help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martin, C.
Right arrow Articles by Habib, F. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martin, C.
Right arrow Articles by Habib, F. K.
The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 6 2928-2935
Copyright © 2004 by The Endocrine Society

CYP7B Generates a Selective Estrogen Receptor ß Agonist in Human Prostate

Cécile Martin, Margaret Ross, Karen E. Chapman, Ruth Andrew, Prasad Bollina, Jonathan R. Seckl and Fouad K. Habib

Endocrinology Unit (C.M., M.R., K.E.C., R.A., J.R.S.), The Prostate Research Group (C.M., M.R., F.K.H.), Department of Oncology, University of Edinburgh Western General Hospital, and Department of Urology (P.B.), Western General Hospital, Edinburgh EH4 2XU, United Kingdom

Address all correspondence and requests for reprints to: Dr. Cécile Martin, Endocrinology Unit, Molecular Medicine Centre, Western General Hospital, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom. E-mail: cecile.martin{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
In human prostate, dehydroepiandrosterone (DHEA) is a substrate for two major metabolic pathways that produce functionally opposing sex steroids. In one pathway, DHEA is converted into potent androgens such as testosterone and 5{alpha}-dihydrotestosterone. In the other, DHEA is metabolized to 7{alpha}-hydroxy-DHEA (7HD). Recently, CYP7B, a novel P450 enzyme originally characterized in mouse brain and expressed in rodent prostate, has been found to be responsible for all extrahepatic 7{alpha}-hydroxylase activity. In this study, we have investigated the expression and function of this novel enzyme in human prostate.

We have used reverse transcription combined with PCR and mRNA in situ hybridization to determine and localize the expression of CYP7B mRNA in human benign prostatic hyperplasia. High levels of CYP7B mRNA were localized in the epithelial cells together with estrogen receptor ß (ERß). 7{alpha}-Hydroxylation was the major metabolic fate of DHEA in human prostate. Furthermore, we have shown that human prostate epithelial cells in primary culture maintain a high level of 7{alpha}-hydroxylase activity, which was enhanced by coculture with stroma cells. To investigate the functional relevance of CYP7B expression to sex-steroid action in prostate, we used transient transfections and ligand binding assay to determine the ability of 7HD to bind and activate the sex-steroid receptors: androgen receptor, ER{alpha}, and ERß. 7HD specifically activates ERß-mediated transcription, mimicking the effects of 17ß-estradiol, but has no impact on ER{alpha} and androgen receptor. Given that DHEA, and its sulfate, circulate at micromolar concentrations, there is a clear possibility that CYP7B generates sufficient 7HD to activate ERß over and above that achieved with very low concentrations of intraprostatic 17ß-estradiol. In conclusion, our study suggests that CYP7B catalyzes oxysterol 7{alpha}-hydroxylation within the human prostate epithelium. By this reaction, an ERß-specific agonist, 7HD, is produced. Therefore, CYP7B may be a novel regulator of the androgens/estrogenic balance within the prostate.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
ANDROGENS PLAY AN important role in the development and function of the prostate and are also involved in the initiation and maintenance of benign prostatic hyperplasia (BPH) and prostate cancer (1). Recent evidence in animal models and humans have suggested that other steroid hormones, including estrogens, may also be involved both in the growth of the gland and in its disorders (2, 3). With age, declining testicular function leads to lower levels of plasma testosterone (4). However, estrogen (17ß-estradiol, E2) levels are maintained by enhanced aromatization of adrenal androgens, notably DHEA (5) (Fig. 1Go), in peripheral tissue such as adipose tissue (6). Therefore, in elderly men, the ratio between free E2 and free testosterone may increase by 40% (7). The endocrine changes at midlife in men have long been observed and might account for the pathologies seen in the prostate after the fifth decade (1, 8, 9).



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 1. The steroid pathway of DHEA. A/enedione, 5{alpha}-Androstenedione; E1, estrone.

 
In the prostate, estrogens interact with two forms of estrogen receptor (ER), ER{alpha} and ERß. The two receptors differ in their binding affinity for a variety of estrogenic compounds and in their sublocalization in the human prostate (10, 11, 12, 13). ER{alpha} is expressed only at low levels and is confined to the stroma, where it may influence epithelial growth in a paracrine manner (14). In contrast, ERß is highly expressed in prostatic epithelium (13). The role of ERß in human prostate is not clear. In one study, ERß knockout mice have been reported to develop prostatic hyperplasia with age (15), suggesting antiproliferative functions. However, other ERß knockout models do not have this prostate phenotype. Although these studies show distinct roles for ER{alpha} and ERß, they do not establish the identity of the ligand responsible for these estrogenic actions. Whereas E2 may be the major ligand for ERß in most target cells, the levels of E2 in human prostate are probably too low to activate the receptors (16). This raises the possibility that other estrogenic ligands produced locally in the prostate might be responsible for ERß estrogenic activity. Indeed, local steroid metabolism is an important determinant of steroid action in various organs (17). Thus, local metabolism in prostate may be a key to steroid biological activity.

