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Endocrinology, doi:10.1210/en.2006-0358
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Endocrinology Vol. 147, No. 8 3843-3850
Copyright © 2006 by The Endocrine Society

Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells

William R. Harrington1, Surojeet Sengupta1 and Benita S. Katzenellenbogen

Departments of Molecular and Integrative Physiology (W.R.H., S.S., B.S.K.) and Cell and Developmental Biology (B.S.K.), University of Illinois, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: Dr. Benita S. Katzenellenbogen, University of Illinois, Department of Molecular and Integrative Physiology, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801-3704. E-mail: katzenel{at}uiuc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogens and androgens influence many properties of breast cancer cells; hence, regulation of local estrogen and androgen levels by enzymes involved in steroid hormone biosynthesis and metabolism would impact signaling by these hormones in breast cancer cells. In this study, we show that the UDP-glucuronosyltransferase (UGT) enzyme UGT2B15, a member of the UGT family of phase II enzymes involved in the glucuronidation of steroids and xenobiotics, is a novel, estrogen-regulated gene in estrogen receptor (ER)-positive human breast cancer cells (MCF-7, BT474, T47D, and ZR-75). UGT2B15 is the only UGT2B enzyme up-regulated by estrogen, and marked estradiol stimulation of UGT2B15 mRNA levels is observed, in a time- and dose-dependent manner. UGT2B15 stimulation by estradiol is blocked by the antiestrogen ICI182,780, but not by the translational inhibitor cycloheximide, indicating that UGT2B15 is likely a primary transcriptional response mediated through the ER. UGT2B15 up-regulation is also evoked by other estrogens (propylpyrazoletriol, genistein) and by the androgen 5{alpha}-dihydrotestosterone working through the ER, but not by other steroid hormone receptor ligands. Western blot and immunocytochemical analyses with several UGT2B-specific antibodies we have designed and steroid glucuronidation assays indicate a large increase in both cellular UGT2B15 protein and enzyme activity after estrogen treatment. Due to the important role of UGT enzymes in forming conjugates between steroids and glucuronic acid, thereby inactivating them and targeting them for removal, the estrogen-induced up-regulation of UGT2B15 might have a significant moderating effect on estrogen and androgen concentrations, thereby reducing their signaling in breast cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENIC HORMONES ARE involved in regulating a number of physiological processes in estrogen receptor (ER)-positive breast cancer cells, including proliferation, invasiveness, and changes in cytoarchitecture (1, 2, 3, 4, 5). Many of the effects of estrogens are mediated through binding to ER, a ligand-inducible transcription factor. Upon binding to estrogenic ligands, ER is thought to dissociate from chaperone proteins, dimerize, and bind to specific regulatory regions of target genes (6, 7, 8), nucleating the recruitment of transcriptional coregulators and altering local chromatin architecture resulting in the stimulation or repression of transcription (9). To better understand the role of estrogen-regulated gene expression in the physiological actions of estrogens in ER-positive breast cancer, we have used microarray transcriptional profiling to identify estrogen-regulated genes in MCF-7 breast cancer cells. These studies revealed the UDP-glucuronosyltransferase (UGT) enzyme, UGT2B15, as a putative estrogen-regulated gene. Because of the important role that UGT2B15 could play in regulation of estrogen and androgen signaling, through enzymatic inactivation of these steroid hormones by conjugation with glucuronic acid, we have in this report characterized in detail the regulation of UGT2B15 by estrogens.

UGTs encompass a family of enzymes that catalyze the formation of water-soluble metabolites of many biologically active substrates through the transfer of glucuronic acid from the cofactor UDP-glucuronic acid (UDPGA) to target substrates. These membrane-associated proteins are localized to the endoplasmic reticulum, and their target substrates are endogenous biomolecules and xenobiotics, including steroids, bile acids, bilirubin, dietary constituents, drugs, environmental toxicants, and carcinogens (10). The conjugation of glucuronic acid leads to substrates that are generally less biologically active and more hydrophilic and acidic, facilitating their excretion through bile and urine. To date, 15 different UGTs have been identified in the human, and these UGT enzymes have been shown to glucuronidate over 350 target substrates (10, 11). UGT enzymes have a highly conserved structure, with the C-terminal 250 amino acids being nearly identical for all UGT enzymes (12). This conserved domain is thought to be involved in the binding of the cofactor UDPGA, whereas the more divergent N-terminal portion of the protein (~280 amino acids) is thought to be involved in substrate binding. Many UGT enzymes also contain an N-terminal signal peptide, which is involved in membrane targeting.

In humans, the UGT enzyme family is composed of two subclasses: UGT1 and UGT2 enzymes. The eight UGT1A enzymes are encoded by the same gene and arise as the result of different transcriptional start sites and differential splicing. UGT1A enzymes are primarily expressed in the liver and gastrointestinal tract, and they glucuronidate a range of substrates, including bilirubin (10). Unlike the UGT1A enzymes, each of the UGT2 enzymes is encoded by a distinct genetic locus. The enzyme UGT2A1 is localized to the nasal epithelium, is capable of glucuronidating a broad range of substrate molecules, and is thought to play a major role in terminating the response to odorants. The other UGT2 enzymes are classified as UGT2B enzymes and are highly expressed in the liver, as well as in extrahepatic steroid target tissues such as the prostate and breast. The UGT2B enzymes are believed to be responsible for the majority of steroid glucuronidation in humans. As such, it has been proposed that UGT2B enzymes play a role in the regulation of steroid signaling through targeted inactivation of the steroid molecules themselves (13).

