Estrogen receptor β and 17β-hydroxysteroid dehydrogenase type 6, a growth regulatory pathway that is lost in prostate cancer
Effect of 17βHSD6 on ERβ Transactivation by 5α-Reduced Steroids.
Because DHT, 3β-Adiol, and 3α-Adiol are present at ∼20, 10, and 2 nM concentrations, respectively, in the human prostate (5), coupled with the fact that 3β-Adiol is a potent ligand of ERβ in this tissue, we asked the question whether 17βHSD6 has the capacity to form sufficient amounts of 3β-Adiol from DHT and 3α-Adiol to activate ERβ in living cells. A transactivation assay in which human ERβ was expressed in human embryonic kidney 293 cells (HEK-293 cells) harboring an ERβ-responsive estrogen response element (ERE) containing promoter/reporter plasmid was used. Fig. 1 shows that coexpression of 17βHSD6 resulted in ERβ activation when DHT (20 nM) was added to the cells; 3α-Adiol also caused a robust activation of ERβ in a 17βHSD6-dependent fashion. ERβ activation by estradiol and 3β-Adiol was not affected by coexpressing 17βHSD6. Fig. 2 shows dose–response curves for ERβ activation by DHT and 3α-Adiol in the presence of recombinant 17βHSD6; the EC50 values were determined to be ∼20–30 nM for both steroids. Cells transfected with empty vector also demonstrated significant ERβ activation; the presence of an endogenous epimerase activity in HEK-293 cells has previously been reported (14).
Steroid Metabolism by 17βHSD6.
To confirm that 17βHSD6 produces 3β-Adiol from DHT, we expressed 17βHSD6 in HEK-293 cells and performed a time-course experiment. Forty-eight hours after transfection, [3H]-DHT was added to the cell medium at a physiological concentration of 20 nM. Samples were collected after 0, 1, 2, 4, and 8 h of incubation. Thereafter steroids were extracted from the medium and separated by TLC on silica gel plates followed by quantitative analysis. Fig. 3 shows that ∼13% of the DHT was converted to 3β-Adiol after 4 h of incubation. As previously reported (11), 17-keto metabolites accumulate over time in 17βHSD6-expressing cells. Mock-transfected cells demonstrated significantly lower DHT metabolism at the 4 h time point. At the end of 8 h, ∼15–20% of DHT was remaining in the medium from cells transfected with 17βHSD6 compared with ∼65–75% in the medium from cells transfected with pCMV vector.
Immunohistochemical Analysis of 17βHSD6 in Human Prostate.
In an effort to assess the relevance of the enzyme in the human prostate, we determined expression of 17βHSD6 and ERβ in human prostate cancers of different Gleason grades (15) and in benign prostatic hyperplasia (BPH) (16). The immunostaining results from the antibody raised against the N-terminal amino acids 1–50 (Fig. 4 A and B) were confirmed with the antibody raised against the full-length protein. Immunohistochemical analyses of human prostate sections demonstrated perinuclear staining of 17βHSD6 protein in the epithelial cells of benign prostatic hyperplasia, especially in the basal epithelial cells (Fig. 4 A and B). Slides incubated without primary antibody demonstrated no staining. As expected (17), sections stained with an antibody against ERβ showed nuclear staining for ERβ protein in the epithelial cells (Fig. 4 C and D).
We found that, compared with BPH prostate, expression of ERβ decreased in prostate cancers of Gleason grade 3 (Fig. 5A) and was undetectable in grades 4 (Fig. 5 B and C) and 5 (Fig. 5D). Expression of 17βHSD6 paralleled that of ERβ in these prostate cancers with a reduction of the immunoreactivity in Gleason grade 3 (Fig. 6 A and B) and loss of immunoreactivity in grades 4 (Fig. 6 C and D) and 5 (Fig. 6 E and F).
Human Samples.