Recently, we have shown that CYP7B, a novel cytochrome P450 identified in rodent hippocampus (18, 19) that catalyzes the 7{alpha}-hydroxylation of DHEA to 7{alpha}-hydroxy-DHEA (7HD), is highly expressed in rodent prostate (20). 7{alpha}-Hydroxylase (CYP7B) is the only route for the 7{alpha}-hydroxylation of DHEA, as confirmed by the CYP7B knockout animals (21, 22, 23), which show no residual DHEA 7{alpha}-hydroxylation in prostate and brain (22). Moreover, Gustafsson and colleagues (23) have found that the prostate of CYP7B knockout mice are smaller than those of their wild-type littermates, suggesting that CYP7B is an important element regulating prostatic growth. CYP7B is unusual among P450s in being much more highly expressed in specific extrahepatic tissues, notably hippocampus and prostate (19), than in the liver. 7{alpha}-Hydroxylase activity has also been reported in humans (24). But the enzyme(s) responsible for this reaction in most tissues is unknown.

In the present study, we have investigated the expression of CYP7B in human prostate and demonstrated that 7{alpha}-hydroxylation is a major route for DHEA metabolism in human prostate producing 7HD. Furthermore, reporter gene experiments have shown that 7HD specifically activates ERß.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Experimental subjects

Paraffin-embedded archival prostate tissues from BPH patients were provided by the Department of Pathology (Western General Hospital, Edinburgh, UK). Fresh prostate samples for CYP7B activity measurements and cell culture were obtained from patients (aged 56–70 yr) undergoing transurethral resection of the prostate who have not been treated with hormone ablation. No biopsy samples were used in this study. Randomly selected prostate chips from each specimen were evaluated histopathologically to establish their benign status and the presence of hyperplasia. Only samples taken with informed consent were studied, and our protocol was approved by the local Research Ethics Committee.

Steroids

[1,2,6,7-3H]4-DHEA (60 Ci/mmol), [4-14C]-DHEA (53.8 mCi/mmol), and [1,2-3H]2-5{alpha}-androstenediol (A/enediol) (42 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA), and [2, 4, 6, 7-3H]4-E2 was purchased from Perkin-Elmer Life Sciences (Boston, MA). Nonradioactive steroids and clotrimazole were obtained from Sigma-Aldrich (Poole, UK). 7HD was purchased from Steraloids, Inc. (Newport, RI). Trilostane was kindly provided by Sanofi Winthrop Development Centre (Newcastle Upon Tyne, UK), and ICI 182,780 was purchased from Tocris (Bristol, UK).

7{alpha}-Hydroxylase activity

7{alpha}-Hydroxylase activity was measured in whole human prostate pieces. Surgical BPH samples were incubated at 37 C for up to 48 h in RPMI 1640 medium supplemented with 5% dextran-coated charcoal-stripped fetal calf serum and the radiolabeled steroid substrates at a concentration of 0.3 µM. Steroids were extracted from the medium with ethyl acetate, dried, and stored at –20 C until analysis. Recovery was approximately 90% (20). The DHEA-to-7HD conversion was assessed by TLC (thin-layer chromatography) as previously described (20) and quantified using a phosphor imager (FLA-2000, Fujifilm, Raytek Scientific, Ltd., Sheffield, UK).

HPLC

HPLC (Waters, Herts, UK) with on-line scintillation counting (Berthold, Leeds, UK) was carried out with a reverse-phase C18 column (Luna, Phenomenex, Cheshire, UK) using a mobile phase (H2O, methanol and acetonitrile, 55:25:20, by volume at 1 ml/min) that gave retention times for 7HD and DHEA at 7 min and 24 min, respectively (20).

RNA extraction

Total RNA was isolated from human prostate tissue and cells as described previously (25), resuspended in ribonuclease (RNase)-free H2O, and stored at –70 C. All samples had intact 18S and 28S RNAs, as judged by ethidium bromide staining after agarose gel electrophoresis.

Oligonucleotide primers, reverse transcription (RT), and PCR amplification of CYP7B cDNA

5' and 3' primers for PCR (Oswel DNA Service, Southampton, UK) were 5'-dAAGCCTAAATGATGTGCTCC-3' and 5'-dGAGTGGTCCTGAACTTACG-3', corresponding to nucleotides 329–347 and 1006–1025, respectively, of the human CYP7B cDNA (26). RT was carried out in 20 µl containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl2, 0.1% (wt/vol) Triton X-100, 1 mM deoxynucleotide 5'-triphosphates, 10 U of RNase inhibitor (Promega, Southampton, UK), 1 µg total RNA, 12 U avian myeloblastosis virus reverse transcriptase (Promega), and 0.1 nmol 3' PCR primer. Reactions were incubated for 10 min at room temperature, followed by 30 min at 42 C, then 95 C for 5 min (to inactivate the reverse transcriptase). Subsequent PCR amplification was carried out by adding 80 µl of buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 2.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 0.1 nmol of 5' PCR primer, and 2.5 U Taq polymerase (Promega, Southampton, UK). After a "hot start" of 5 min at 94 C, 30 cycles of PCR were carried out: 94 C for 1 min, 56 C for 1 min, and 72 C for 1 min followed by 72 C for 10 min. Amplified products were analyzed by electrophoresis on 1% (wt/vol) agarose gels. No products were detected in reactions from which reverse transcriptase had been omitted.

Cloning and sequencing of RT-PCR products

PCR products were subcloned into pGEM-(T) Easy (Promega) and sequenced on both strands.

CYP7B probe for mRNA in situ hybridization

CYP7B PCR fragment subcloned into pGEM-(T) Easy was used as a template for SP6 or T7 RNA polymerase to generate the antisense and sense cRNA probes as previously described (27).

