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Carcinogenesis Advance Access originally published online on November 20, 2006
Carcinogenesis 2007 28(5):916-921; doi:10.1093/carcin/bgl222
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Characterization of androgen-regulated expression of CYP3A5 in human prostate

Anne-Mari Moilanen, Jukka Hakkola2, Markku H. Vaarala1,*, Saila Kauppila, Pasi Hirvikoski, Jussi T. Vuoristo3, Robert J. Edwards4 and Timo K. Paavonen5

Department of Pathology, University of Oulu and University Hospital of Oulu, FIN-90014 Oulu, Finland
1 Department of Surgery, University of Oulu and University Hospital of Oulu, PO Box 21, FIN-90029 OYS, Oulu, Finland
2 Department of Pharmacology and Toxicology, University of Oulu, FIN-90014 Oulu, Finland
3 Biocenter Oulu, FIN-90014 Oulu, Finland
4 Section on Experimental Medicine and Toxicology, Imperial College London, London, WC2A 3PX, UK
5 Department of Pathology, University of Tampere, FIN-33014 Tampere, Finland

* To whom correspondence should be addressed. Tel: +358 8 315 2840; Fax: +358 8 315 2004; Email: markku.vaarala{at}oulu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Testosterone is needed for the growth and development of the prostate. Androgen deprivation therapy is used for the treatment of prostate cancer. CYP3A5 is a human drug-metabolizing cytochrome P450 enzyme that metabolizes testosterone to the inactive 6ß-hydroxylated metabolite. We identified CYP3A5 as a novel androgen-regulated gene in human prostate by GeneChip analysis of human prostate tissues obtained from patients 3 days after therapeutic castration and from control patients. We further showed androgen induction of CYP3A5 messenger RNA (mRNA) in LNCaP prostate cancer cell line. Immunoblotting studies revealed CYP3A5 protein expression in all prostate samples studied. Immunohistochemistry and in situ hybridization was used for localization of CYP3A5 expression in prostate tissue. CYP3A5 was detected both in luminal and in basal epithelial cells of human prostate. Androgen response element was identified in the CYP3A5 proximal promoter and in electrophoretic mobility shift assay androgen receptor was found to bind this element. Androgen induction was abolished by mutation of the response element. We suggest that CYP3A5 is a part of an autoregulatory feedback loop controlling prostate cell exposure to androgens.

Abbreviations: AR, androgen receptor; ARE, androgen response element; CYP, cytochrome P450; mRNA, messenger RNA; RT–PCR, reverse transcription–polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Prostate cancer is the most common malignancy amongst men in many Western industrialized countries. Testosterone is essential for the development of prostate cancer. The action of testosterone is mediated via the androgen receptor (AR) (1). AR belongs to a superfamily of nuclear receptors that function as ligand-activated transcription factors. The AR is a type 1 receptor that forms homodimers after ligand binding and binds inverted repeat-type DNA response elements in the regulatory regions of the target genes (2). Androgen ablation therapy by chemical or surgical castration is commonly used for the treatment of advanced prostate cancer to reduce circulating testosterone levels. However, the success of the therapeutic response is limited, as prostate cancer often subsequently emerges with a hormone refractory phenotype.

Cytochrome P450 (CYP) enzymes form a large superfamily of heme-containing monooxygenases (3). CYP3A subfamily enzymes are the most abundantly expressed CYP enzymes in the liver and are responsible for the metabolism of over 50% of all clinically used drugs, and also metabolize several toxins and endogenous compounds (4). The human subfamily is encoded by four genes: CYP3A4, CYP3A5, CYP3A7 and CYP3A43 that are arranged as a cluster at chromosome 7 (4,5). In addition to liver, CYP3A5 protein is expressed in many extrahepatic tissues, for example, lung, small intestine and prostate (6–8). In one study, it was reported that CYP3A5 has a unique 5'-untranslated region in prostate (9), suggesting a different mechanism of regulation of CYP3A5 in prostate compared with that in liver. However, no confirmation of these findings has been published to date. In liver, CYP3A5 expression is bimodal and correlated with an intron 3 polymorphism (5). The CYP3A5*3 allele encodes a deviate spliced messenger RNA (mRNA) with a premature stop codon. Further, CYP3A5*3 mRNA is more unstable and rapidly degraded than wild-type CYP3A5*1 mRNA (5). The effect of this polymorphism is, however, not absolute and also some correctly sliced mRNA is produced (10). The prevalence of the CYP3A5 polymorphism is different in different ethnic groups. CYP3A5*1 genotype is present in 10% of Caucasians, 30% of Japanese and ~50% of Afro-Americans (5,11). Individuals with high hepatic CYP3A5 level have a heterozygous genotype CYP3A5*1/*3, but also CYP3A5*3/*3 individuals express low level of CYP3A5.

