive response to androgen signaling (Fig. 6AGo) and would be consistent with the amplification of AR signaling predicted by changes in the immunohistochemical staining of the receptors in CaP (40).

In CaP PEC, AKR1C2 and AKR1C3 transcript levels remained at the same high levels as seen in normal PEC. These data would be inconsistent with the down-regulation of AKR1C2 noted by real-time RT-PCR in whole CaP tissue vs. adjacent normal tissue reported by Stolz and colleagues (10). These differences may exist for several reasons. First, we examined the expression of AKR1C isoforms in individual cell types as opposed to whole prostate. Second, we examined expression levels under primary culture conditions. Third, expression may be dependent upon the Gleason grade of CaP. Our data on the high level of AKR1C3 expression in PEC may precede the high expression of AKR1C3 observed in late stage CaP (43).

In BPH PEC, an increase in the ERß signal may exist due to a decrease in the expression of AR and an increase in AKR1C1 compared with normal PEC. These changes could increase signaling via 3ß-diol (Fig. 6AGo). In BPH, proliferative properties of epithelial cells appear to be dependent upon growth factors rather than androgens; thus, large changes in transcripts for steroid transforming enzymes and steroid receptors involved in androgen signaling were not expected (44, 45, 46).

In CaP PSC, an increase in RODH 4 expression vs. normal PSC was noted (Fig. 6BGo). RODH 4 has been implicated in the oxidation of 3{alpha}-diol back to DHT but RL-HSD appears to be the more relevant enzyme for this reaction (17). Elevated RODH 4 may instead be involved regulating ligands for RXR and RAR. Activation of these nuclear receptors may be antiproliferative because these liganded receptors heterodimerize with peroxisome proliferative-activated receptor-{gamma}.

In BPH PSC, some of the most dramatic differences were observed. There was a coordinated increase in ERAB, AKR1C1, AKR1C2, AKR1C3, RL-HSD, AR, whereas no change in type 2 5{alpha}-reductase expression was noted (Fig. 6BGo). The ratio of AR:ERß which is 280 in normal PSC increased further in BPH PSC to 820. These changes would support an increase in androgen signaling via DHT not only by the reduction of testosterone by type 2 5{alpha}-reductase but also by the oxidation of 3{alpha}-diol by RL-HSD. Elevated AKR1C3 expression would produce more prostatic testosterone, and elevated AKR1C1 and AKR1C2 would provide a mechanism for the clearance of excess androgen. When combined, these data support the concept that BPH PSC have become more androgen dependent than normal PSC.

The ratios of AKR1C1:AKR1C2 transcripts may regulate normal prostate growth by altering the amount of 3ß-diol:3{alpha}-diol produced from DHT. In normal PEC, this ratio is 1.9 and increases to 22 in normal PSC, indicating that either stromal cell 3ß-diol may regulate the growth of epithelial cells in a paracrine manner or it may mediate its effects through stromal ERß signaling. The ratio of AKR1C1:AKR1C2 transcripts decreased in CaP PSC and BPH PSC compared with normal PSC, suggesting that less 3ß-diol is produced. Thus, the growth constraint seen with 3ß-diol would be attenuated.

Estrogen signaling will also be determined by the ER{alpha}:ERß ratio. In rodent and humans, ER{alpha} is predominantly located in the stromal cells and ERß is found in the basal and luminal epithelial cells (40, 47). We find that the ER{alpha}:ERß ratios are close to 1.0 in normal, CaP and BPH PEC, and that this value increases to 6.0 in normal, CaP, and BPH PSC, and this is consistent with the distribution of ER{alpha} and ERß seen by others. These data would support the use of traditional SERMs, e.g. tamoxifen to limit the growth of BPH and SERMS that selectively target ERß to be chemopreventive for CaP (47). Toremifene has been shown to reduce the incidence of PIN in phase II clinical trials, and raloxifene has been found effective in inhibiting the growth of human CaP xenografts (47, 48).

In summary, we have measured levels of transcripts that regulate androgen signaling in normal prostate, and PEC and PSC from normal prostate, BPH and CaP. We find that the processing of the androgen signal differs based on cell type and prostate disease. Our data suggest that ERß signaling may be an important growth constraint in epithelial cells and that androgen signaling is an important component of stromal cell response. Our data support the concept that SERMs targeting ERß may be chemopreventive for CaP and that androgen ablative therapy may be better suited for BPH.


   Acknowledgments
 
We would like to thank Dr. S. Bruce Malkowicz (Department of Urology, University of Pennsylvania School of Medicine) for discussion of the manuscript and Dr. Andrea Troxel (Center of Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine) for statistical advice.


   Footnotes
 
This work was supported by the National Institutes of Health (NIH) (1R01-CA-90744 and P30-ES013508 awarded to T.P.); and Department of Defense Grant DAMD (PC-040420 awarded to D.P.). D.B. was supported in part by a NIH predoctoral fellowship (1R25 CA-101871).

Disclosure Statement: D.M.P., D.R.B., and S.S., have nothing to declare. T.M.P. consults for Organon and Syrrx.

First Published Online September 7, 2006

Abbreviations: AKR, Aldo-keto reductase; AKR1C1, 20{alpha}-HSD; AKR1C2, type 3 3{alpha}-HSD; AKR1C3, type 2 3{alpha}-HSD/type 5 17ß-HSD; AKR1C4, type 1 3{alpha}-HSD; AR, androgen receptor; BPH, benign prostatic hyperplasia; CaP, prostate adenocarcinoma; DHT, 5{alpha}-dihydrotestosterone; 3{alpha}-diol, 5{alpha}-androstane-3{alpha},17ß-diol; 3ß-diol, 5{alpha}-androstane-3ß,17ß-diol; ER, estrogen receptor; ERAB, L-3-hydroxyacyl coenzyme A dehydrogenase/type 10 17ß-HSD; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSD, hydroxysteroid dehydrogenase; KO, knockout; NT 3{alpha}-HSD novel type of human microsomal 3{alpha}-HSD; P4507B1, cytochrome P450 7B1; PBGD, porphobilinogen deaminase; PEC, primary epithelial cells; PG, prostaglandin; PIN, prostatic intraepithelial neoplasia; PSC, primary stromal cells; RL-HSD, retinol dehydrogenase-like 3{alpha}-HSD; RODH 4, retinol dehydrogenase 4; RODH 5, 11-cis retinol dehydrogenase; SERM, selective ER modulator.

Received May 10, 2006.

Accepted for publication August 30, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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