CYP7B mRNA in situ hybridization

Paraffin-embedded sections (5 µm) were deparaffinized in xylene, soaked in PBS, and treated with proteinase K treatment in PBS (20 mg/ml) for 10 min at 37 C. Sections were fixed in 4% paraformaldehyde (vol/vol) for 20 min, then treated with 0.25% acetic anhydride (vol/vol) in 0.1 M triethanolamine for 10 min. Hybridization with digoxigenin (DIG)-labeled riboprobes at 50 C for 14–16 h in moist chamber, RNase A treatment, and washing were as previously described (27). After hybridization, DIG-labeled riboprobes were visualized using DIG-alkaline phosphatase-conjugated antibody at 1:2500 (Roche Molecular Biochemicals, Lewes, UK) for 30 min at room temperature, washed, and developed overnight using a Roche Molecular Biochemicals developing reagent. Nonspecific hybridization was determined by incubation with a DIG-labeled sense probe under identical conditions.

ERß immunohistochemistry

Immunohistochemistry study was carried out as previously described (28) using a commercial monoclonal antibody against human ERß (hERß) (Serotec, Oxford, UK).

Primary cell cultures of prostate

BPH chips were used to establish primary cultures of separated stroma and epithelial cells (29, 30). Cocultures of epithelial and stroma cells was as previously described (30).

Luciferase reporter assays

COS-1 cells and HepG2 cells were maintained in high-glucose DMEM containing penicillin (25 U/ml), streptomycin (25 µg/ml), and 10% fetal calf serum (vol/vol). Cells were seeded at a density of 5 x 105 cells/dish and left to adhere overnight. On the day of the transfection, the medium was replaced with DMEM lacking phenol red supplemented with 10% dextran-coated charcoal-stripped fetal calf serum (vol/vol). Transfections were carried out using the calcium phosphate procedure according to standard protocols with 10 µg DNA [1 µg expression plasmid, 1–5 µg reporter plasmid, 1 µg pCH110 encoding ß-galactosidase used as internal control (Pharmacia, Milton Keynes, UK), and 3–7 µg pGEM3]. Expression plasmids were as follows: the mouse ER{alpha} [mER{alpha}; gift from Professor M. Parker, Imperial College London, London, UK (31)], hERß [from Dr. R. White, Imperial College London, London, UK (32)], and human androgen receptor (AR) (pSVARo, from Professor A. Brinkmann, Erasmus University, Rotterdam, Holland). mER{alpha} shows 88% identity with the hERß, and both species have the same selectivity for the majority of the steroids. Reporter plasmids were as follows: estrogen response element (ERE)-TK-Luc (gift From Dr. V. Giguere, McGill University, Montreal, Canada) for estrogen responsivity and prostate-specific antigen (PSA) (PSA61-luc, Professor J. Trapman, Erasmus University, Rotterdam, Holland) for androgen responsivity. After 24 h, the medium was changed, and the cells were treated with steroid [E2, 5{alpha}-dihydrotestosterone (DHT), 7HD, or an appropriate concentration of ethanol]. After cell lysis, luciferase and ß-galactosidase activities were measured as described (33). Data are expressed as relative luciferase/ß-galactosidase and are means ± SEM from at least three independent experiments.

Ligand-competition studies

hERß clone was synthesized in vitro using the TnT-coupled reticulocyte lysate system following manufacturer instructions (Promega). Translation reaction mixtures were diluted five times with TEDGMo buffer (40 mM Tris/Hcl, pH 7.4/1 mM EDTA/10% (vol/vol) glycerol/10 mM Na2MoO4/10 mM dithiothreitol), and 0.1-ml aliquots were incubated for 16 h at 4 C with 0.5 nM [2,4,6,7-3H]4-E2 (specific radioactivity, 89 Ci/mmol) in presence of either 0, 1, 5, 10, 20, 50, or 250 µM 7HD. Bound and unbound steroids were separated by filtration.

Statistics

Statistical comparisons (SigmaStat, SPSS, Inc., Chicago, IL) were by ANOVA and the rank sum test. Significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
P450-dependent production of 7HD from DHEA by human prostate

To determine whether CYP7B activity is present in human prostate, we measured DHEA metabolism in whole prostate chips. DHEA has been reported previously to be the best substrate for recombinant CYP7B in vitro (19). 7{alpha}-Hydroxylation of DHEA and A/enediol was clearly detectable in chips of whole human prostate and was time-dependent (Fig. 2Go). The radioactive compound marked "E" (Fig. 2Go) comigrated in an identical manner with unlabeled commercial reference compound, 7{alpha}-hydroxy-DHEA, with the major product of DHEA metabolism by protein extracts from HeLa cells transfected with recombinant CYP7B and also with the rat prostate product of DHEA metabolism, as previously described (20) (data not shown). A/enediol and 5{alpha}-androstenedione (both in spot "B") were also produced from DHEA, as previously reported in prostate tissue (34). Of all the new radioactive compounds produced from DHEA, the products of CYP7B represent 50% (of which 9% are 3ß-7{alpha}-17ß-androstenetriol), and only 37% were products of 3ß-hydroxysteroid dehydrogenase (HSD) and 17ß-HSD. For a better separation of the different labeled compounds produced during the reaction, we analyzed them by HPLC using cold steroid standards. 7{alpha}-Hydroxylation of DHEA by human prostate chips was inhibited by clotrimazole (1 µM), confirming that the production of 7HD was P450 dependent (Fig. 2Go). Production of the minor products "C" and "D" was also reduced in the presence of clotrimazole, suggesting that they are also the products of a P450 enzyme. Trilostane, which specifically inhibits 3ß-HSD activity, increased radioactive 7HD production by 49 ± 23%, P < 0.05 (Fig. 2Go), although it successfully blocked the production of A/anedione. Minor products "C" and "D" were not affected by trilostane. The 7{alpha}-hydroxylation of DHEA observed in human prostate was consistent with it being the product of CYP7B metabolism.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 2. 7HD is produced from DHEA in chips of human prostate. A, TLC analysis of products of DHEA in human prostate chips after 24-h incubation in medium in the absence (lane 1) or in the presence (lane 2) of 1 µM trilostane, 1 µM clotrimazole (lane 3), or 1 µM clotrimazole plus 1 µM trilostane (lane 4). B, Time course of production of [14C]7HD by human prostate samples (n = 2–7).