From the beginning of embryonic differentiation to pubertal maturation and beyond, androgens are a prerequisite for the normal development and physiological control of the prostate (12). Androgens also help maintain the normal metabolic and secretory functions of the prostate. Exposure of prostate cells to testosterone is regulated by the rates of synthesis and metabolism of the hormone. CYP3A5 catalyzes the 6ß-hydroxylation of testosterone producing a metabolite that is less biologically active and more readily eliminated and thus inhibits testosterone metabolism to more biologically active forms of androgens (9,13). Variation in the expression of CYP3A5 in the prostate could be an effective way to regulate local levels on testosterone and to control tissue-specific androgen effects. The regulation of CYP3A5 is not well understood at present, but nuclear receptors pregnane X receptor, constitutively activated receptor and glucocorticoid receptor participate in regulation in liver and lung (10,14). There is little information on regulation of CYPs in human prostate. However, in rat prostate some CYP forms have been reported to be expressed androgen dependently (15,16).

In the present study, we used a GeneChip array to search for novel genes regulated by androgens in human prostate. CYP3A5 was identified as being downregulated by castration indicating androgen-dependent expression. CYP3A5 androgen induction was detected also in prostate-derived cell line LNCaP. We studied expression and tissue localization of CYP3A5 in prostate and show both at mRNA and at protein levels expression of CYP3A5 in human prostate. We studied the mechanism of CYP3A5 regulation by androgens and present evidence showing that androgens regulate CYP3A5 by classical AR-mediated mechanism involving binding of AR to the CYP3A5 5' regulatory region. These results establish CYP3A5 as a novel androgen regulated, steroid and drug-metabolizing enzyme in human prostate.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patient samples
Prostate samples were collected from patients undergoing prostatectomy or transurethral resection of prostate for the treatment of prostate cancer, benign prostatic hyperplasia or bladder outlet obstruction. Androgen-regulated genes in human prostate were studied from prostate biopsy samples taken 3 days after surgical castration performed as the treatment of prostate cancer. These men had freshly diagnosed prostate cancer with no previous hormonal treatments. Biopsies were taken using a biopty gun technique and 18G needles. Six biopsies were taken and prophylactic ciprofloxacin 500 mg single dose was given. A written consent was obtained from every patient giving tissue samples for the study. The Ethics Council of the Northern Ostrobothnia Hospital District has accepted the research plan. RNA from human samples were isolated using QuickPrep Total RNA Extraction Kit (Amersham Biosciences, Piscataway, NJ) according to manufacturer's instructions. RNAs from six patient's samples were individually labeled and used for the GeneChip array. Three samples were benign prostate tissue and three samples from biopsies taken after surgical castration performed as a therapeutic procedure for prostate cancer.

Cell culture
Prostate cancer cell line LNCaP (CRL-1740) cells and African green monkey kidney fibroblast-like cell line COS-7 (CRL-1651) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). LNCaP and COS-7 cell cultures were maintained in RPMI 1640 (Sigma-Aldrich, St Louis, MO) supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 2.5 g/l D-glucose or DMEM (Sigma-Aldrich) supplemented with 4500 mg/l glucose, L-glutamine and 1% penicillin–streptomycin (Invitrogen-Gibco, Carlsbad, CA), respectively, and were added with 10% fetal bovine serum (HyClone, Logan, UT) at 37°C in a humified atmosphere of 5% CO2. Fetal bovine serum was substituted with charcoal-treated fetal bovine serum in the hormone-induction experiments. Seventy-two hours prior to experiments the LNCaP cells were plated with 1 x 106 cells per plate. Cells were treated with 10 nM R1881 (PerkinElmer, Boston, MA) or an equal volume of ethanol for 0, 6, 24 or 48 h. After the incubation, cells were harvested, washed with phosphate-buffered saline and used directly for isolation of RNA. For transfection, COS-7 cells were grown to 80% confluency before transfection.