UGT2B15 is highly expressed in a number of steroid target tissues, including the liver, prostate, kidney, testis, mammary gland, placenta, adipose, and uterus (14). UGT2B15 is highly homologous to other UGT2B enzymes and shares 95% amino acid identity with the androgen-specific UGT2B17 (10, 15). UGT2B15 is known to glucuronidate a wide range of endogenous estrogens and androgens, including catechol estrogens and the androgens testosterone, 5{alpha}-dihydrotestosterone (DHT) and 5{alpha}-androstane-3{alpha},17ß-diol, as well as some exogenous compounds (16, 17). As such, it is possible that UGT2B15 may play a role in the regulation of steroid signaling through targeted inactivation of androgens and estrogens. Thus, the estrogen up-regulation of UGT2B15 in ER-positive breast cancer cells that we document in this study may represent a novel negative feedback mechanism on estrogen signaling or a means of cross-talk between the androgen and estrogen signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Cell culture media were purchased from Life Technologies, Inc. (Grand Island, NY). Calf serum (CS) was obtained from HyClone Laboratories (Logan, UT), and fetal CS (FCS) was obtained from Atlanta Biologicals (Atlanta, GA). The MCF-7 cell line was routinely maintained in MEM media supplemented with 5% CS. Six days before harvesting, cells were plated in 10-cm plates and switched to phenol red-free MEM containing 5% charcoal dextran (CD)-treated CS. Media was changed on d 2 and 4 of culture. For the final 48 h of culture, cells were treated with ligand as described in figure legends. All treatment groups were harvested at the same time, and total RNA was prepared using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

BT474, T47D, and ZR-75 cell lines were maintained in a manner similar to MCF-7 cells with the following changes. BT474 cells were routinely maintained in Improved MEM supplemented with 10% FCS. Media was changed to phenol red-free Improved MEM containing 10% CD-treated FCS before treatment. T47D cells were routinely maintained in MEM supplemented with 5% FCS and bovine insulin (6 ng/ml, Sigma, St. Louis, MO). Media was changed to phenol red-free MEM supplemented with 5% CD-FCS before treatment. ZR-75 cells were routinely maintained in DMEM/nutrient mix F12 media supplemented with 10% FCS. Media were changed to DMEM/nutrient mix F12 supplemented with 10% CD-FCS before treatment. All experiments were performed at least three times. Values shown are the mean ± SEM.

Microarray analysis and real-time PCR
Affymetrix GeneChip microarray analysis of estrogen-treated vs. control vehicle-treated MCF-7 cells was performed as described previously (18). MCF-7 cells were maintained and treated as described above before real-time PCR analyses. To generate cDNA for each sample, 1 µg total RNA was reverse transcribed in a total volume of 20 µl using 200 U reverse transcriptase, 50 pmol random hexamer, and 1 mM dNTP (New England Biolabs, Beverly, MA). The resulting cDNA was then diluted to a total volume of 100 µl with sterile H2O. Each real-time PCR consisted of 1 µl diluted RT product, 1x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), and 50 nM forward and reverse primer. Reactions were carried out in an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems) for 40 cycles (95 C for 15 sec, 60 C for 1 min) after an initial 10-min incubation at 95 C. The fold change in expression of UGT2B15 was calculated using the {Delta}{Delta}Ct method, with the ribosomal protein 36B4 mRNA as the internal control (19). 36B4 and UGT2B-specific primer sets used in this study for real-time PCR have been previously described (18, 20).

Antibody design and characterization
To develop antibodies against UGT2B15, antigenic peptides within the protein were determined using Mac Vector software (Oxford Molecular Group, Campbell, CA). Peptides were scored for their antigenicity (Parker antigenicity, Protrusion Index antigenicity, Welling antigenicity), hydrophilicity (Kyte/Doolittle hydrophilicity, Hopp/Woods hydrophilicity, Argos transmembrane, von Heijne transmembrane), and structure (protein flexibility, amphiphilic helix, amphiphilic sheet). Based on this analysis, three peptides were chosen for immunization and antibody production: peptide 1, LWRFDGKKPNTLGSNTRL (amino acids 337–354); peptide 2, SAIKLEVYPTSLTKNDLEDS (amino acids 70–89); and peptide 3, YYDYSNKLCKDAVLNKKLM (amino acids 120–138). Peptides were synthesized and conjugated to KLH by American Peptide Company (Sunnyvale, CA). Due to the high homology of UGT enzymes across mammalian species, we elected to use chickens as host animals. Immunizations of host animals and collection of preimmune and immune serum were conducted by Pocono Rabbit Farms (Canadensis, PA).

ELISA antibody titration assays were performed to demonstrate specific binding of immune serum to the antigenic peptide. Antigen conjugated to BSA (100 ng/well) was plated in a NUNC Maxi-Sorb 96-well plate (Nalge Nunc International, Naperville, IL). After a 2-h incubation at room temperature, a blocking step was performed by adding PBS + 3% BSA and incubating overnight at room temperature. The wells were then washed twice with PBS, and either preimmune or immune serum dilutions were added to the wells and allowed to incubate for 2 h. Wells were then washed four times with PBS before incubation with secondary antibody [goat {alpha}-chicken-horseradish peroxidase (HRP); Pocono Rabbit Farms] at a 1:5000 dilution for 2 h at room temperature. Wells were again washed four times with PBS before incubation with tetramethylbenzidine substrate (Sigma). Reaction with substrate was terminated by the addition of 1 M H2SO4. Intensity of reaction products was determined by measuring the absorbance at 470 nm.