 
CYP7B is expressed in human prostate

To verify the expression of CYP7B in prostate, RT-PCR was carried out on RNA from four different human prostate samples. CYP7B-specific primers amplified the expected 696-bp fragment (Fig. 3Go). The identity of the PCR product was verified by digestion with HindIII, Pst1, and Ssp1, which produced the predicted fragments (Fig. 3Go). Sequencing of the subcloned PCR product confirmed its identity as human CYP7B (26).



View larger version (73K):
[in this window]
[in a new window]
  		FIG. 3. RT-PCR analysis of CYP7B mRNA in human prostate. The identity of the PCR product (696 bp) (lane 1) was verified by enzymatic restriction with HindIII (lane 2), Pst1 (lane 3), and Ssp1 (lane 4), which cut the PCR product at 158, 384, and 394 bp, respectively. The nucleic acid size markers (M) are indicated.

 
CYP7B mRNA is colocalized with ERß immunoreactivity in human prostate

To determine the site of CYP7B mRNA expression in the human prostate, in situ mRNA hybridization was carried out on paraffin-embedded sections of prostate using cRNA probe generated from the subcloned PCR product. CYP7B mRNA was highly expressed in the epithelium with very little expression in the stroma and in the vasculature (Fig. 4AGo). Control sections hybridized to sense RNA probe showed low background levels of hybridization (Fig. 4BGo). We also determined the localization of ERß in human prostate samples using a specific ERß antibody (Fig. 4CGo). Interestingly, ERß was also expressed in the epithelial cells, predominantly in basal regions of the epithelium as confirmed by high-molecular-weight cytokeratins labeling. This result suggests a coexpression of ERß with CYP7B.



View larger version (112K):
[in this window]
[in a new window]
  		FIG. 4. CYP7B mRNA and ERß are colocalized in human prostate epithelium. Representative high-resolution views of mRNA in situ hybridization encoding CYP7B (A) and immunostaining of ERß (C) in BPH sections. Representative sense control sections for CYP7B mRNA and control sections (without primary antibody) for ERß are shown in B and D, respectively.

 
CYP7B mRNA expression is maintained in primary epithelial cells culture and is increased by coculture of stroma with epithelial cells

Both CYP7B mRNA and 7{alpha}-hydroxylase activity were detected in primary culture of human prostate epithelial cells (Fig. 5Go). Moreover, epithelial CYP7B activity was enhanced after 5 d of coculture of epithelial cells with stroma cells (P < 0.001; Fig. 5CGo), suggesting that high epithelial expression of CYP7B is dependent on a diffusible factor produced by coculture of stroma and epithelial cells. No CYP7B mRNA was found in the stroma cells alone, consistent with the in situ hybridization findings that CYP7B mRNA is restricted to the epithelium.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 5. CYP7B is expressed in primary culture of epithelial cells and is increased when cocultured with stroma cells. A, RT-PCR detection of CYP7B mRNA in whole human prostate (WP, lane 1), primary stromal cells (St, lane 2), and epithelial cells (Ep, lane 3). M, Molecular weight markers. B, TLC resolution of products generated by 24-h incubation with [14C]DHEA of primary culture of stroma cells (St), epithelial cells (Ep), or coculture of epithelial and stromal cells (St+Ep). Arrow indicates 7HD. C, Production of [14C]7HD from DHEA in 24 h by epithelial cells (Ep), stroma cells (St), and coculture of epithelial and stromal cells. *, P < 0.001, Ep vs. St+Ep.