GeneChip protocol
Experimental procedures for GeneChip were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual following the minimum information about a microarray experiment guidelines. In essence, using 8 µg of total RNA as template, double-stranded DNA was synthesized by means of the One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA) and T7-(dT)24 primer. The DNA was purified using GeneChip Sample Cleanup Module (Qiagen, Venlo, The Netherlands). In vitro transcription was performed to produce biotin-labeled cRNA using an in vitro transcription labeling kit (Affymetrix) according to manufacturer's instructions. Biotinylated cRNA was cleaned with a GeneChip Sample Cleanup Module (Qiagen), fragmented to 35–200 nucleotide and hybridized to Affymetrix Human Genome U133 Plus 2 arrays that contain ~55 000 human transcripts. After being washed, the array was stained with streptavidin–phycoerythrin (Molecular Probes, Eugene, OR). Staining signal was amplified using biotinylated antistreptavidin (Vector Laboratories, Burlingame, CA) and second staining with streptavidin–phycoerythrin, and then scanned on a GeneChip Scanner 3000. The expression data were analyzed using Affymetrix GeneChip Operating System Software. Signal intensities of all probe sets were scaled to the target value of 500. Values presented represent numeric values obtained from the scanner after this scaling.

Plasmids
CYP3A5 promoter constructs were prepared as described previously (17). The CYP3A5 promoter fragments were cloned to pGL3-Basic (Promega, Madison, WI) in front of the luciferase reporter gene. Potential androgen response elements (AREs) were identified using the fuzznuc program (http://bioweb.pasteur.fr/docs/EMBOSS/fuzznuc.html). Mutagenesis reactions were performed using GeneTailor Site-Directed Mutagenesis System (Invitrogen-Gibco) according to manufacturer's protocol. Oligonucleotides 5'-CTTTATTAGTTGAAATACAGTTTGACTGCATTTGAGTCCC-3' and 5'-GGGACTCAAATGCAGTCAAACTGTATTTCAACTAATAAAG-3' were used for mutation of the putative ARE at –738 to –724.

Transfections
The COS-7 cells were seeded into 24-well plates at 30 000 cells per well 24 h before transfection. The cells were transfected with Dotap transfection reagent (Roche Diagnostics GmbH, Penzberg, Germany) according to manufacturer's protocol, using 0.65 µg of plasmid and 3 µg of transfection reagent and DMEM media (Sigma-Aldrich). AR cDNA in pcDNA3.1 vector (a gift from Prof. T.Visakorpi, University of Tampere, Tampere, Finland) was cotransfected. Twenty-four hours after transfection, the media was replaced with DMEM and 100 nM R1881 (PerkinElmer) was added when appropriate for 48 h. To provide an internal control for transfection efficiency, a second reporter plasmid pRL-TK (Promega) was transfected. The luciferase activities for COS-7 cells were measured using the Dual-luciferase reporter assay system (Promega). Mouse mammary tumor virus promoter in pGL3-Basic (a gift from Prof. J.Palvimo, University of Kuopio, Kuopio, Finland) was used as a control for androgen response.

Quantitative reverse transcription–polymerase chain reaction
Total RNA from LNCaP cells for quantitative reverse transcription–polymerase chain reaction (RT–PCR) measurements was isolated with TriZol reagent (Invitrogen-Gibco) according to manufacturer's protocol. RNA isolated from prostate samples was also used. The first-strand cDNA was synthesized with the First-Strand cDNA synthesis kit (Amersham Biosciences) using 1 µg of RNA and pd(N)6 random hex deoxynucleotides according to manufacturer's instructions. mRNA levels for LNCaP cells and human prostate samples were measured by quantitative RT–PCR analysis (ABI 7700, Applied Biosystems, Foster City, CA) as described (18). Forward and reverse primers for CYP3A5 mRNA detection were 5'-AAGGAAGACTCACAGAACACAGTTGA-3' and 5'-GGTTTCCACCGCCAAATTT-3', respectively. Amplicon was detected using fluorogenic probe 5'-FAM-AAGGAAAGTGGCGATGGACCTCATCC-TAMRA-3'. The primers and the probe for the 18S amplicon were 5'-TGGTTGCAAAGCTGAAACTTAAAG-3', 5'-AGTCAAATTAAGCCGCAGGC-3' and 5'-VIC-CCTGGTGGTGCCCTTCCGTCA-TAMRA-3', respectively.