Western blotting
Whole-cell lysates of MCF-7 cells were prepared using 1x Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA) in the presence of Complete Mini protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Protein concentration of whole-cell lysate was determined by BCA Protein Assay (Pierce, Rockford, IL). Proteins (35 µg) were separated by electrophoresis using 10% SDS-PAGE at 150 V for 50 min and were then transferred to a nitrocellulose membrane (Pall Corp., Pensacola, FL), using the wet transfer method, at 100 V for 90 min. Membranes were blocked with 5% milk in Tris-buffered saline. Anti-UGT2B15 primary antibody (antibody no. 679, raised against peptide no. 2, as described above) was incubated with blocked membrane at a 1:2000 dilution overnight at 4 C. The blot was then washed with Tris-buffered saline containing 0.1% Tween 20 before secondary antibody incubation with HRP-labeled bovine antichicken IgY (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:10,000 dilution for 1 h at room temperature. The blot was incubated with Super Signal West Femto ECL reagents (Pierce) and exposed to film to observe protein bands. ß-Actin protein levels were also monitored to assess equal protein loading (ß-actin antibody, Sigma).

Glucuronidation assays
Microsomal protein (50–200 µg) from MCF-7 cells treated with vehicle or estradiol (E2) for 72 h was incubated with 1 mM C-14-labeled UDPGA (specific radioactivity, 2 µCi/µmol) in the presence of 8.5 mM saccahrolactone, 0.25 mg/ml alamethicin, and 500 µM DHT as aglycone. Tris-Cl (pH 7.4; 50 mM) and 10 mM MgCl2 were used as assay buffer, and incubation was carried out at 37 C for 16 h. Assays were terminated by adding 100 µl absolute ethanol and centrifuging at 12,000 x g for 10 min. Supernatant (190 µl) was separated in a fresh 1.5-ml tube and was vacuum dried. The dried contents were reconstituted in 12 µl water and chromatographed on thin-layer chromatography (TLC) plates (EMD Chemicals, Gibbstown, NJ) in butanol, acetone, acetic acid, and water in the ratio of 7:7:2:4. After the run was complete, the TLC plate was dried and exposed to x-ray film at –80 C for 7 d before quantification using ImageQuant TL software (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Quantitation of the increase in enzymatic activity after estrogen treatment was also done by scintillation counting of TLC silica gel regions that were excised from the plates after development and plate drying.

Immunocytochemistry
Cells grown in phenol red-free MEM containing 5% CD-CS for 4 d were plated at 12,000 cells per well of a 24-well plate, with each well containing a coverslip. Cells were allowed to adhere to the coverslip for 16 h, and treatments with vehicle or estrogen (10 nM) were then started. After treatments, cells were washed with PBS and subsequently fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Cells were next permeabilized with 0.2% Triton X-100 in PBS for 20 min at room temperature, and then endogenous peroxidase activity was quenched by incubation with 1% H2O2 in PBS for 5 min at room temperature. Cells were then washed with PBS and blocked with 3% BSA in PBS for 2 h at room temperature, followed by overnight incubation with anti-UGT2B15 primary antibody (antibody no. 677, raised against peptide no. 1, as described above) at a 1:500 dilution, in a humidified chamber at 4 C. Coverslips with attached cells were washed with PBS containing 0.05% Tween 20 and then incubated with an HRP-labeled bovine antichicken IgY secondary antibody (Santa Cruz Biotechnology), at a 1:400 dilution, for 1 h at room temperature. After extensive washing, color was developed using diaminobenzidine and H2O2, and coverslips were mounted using Permamount (Fisher Scientific, Pittsburgh, PA). Before mounting, cells were counterstained with hematoxylin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Microarray analysis and real-time PCR: time course and dose-response studies
Based on prior microarray transcriptional profiling analysis of E2-treated MCF-7 cells (18), we identified UGT2B15 as a putative estrogen-regulated gene. To investigate in detail and confirm UGT2B15 regulation by estrogen, real-time PCR was performed on cDNA samples from MCF-7 cells treated with E2 over a range of concentrations and treatment times. For the time course study, MCF-7 cells were treated with E2 (10 nM) for 2, 4, 8, 24, and 48 h (Fig. 1AGo). E2-mediated increases in UGT2B15 mRNA were observed as early as 4 h. Maximal induction of UGT2B15 mRNA levels was observed at 8 h, with mRNA levels remaining elevated after 48 h of treatment. A dose-response study was also performed with cells being treated for 8 h with concentrations of E2 ranging from 1 pM to 10 nM (Fig. 1BGo). E2 stimulation of UGT2B15 mRNA levels occurred in a dose-dependent manner. Induction was observed at concentrations as low as 10 pM, with 0.1 nM E2 giving half-maximal stimulation and maximal stimulation being observed at 10 nM. An E2 concentration of 10 nM and a treatment time of 8 h were frequently used in subsequent studies because they yield maximal stimulation of UGT2B15.