 
7HD activates ERß but not ER{alpha} or AR

To further assess the possible function of the CYP7B product 7HD, we analyzed its ability to transactivate ER{alpha}, ERß, and AR in a cotransfection assay with estrogen- and androgen-responsive reporter genes. Both COS-1 cells and HepG2 cells were used, and both these cell lines require exogenous AR, ER{alpha}, and ERß to activate androgen (PSA) or estrogen-responsive reporter genes (ERE). In primary experiments, maximal activation of ER{alpha} by E2 was found with 10 nM E2, whereas maximal activation of ERß was obtained with 50 nM E2. Remarkably, 7HD also activated ERß (Fig. 6Go, A and B). 7HD significantly activated ERß-mediated transcription with an EC50 of 6.2 µM. 7HD transcriptional activity effect was additive to a subminimal dose of E2 (0.1 nM; P < 0.01) (Fig. 6BGo). This effect was specific for ERß because a similar concentration of 7HD caused only a minimal, nonspecific activation of ER{alpha} (Fig. 6CGo). Moreover, 7HD was unable to activate AR-dependent transcription of a PSA-luciferase reporter, whereas DHT clearly produced strong activation (Fig. 6DGo). To confirm that 7HD is activating transcription through ER, we used the specific antiestrogen ICI 182,780. As expected, ICI 182,780 (1 µM) alone could not stimulate ER{alpha} or ERß activity but completely abolished both E2 and 7HD-induced transcription of luciferase in cells cotransfected with ER{alpha} or ERß receptors (Fig. 6BGo).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 6. Transactivation of ERß (A and B), ER{alpha} (C), and AR (D) by 7HD. Values shown are means (±SEM) of three to five independent experiments, each carried out in triplicate. A and B, Transactivation of (ERE)-TK-Luc by ERß in HepG2 cells. C, Transactivation of (ERE)-TK-Luc by ER{alpha} in COS cells. D, Transactivation of PSA-Luc reporter construct in COS-1 cells containing hAR. Data are presented as the percentage of maximal induction obtained with 20 nM E2 (hERß; x4-fold induction over control; A and B), 10 nM E2 (mER{alpha}; x7-fold induction over control; C), and 10 nM DHT (hAR; x17-fold induction over control; D). *, P < 0.001; {phi}, P < 0.05, 7HD vs. control without 7HD; {gamma}, P < 0.01, E2 vs. 7HD + E2.

 
The ability of 7HD to inhibit [3H]E2 binding was also measured by a competition binding assay using hERß receptor synthesized in vitro from our hERß clone using a TnT-coupled reticulocyte lysate system (Fig. 7Go). In our competition study, 7HD interacted with ERß receptor and inhibited [3H]E2 binding dose dependently.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 7. Competition by 7HD for [3H]E2 binding to in vitro synthesized ERß protein. Reticulocyte lysate containing ERß protein was equilibrated for 16 h with 5 nM [3H]E2 and the indicated fold excess of 7HD. Data represent [3H]E2 bound in the presence of 7HD (0–250 µM). [3H]E2 binding in the absence of 7HD was set at 100%. *, P < 0.001; and {gamma}, P < 0.01 for 7HD vs. control without 7HD.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this report, we confirm the expression of oxysterol CYP7B in human prostate and demonstrate that this pathway is responsible for more than 50% of the DHEA metabolism in this organ. In contrast, metabolism of DHEA toward classical androgens and estrogens forms a relatively minor pathway. Furthermore, these studies show that 7HD is a specific agonist for ERß but not for ER{alpha} or AR, suggesting that 7HD may act as an endogenous ligand for ERß in the human prostate. Together, the data support the notion that CYP7B generates active steroids within the prostate that may affect the intracrine estrogen:androgen balance and, potentially, pathogenesis.

7{alpha}-Hydroxylation of DHEA in humans has been known about for many years, initially with the identification of 7HD in urine (35, 36) and subsequently with the detection of 7HD production in skin, brain, mammary tissue, and fetal tissues (37, 38). 7{alpha}-Hydroxylation of 3ß-hydroxysteroids in prostate has been previously described, but the enzyme(s) responsible were hitherto unidentified (24, 39). In the present study, we used a combination of RT-PCR, in situ hybridization, biochemical, and cell culture approaches to show that CYP7B mRNA, along with its functional enzyme activity, is localized to the epithelium. Whereas this report confirms that CYP7B is a prime candidate to catalyze the 7{alpha}-hydroxylation in prostate, other enzymes might conceivably also be involved. However, mice lacking CYP7B show absolutely no residual 7{alpha}- (or 7ß-) hydroxylation of DHEA in prostate (22), confirming that CYP7B is the only enzyme involved.

7{alpha}-Hydroxylation of DHEA is restricted to prostate epithelial cells in vivo and in vitro. This is the first report demonstrating a steroid-metabolizing enzyme associated exclusively with one type of tissue in the prostate, raising the possibility that 7HD activity might be confined exclusively to the epithelium. Our cotransfection assays show that 7HD is able to activate ERß, which is also localized in the epithelium (12, 13). At subminimal concentrations of E2, 7HD’s effect on ERß is additive to E2. Although 7HD was clearly much less potent than E2, it achieved similar maximal activation of ERß. Given that DHEA, and its sulfate, circulate at micromolar concentrations, whereas serum estrogens are at picomolar levels, there is a clear possibility that CYP7B generates sufficient 7HD within the prostatic epithelia to activate ERß over and above that achieved the very low concentrations of intraprostatic E2 (40). Indeed, it is conceivable that 7HD may be a more important ERß ligand than E2 itself. A similar proposal has been advanced for 5{alpha}-androstanediol, which is also an ERß ligand locally produced in the prostate (15, 23, 41). Both 7HD and 5{alpha}-androstanediol can add to the low level of E2 in the prostate to activate ERß. The data reported herein support the notion that DHEA is a prohormone and 7HD is its active metabolite. In the presence of CYP7B, DHEA is metabolized to an estrogenic steroid acting as an ERß agonist, 7HD, which may influence the prostatic growth and pathogenesis.

Interestingly, epithelial CYP7B activity was enhanced by coculture of epithelial and stroma cells. Whether this reflects a differentiation effect in epithelia in cocultures or is a result of a cross-talk signaling between stromal and epithelial cells is uncertain. Previous characterizations of prostate cocultures suggest the presence of diffusible factors produced by one cell type, which in turn influences the differentiation and gene expression of the other (30, 42, 43). It is conceivable that the cross-talk between stromal and epithelial components of the prostate is an important regulator of DHEA metabolism and may therefore modulate the hormonal status of the gland.