Immunochemistry
Microsomal fractions of prostate samples were isolated and samples were transferred to nitrocellulose filters after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using standard methods. Immunoblotting was then carried out as described earlier (19) using a CYP3A5 peptide antibody (20) and an actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Prostate tissue slides containing 4 µm sections were used for immunohistochemistry with CYP3A5 peptide antibody using routine methods.

In situ hybridization
Poly-L lysine coating slides containing 4 µm prostate tissue sections were used. In situ hybridizations were carried out as described (21). [{alpha}-35S]CTP-labeled RNA probes were synthesized using CYP3A5 cDNA fragment 1191–1536 (NM_000777 [GenBank] .2) in pCRII (Invitrogen-Gibco).

Genotyping
The prevalence of CYP3A5*1 and CYP3A5*3 genotypes in human prostate samples was studied using formalin-fixed, paraffin-embedded prostate tissue samples. Genomic DNA was isolated as described (22). Oligonucleotides 5'-ACCACCCAGCTTAACGAATG-3' and 5'-TTGTACGACACACAGCAACCT-3' were used for direct sequencing of intron 3 for single nucleotide polymorphism in nucleotide 6986 (CYP3A5*3 A->G). PCRs and sequencing were done as described (21). Annealing temperature was 58°C.

Electrophoretic mobility shift assay
Double-stranded oligonucleotides were prepared by annealing the desired sense and antisense oligonucleotides. Only the sense strands of the oligos are presented: wild-type CYP3A5 5' –749, 5'-CTTTATTAGTTGGGACACAGTGTGGCTGCATTTGAGTCCC-3'; mut CYP3A5 5' –749 5'-CTTATTAGTTGAAATACAGTTTGACTGCATTTGAGTCCC-3' and prostate-specific antigen–ARE 5' 5'-TTGCAGAACAGCAAGTGCTAGCTC-3'. Single-stranded oligonucleotides were purchased from Sigma-Genosys (The Woodlands, TX). Double-stranded probes were end labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase and then purified using the QIAquick nucleotide removal kit (Qiagen). The full-length AR coding region in pSP64poly(A) plasmid was transcribed and translated in vitro to produce a non-radioactive protein using the TNT Quick coupled transcription–translation system according to manufacturer's protocol (Promega). Seven microliters of the in vitro transcribed AR protein was incubated with the labeled, double-stranded oligonucleotide in a binding buffer containing 10 mM Tris–HCl, pH 8.0, 50 mM NaCl, 10% glycerol, 1 mM 1,4-dithiothreitol, 0.5 mM EDTA, 1 mM KCl, 100 nM R1881 and 3 µg of poly(dI-dC). The samples were subjected to electrophoresis on a 5% non-denaturing polyacrylamide gel in 1x TGE buffer (25 mM Tris–base, 188 mM glycine and 1 mM EDTA) for 2.5 h. For competition experiments, an excess of unlabeled oligonucleotides was incubated with the AR protein for 15 min on ice prior to the addition of the labeled oligo. For antibody supershift experiments, 5 µl of AR antibody (N-20: SC-816, Santa Cruz Biotechnology) was added to the reaction mixture and incubated for 20 min at room temperature prior to the addition of labeled oligo. All protein–DNA interactions were performed in a 20 µl reaction volume for 60 min at room temperature.