Figure 1
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  		FIG. 1. UGT2B15 mRNA levels are stimulated by E2 in a time- and dose-dependent manner in MCF-7 breast cancer cells. A, Time course for E2 treatment. Cells were treated with E2 (10 nM) for 2, 4, 8, 24, or 48 h. B, Dose response for E2 treatment. MCF-7 cells were treated with various concentrations of E2 (1 pM to 10 nM) for 8 h. RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods. Values are the mean ± SEM of three experiments.

 
Effects of antiestrogen and cycloheximide (CHX) treatment on E2-mediated increases in UGT2B15 mRNA
The antiestrogen ICI182,780 (ICI) is able to inhibit estrogen-induced transcriptional activation by competing with E2 for binding to ER and also by enhancing ER degradation (21). As such, cotreating cells with estrogen and antiestrogen provides a means of confirming that estrogen effects are mediated through ER. We treated MCF-7 cells for 8 h with E2 (10 nM) alone or after a 2-h pretreatment with ICI (1 µM; Fig. 2AGo). ICI alone did not stimulate UGT2B15 mRNA production. However, ICI almost completely reversed the increase in UGT2B15 mRNA levels evoked by E2. An ER{alpha}-selective agonist, propylpyrazoletriol (PPT; 100 nM), and the phytoestrogen genistein (1 µM) also induced increases in UGT2B15 (Fig. 2BGo), and the stimulation by both ligands was also completely reversed by cotreatment with ICI (data not shown). This indicates that the stimulation of UGT2B15 gene transcription by E2 and other estrogenic ligands is likely mediated through ER.


Figure 2
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  		FIG. 2. E2-induced increase in UGT2B15 mRNA levels is inhibited by ICI but not by CHX. A, MCF-7 cells were treated with ethanol vehicle, E2 (10 nM), ICI (1 µM), or a combination of E2 and ICI for 8 h. B, Cells were treated with ethanol vehicle (control), E2 (10 nM), PPT (100 nM), or genistein (Gen; 1 µM) alone or in combination with CHX (10 µM). RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods. Values are the mean + SEM of three experiments.

 
To determine whether the transactivation of UGT2B15 is a primary or secondary transcriptional response to estrogenic ligands, we used CHX, a translational inhibitor, which blocks new protein synthesis and thus prevents secondary transcriptional responses from occurring. MCF-7 cells were pretreated for 2 h with CHX (10 µM) before an 8-h treatment with E2 (10 nM), PPT (100 nM), and genistein (1 µM) in the absence or presence of CHX (Fig. 2BGo). Treatment with CHX alone increased UGT2B15 mRNA levels to about 25% of E2-stimulated levels. This increase by CHX might be due to inhibition of the synthesis of proteins involved in UGT2B15 promoter repression or mRNA stability. CHX had little if any effect on the estrogen-induced increase of UGT2B15 mRNA levels for any of the three estrogenic ligands tested. Thus, it appears that the estrogen-induced up-regulation of UGT2B15 is a primary transcriptional response.

Evaluation of steroid specificity in regulation of UGT2B15
We next wanted to determine whether the stimulation of UGT2B15 transcription was unique to estrogenic hormones or if other steroid hormones were also capable of stimulating transcription of this gene. We therefore treated MCF-7 cells for 8 h with a 10 nM concentration of an androgen (5{alpha}-DHT), a glucocorticoid (hydrocortisone), a progestin (R5020), or E2, for comparison. Hydrocortisone and R5020 failed to stimulate an increase in UGT215 mRNA levels (Fig. 3AGo). 5{alpha}-DHT, however, stimulated UGT2B15 mRNA levels to approximately 25% of E2-stimulated levels. In time-course studies, we observed that UGT2B15 mRNA levels were increased over time with 5{alpha}-DHT treatment (Fig. 3BGo), yielding stimulation approximately 25–35% that of E2 over the duration of the time course. As shown in Fig. 3CGo, the E2 and DHT stimulated increases in UGT2B mRNA were both blocked by the antiestrogen ICI but were unaffected by the antiandrogen hydroxyflutamide (HFLT), indicating mediation through the ER for both ligands.


Figure 3
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  		FIG. 3. UGT2B15 regulation by steroid hormone receptor ligands. A, Cells were treated with ethanol vehicle (control), 5{alpha}-DHT (10 nM), hydrocortisone (HC; 10 nM), R5020 (10 nM), or E2 (10 nM) for 8 h. B, Time course for E2 or DHT treatment. Cells were treated with E2 (10 nM) or DHT (10 nM) for 2, 4, 8, 24, and 48 h. C, Effect of antiestrogen (1 µM ICI) or antiandrogen (1 µM HFLT) on the stimulation of UGT2B15 mRNA by E2 (10 nM) or DHT (10 nM). Cells were treated with vehicle or ligand alone, or with ligand plus ICI or HFLT for 8 h. RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods.