The possibility of a biological role for 7HD, in prostate and elsewhere, has so far been unconfirmed, and any attempt to elucidate this problem has been hampered by the lack of an established receptor for 7HD. The findings reported here might overcome this impasse and establish a function for 7HD in the prostate. Recent studies have shown that CYP7B expression decreases during development in rodents (44) and is also altered by stress and in Alzheimer’s disease (45, 46), suggesting that the expression of CYP7B may change in response to environmental signals and during aging, at least in the brain. Loss of prostatic CYP7B may alter the balance between estrogens and androgens, favoring androgenic over estrogenic pathways, by reducing synthesis of the selective ERß agonist. Concomitantly, any decrease of CYP7B expression increases the availability of native DHEA within the prostate for synthesis of potent androgens. The exact effects of 7HD binding to ERß on human prostate epithelium and whole prostate are still unknown. One possible role for ERß, as shown in bone, is to modulate ER{alpha}-mediated gene transcription (47). Reporter gene assays have demonstrated that ERß has the capacity to repress the transcriptional activity of ER{alpha} (48). By binding to ERß, 7HD can modulate ER{alpha} activity in the stroma compartment and therefore can control the growth of the stroma cells. Also, ERß is suggested to play a role in the differentiation and proliferation of the prostate cells as well as to modulate both the initial phases of prostate carcinogenesis and androgen-dependent tumor growth (49). Thus, CYP7B may have a significant role in the regulation of the intraprostatic concentration of active steroids and may be a useful tool in the prevention or clinical management of prostate diseases.


   Acknowledgments
 
We are most grateful to Ms. Val Lyons for her technical assistance and Dr. Chris Kenyon for the statistical analysis of the data and helpful discussions.


   Footnotes
 
This work was supported by a grant from the Association for International Cancer Research (to C.M.).

Abbreviations: A/enediol, [1,2-3H]2-5{alpha}-Androstenediol; AR, androgen receptor; BPH, benign prostatic hyperplasia; DHEA, dehydroepiandrosterone; DHT, 5{alpha}-dihydrotestosterone; DIG, digoxigenin; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; h, human; 7HD, 7{alpha}-hydroxy-DHEA; HSD, hydroxysteroid dehydrogenase; m, mouse; PSA, prostate-specific antigen; RNase, ribonuclease; RT, reverse transcription; TLC, thin-layer chromatography.

Received October 22, 2003.