Statistical analyses
Statistical analyses were performed using SPSS version 12.0.1 (SPSS, Chicago, IL). Student's t-test was used for comparison between two groups and the difference was considered to be statistically significant when the P value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Androgen regulation of CYP3A5 mRNA in human prostate tissue and LNCaP prostate cancer cell line
In search for novel androgen-regulated genes in human prostate, we analyzed relative gene expression levels in benign prostate tissue samples and in prostate samples taken after castration by using a GeneChip array. Relative expression values of CYP3A5 mRNA in three benign prostate tissue samples and in three prostate samples taken after castration were 2566, 1366, 2219 and 614, 173, 488, respectively. Respective values for the well-documented androgen-regulated gene, PART1 were 414, 1026, 808 and 212, 172, 134. For the other CYP3A genes detected by the chip, no evidence of androgen regulation was found. Respective values for CYP3A4 were 215, 427, 535 and 58, 379, 331; for CYP3A43 256, 317, 207 and 277, 392, 200 and for CYP3A7 116, 11, 114 and 100, 34, 66.

CYP3A5 expression and androgen regulation were then analyzed in the LNCaP cell line using quantitative RT–PCR. A 41/2-fold induction of CYP3A5 mRNA expression was detected after 48 h treatment with 10 nM R1881 (Figure 1). Androgen induction seen with CYP3A5 mRNA appeared at approximately the same time as another well-known androgen-regulated gene, prostate-specific antigen (23).


Figure 1
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  		Fig. 1. Response of CYP3A5 mRNA in LNCaP cells after treatment with 10 nM synthetic androgen R1881. Evident, statistically significant induction of CYP3A5 mRNA was seen after 48 h. * P = 0.02 Student's paired samples t-test, compared with 48 h control. The values represent means ± standard error of means of three individual samples.

 
Identification of ARE in CYP3A5 promoter
To study the involvement of transcriptional regulation in androgen regulation of CYP3A5 and to locate the putative androgen responsive regulatory region, a series of constructs containing the CYP3A5 promoter region starting from positions –208, –433, –637, –1140 or –1355 to +40 (positions numbered from the transcription initiation start site) were transfected into COS-7 cells together with AR expression vector and the cells were treated with androgen or vehicle only. Results for transient transfections are shown in Figure 2A. Androgen induction was detected for the constructs –1140 to +40 and –1355 to +40, but not for the constructs with shorter promoter regions, indicating that the promoter region –1140 to –637 contains sequence elements critical for CYP3A5 androgen induction. This region was screened for putative AREs (24) using the fuzznuc program and one ARE-like element (GGGACACAGTGTGGC) was detected at position –738 to –724. Based on sequence analysis, the identified ARE is not conserved among the other human CYP3A genes. Using site-directed mutagenesis, the potential ARE in the –1140 to +40 promoter construct was mutated to lose AR-binding ability and the construct was transfected into COS-7 cells. This mutation led to the disappearance of the androgen induction detected with the wild-type construct (Figure 2B). The same constructs were studied in the LNCaP cell line. Similar results were seen in transient transfections to LNCaP cells as to COS-7 cells (data not shown).


Figure 2
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  		Fig. 2. (A) Results from transient transfections of different CYP3A5 promoter constructs to COS-7 cells together with AR expression vector. Statistically significant induction of luciferase activity by 100 nM R1881 was detected with constructs –1140 to +40 and –1355 to +40. With shorter constructs no induction was seen. Mouse mammary tumor virus promoter in pGL3-Basic was used as positive control. * Statistically significant induction of luciferase activity by R1881 compared with the respective control, P < 0.05 Student's paired samples t-test. (B) Mutation of the CYP3A5 5' ARE leads to loss of androgen induction. CYP3A5 promoter constructs –1140 to +40 with wild-type (WT) ARE or mutated ARE at position –738 to –724 were transfected to COS-7 cells and the cells were treated with 100 nM R1881. * Statistically significant androgen induction of the –1140 to +40 (P = 0.021) construct was abolished by the ARE mutation. The mutated nucleotides in the ARE sequence are underlined. The values represent means ± standard error of means of four samples in three independent experiments (A and B).

 
Binding of AR to CYP3A5 promoter ARE
Binding of AR to the CYP3A5 promoter androgen responsive region was studied with electrophoretic mobility shift assay (Figure 3). In vitro produced AR was incubated with double-stranded, end labeled oligo containing the CYP3A5 ARE sequence and the formed complexes were separated by polyacrylamide gel electrophoresis. Two retarded complexes were formed. However, the lower complex was produced also in control reactions without AR (Figure 3B) indicating that it is unspecific by nature. These complexes were strongly reduced by competition with excess unlabeled oligonucleotides containing the CYP3A5 ARE or prostate-specific antigen gene ARE, but not by the mutated CYP3A5 ARE sequence. A human AR antibody was able to supershift the upper band indicating that this complex represents DNA-bound AR, whereas the lower represents a non-specific complex.


Figure 3
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  		Fig. 3. Binding of AR to the CYP3A5 promoter ARE. Electrophoretic mobility shift assays were performed using double-stranded, end labeled oligos containing CYP3A5 ARE sequence (A) or control electrophoretic mobility shift assay reactions with rabbit reticulosyte lysate system only or with empty expression vector (B). The labeled probes were incubated with in vitro produced AR protein. Two complexes were formed with CYP3A5 ARE (A). The lower band is present also in control reactions without AR indicating that this is unspecific by nature (B). The formed complexes were strongly reduced by competition by excess of unlabeled oligonucleotides containing CYP3A5 ARE, but not by mutated CYP3A5 ARE sequence (A). Human AR antibody was able to supershift the upper CYP3A5 ARE complex and the prostate-specific antigen–ARE complex indicating that these complexes represent DNA-bound AR (indicated with arrows).

 
CYP3A5 protein and mRNA expression in human prostate
Immunoblotting studies revealed the presence of the CYP3A5 protein in all prostate samples studied. The CYP3A5 protein level was 10- to 20-fold lower than in a liver sample used as control. This liver sample was chosen based on our previous studies and expresses high level of CYP3A5 protein based on genetic polymorphism (19). The first series of prostate samples examined by immunoblotting were from patients homozygous for CYP3A5*3 allele and thus expected to express low level of CYP3A5 protein. All these samples expressed similar amounts of CYP3A5 (Figure 4, panel A). In the second series of samples, three individuals genotyped as CYP3A5*1/*3 (expected to express high level of CYP3A5) and three with the CYP3A5*3/*3 genotype were assessed. One of these samples was found to express higher level of CYP3A5 than the others, but there was no apparent relationship between genotype and CYP3A5 protein level (data not shown). Also, CYP3A5 mRNA levels were measured by quantitative real-time RT–PCR in five of these samples available. CYP3A5 mRNA level did correlate with the genotype and CYP3A5*1/*3 samples had in average 4-fold higher level compared with the CYP3A5*3/*3 samples (Figure 4, panel B).


Figure 4
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  		Fig. 4. Detection of CYP3A5 protein in human prostate samples by immunoblotting. Panel (A): lane 1, human liver microsomes with genotype CYP3A5*1/*3 (2.5 µg); lanes 2–5, prostate microsomal samples homozygous for CYP3A5*3 allele (25 µg); lane 6, the same prostate sample as lane 5 (25 µg) plus the liver sample identical to lane 1 (2.5 µg) and lanes 7–10, prostate microsomal samples homozygous for CYP3A5*3 allele (25 µg). Panel (B): CYP3A5 mRNA levels measured by quantitative RT–PCR. The genotype of each prostate sample is indicated.

 
Expression and localization of CYP3A5 in the prostate was further studied by in situ hybridization and immunohistochemistry (Figure 5). In situ hybridization of human prostate samples with CYP3A5 antisense probe detected mRNA in both luminal and basal epithelial cells. In immunohistochemistry, the CYP3A5 antibody stained the luminal and basal epithelial cells strongly, and there was only weak staining in prostatic stroma. In genotyped human prostate samples, four individuals with a CYP3A5*1/*3 genotype and four with a CYP3A5*3/*3 genotype showed no difference in CYP3A5 expression (data not shown). Expression of CYP3A5 was also examined in prostate cancer samples, and these showed expression in epithelial cells at a similar level to that found in benign prostate tissue (Figure 5).


Figure 5
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  		Fig. 5. CYP3A5 mRNA and protein expression in human prostate. In situ hybridization (A and B) of human prostate samples with CYP3A5 antisense probe detected mRNA in both luminal and basal epithelial cells of human prostate. Normal prostate (A) and prostate cancer (B). Sense probe in situ hybridizations shows no specific labeling (C). Immunohistochemistry (D and E) of prostate tissue slides detected CYP3A5 protein (brown) both in luminal and in basal epithelial cells. Only weak staining is seen in stroma. Normal prostate (D) and prostate cancer (E). In (F), same immunohistochemical staining procedure as in (D) and (E) but no CYP3A5 antibody. Original magnification x40 (A-F).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In search for novel androgen-regulated genes in prostate, we identified CYP3A5. CYP3A5 is a drug-metabolizing enzyme expressed at the highest level in liver, but known to be expressed also in several extrahepatic tissues including prostate (25). Little is, however, known about its regulation outside of the liver. In the current study, we show for the first time that androgens upregulate CYP3A5 expression in human prostate and also in a prostate-derived cell line LNCaP. We identified the ARE in the 5' regulatory region of the CYP3A5 gene and showed that the AR is able to directly bind to this sequence. Furthermore, we studied extent and localization of CYP3A5 expression in human prostate both at the mRNA and at the protein level.

In liver, the expression of CYP3A enzymes is dominated by CYP3A4 that on average accounts for 95% of CYP3A mRNA (8). However, individual variation is large and in some individuals polymorphic CYP3A5 and CYP3A7 expression accounts for up to 20% of CYP3A transcripts (8). The predominant expression of CYP3A4 is limited to liver and small intestine (25) and in several other tissues including lung, adrenal gland, kidney and prostate CYP3A5 is the major CYP3A form with little expression of the other CYP3A enzymes (8).

Expression of CYP3A5 in prostate tissue has been previously studied only at the mRNA level (9,26,27) although CYP3A-related protein has been detected by immunohistochemistry from prostate tumor samples (28,29). In the current study, we detected expression of CYP3A5 both at mRNA and at protein levels in all prostate samples studied although the prostatic protein level was substantially lower than that in the liver sample used as a control. For protein studies, we used a peptide antibody that specifically detects only CYP3A5 and not the other CYP3A species indicating that the protein detected indeed represent CYP3A5 (20). In liver, high-level CYP3A5 expression is strongly associated with CYP3A5*1 allele. In the present study, we found higher prostatic CYP3A5 mRNA level in heterozygous CYP3A5*1/*3 samples than in the homozygous CYP3A5*3/*3 samples as expected. However, at the protein level there was no correlation. Although the number of samples analyzed for CYP3A5 expression was small, the obvious lack of correlation suggests that CYP3A5 may be differentially regulated in prostate than in liver at translational or posttranslational level. Unfortunately, we could not examine the CYP3A5 protein levels in prostate and liver from the same individuals as such fresh samples may be impossible to obtain for ethical reasons.

In immunohistochemistry, CYP3A5 expression was localized particularly to the epithelial cells. These cells are probably to be the metabolically more active component of the prostate compared with the stroma, as epithelial cells are active in prostatic fluid formation (30). In addition, adenocarcinoma of prostate arises from these cells. Further, after castration, effect of apoptosis is seen predominantly in the epithelial cells (31). In the normal prostate, the function of stroma is mainly contraction during ejaculation. However, in benign prostatic hyperplasia, there is often overgrowth of stroma. CYP3A5 expression is localized to functionally active cells of the prostate that are also androgen responsive. Expression of AR is also detectable in the nuclei of both benign and malignant prostatic epithelial cells (32). Stromal–epithelial interaction and its significance in prostate development and prostate diseases has been the subject of research for over a decade. Prostate epithelial cells secrete growth factors and cytokines regulating adjacent stroma. Stromal cells in turn act similarly in a paracrine manner to regulate epithelial cells. Prostate development is also under hormonal control and it is supposed that the influence of androgen is primarily mediated by the stromal cells that release soluble paracrine factors that are important in the growth and development of the prostate epithelium (33). These paracrine pathways may be critical in regulation of the balance between proliferation and apoptosis of prostate epithelial cells in the adult (34). Dysregulation of androgen-inactivating function of CYP3A5 could thus act both on epithelial cells themselves and on adjacent stroma resulting in increased local testosterone concentrations leading to interference of stromal–epithelial interactions that are even susceptible to malignant transformations.

Characterization of CYP3A5 regulation has been limited so far. Recently, CYP3A5 was shown to be under the control of nuclear receptors pregnane X receptor and constitutively activated receptor in liver and in intestine (10). In lung-derived A549 cells, CYP3A5 is induced by glucocorticoids by a glucocorticoid receptor-mediated mechanism (14). Our present study shows that CYP3A5 is upregulated by AR and androgens appear to play significant role in maintenance of CYP3A5 transcription in prostate as CYP3A5 mRNA levels were clearly lower in the castrated men compared with the men having normal androgen level. There are some suggestions from animal studies that AR could play role in the regulation of drug metabolism (35,36). However, to our knowledge, this is the first study to report AR regulation of a human drug-metabolizing CYP. We also show that the CYP3A5 androgen regulation follows the classical nuclear receptor-mediated mechanism involving direct binding of activated AR to the cognate binding site at the CYP3A5 5' regulatory region. However, our results do not exclude involvement of also posttranscriptional mechanism. AR is much more abundant in prostate compared with the other tissues expressing CYP3A5 such as liver and it remains to be studied if the CYP3A5 androgen regulation is limited to prostate tissue or if this regulatory mechanism plays a role in other tissues as well. In extrahepatic tissues with low CYP3A4 levels, there could be significant local effects and surgical or chemical castration therapy of men having prostate cancer could change their CYP3A5 status. Furthermore, androgen levels decline in aging males that could also decrease CYP3A5 expression.

Drug-metabolizing CYP enzymes also catalyze the synthesis and catabolism of numerous endogenous compounds such as steroids, arachidonic acid and retinoids, and an increasing body of knowledge suggests that CYP enzymes control amount and effects of many of these compounds and affect vital functions such as vascular and endocrine homeostasis (37,38). CYP3A5 catalyzes the 6ß-hydroxylation of testosterone leading to a loss of the androgenic effect of the molecule (9,39). Therefore, the CYP3A5 enzyme may control the local exposure of prostate cells to testosterone. Our current finding that androgens upregulate CYP3A5 expression in the prostate suggests that CYP3A5 serves as part of a negative feedback mechanism in which androgens restrict their own effects by inducing testosterone inactivation.

In conclusion, we describe for the first time androgen regulation of CYP3A5 in human prostate and show that this regulation involves classical mechanism via AR. Considering that CYP3A5 is a testosterone-inactivating enzyme, these findings suggest that regulation of CYP3A5 has evolved to control testosterone exposure of prostate cells as part of an autoregulatory loop. This may affect pathogenesis of prostate disorders via stromal–epithelial interaction.


   Acknowledgments
 
We are grateful to Mrs Annikki Huhtela and Mrs Heli Ylisuutari for performing in situ hybridizations, Mrs Erja Tomperi and Mrs Mirja Vahera for doing immunohistochemistry, Mrs Marja Tolppanen for help in cell culturing, Mrs Marjaana Vuoristo for technical guidance, and Hannu Wäänänen for assistance with creating figures. Mrs Tarja Piispanen assisted with RNA isolations. Dr Mika Ilves performed the quantitative RT–PCR experiments. Dr A.Domanskyi is acknowledged for sharing his experience with electrophoretic mobility shift assay. The computations presented in this document have been made with center for scientific computing computing environment. CSC is the Finnish IT center for science and is owned by the Ministry of Education. This work was supported by grants from Päivikki and Sakari Sohlberg Foundation, Lilly Foundation, Finland; Urologic Research Foundation, Finland, and State subsidy for the University Hospital of Oulu. J.H. was supported by The Academy of Finland (contract 110591).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 11, 2006; revised November 6, 2006; accepted November 6, 2006.


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S Leskela, E Honrado, C Montero-Conde, I Landa, A Cascon, R Leton, P Talavera, J M Cozar, A Concha, M Robledo, et al.
Cytochrome P450 3A5 is highly expressed in normal prostate cells but absent in prostate cancer
Endocr. Relat. Cancer, September 1, 2007; 14(3): 645 - 654.
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