Prostate biopsies were obtained from the Department of Urology at Danderyd Hospital, Stockholm, Sweden. Biopsies were taken by Linda Waage, and the samples categorized for their Gleason grade (15) by Ulf Bergerheim. Samples were fixed in buffered paraformaldehyde, dehydrated, and imbedded in wax at Danderyd Hospital. We obtained the sections in a blind fashion. Samples from seven different patients with BPH (16), two patients with prostate cancer Gleason grade 3, three patients with prostate cancer Gleason grade 4, and two patients with prostate cancer Gleason grade 5 were used for this study.
Steroids.
DHT (112 Ci/mmol) was purchased from Perkin-Elmer. E2, estradiol-17β; DHT, 17β-hydroxy-5α-androstan-3-one; 3α-Adiol, 5α-androstane-3α,17β-diol; and 3β-Adiol, 5α-androstane-3β,17β-diol were obtained from Steraloids.
ERβ Transactivation Assay.
HEK-293 cells (ATCC CRL no. 1573) were maintained in DMEM with 10% FBS, 50 μg/mL kanamycin, and 10 mM Hepes, pH 7. On day 0, HEK-293 cells were seeded in 24-well Costar plates containing 0.5 mL phenol red-free DMEM, 10% dextran-treated charcoal/dextran-treated FBS (HyClone), 50 μg/mL kanamycin, 2% glutamine, and 10 mM Hepes, pH 7. For incubation with 5α-reduced steroids, on day 1, the cells were transfected with pCMV–human ERβ1 (25) (40 ng/well), p3 × (ERE)–TATA–luciferase (90 ng/well), pRSV–β-galactosidase (20 ng/well), and either pCMV–human 17βHSD6 (11) or pCMV vector (150 ng/well) using Fugene 6 (Roche) at a Fugene-to-DNA ratio of 4:1. Forty-eight hours after transfection, DHT, 3α-Adiol, 3β-Adiol, or E2 (1,000 × stock solutions in DMSO) were added to final concentrations of 20 nM. After 16 additional hours of incubation, the cells were lysed and assayed for luciferase activity using a luciferase assay kit (Biovision) and β-galactosidase activity according to manufacturer's instructions using a PerkinElmer Victor ×4 plate reader (PerkinElmer). Relative level of transactivation was calculated by dividing luciferase units by β-galactosidase units. For dose–response curves with DHT or 3α-Adiol, cells were transfected as described above, and 48 h after transfection steroids were added to final concentrations of 1 nM to 10 μM. After an additional 8 h incubation, the cells were lysed and assayed for luciferase and β-galactosidase activities as described above. EC50 values were generated by fitting the data to a sigmoidal dose–response curve using the GraphPad Prism 6.0 software (GraphPad Software).
Enzyme Assay for 17βHSD6 in Transfected HEK-293 Cells.
HEK-293 cells were seeded in 24-well Costar plates on day 0 as described above. On day 1, cells were transfected with pCMV–17βHSD6 plasmid (270 ng/well) and pRSV–β-galactosidase (30 ng/well). Forty-eight hours after transfection, radioactive DHT was added to the individual wells (20 nM final concentration). One hundred microliters of medium was collected at different time points, and total lipids were extracted with 400 μL Folch reagent (chloroform/methanol: 2:1 vol/vol), vortexing and spinning at 14,000 × g for 5 min in a microcentrifuge. The organic phase was collected and evaporated in a speed-vac. The dried extract was redissolved in 40 μL Folch and spotted onto Partisil LK5D TLC plates (catalog no. 4855–821;Whatman). The plates were developed twice in chloroform–ethyl acetate (4:1 vol/vol) (8). Steroid metabolites were quantified using a BioScan AR-2000 Imaging System (Bioscan). After the final time point, cells were lysed and assayed for β-galactosidase activity to normalize for transfection efficiency.
Immunohistochemistry.
For immunohistochemistry, two antibodies against 17βHSD6 were used: a rabbit polyclonal antibody (cat. no. ab62221; Abcam) raised against N-terminal amino acids 1–50 of human 17βHSD6 and a mouse polyclonal antibody (catalog no. H8630-B01P; Novus Biologicals) raised against the full-length human protein. For detection of ERβ, an antibody raised in chickens (ERβ 503 IgY) was used (26). Slides were deparaffinized with xylene and rehydrated through graded ethanol. For 17βHSD6, antigens were retrieved by boiling the slides in Tris⋅EDTA (Tris base 10 mM, EDTA 1 mM, 0.05% Tween 20, pH 9) buffer for 20 min in microwave oven. For ERβ, antigen was retrieved by heating at 97 °C for 15 min in citrate buffer (10 mM, pH 6) using a Pre Treatment module (ThermoScientific). After blocking endogenous peroxidase activity with 3% H2O2 in 50% methanol for 30 min, the slides were incubated in 3% BSA in PBS to block nonspecific binding followed by incubation with primary antibodies diluted in 3% BSA in PBS (17βHSD6, 1:300; ERβ, 1:200) overnight at room temperature. Slides were then washed with 0.1% Nonidet P-40 in PBS for 30 min and incubated with secondary antibodies. For ERβ, slides were incubated for 1 h with biotinylated goat anti-chicken antibody (Abcam) diluted to 1:200 in 3% BSA in PBS, washed in 0.1% Nonidet P-40 in PBS for 30 min, and incubated with Vectastain ABC (Vector Labs) for 1 h. For 17βHSD6, the slides were first incubated with Rabbit on Rodent HRP Polymer (Biocare) for 30 min followed by washing with 0.1% Nonidet P-40 in PBS for 30 min. After incubating with diaminobenzidine for 30 s and counterstaining with hematoxylin, the slides were dehydrated and mounted.
Author contributions: S.A. and J.-Å.G. designed research; S.M., H.-J.K., and R.B. performed research; L.W. and U.B. contributed new reagents/analytic tools; S.M., S.A., H.-J.K., R.B., and J.-Å.G. analyzed data; and S.M., S.A., and J.-Å.G. wrote the paper.
Abstract
Estrogen receptor β (ERβ) is activated in the prostate by 5α-androstane-3β,17β-diol (3β-Adiol) where it exerts antiproliferative activity. The proliferative action of the androgen receptor is activated by 5α-dihydrotestosterone (DHT). Thus, prostate growth is governed by the balance between androgen receptor and ERβ activation. 3β-Adiol is a high-affinity ligand and agonist of ERβ and is derived from DHT by 3-keto reductase/3β-hydroxysteroid dehydrogenase enzymes. Here, we demonstrate that, when it is expressed in living cells containing an estrogen response element-luciferase reporter, 17β-hydroxysteroid dehydrogenase type 6 (17βHSD6) converts the androgen DHT to the estrogen 3β-Adiol, and this leads to activation of the ERβ reporter. This conversion of DHT occurs at concentrations that are in the physiological range of this hormone in the prostate. Immunohistochemical analysis revealed that 17βHSD6 is expressed in ERβ-positive epithelial cells of the human prostate and that, in prostate cancers of Gleason grade higher than 3, both ERβ and 17βHSD6 are undetectable. Both proteins were present in benign prostatic hyperplasia samples. These observations reveal that formation of 3β-Adiol via 17βHSD6 from DHT is an important growth regulatory pathway that is lost in prostate cancer.
Estrogen receptor β (ERβ) is a member of the nuclear receptor superfamily of transcription factors (1). ERβ is activated by its endogenous ligands estradiol-17β (E2) and 5α-androstane-3β,17β-diol (3β-Adiol). Activated ERβ is known to antagonize the proliferative actions of ERα (2–4). It has been shown that, in the prostate, 3β-Adiol is the physiological ligand of ERβ (2). This notion is supported by the fact that the intraprostatic 3β-Adiol level (10 nM) is 100-fold higher than that of E2 (0.1 nM) (5). 3β-Adiol is a metabolite of the androgen receptor (AR) agonist 5α-dihydrotestosterone (DHT).
In vitro experiments using membrane preparations or purified enzymes have demonstrated that 3β-Adiol can be formed from DHT in two ways: (i) directly by 3-keto reduction of DHT to 3β-Adiol or (ii) by a two-enzyme process that entails a 3-keto reduction to 3α-Adiol followed by 3α- to 3β-hydroxysteroid epimerization.
The most likely candidate enzyme for direct reduction is AKR1C1, a member of the aldo-keto reductase (AKR) family (6, 7). This cytosolic enzyme predominantly catalyzes the conversion of DHT to 3β-Adiol (8). A candidate for the first step in the two-enzyme pathway is AKR1C2, which converts DHT to 3α-Adiol (9). The mRNAs for both enzymes have been quantified in the human prostate by quantitative PCR (10); however, their cell type-specific protein expression in the prostate has not been reported.
The epimerase reaction has been shown to be catalyzed by 17β-hydroxysteroid dehydrogenase type 6 (17βHSD6) (11, 12), also termed retinol dehydrogenase-like 3α-hydroxysteroid dehydrogenase (13, 14). 17βHSD6 is a microsomal enzyme that belongs to the short-chain dehydrogenase superfamily whose mRNA is expressed in the human and rat prostate (11); however, the protein's cell type-specific expression has not been described. We show here that 17βHSD6 has the capacity to directly convert physiological concentrations of DHT to 3β-Adiol with a concomitant activation of ERβ. The biological significance of 17βHSD6 acting as a 3β-Adiol synthase is supported by the fact that the enzyme colocalizes with ERβ protein in epithelial cells of the human prostate.
Acknowledgments
We thank Chris Brooks for technical assistance. This work was supported by National Institutes of Health Grant HD60541 (to S.A.), by Cancer Prevention and Research Institute of Texas Grant RP110444-P1 (to J.-Å.G.), and by the Swedish Cancer Society (J.-Å.G.).
Footnotes
The authors declare no conflict of interest.
References
- 1. Heldring N, et al Estrogen receptors: How do they signal and what are their targets. Physiol Rev. 2007;87:905–931.[PubMed][Google Scholar]
- 2. Weihua Z, et al A role for estrogen receptor β in the regulation of growth of the ventral prostate. Proc Natl Acad Sci USA. 2001;98:6330–6335.[Google Scholar]
- 3. Matthews J, et al Estrogen receptor (ER) β modulates ERalpha-mediated transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-responsive promoters. Mol Endocrinol. 2006;20:534–543.[PubMed][Google Scholar]
- 4. Ricke WA, et al Prostatic hormonal carcinogenesis is mediated by in situ estrogen production and estrogen receptor alpha signaling. FASEB J. 2008;22:1512–1520.[PubMed][Google Scholar]
- 5. Bélanger A, Couture J, Caron S, Roy RDetermination of nonconjugated and conjugated steroid levels in plasma and prostate after separation on C-18 columns. Ann N Y Acad Sci. 1990;595:251–259.[PubMed][Google Scholar]
- 6. Penning TM, et al Human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: Functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. Biochem J. 2000;351:67–77.[Google Scholar]
- 7. Hyndman D, Bauman DR, Heredia VV, Penning TMThe aldo-keto reductase superfamily homepage. Chem Biol Interact. 2003;143–144:621–631.[PubMed][Google Scholar]
- 8. Steckelbroeck S, Jin Y, Gopishetty S, Oyesanmi B, Penning TMHuman cytosolic 3α-hydroxysteroid dehydrogenases of the aldo-keto reductase superfamily display significant 3β-hydroxysteroid dehydrogenase activity: Implications for steroid hormone metabolism and action. J Biol Chem. 2004;279:10784–10795.[PubMed][Google Scholar]
- 9. Rizner TL, et al Human type 3 3α-hydroxysteroid dehydrogenase (aldo-keto reductase 1C2) and androgen metabolism in prostate cells. Endocrinology. 2003;144:2922–2932.[PubMed][Google Scholar]
- 10. Bauman DR, Steckelbroeck S, Peehl DM, Penning TMTranscript profiling of the androgen signal in normal prostate, benign prostatic hyperplasia, and prostate cancer. Endocrinology. 2006;147:5806–5816.[PubMed][Google Scholar]
- 11. Biswas MG, Russell DWExpression cloning and characterization of oxidative 17β- and 3α-hydroxysteroid dehydrogenases from rat and human prostate. J Biol Chem. 1997;272:15959–15966.[PubMed][Google Scholar]
- 12. Huang X-F, Luu-The VMolecular characterization of a first human 3(α→β)-hydroxysteroid epimerase. J Biol Chem. 2000;275:29452–29457.[PubMed][Google Scholar]
- 13. Chetyrkin SV, Hu J, Gough WH, Dumaual N, Kedishvili NYFurther characterization of human microsomal 3α-hydroxysteroid dehydrogenase. Arch Biochem Biophys. 2001;386:1–10.[PubMed][Google Scholar]
- 14. Belyaeva OV, et al Role of microsomal retinol/sterol dehydrogenase-like short-chain dehydrogenases/reductases in the oxidation and epimerization of 3α-hydroxysteroids in human tissues. Endocrinology. 2007;148:2148–2156.[Google Scholar]
- 15. Gleason DF, Mellinger GTPrediction of prognosis for prostatic adenocarcinoma by combined histological grading and clinical staging. J Urol. 1974;11:58–64.[PubMed][Google Scholar]
- 16. Lee KL, Peehl DMMolecular and cellular pathogenesis of benign prostatic hyperplasia. J Urol. 2004;172:1784–1791.[PubMed][Google Scholar]
- 17. Leav I, et al Comparative studies of the estrogen receptors β and α and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma. Am J Pathol. 2001;159:79–92.[Google Scholar]
- 18. Andersson S, Berman DM, Jenkins EP, Russell DWDeletion of steroid 5 α-reductase 2 gene in male pseudohermaphroditism. Nature. 1991;354:159–161.[Google Scholar]
- 19. Russell DW, Wilson JDSteroid 5 α-reductase: Two genes/two enzymes. Annu Rev Biochem. 1994;63:25–61.[PubMed][Google Scholar]
- 20. Bauman DR, Steckelbroeck S, Williams MV, Peehl DM, Penning TMIdentification of the major oxidative 3α-hydroxysteroid dehydrogenase in human prostate that converts 5α-androstane-3α,17β-diol to 5α-dihydrotestosterone: A potential therapeutic target for androgen-dependent disease. Mol Endocrinol. 2006;20:444–458.[PubMed][Google Scholar]
- 21. Stiles AR, McDonald JG, Bauman DR, Russell DWCYP7B1: One cytochrome P450, two human genetic diseases, and multiple physiological functions. J Biol Chem. 2009;284:28485–28489.[Google Scholar]
- 22. Weihua Z, Lathe R, Warner M, Gustafsson J-ÅAn endocrine pathway in the prostate, ERbeta, AR, 5α-androstane-3β,17β-diol, and CYP7B1, regulates prostate growth. Proc Natl Acad Sci USA. 2002;99:13589–13594.[Google Scholar]
- 23. Huang X-F, Luu-The VGene structure, chromosomal localization and analysis of 3-ketosteroid reductase activity of the human 3(α→β)-hydroxysteroid epimerase. Biochim Biophys Acta. 2001;1520:124–130.[PubMed][Google Scholar]
- 24. Saijo K, Collier JG, Li AC, Katzenellenbogen JA, Glass CKAn ADIOL-ERβ-CtBP transrepression pathway negatively regulates microglia-mediated inflammation. Cell. 2011;145:584–595.[Google Scholar]
- 25. Ogawa S, et al The complete primary structure of human estrogen receptor β (hER β) and its heterodimerization with ERα in vivo and in vitro. Biochem Biophys Res Commun. 1998;243:122–126.[PubMed][Google Scholar]
- 26. Saji S, et al Estrogen receptors α and β in the rodent mammary gland. Proc Natl Acad Sci USA. 2000;97:337–342.[Google Scholar]