Accepted February 23, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Bosland MC 2000 The role of steroid hormones in prostate carcinogenesis. J Natl Cancer Inst Monogr 27:39–66
  2. Schweikert HU, Tunn UW, Habenicht UF, Arnold J, Senge T, Schulze H, Schoder FH, Blom JHM, Ennemoser O, Horniger W, Bartsch G 1993 Effects of estrogen deprivation on human benign prostatic hyperplasia. J Steroid Biochem Mol Biol 44:573–576[CrossRef][Medline]
  3. Jarred RA, Cancilla B, Prins GS, Thayer KA, Cunha GR, Risbridger GP 2000 Evidence that estrogens directly alter androgen-regulated prostate development. Endocrinology 141:3471–3477[Abstract/Free Full Text]
  4. Ferrini RL, Barett-Connor E 1998 Sex hormones and age: a cross-sectional study of testosterone and estradiol and their bioavailable fractions in community-dwelling men. Am. J Epidemiol 147:750–754
  5. Labrie C, Belanger A, Labrie F 1988 Androgenic activity of dehydroepiandrosterone and androsterone in the rat ventral prostate. Endocrinology 123:1412–1417[Abstract]
  6. Nelson LR, Bulun SE 2001 Estrogens production and actions. J Am Acad Dermatol 45:S116–S124
  7. Ekman P 2000 The prostate as an endocrine organ: Androgens and estrogens. Prostate 10:14–18[CrossRef]
  8. Risbridger G P, Bianco JJ, Ellem SJ, McPherson SJ 2003 International congress on hormonal steroids and hormones ans cancer: estrogens and prostate cancer. Endocr Relat Cancer 10:187–191[Abstract]
  9. Leav I, Ho SM, Ofner P, Merk FB, Kwan PW, Damassa D 1988 Biochemical alterations in sex hormone-induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats. J Nat Cancer Inst 80:1045–1053[Abstract/Free Full Text]
  10. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptor {alpha} and ß. Endocrinology 138:863–870[Abstract/Free Full Text]
  11. Brolin J, Skoog L, Ekman P 1992 Immunohistochemistry and biochemistry in detection of androgen, progesterone, and estrogen receptors in benign and malignant human prostatic tissue. Prostate 20:281–295[Medline]
  12. Horvath LG, Henshall SM, Lee CS, Head DR, Quinn DI, Makela S, Delprado W, Golovsky D, Brenner PC, O’Neill G, Kooner R, Stricker PD, Grygiel JJ, Gustafsson JA, Sutherland RL 2001 Frequent loss of estrogen receptor-ß expression in the prostate cancer. Cancer Res 61:5331–5335[Abstract/Free Full Text]
  13. Leav I, Lau KM, Adams JY, McNeal JE, Taplin ME, Wang J, Singh H, Ho SM 2001 Comparative studies of the estrogen receptors ß and {alpha} and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma. Am J Pathol 159:79–92[Abstract/Free Full Text]
  14. Risbridger G, Wang H, Young P, Kurita T, Wang YZ, Lubahn D, Gustafsson JA, Cunha G, Wong YZ 2001 Evidence that epithelial and mesenchymal estrogen receptor-{alpha} mediates effects of estrogen on prostatic epithelium. Dev Biol 229:432–442[CrossRef][Medline]
  15. Weihua Z, Makela S, Andersson LC, Salmi S, Saji S, Webster JI, Jensen EV, Nilsson S, Warner M, Gustafsson JA 2001 A role for estrogen receptor ß in the regulation of growth of the ventral prostate. Proc Natl Acad Sci USA 98:6330–6335[Abstract/Free Full Text]
  16. Ghanadian R, Puah CM 1981 Relationships between oestradiol-17 ß, testosterone, dihydrotestosterone and 5 {alpha}-androstane-3 {alpha}, 27 ß-diol in human benign hypertrophy and carcinoma of the prostate. J Endocrinol 88:255–262[Abstract]
  17. Seckl JR, Walker BR 2001 Steroid metabolism. Baillière’s clinical endocrinology and metabolism. Vol 15.1. London: Baillière Tindall; 122
  18. Stapleton G, Steel M, Richardson M, Mason JO, Rose KA, Morris RG, Lathe R 1995 A novel cytochrome P450 expressed primarily in the brain. J Biol Chem 270:29739–29745[Abstract/Free Full Text]
  19. Rose KA, Stapleton G, Dott K, Kieny MP, Best R, Schwarz M, Russell DW, Bjoorkheim I, Seckl JR, Lathe R 1997 Cyp7b, a novel brain cytochrome P450, catalyses the synthesis of neurosteroids 7{alpha}-hydroxy-DHEA and 7{alpha}-hydroxy-PREG. Proc Natl Acad Sci USA 94:4925–4930[Abstract/Free Full Text]
  20. Martin C, Bean R, Rose K, Habib FK, Seckl JR 2001 CYP7B1 catalyses the 7{alpha}-hydroxylation of dehydroepiandrosterone and 25-hydroxycholesterol in rat prostate. Biochem J 355:509–515[CrossRef][Medline]
  21. Li-Hawkins J, Lund EG, Turley SD, Russell DW 2000 Disruption of the oxysterol 7{alpha}-hydroxylase gene in mice. J Biol Chem 275:16536–16542[Abstract/Free Full Text]
  22. Rose K, Allan A, Gauldie S, Stapleton G, Dobbie L, Dott K, Martin C, Wang L, Hedlund E, Gustafsson JA, Seckl J, Lathe R 2001 Targeting the gene encoding mouse neurosteroid hydroxylase CYP7B: vivid reporter activity in dentate gyrus and abolition of a major pathway of steroid and oxysterol hydroxylation. J Biol Chem 276:23937–23944[Abstract/Free Full Text]
  23. Weihua Z, Lahe R, Warner M, Gustafsson JA 2002 An endocrine pathway in the prostate, ERß, AR, 5{alpha}-androstane-3ß, 17ß-diol, and CYP7B1, regulates prostate growth. Proc Natl Acad Sci USA 99:13589–13594[Abstract/Free Full Text]
  24. Li K Foo T, Adams JB 1978 Products of dehydroepiandrosterone metabolism by human mammary tumors and their influence on estradiol receptor binding. Steroids 31:113–127[CrossRef][Medline]
  25. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  26. Wu Z, Martin KO, Javitt NB, Chiang JYL 1999 Structure and functions of human oxysterol 7{alpha}-hydroxylase cDNAs and gene CYP7B1. J Lipid Res 40:2195–2203[Abstract/Free Full Text]
  27. Habib FK, Ross M, Bayne CW, Grigor K, Buck AC, Bollina P, Chapman K 1998 The localisation and expression of 5{alpha}-reductase types I and II mRNAs in human hyperplastic prostate and in prostate primary cultures. J Endocrinol 156:509–517[Abstract]
  28. Saunders PT, Millar MR, Williams K, Macpherson S, Harkiss D, Anderson RA, Orr B, Groome NP, Scobie G, Fraser HM 2000 Differential expression of estrogen receptor-{alpha} and -ß and androgen receptor in the ovaries of marmosets and humans. Biol Reprod 63:1098–1105[Abstract/Free Full Text]