 
Estrogen regulation of UGT2B15 in ER-positive breast cancer cell lines
To determine whether E2 caused an up-regulation of UGT2B15 in other breast cancer cell lines, we performed E2 time course experiments in the ER-positive breast cancer cell lines BT474, T47D, and ZR-75 (Fig. 4Go). In BT474 and T47D cells, maximal (~10-fold) increases in UGT2B15 mRNA levels were observed by 24 h of E2 treatment, and mRNA levels remained elevated at 48 h. In ZR-75 cells, E2 stimulated an increase in UGT2B15 mRNA, but at a lower magnitude. A slight increase in UGT2B15 mRNA levels was observed as early as 2 h of treatment, and maximal induction was observed at 48 h (approximately 4-fold). This lower magnitude of stimulation of an estrogen-responsive gene is not limited to UGT2B15 because the estrogen-stimulated gene pS2 was also less robustly stimulated in ZR-75 cells than in MCF-7 cells (data not shown). Hence, the E2-induced increase in UGT2B15 mRNA levels was not limited to the MCF-7 cell line but was also observed in other ER-positive breast cancer cell lines.


Figure 4
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  		FIG. 4. UGT2B15 is an estrogen-regulated gene in several ER-positive human breast cancer cell lines. BT474, T47D, and ZR-75 cells were treated with E2 (10 nM) for 2, 4, 8, 24, or 48 h. RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods.

 
Assessment of the E2 responsiveness of other UGT2B enzymes
Because there are six UGT2B enzymes that have been described in the human, we were interested to determine whether or not other UGT2B enzymes were regulated by estrogen. To differentiate between the highly homologous UGT2B enzymes, we used enzyme-specific real-time PCR primers that have been previously described (20). We were unable to detect mRNA for the enzymes UGT2B4, UGT2B7, and UGT2B11 in MCF-7 cells using real-time PCR, even after estrogen stimulation. UGT2B10 and UGT2B17 mRNA were detected in MCF-7 cells, but the mRNAs for these enzymes were not stimulated after E2 treatment (Fig. 5Go). By contrast, UGT2B15 was robustly up-regulated. Therefore, it appears that UGT2B15 is the only UGT2B enzyme that is regulated by E2 in these cells.


Figure 5
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  		FIG. 5. UGT2B15 is the only UGT2B enzyme regulated by estrogen in MCF-7 cells. Cells were treated with E2 (10 nM) for up to 48 h. RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods. UGT2B4 and UGT2B7 mRNA were not detectable in MCF-7 cells using real-time PCR.

 
Regulation of UGT2B15 protein and enzymatic activity by E2
To determine the effect of E2 treatment on UGT2B15 protein levels, we generated antibodies against UGT2B15. Three distinct peptides were determined by our analysis to likely be strongly antigenic, and these were selected as antigens for the immunization procedure. All of the antisera tested proved to bind their respective antigen with high affinity, as demonstrated by ELISA antibody titration assay (Fig. 6AGo). In contrast, nonimmune serum did not specifically bind antigen. Western blot and IHC findings were similar using antibodies to peptides 1 or 2. Antibody to peptide 3, although of similar high titer, gave considerable background and therefore was not further used.


Figure 6
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  		FIG. 6. UGT2B15 protein levels increase after E2 treatment in MCF-7 breast cancer cells. A, ELISA antibody titration assay. Preimmune (solid line) or immune (dashed line) antisera were incubated with BSA-conjugated antigen, and amount of antibody bound was determined by performing a colorimetric assay as described in Materials and Methods. Similar curves were obtained for immune antisera made to peptides 1 or 2. B, Cells were treated for 4, 8, 24, 48, or 72 h with E2 (10 nM). The blot was probed with anti-UGT2B15 antibody to peptide 2. ß-Actin protein levels were also monitored to assess protein loading. Similar findings were observed in several repeat studies.

 
To investigate whether E2 treatment causes an increase in UGT2B15 protein levels, we performed Western blots using whole-cell lysates from MCF-7 cells treated with E2 (10 nM) for varying times up to 72 h. UGT2B15 protein was very low in vehicle-treated cells and was increased over time in cells treated with E2 (Fig. 6BGo). The increase in UGT2B15 protein level was observed as early as 4 h after E2 and was further elevated over the 72 h studied. We also used immunocytochemistry (Fig. 7Go) to investigate the cellular regulation of UGT2B15 protein by E2. Staining for UGT2B15 protein was very low in vehicle-treated cells (Fig 7BGo) but was found to increase over the duration of the E2 treatment (Fig. 7Go, C and D). UGT2B15 was cytoplasmic and was present in a meshwork throughout the cytoplasm, as would be expected for a protein known to localize to the endoplasmic reticulum. No staining was observed in estrogen-treated cells in the absence of anti-UGT2B15 primary antibody (Fig. 7AGo), demonstrating the specificity of the staining.


Figure 7
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  		FIG. 7. Immunocytochemistry showing the increase in UGT2B15 protein in MCF-7 cells treated with E2. Cells were treated with vehicle for 72 h (B) or with E2 (10 nM) for 48 (C) or 72 (D) h. After treatment, cells were fixed and processed for immunocytochemistry as described in Materials and Methods. To demonstrate specificity of staining, a no primary antibody control (A) was performed with cells treated for 72 h with E2 (10 nM). Magnification, x40. For the immunocytochemistry shown, antibody against peptide 1 was used, but similar results were obtained using antibodies against peptides 2 and 3.

 
The increase in UGT2B15 protein observed with E2 treatment was also associated with a concomitant increase in UGT2B15 enzymatic activity. As shown in Fig. 8Go, assessment of the glucuronidation of DHT by quantitation of glucuronidated DHT using the microsomal fraction of control vehicle or E2-treated MCF-7 cells revealed an almost 6-fold increase in enzymatic activity after E2 treatment for 72 h. This increase in enzymatic activity, determined by ImageQuant analysis of spots after TLC separation of glucuronidated DHT (Fig. 8Go), was also verified by scintillation counting of 14C-glucuronidated DHT after TLC separation and quantification (data not shown).


Figure 8
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  		FIG. 8. Assessment of UGT2B15 glucuronidation enzymatic activity of MCF-7 cells after E2 treatment. [C-14]Glucuronidated DHT was quantitated using microsomal fractions of vehicle or 10 nM E2-treated (for 72 h) MCF-7 cells after TLC and ImageQuant analysis. Values are mean ± SEM of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The observation that the glucuronidation enzyme UGT2B15 is up-regulated in human breast cancer cells by estrogens may have implications for both steroid signaling and breast cancer physiology. UGT enzymes catalyze the glucuronidation of a number of target substrates, including drugs, exogenous compounds, steroids, and other hormones (10), resulting in inactive and more water-soluble conjugate that is facilely eliminated from the body via the urine or bile. UGT2B15 is specifically known to glucuronidate both endogenous androgens and estrogens and through this action generates inactive, more water-soluble compounds. Hence, UGT1B15 activity brings about the elimination of active hormones and thereby may influence the level and activity of these steroids in cells in which it is expressed (17). Thus, the amount of UGT2B15 enzyme in breast cancer cells may impact the level of estrogens and androgens present locally in breast tumor tissue.

The occurrence of breast cancer in women is highest after menopause, at which point the ovaries cease to be functional, and a drop in circulating estrogen levels is observed. However, estrogen levels in breast tumor tissue are several times higher than serum estrogen levels, supporting the importance of local synthesis of estrogens in breast tumor tissue (22, 23). One of the major pathways of local estrogen synthesis is through the conversion of aromatizable androgens to estrogens via the aromatase P450 enzyme. The importance of this pathway is demonstrated by the fact that aromatase inhibitors have been successfully used as a means of decreasing estrogen levels for breast cancer treatment (24). It has been proposed recently that local E2 levels may be regulated by a balance between the activity of local estrogen producing enzymes (such as aromatase) and those enzymes (such as UGTs) involved in the inactivation and elimination of E2 and E2 precursors (25). The aromatizable androgen testosterone, which is converted to E2 by aromatase, is a substrate of UGT2B15; thus, by decreasing testosterone availability, local estrogen production may be affected. Also, human breast cancer cell lines are known to have the ability to form E2 glucuronides (26). Interestingly, a recent study found that plasma E2 levels varied depending on the identity of the polymorphic variant of UGT2B15 found in breast cancer patients (27).

There are multiple mechanisms by which estrogens can limit their own signaling, acting in a negative feedback loop. First, estrogens are classically known to act on the pituitary to depress gonadotropin secretion (28). This effect of estrogen results in a decrease in the production of testosterone, which is then converted to estrogen via aromatase. Second, estrogen-bound ER{alpha} has been shown to depress aromatase promoter activity by directly binding to a silencer element in the aromatase promoter (29). This E2-mediated down-regulation of aromatase activity has been observed in cell culture systems and also in a primate (baboon) mammary gland model (30, 31). Third, E2 binding to ER results in a decrease in ER protein levels by altering receptor half-life from approximately 4–5 h for apo-ER to 2–3 h for ligand-bound ER (32, 33). Binding of estrogen results in the ubiquitination of ER and its subsequent degradation by the 26S proteasome (34). Finally, E2 is known to inhibit the enzymatic activity of sulfatase in breast cancer cells (22). Sulfatase is the enzyme that converts estrone-sulfate (a major circulating form of estrogen in postmenopausal women) to estrone, which is then converted to E2 by 17ß-hydroxysteroid dehydrogenase. All of these mechanisms may combine to provide a fine-tuning regulation of estrogen signaling by limiting the magnitude and duration of the estrogenic response. UGT2B15 has been shown to have the ability to glucuronidate both endogenous estrogens and their precursors, aromatizable androgens such as testosterone. As such, the estrogen-mediated up-regulation of UGT2B15 described in this paper may be part of a novel negative feedback loop that limits local levels of estrogens and estrogen precursors in breast cancer cells.

In addition to its potential effects on estrogen signaling, an increase in UGT2B15 may also affect several other hormone signaling pathways. UGT2B15 is known to glucuronidate the potent androgen 5{alpha}-DHT (17). The specific role for androgens in the progression of breast cancer is not well understood because both proliferative and antiproliferative effects have been observed after androgen treatment of breast cancer cells (35). It is interesting to hypothesize that UGT2B15 up-regulation by estrogen may provide a means of limiting local androgen levels in the breast and thus may influence androgen receptor signaling in breast cancer cells. Of note, another substrate of UGT2B15, the DHT metabolite 5{alpha}-androstane-3ß,17ß-diol, has been characterized recently as a ligand for ERß (17, 36). Also, UGT2B15 is known to catalyze the glucuronidation of a number of catechol and other estrogens, including 4-hydroxy-estrone, 16{alpha}-hydroxy-estrone, and 4-hydroxy-E2 (17). These catechol estrogens and 16{alpha}-hydroxy-estrone display estrogenic activity and are present at high concentrations in breast tumors (37, 38, 39, 40). Quinone intermediates derived from the 4-hydroxy catechol estrogens in particular have been implicated in mutagenesis and the induction of cancer (41). Thus, the clearance of these compounds by UGT2B15 may serve, in part, as a protective mechanism against the stimulation or further progression of breast cancer. In summary, our report that UGT2B15, which encodes an enzyme involved in the glucuronidation of endogenous androgens and estrogens, is a novel estrogen-responsive gene, suggests that its up-regulation by estrogen might play a significant role in modulation of the local concentration of estrogen and androgen and their signaling in breast cancer.


   Acknowledgments
 
We thank Dr. Jonna Frasor for valuable suggestions and for discussing her original microarray findings with us.


   Footnotes
 
This work was supported by National Institutes of Health Grant CA18119 and by a grant from The Breast Cancer Research Foundation (to B.S.K.). W.R.H. was a predoctoral trainee supported by National Institutes of Health Grant T32 HD07028.

All authors (W.R.H., S.S., and B.S.K.) indicate that they have nothing to declare.

First Published Online May 11, 2006

1 W.R.H. and S.S. are equal first authors. Back

Abbreviations: CD, Charcoal dextran; CHX, cycloheximide; CS, calf serum; DHT, dihydrotestosterone; E2, estradiol; ER, estrogen receptor; FCS, fetal CS; HFLT, hydroxyflutamide; HRP, horseradish peroxidase; ICI, ICI182,780; PPT, propylpyrazoletriol; UDPGA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase.

Received March 20, 2006.

Accepted for publication May 4, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hall JM, Couse JF, Korach KS 2001 The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 276:36869–36872[Free Full Text]
  2. Katzenellenbogen BS, Frasor J 2004 Therapeutic targeting in the estrogen receptor hormonal pathway. Semin Oncol 31:28–38[Medline]
  3. Katzenellenbogen BS, Montano MM, Ediger TR, Sun J, Ekena K, Lazennec G, Martini PG, McInerney EM, Delage-Mourroux R, Weis K, Katzenellenbogen JA 2000 Estrogen receptors: selective ligands, partners, and distinctive pharmacology. Recent Prog Horm Res 55:163–193; discussion 194–195[Medline]
  4. Osborne CK 1998 Tamoxifen in the treatment of breast cancer. N Engl J Med 339:1609–1618[Free Full Text]
  5. Vic P, Vignon F, Derocq D, Rochefort H 1982 Effect of estradiol on the ultrastructure of the MCF7 human breast cancer cells in culture. Cancer Res 42:667–673[Abstract/Free Full Text]
  6. Kumar V, Chambon P 1988 The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145–156[CrossRef][Medline]
  7. Murdoch FE, Gorski J 1991 The role of ligand in estrogen receptor regulation of gene expression. Mol Cell Endocrinol 78:C103–C108
  8. Pratt WB, Toft DO 1997 Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr Rev 18:306–360[Abstract/Free Full Text]
  9. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108:465–474[CrossRef][Medline]
  10. Tukey RH, Strassburg CP 2000 Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 40:581–616[CrossRef][Medline]
  11. Thibaudeau J, Lepine J, Tojcic J, Duguay Y, Pelletier G, Plante M, Brisson J, Tetu B, Jacob S, Perusse L, Belanger A, Guillemette C 2006 Characterization of common UGT1A8, UGT1A9, and UGT2B7 variants with different capacities to inactivate mutagenic 4-hydroxylated metabolites of estradiol and estrone. Cancer Res 66:125–133[Abstract/Free Full Text]
  12. Tukey RH, Strassburg CP 2001 Genetic multiplicity of the human UDP-glucuronosyltransferases and regulation in the gastrointestinal tract. Mol Pharmacol 59:405–414[Abstract/Free Full Text]
  13. Mackenzie PI, Mojarrabi B, Meech R, Hansen A 1996 Steroid UDP glucuronosyltransferases: characterization and regulation. J Endocrinol 150(Suppl):S79–S86
  14. Levesque E, Beaulieu M, Green MD, Tephly TR, Belanger A, Hum DW 1997 Isolation and characterization of UGT2B15(Y85): a UDP-glucuronosyltransferase encoded by a polymorphic gene. Pharmacogenetics 7:317–325[Medline]
  15. Beaulieu M, Levesque E, Hum DW, Belanger A 1996 Isolation and characterization of a novel cDNA encoding a human UDP-glucuronosyltransferase active on C19 steroids. J Biol Chem 271:22855–22862[Abstract/Free Full Text]
  16. Chen F, Ritter JK, Wang MG, McBride OW, Lubet RA, Owens IS 1993 Characterization of a cloned human dihydrotestosterone/androstanediol UDP-glucuronosyltransferase and its comparison to other steroid isoforms. Biochemistry 32:10648–10657[CrossRef][Medline]
  17. Green MD, Oturu EM, Tephly TR 1994 Stable expression of a human liver UDP-glucuronosyltransferase (UGT2B15) with activity toward steroid and xenobiotic substrates. Drug Metab Dispos 22:799–805[Abstract]
  18. Frasor J, Danes JM, Komm B, Chang KC, Lyttle CR, Katzenellenbogen BS 2003 Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144:4562–4574[CrossRef][Medline]
  19. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2–{Delta}{Delta}CT method. Methods 25:402–408[CrossRef][Medline]
  20. Congiu M, Mashford ML, Slavin JL, Desmond PV 2002 UDP glucuronosyltransferase mRNA levels in human liver disease. Drug Metab Dispos 30:129–134[Abstract/Free Full Text]
  21. Dauvois S, Danielian PS, White R, Parker MG 1992 Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by increasing its turnover. Proc Natl Acad Sci USA 89:4037–4041[Abstract/Free Full Text]
  22. Pasqualini JR, Chetrite GS 2005 Recent insight on the control of enzymes involved in estrogen formation and transformation in human breast cancer. J Steroid Biochem Mol Biol 93:221–236[CrossRef][Medline]
  23. Suzuki T, Moriya T, Ishida T, Ohuchi N, Sasano H 2003 Intracrine mechanism of estrogen synthesis in breast cancer. Biomed Pharmacother 57:460–462[CrossRef][Medline]
  24. Howell A, Cuzick J, Baum M, Buzdar A, Dowsett M, Forbes JF, Hoctin-Boes G, Houghton J, Locker GY, Tobias JS 2005 Results of the ATAC (Arimidex, Tamoxifen, alone or in combination) trial after completion of 5 years’ adjuvant treatment for breast cancer. Lancet 365:60–62[CrossRef][Medline]
  25. Guillemette C, Belanger A, Lepine J 2004 Metabolic inactivation of estrogens in breast tissue by UDP-glucuronosyltransferase enzymes: an overview. Breast Cancer Res 6:246–254[CrossRef][Medline]
  26. Adams JB, Phillips NS, Young CE 1989 Formation of glucuronides of estradiol-17 ß by human mammary cancer cells. J Steroid Biochem 33:1023–1025[CrossRef][Medline]
  27. Sparks R, Ulrich CM, Bigler J, Tworoger SS, Yasui Y, Rajan KB, Porter P, Stanczyk FZ, Ballard-Barbash R, Yuan X, Lin MG, McVarish L, Aiello EJ, McTiernan A 2004 UDP-glucuronosyltransferase and sulfotransferase polymorphisms, sex hormone concentrations, and tumor receptor status in breast cancer patients. Breast Cancer Res 6:R488–R498
  28. Cox RL, Crawford ED 1995 Estrogens in the treatment of prostate cancer. J Urol 154:1991–1998[CrossRef][Medline]
  29. Yang C, Yu B, Zhou D, Chen S 2002 Regulation of aromatase promoter activity in human breast tissue by nuclear receptors. Oncogene 21:2854–2863[CrossRef][Medline]
  30. Nakamura J, Lu Q, Aberdeen G, Albrecht E, Brodie A 1999 The effect of estrogen on aromatase and vascular endothelial growth factor messenger ribonucleic acid in the normal nonhuman primate mammary gland. J Clin Endocrinol Metab 84:1432–1437[Abstract/Free Full Text]
  31. Pasqualini JR, Chetrite GS 2006 Estradiol as an anti-aromatase agent in human breast cancer cells. J Steroid Biochem Mol Biol 98:12–17[CrossRef][Medline]
  32. Eckert RL, Mullick A, Rorke EA, Katzenellenbogen BS 1984 Estrogen receptor synthesis and turnover in MCF-7 breast cancer cells measured by a density shift technique. Endocrinology 114:629–637[Abstract/Free Full Text]
  33. Monsma Jr FJ, Katzenellenbogen BS, Miller MA, Ziegler YS, Katzenellenbogen JA 1984 Characterization of the estrogen receptor and its dynamics in MCF-7 human breast cancer cells using a covalently attaching antiestrogen. Endocrinology 115:143–153[Abstract/Free Full Text]
  34. Wijayaratne AL, McDonnell DP 2001 The human estrogen receptor-{alpha} is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators. J Biol Chem 276:35684–35692[Abstract/Free Full Text]
  35. Liao DJ, Dickson RB 2002 Roles of androgens in the development, growth, and carcinogenesis of the mammary gland. J Steroid Biochem Mol Biol 80:175–189[CrossRef][Medline]
  36. 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]
  37. Fishman J, Martucci C 1980 Biological properties of 16 {alpha}-hydroxyestrone: implications in estrogen physiology and pathophysiology. J Clin Endocrinol Metab 51:611–615[Abstract/Free Full Text]
  38. Hershey RM, Weisz J, Katzenellenbogen BS 1982 Estrogenic potency, receptor interactions and metabolism of catechol estrogens in the immature rat uterus in vitro. Endocrinology 111:896–903[Abstract/Free Full Text]
  39. Liehr JG, Ricci MJ 1996 4-Hydroxylation of estrogens as marker of human mammary tumors. Proc Natl Acad Sci USA 93:3294–3296[Abstract/Free Full Text]
  40. Schutze N, Vollmer G, Knuppen R 1994 Catecholestrogens are agonists of estrogen receptor dependent gene expression in MCF-7 cells. J Steroid Biochem Mol Biol 48:453–461[CrossRef][Medline]
  41. Cavalieri E, Frenkel K, Liehr JG, Rogan E, Roy D 2000 Estrogens as endogenous genotoxic agents: DNA adducts and mutations. J Natl Cancer Inst Monogr 75–93



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