  29. Chaproniere DM, McKeelan WL 1986 Serial culture of single adult human prostatic epithelial cells in serum-free medium containing low calcium and a new growth factor from bovine brain. Cancer Res 46:819–824[Abstract/Free Full Text]
  30. Habib FK, Ross M, Bayne CW 2000 Development of a new in vitro model for the study of benign prostatic hyperplasia. Prostate 9:15–20
  31. White R, Lees JA, Ham J, Parker M 1987 Structural organization and expression of the mouse estrogen receptor. Mol Endocrinol 1:735–744[Abstract]
  32. Cowley SW, Parker MJ 1999 A comparison of transcriptional activation by ER {alpha} and ER ß. J Steroid Biochem Mol Biol 69:165–175[CrossRef][Medline]
  33. Leckie C, Chapman KE, Edwards CR, Seckl JR 1995 LLC-PK1 cells model 11 ß-hydroxysteroid dehydrogenase type 2 regulation of glucocorticoid access to renal mineralocorticoid receptors. Endocrinology 136:5561–5569[Abstract]
  34. Mitamura K, Nakagawa T, Shimada K, Namiki M, Koh E, Mizokami A, Honma S 2002 Identification of dehydroepiandrosterone metabolites formed from human prostate homogenate using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. J Chromatogr A 961:97–105[CrossRef][Medline]
  35. Schneider JJ, Lewbart ML 1959 Fractionation and isolation of steroid conjugates. Recent Prog Horm Res 15:201–230
  36. Okada M, Fukushima DK, Gallagher TF 1959 Isolation and characterization of 3ß-hydroxy{Delta}5steroids in adrenal carcinoma. J Biol Chem 234:1688–1692[Free Full Text]
  37. Faredin I, Fazekas AG, Toth I, Kokai K, Julesz M 1969 Transformation in vitro of [4-14C]-dehydroepiandrosterone into 7-oxygenated derivatives by normal human male and female skin tissue. J Invest Derm 52:357–361[Medline]
  38. Sulcova J, Capkova A, Jirasek JE, Starka L 1968 7-hydroxylation of dehydroepiandrosterone in human foetal liver, adrenals and chorion in vitro. Acta Endocrinol (Copenh) 7:591–599
  39. Sundin M, Warner M, Haaparanta T, Gustafsson JA 1987 Isolation and catalytic activity of cytochrome P-450 from ventral prostate of control rats. J Biol Chem 262:12293–12297[Abstract/Free Full Text]
  40. Voigt KD, Bartsch W 1986 Intratissular androgens in benign prostatic hyperplasia and prostatic cancer. J Steroid Biochem 25:749–757[CrossRef][Medline]
  41. Weihua Z, Andersson S, Cheng G, Simpson ER, Warmer M, Gustafsson JA 2003 Update on estrogen signalling. FEBS Lett 546:17–24[CrossRef][Medline]
  42. Bayne WC, Donnelly F, Chapman K, Bollina P, Buck C, Habib FK 1998 A novel co-culture model for benign prostatic hyperplasia expressing both isoforms of 5{alpha}-reductase. J Clin Endocrinol Metab 83:206–213[Abstract/Free Full Text]
  43. Bayne CW, Ross M, Inglis NF 2003 Induction of 5{alpha}-reductase type II mRNA transcription in primary cultured prostate epithelial cells by a soluble factor produced by primary cultured prostate fibroblast cells. Eur J Cancer 39:1004–1011
  44. Bean R, Seckl JR, Lathe R, Martin C 2001 Ontogeny of the neurosteroid enzyme Cyp7b in the mouse. Mol Cell Endocrinol 174:137–144[CrossRef][Medline]
  45. Graham M, Noble J, Andrew R, Rasmuson S, Olsson T, Seckl J, Yau J 2000 Regulation of Cyp7b expression during stress and ageing in rats and Alzheimer’s disease in humans. Eur J Neuroscience 12:184.01 (Abstract)
  46. Yau J, Rasmuson S, Andrew R, Graham M, Noble J, Olsson T, Fuchs E, Lathe R, Seckl JR 2004 Dehydroepiandrosterone 7-hydroxylase CYP7B: predominant expression in primate hippocampus and reduced expression in Alzheimer’s disease. Neuroscience 121:307–314
  47. Lindberg MK, Moverare S, Skrtic S, Gao H, Dahlman-Wright K, Gustafsson JA, Ohlsson C 2003 Estrogen receptor (ER)-ß reduces ER{alpha}-regulated gene transcription, supporting a "ying yang" relationship between ER{alpha} and ERß in mice. Mol Endocrinol 17:203–208[Abstract/Free Full Text]
  48. Hall JM, McDonnell DP 1999 The estrogen receptor ß-isoform (ERß) of the human estrogen receptor modulates ER{alpha} transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578[Abstract/Free Full Text]
  49. Signoretti S, Loda M 2001 Estrogen receptor ß in prostate cancer: brake pedal or accelerator? Am J Pathol 159:13–16[Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
D. R. Bauman, S. Steckelbroeck, D. M. Peehl, and T. M. Penning
Transcript Profiling of the Androgen Signal in Normal Prostate, Benign Prostatic Hyperplasia, and Prostate Cancer
Endocrinology, December 1, 2006; 147(12): 5806 - 5816.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. T. Arnold and M. R. Blackman
Does DHEA Exert Direct Effects on Androgen and Estrogen Receptors, and Does It Promote or Prevent Prostate Cancer?
Endocrinology, November 1, 2005; 146(11): 4565 - 4567.
[Full Text] [PDF]

Home page
 EndocrinologyHome page
F. Chen, K. Knecht, E. Birzin, J. Fisher, H. Wilkinson, M. Mojena, C. T. Moreno, A. Schmidt, S.-i. Harada, L. P. Freedman, et al.
Direct Agonist/Antagonist Functions of Dehydroepiandrosterone
Endocrinology, November 1, 2005; 146(11): 4568 - 4576.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
J. T. Arnold, H. Le, K. K. McFann, and M. R. Blackman
Comparative effects of DHEA vs. testosterone, dihydrotestosterone, and estradiol on proliferation and gene expression in human LNCaP prostate cancer cells
Am J Physiol Endocrinol Metab, March 1, 2005; 288(3): E573 - E584.
[Abstract] [Full Text] [PDF]

Home page
 Ann. N. Y. Acad. Sci.Home page
H L. BRADLOW and D. W. SEPKOVIC
Steroids as Procarcinogenic Agents
Ann. N.Y. Acad. Sci., December 1, 2004; 1028(1): 216 - 232.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martin, C.
Right arrow Articles by Habib, F. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martin, C.
Right arrow Articles by Habib, F. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals