Abstract
Androgens and estrogens play major roles in cell differentiation, cell growth, and peptide secretion in steroid target tissues. In addition to the binding of these hormones to their receptors, formation and metabolism are important in the action of steroids. Metabolism of the potent steroid hormones includes glucuronidation, a major pathway of steroid elimination in liver and several steroid target tissues. Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs), which transfer the polar moiety from UDP-glucuronic acid to a wide variety of endogenous compounds, including steroid hormones. The UGT superfamily of enzymes is subdivided into two families, UGT1 and UGT2, on the basis of sequence homology. To date, six UGT2B proteins have been isolated, namely UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, all of which have been demonstrated to be active on steroid molecules, except for UGT2B10 and UGT2B11, for which no substrate was found. The relative activity of these enzymes on steroidal compounds remains unknown due to variable levels of UGT2B expression in different in vitro cell line models and various conditions of the enzymatic assays. Comparison of the glucuronidation rates of these enzymes requires a unique system for UGT2B protein expression, protein normalization, and enzymatic assays. In this study we have stably expressed UGT2B4, UGT2B7, UGT2B15, and UGT2B17 in the HK293 cell line, which is devoid of steroid UGT activity; characterized their kinetic properties relative to UGT protein expression; determined their transcript and protein stabilities; and established extensively their tissular distributions. UGT2B7 was demonstrated to glucuronidate estrogens, catechol estrogens, and androstane-3α,17β-diol more efficiently than any other human UGTB isoform. UGT2B15 and UGT2B17 showed similar glucuronidation activity for androstane-3α,17β-diol (30% lower than that of UGT2B7), whereas UGT2B17 demonstrated the highest activity for androsterone, testosterone, and dihydrotestosterone. UGT2B4 demonstrates reactivity toward 5α-reduced androgens and catechol estrogens, but at a significantly lower level than UGT2B7, 2B15, and 2B17. Cycloheximide treatment of stably transfected HK293 cells demonstrated that the UGT2B17 protein is more labile than the other enzymes; the protein levels decrease after 1 h of treatment, whereas other UGT2B proteins were stable for at least 12 h. Treatment of stable cells with actinomycin D reveals that UGT2B transcripts are stable for 12 h, except for the UGT2B4 transcript, which was decreased by 50% after the 12-h incubation period. Tissue distribution of the UGT2B enzymes demonstrated that UGT2B isoforms are expressed in the liver as well as in several extrahepatic steroid target tissues, namely, kidney, breast, lung, and prostate. This study clearly demonstrates the relative activities and the major substrates of human steroid-metabolizing UGT2B enzymes, which are expressed in a wide variety of steroid target tissues.
THE FORMATION OF dihydrotestosterone (DHT) from adrenal and testicular C19 steroids in target tissues requires the actions of several steroidogenic enzymes, including 5α-reductase and hydroxysteroid dehydrogenases (HSDs) (1, 2, 3, 4). The transformation of DHT by HSD enzymes in these tissues also leads to metabolites, namely androstane-3α,17β-diol (3α-Diol) and androsterone (ADT), which have a lower affinity for the androgen receptor (5), but these changes are reversible and do not lead to the termination of the androgenic signal (6). Conjugation of 3α-Diol and ADT to polar cofactors, such as UDP-glucuronic acid, however, is an irreversible step that causes steric hindrance of the parental molecule, abolishing its affinity for the steroid receptor (7, 8).
Glucuronidation reaction is catalyzed by UDP-glucuronosyltransferases (UGTs), and this metabolic pathway is responsible for the clearance of phenolic compounds, medicinal drugs, environmental pollutants, bile acids, bilirubin, and steroid hormones (9, 10, 11, 12). UGT enzymes are regrouped into two families based on their evolutionary divergence (13). The members of the UGT1 family are encoded by a single gene, share exons 2–5, and are distinct by their 5′-upstream exon 1 (14). The UGT1 enzymes are generally known to conjugate bilirubin, phenols, and, more recently, estrogens (15). In humans, the members of the UGT2 family are products of separate genes and are subdivided into two subfamiles: UGT2A, which is found in the olfactive epithelium (16), and UGT2B, which had been detected in human hepatocytes and extrahepatic tissues (17, 18, 19, 20). The UGT2B subfamily is composed of 11 members: UGT2B4 (19, 21), UGT2B7 (22, 23), UGT2B10 (24), UGT2B11 (20), UGT2B15 (18, 25), UGT2B17 (17), UGT2B24P (26), UGT2B25P (26), UGT2B26P (26), UGT2B27P (26), and UGT2B29P (26). With the exception of UGT2B24P, UGT2B25P, UGT2B26P, UGT2B27P, and UGT2B29P, which are pseudogenes and do not encodes isoenzymes (26), the UGT2B proteins encoded are steroidmetabolizing enzymes with distinct but overlapping substrate specificities. The UGT2B4 conjugates 3α-Diol, estriol, and 4hydroxyestrone (4-OH-E1) (19), whereas UGT2B7, UGT2B15, and UGT2B17 catalyze the conjugation of the 17β-hydroxy position of DHT, testosterone (Testo), and 3α-Diol. In addition, UGT2B7 and UGT2B17 glucuronidate ADT (17, 18, 25). To date, no substrates have been found for UGT2B10 and UGT2B11, which are considered orphan UGT enzymes (20, 24).
The tissue distributions of UGT2B4, 2B15, and 2B17 were reported and revealed the expression of their transcripts in extrahepatic tissues, including kidney, prostate, mammary gland, and ovary. It was suggested that the presence of these UGT2B proteins in peripheral tissues may contribute to the high plasma levels of circulating 5α-reduced C19 steroid glucuronides observed in humans (7). The localization of UGT2B15 and UGT2B17 in the human prostate is also consistent with the high concentrations of 3α-Diol-G and ADT-G found in this tissue (17).
Despite characterization of the enzymatic specificity of UGT2B proteins and the evidence of overlapping specificities between isoforms, the relative activities of UGT2B isoform on androgens, estrogens, and their metabolites remain speculative, and the tissue distribution was not completely elucidated (27, 28, 29). The activities of UGT2B enzymes obtained in different laboratories with different cell lines, expression systems, and enzymatic assays did not provide accurate information concerning the relative velocity of each isoform on steroids. Here, we report the characterization of all of the human UGT2B enzymes stably transfected in the HK293 cell line, which is devoid of endogenous steroid transferase activity. The relative glucuronidation activity and the kinetic properties were assessed under identical conditions, and UGT activity was normalized according to the amount of UGT2B protein expressed.
Materials and Methods
Materials
UDP-glucuronic acid and all aglycon substrates were obtained from Sigma (St. Louis, MO) and ICN Pharmaceuticals, Inc. (Montréal, Canada). Radioinert steroids were purchased from Steraloids, Inc. (Wilton, NH).[ 14C]UDP-glucuronic acid (285 mCi/mmol) and[α -32P]deoxy (d)-CTP (3000 Ci/mmol) were obtained from NEN Life Science Products-DuPont (Boston, MA) and Amersham Pharmacia Biotech (Oakville, Canada), respectively. Geneticin (G418) and lipofectin were obtained from Life Technologies, Inc. (Burlington, Canada). Protein assay reagents were obtained from Bio-Rad Laboratories, Inc. (Richmond, CA). Restriction enzymes and other molecular biology reagents were purchased from Pharmacia LKB (Milwaukee, WI), Life Technologies, Inc., Stratagene (La Jolla, CA), and Roche Molecular Biochemicals (Indianapolis, IN). The mammalian expression vector pcDNA6/blasticidin and blasticidin were purchased from Invitrogen (San Diego, CA), and Pwo polymerase was obtained from Roche Molecular Biochemicals. Human embryonic kidney 293 (HK293), MCF-7, HepG2, ZR-75–1, T47-D, and LNCaP cells were obtained from American Type Culture Collection (Manassas, VA). Total RNA from human liver, kidney, prostate, adrenal, testis, mammary gland, adipose, uterus, spleen, stomach, small intestine, lung, and brain was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA).
Stable expression of UGT2B enzymes
HK293 cells were grown in DMEM containing 4.5 g/liter glucose, 10 mm HEPES, 110 μg/ml sodium pyruvate, 100 IU penicillin/ml, 100 μg/ml streptomycin, and 10% FBS in a humidified incubator with an atmosphere of 5% CO2 at 37 C. The HK293 cell line stably expressing UGT2B7(H268) was provided by Dr. Thomas R. Tephly (30). HK293 cell lines stably expressing UGT2B4(D458), UGT2B15(D85), and UGT2B17 were obtained as previously published (17, 18, 19).
Northern blot analysis
Total RNA was isolated from human embryonic cells (HK293) untransfected and stably expressing UGT2B4, 2B7, 2B15, and 2B17 according to the Tri-reagent acid phenol protocol specified by the supplier (Molecular Research Center, Inc., Cincinnati, OH). Twenty micrograms of total RNA were electrophoresed on a 1.0% agarose gel and transferred to a GeneScreen Plus membrane (Boston, MA). UGT2B4, UGT2B7, UGT2B15, UGT2B17, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNAs (cDNAs; 37.5 × 10−3 pmol) were radiolabeled with the random primed DNA labeling kit (Roche Molecular Biochemicals) in the presence of[α -32P]dCTP. The radiolabeled cDNAs were pooled and used as probe. The Northern blot was prehybridized in 40% formamide, 5 × Denhardt’s solution, 5 × sodium chloride-sodium phosphate-EDTA, 50 mm Tris-HCl (pH 8.0), 1% SDS, and 100 μg/ml salmon sperm DNA for 4 h at 42 C. Hybridization was performed with 5.0 × 106 cpm/ml cDNA probe in the same solution for 16 h at 42 C. The blot was washed twice in 0.5 × SSC (standard saline citrate)-0.1% SDS at 42 C for 10 min and exposed for 36 h at −80 C on XAR Hyperfilm with an intensifying screen (Eastman Kodak Co., Rochester, NY).
Western blot analysis
Microsomal proteins from HK293 cells and HK293 cells stably expressing UGT2B4, 2B7, 2B15, and 2B17 were separated by 10% SDS-PAGE. The gel was transferred onto a nitrocellulose membrane and probed with the EL-93 anti-UGT2B17 antisera (1:2000 dilution), which was demonstrated to recognize all human UGT2B enzymes (18, 31). An antirabbit IgG horse antibody conjugated with peroxidase (Amersham Pharmacia Biotech, Oakville, Canada) was used as the second antibody, and the resulting immunocomplexes were visualized using a chemiluminescence kit (Renaissance, Québec, Canada) and exposed on Hyperfilm for 30 sec (Eastman Kodak Co.). The expression of each isoform was quantified by BioImage Visage 110s from Genomic Solutions, Inc. (Ann Harbor, MI), and UGT2B7 was arbitrary designated the basal UGT2B protein expression level. Comparison of the protein expression level relative to UGT2B7 allowed determination of the exogenous UGT2B protein expression ratios. These ratios where subsequently used as multiplicative factors to obtain the relative glucuronidation rate for each isoform.
Glucuronidation assay using cell homogenates
HK293 cells expressing exogenous UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were suspended in Tris-buffered saline (32) containing 0.5 mm dithiothreitol and homogenized using a Polytron (Brinkmann Instruments, Inc., Westbury, NY). As identical results are obtained with the microsomal fraction and the whole cell homogenate, the latter preparation was used in this experiment because it is a more convenient method. Enzymatic assays were performed using [14C]UDP-glucuronic acid (UDPGA), 200 μm of the various aglycons, and 170 μg protein from cell in 50 mm Tris-HCl (pH 7.6), 10 mm MgCl2, 100 μg/ml phosphatidylcholine, and 8.5 mm saccharoloactone in a final volume of 100 μl. The assays were terminated by adding 100 μl methanol and were centrifuged at 14,000 × g for 1 min. One hundred microliters of the aqueous phases were loaded onto TLC plates (0.25-mm-thick silica gel 60 F254 S, EM Science, Gibbstown, NJ) and chromatographed in the solvent toluene-methanol-acetic acid (7:3:1). The TLC plates were exposed for 1–4 days, and the levels of glucuronidation were assessed by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). To screen for the substrates that react with UGT2B4 and UGT2B7, the assays were performed using 15 μm[ 14C]UDPGA and 100 μm unlabeled UDPGA for 16 h at 37 C. Compounds that demonstrated reactivity with UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were subsequently reassayed in the presence of 15 μm[ 14C]UDPGA and 500 μm unlabeled UDPGA for 2 h for UGT2B4, 2B7, 2B15, and for 30 min for 2B17 at 37 C to obtain linear reaction rates. Protein expression levels determined by Western blot were used to convert gross glucuronidation rates (pmol/min·mg protein) into values normalized to the UGT2B protein expression, and these were termed relative glucuronidation rates (pmol/min·mg protein). The limit of detection was 0.2 pmol/min·mg protein.
Km determination
Determination of the Km for UGT2B7 to 3α-diol was performed by incubating 15 μg microsomal protein from HK293 cells stably expressing UGT2B7 with from 0.1–1.0μ m of the aglycone in the presence of 15 μm[ 14C]UDPGA and 500 μm unlabeled UDPGA for 2 h, as previously described (17).
Protein stability analysis
Stable HK293-UGT2B cell lines were treated with 20 μg/ml cycloheximide for 0, 1, 3, 6, and 12 h. After treatments, Western blot analyses were performed with microsomal proteins using the polyclonal EL-93 antibody. The signal intensity of the immunocomplexes was determined as described in the previous Western blot analysis section. To measure transferase activity after cycloheximide treatment, microsomal fractions were incubated with 15 μm[ 14C]UDPGA, 500 μm unlabeled UDPGA, and 200 μm Eugenol (Sigma, St. Louis, MO) during 2 h for UGT2B4, UGT2B7, UGT2B15 and during 30 min for UGT2B17 at 37 C. Total RNA of stable cell lines was extracted after cycloheximide treatments to perform Northern blot analyses.
Stability of the UGT2B transcripts
HK293 cells stably transfected with UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were treated with 1 μg/ml actinomycin D for 0, 1, 3, 6, and 12 h. Total RNA was extracted following the Tri-Reagent protocol. Northern blot analyses were performed as described above. GAPDH was used to normalize the amount of messenger RNA (mRNA) in each lane.
RT-PCR analyses
The tissue distributions of UGT2B4, 2B7, 2B10, 2B11, 2B15, and 2B17 were determined using a specific RT-PCR technique as previously reported (17). Antisense primer specific for UGT2B7 and UGT2B10 was used to perform the RT reaction (5′-CTT-TTC-TAG-CAA-ACT-TCC-AGA-AAC-AAA-ACA-GA-3′). To obtain the UGT2B7 681-bp amplicon, the PCR reaction was carried out by adding 100 pmol of the specific sense primer 5′-GAA-AAG-CAT-AGT-GGA-GGA-TTT-ATT-TTC-CCT-CTC-C-3′ and 100 pmol of the specific antisense primer 5′-CCT-TCA-TGT-GAG-CAA-TGT-TAT-CAG-GTT-GAT-CG-3′. To amplify the UGT2B10 793-bp DNA fragment, 100 pmol of the specific sense primer 5′-AGA-TTT-GAC-ATC-GTT-TTT-GCA-GAT-GCT-TA-3′ and 100 pmol of the specific antisense primer 5′-CCT-TCA-TGT-GAG-CAA-TAT-TAT-CAG-GTT-GAT-CAA-A-3′ were used to perform the PCR reaction. All RT-PCR reactions were controlled using specific oligonucleotides for GAPDH. The identity of each PCR product was verified by direct sequencing (33).
Results
Expression of UGT2B4, UGT2B7, UGT2B15, and UGT2B17 in HK293 cells
To characterize the enzymatic properties of UGT2B4, UGT2B7, UGT2B15, and UGT2B17, each cDNA was stably transfected in the HK293 cell line. Total RNA was isolated from each stable cell line and hybridized with a pool of UGT2B cDNA probes to confirm the expression of exogenous transcripts in the cell lines transfected with the corresponding UGT2B4, 2B7, 2B15, and 2B17 cDNAs (Fig. 1A). The sizes of the transcripts corresponded to those previously reported (17, 18, 22, 34). The intensity of each band was quantified by BioImage, and relative expression of UGT2B messengers was determined using UGT2B7 mRNA as a reference (Fig. 1C). Transcript expression of UGT2B4 represents 40% of the UGT2B7 expression level; however, UGT2B15 and UGT2B17 are, respectively, 1.75- and 1.89-fold more expressed than UGT2B7 (Fig. 1C). To ascertain the quantity of mRNA in each lane, a GAPDH cDNA probe was used to hybridize GAPDH transcripts.
A, Northern blot analysis of HK293 cells stably expressing UGT2B4, UGT2B7, UGT2B15, and UGT2B17. Total RNA was isolated from HK293, HK293-UGT2B4, HK293-UGT2B7, HK293-UGT2B15, and HK293-UGT2B17 cells. Twenty micrograms of each RNA were separated on a 1.0% agarose gel and transferred to nylon membrane. The blot was hybridized with a pool of full-length cDNA encoding for UGT2B4, UGT2B7, UGT2B15, and UGT2B17. The signal intensities were quantitated with BioImage Visage 110s. GAPDH was used as an internal control to evaluate the expression of each transcript. B, Immunoblot analysis of HK293 cells stably expressing UGT2B4, UGT2B7, UGT2B15, and UGT2B17. Ten micrograms of microsomal proteins isolated from HK293, HK293-UGT2B4, HK293-UGT2B7, HK293-UGT2B15, and HK293-UGT2B17 cells were chromatographed on a 10% SDS-polyacrylamide gel. The gel was transferred onto a nitrocellulose membrane and probed with EL-93 UGT2B-specific antibody. The signal intensities were quantitated with BioImage Visage 110s. C, Relative expressions of UGT2B4, UGT2B7, UGT2B15, and UGT2B17 transcripts and proteins.
The expression of UGT2B proteins in the transfected cell lines was determined by hybridization of microsomal preparation from each stable cell line with a polyclonal antibody that recognizes all UGT2B proteins (EL-93) (18, 31). Expression of exogenous UGT2B proteins was demonstrated in the HK293 cell lines expressing UGT2B4, UGT2B7, UGT2B15, and UGT2B17. The 53-kDa proteins were also expressed in liver, but were absent in the untransfected HK293 cells (Fig. 1B). The expression levels of UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were quantified by BioImage, and the relative expression of proteins was established using UGT2B7 as the reference expression level (Fig. 1C). UGT2B4, UGT2B15, and UGT2B17 protein levels were 10%, 28%, and 50% the level of UGT2B7, respectively. The highest mRNA/protein ratio (compared with UGT2B7) was obtained with the HK293-UGT2B15 stable cells, which demonstrated 6 times more messenger RNA than the corresponding UGT2B15 protein. The UGT2B4 and the UGT2B17 stable cells expressed, respectively, 3.9- and 3.7-fold more transcripts than the respective proteins (Fig. 1C).
To ascertain protein and mRNA stabilities, stable cell lines expressing exogenous UGT2B enzymes were treated for 0, 1, 3, 6, and 12 h with cycloheximide and actinomycin D. After 12 h of cycloheximide treatment of HK293 cells stably expressing UGT2B, protein expression levels of UGT2B4, UGT2B7, and UGT2B15 remained unchanged; however, a significant decrease in UGT2B17 protein expression was observed after only 1 h of treatment (Fig. 2). Glucuronidation velocities were also determined in the cycloheximide-treated microsomal fraction, and results showed correlations between UGT expression and transferase activities (Fig. 2). No changes in UGT2B mRNA expression were noted after cycloheximide treatment, clearly indicating no posttranscriptional changes in protein levels. On the other hand, actinomycin D treatment slightly affected the transcript levels of UGT2B4 after a 12-h incubation period, as indicated by a 2-fold transcript decrease compared with the untreated control value (Fig. 2). No alteration in transcript expression was observed with UGT2B7, UGT2B15, and UGT2B17 stable cells after actinomycin D treatment (Fig. 2).
Stabilities of the UGT2B4, UGT2B7, UGT2B15, and UGT2B17 transcripts and proteins. Stable cell lines expressing UGT2B4 (A), UGT2B7 (B), UGT2B15 (C), and UGT2B17 (D) were treated with 20μ g/ml cycloheximide and 1 μg/ml actinomycin D for 1, 3, 6, and 12 h, after which total RNA and microsome preparations were extracted. Western blot analysis and kinetic analyses were performed on microsome preparations to evaluate the stabilities of the UGT2B proteins. The stabilities of mRNAs were assessed by Northern blot analysis after each treatment. The intensities of the signals were quantitated with BioImage Visage 110s.
Kinetic properties of UGT2B4, UGT2B7, UGT2B15, and UGT2B17 expressed in HK293 cells
To determine the relative activities of UGT2B4, 2B7, 2B15, and 2B17 on androgens and estrogens, glucuronidation assays were performed using total cell homogenates from stable cell lines. The maximal rate of conjugation was established for each stable cell line, and kinetic conditions were determined with respect to enzyme stability, cofactor concentration, and expression of exogenous UGT2B proteins. Determination of the substrate specificities of UGT2B4, 2B7, 2B15, and 2B17 are in agreement with previous observations (17, 18, 19, 30). UGT2B4 conjugates 3α-diol (12.0 pmol/min·mg protein), estriol (12.3 pmol/min·mg protein), 4-OH-E1 (12.0 pmol/min·mg protein), and ADT (3.9 pmol/min·mg protein). Under the same conditions, glucuronidation rates of UGT2B4 on these steroid molecules were lower compared with the other human isoforms (Table 1). On the other hand, UGT2B7 had the broadest substrate specificity, glucuronidating 17 of the 18 androgens and estrogens tested. Indeed, UGT2B7 catalyzed the glucuronidation of 4-OH-E1 and estriol at velocities of 364.6 and 171.7 pmol/min·mg protein, respectively. UGT2B7 showed a 6- to 20-fold higher glucuronidating activity for the catechol estrogen 4-OH-E1 than UGT2B4 or UGT2B15 (Table 1). Moreover, UGT2B7 catalyzed glucuronidation of ADT at a velocity of 97 pmol/min·mg protein, which was 5-fold more elevated than that of UGT2B17 (21.7 pmol/min·mg protein). UGT2B15 has been demonstrated to conjugate 3α-diol with the highest glucuronidation rate, with a normalized Vmax value of 125.4 pmol/min·mg protein, as opposed to UGT2B7 and UGT2B17, which conjugate this substrate with Vmax values of 46.3 and 70.8 pmol/min·mg protein, respectively (Table 1). The present study demonstrated that UGT2B17 was highly active toward the two potent androgens, namely Testo and DHT (Table 1). Indeed, UGT2B17 transformed Testo and DHT to their glucuronide derivatives with velocities of 36.7 and 63.1 pmol/min·mg, respectively, which were 10- and 6-fold more active on Testo and DHT than UGT2B15 (Table 1). However, UGT2B17 was unable to catalyze the conjugation of C18 steroids, as shown by its inability to inactivate estrogens and catechol estrogens. The maximum velocity (Vmax)/Km ratio demonstrated that UGT2B17 was the enzyme that conjugates ADT and DHT with the highest efficiency, whereas UGT2B7 was 50% more efficient on 3α-diol than 2B15 and 2B17 (Table 2).
Relative glucuronidation activity of eugenol and steroid hormones catalyzed by expressed UGT2B4, UGT2B7, UGT2B15, and UGT2B17
| Substrates | UGT2B relative Vmax (pmol/min·mg protein) | |||
|---|---|---|---|---|
| UGT2B4 | UGT2B7 | UGT2B15 | UGT2B17 | |
| Eugenol | 52.6 ± 23.1 | 347.3 ± 265.5 | 39.7 ± 5.2 | 50.6 ± 5.1 |
| Testosterone | 0.4 ± 0.1 | 4.4 ± 1.0 | 36.7 ± 5.6 | |
| Dihydrotestosterone | 1.7 ± 0.3 | 10.4 ± 2.7 | 63.1 ± 4.1 | |
| Androsterone | 3.9 ± 1.8 | 97.0 ± 6.1 | 21.7 ± 2.7 | |
| Epiandrosterone | 2.2 ± 0.3 | |||
| Dehydroepiandrosterone | 2.1 ± 0.5 | |||
| Etiocholanolone | 19.5 ± 4.7 | 46.0 ± 8.8 | ||
| Androst-5-ene-3β,17β-diol | 1.8 ± 0.6 | |||
| Androstane-3α,17β-diol | 12.0 ± 5.5 | 46.3 ± 9.2 | 125.4 ± 25.3 | 70.8 ± 18.3 |
| Androstane-3β,17β-diol | 3.0 ± 1.1 | |||
| Estrone | ||||
| Estradiol | 3.0 ± 0.8 | |||
| Estriol | 12.3 ± 4.3 | 171.7 ± 25.6 | ||
| 4-Hydroxyestrone | 12.0 ± 5.9 | 364.6 ± 54.9 | 65.2 ± 15.6 | |
| 2-Hydroxyestrone | 11.8 ± 6.6 | 22.6 ± 7.6 | ||
| 4-Hydroxyestradiol | 52.2 ± 1.7 | 8.1 ± 1.6 | ||
| 2-Hydroxyestradiol | BLD | 4.9 ± 0.8 | ||
| Substrates | UGT2B relative Vmax (pmol/min·mg protein) | |||
|---|---|---|---|---|
| UGT2B4 | UGT2B7 | UGT2B15 | UGT2B17 | |
| Eugenol | 52.6 ± 23.1 | 347.3 ± 265.5 | 39.7 ± 5.2 | 50.6 ± 5.1 |
| Testosterone | 0.4 ± 0.1 | 4.4 ± 1.0 | 36.7 ± 5.6 | |
| Dihydrotestosterone | 1.7 ± 0.3 | 10.4 ± 2.7 | 63.1 ± 4.1 | |
| Androsterone | 3.9 ± 1.8 | 97.0 ± 6.1 | 21.7 ± 2.7 | |
| Epiandrosterone | 2.2 ± 0.3 | |||
| Dehydroepiandrosterone | 2.1 ± 0.5 | |||
| Etiocholanolone | 19.5 ± 4.7 | 46.0 ± 8.8 | ||
| Androst-5-ene-3β,17β-diol | 1.8 ± 0.6 | |||
| Androstane-3α,17β-diol | 12.0 ± 5.5 | 46.3 ± 9.2 | 125.4 ± 25.3 | 70.8 ± 18.3 |
| Androstane-3β,17β-diol | 3.0 ± 1.1 | |||
| Estrone | ||||
| Estradiol | 3.0 ± 0.8 | |||
| Estriol | 12.3 ± 4.3 | 171.7 ± 25.6 | ||
| 4-Hydroxyestrone | 12.0 ± 5.9 | 364.6 ± 54.9 | 65.2 ± 15.6 | |
| 2-Hydroxyestrone | 11.8 ± 6.6 | 22.6 ± 7.6 | ||
| 4-Hydroxyestradiol | 52.2 ± 1.7 | 8.1 ± 1.6 | ||
| 2-Hydroxyestradiol | BLD | 4.9 ± 0.8 | ||
BLD, Vmax could not be evaluated under these kinetic conditions.
Assays were performed by incubating whole cell homogenates with 200μ m substrate in the presence of [14C]UDPGA. The final concentration of UDPGA was 500 μm, and assays were incubated for 2 h at 37 C, except for UGT2B17 cell homogenate, which was incubated for 30 min. Enzymatic rates were normalized to the UGT2B protein expression and are expressed as the mean ± sd of at least two experiments.
Relative glucuronidation activity of eugenol and steroid hormones catalyzed by expressed UGT2B4, UGT2B7, UGT2B15, and UGT2B17
| Substrates | UGT2B relative Vmax (pmol/min·mg protein) | |||
|---|---|---|---|---|
| UGT2B4 | UGT2B7 | UGT2B15 | UGT2B17 | |
| Eugenol | 52.6 ± 23.1 | 347.3 ± 265.5 | 39.7 ± 5.2 | 50.6 ± 5.1 |
| Testosterone | 0.4 ± 0.1 | 4.4 ± 1.0 | 36.7 ± 5.6 | |
| Dihydrotestosterone | 1.7 ± 0.3 | 10.4 ± 2.7 | 63.1 ± 4.1 | |
| Androsterone | 3.9 ± 1.8 | 97.0 ± 6.1 | 21.7 ± 2.7 | |
| Epiandrosterone | 2.2 ± 0.3 | |||
| Dehydroepiandrosterone | 2.1 ± 0.5 | |||
| Etiocholanolone | 19.5 ± 4.7 | 46.0 ± 8.8 | ||
| Androst-5-ene-3β,17β-diol | 1.8 ± 0.6 | |||
| Androstane-3α,17β-diol | 12.0 ± 5.5 | 46.3 ± 9.2 | 125.4 ± 25.3 | 70.8 ± 18.3 |
| Androstane-3β,17β-diol | 3.0 ± 1.1 | |||
| Estrone | ||||
| Estradiol | 3.0 ± 0.8 | |||
| Estriol | 12.3 ± 4.3 | 171.7 ± 25.6 | ||
| 4-Hydroxyestrone | 12.0 ± 5.9 | 364.6 ± 54.9 | 65.2 ± 15.6 | |
| 2-Hydroxyestrone | 11.8 ± 6.6 | 22.6 ± 7.6 | ||
| 4-Hydroxyestradiol | 52.2 ± 1.7 | 8.1 ± 1.6 | ||
| 2-Hydroxyestradiol | BLD | 4.9 ± 0.8 | ||
| Substrates | UGT2B relative Vmax (pmol/min·mg protein) | |||
|---|---|---|---|---|
| UGT2B4 | UGT2B7 | UGT2B15 | UGT2B17 | |
| Eugenol | 52.6 ± 23.1 | 347.3 ± 265.5 | 39.7 ± 5.2 | 50.6 ± 5.1 |
| Testosterone | 0.4 ± 0.1 | 4.4 ± 1.0 | 36.7 ± 5.6 | |
| Dihydrotestosterone | 1.7 ± 0.3 | 10.4 ± 2.7 | 63.1 ± 4.1 | |
| Androsterone | 3.9 ± 1.8 | 97.0 ± 6.1 | 21.7 ± 2.7 | |
| Epiandrosterone | 2.2 ± 0.3 | |||
| Dehydroepiandrosterone | 2.1 ± 0.5 | |||
| Etiocholanolone | 19.5 ± 4.7 | 46.0 ± 8.8 | ||
| Androst-5-ene-3β,17β-diol | 1.8 ± 0.6 | |||
| Androstane-3α,17β-diol | 12.0 ± 5.5 | 46.3 ± 9.2 | 125.4 ± 25.3 | 70.8 ± 18.3 |
| Androstane-3β,17β-diol | 3.0 ± 1.1 | |||
| Estrone | ||||
| Estradiol | 3.0 ± 0.8 | |||
| Estriol | 12.3 ± 4.3 | 171.7 ± 25.6 | ||
| 4-Hydroxyestrone | 12.0 ± 5.9 | 364.6 ± 54.9 | 65.2 ± 15.6 | |
| 2-Hydroxyestrone | 11.8 ± 6.6 | 22.6 ± 7.6 | ||
| 4-Hydroxyestradiol | 52.2 ± 1.7 | 8.1 ± 1.6 | ||
| 2-Hydroxyestradiol | BLD | 4.9 ± 0.8 | ||
BLD, Vmax could not be evaluated under these kinetic conditions.
Assays were performed by incubating whole cell homogenates with 200μ m substrate in the presence of [14C]UDPGA. The final concentration of UDPGA was 500 μm, and assays were incubated for 2 h at 37 C, except for UGT2B17 cell homogenate, which was incubated for 30 min. Enzymatic rates were normalized to the UGT2B protein expression and are expressed as the mean ± sd of at least two experiments.
Kinetic analysis of UGT2B7, UGT2B15, and UGT2B17
| Substrate | Enzyme | Relative Vmax (pmol/min·mg protein) | Km (μm) | Glucuronidation efficiency (L·min−1·mg−1·10−6) | Ref. no. |
|---|---|---|---|---|---|
| 3α-Diol | UGT2B7 | 46.3 | 0.5 | 92.6 | This work |
| UGT2B15 | 125.4 | 2.2 | 57.0 | 18 | |
| UGT2B17 | 70.8 | 1.1 | 64.4 | 17, 31 | |
| ADT | UGT2B7 | 97.0 | 9.7 | 10.0 | 30 |
| UGT2B17 | 21.7 | 0.6 | 36.2 | 17, 31 | |
| DHT | UGT2B15 | 10.4 | 2.8 | 3.7 | 18 |
| UGT2B17 | 63.1 | 0.7 | 90.1 | 17 |
| Substrate | Enzyme | Relative Vmax (pmol/min·mg protein) | Km (μm) | Glucuronidation efficiency (L·min−1·mg−1·10−6) | Ref. no. |
|---|---|---|---|---|---|
| 3α-Diol | UGT2B7 | 46.3 | 0.5 | 92.6 | This work |
| UGT2B15 | 125.4 | 2.2 | 57.0 | 18 | |
| UGT2B17 | 70.8 | 1.1 | 64.4 | 17, 31 | |
| ADT | UGT2B7 | 97.0 | 9.7 | 10.0 | 30 |
| UGT2B17 | 21.7 | 0.6 | 36.2 | 17, 31 | |
| DHT | UGT2B15 | 10.4 | 2.8 | 3.7 | 18 |
| UGT2B17 | 63.1 | 0.7 | 90.1 | 17 |
The value of UGT2B7 apparent Km on 3α-diol was determined using microsome preparation from HK293 stably expressing UGT2B7, as described in Materials and Methods. Result represents the mean of two independent experiments. Other Km values were determined as reported by the references.
Kinetic analysis of UGT2B7, UGT2B15, and UGT2B17
| Substrate | Enzyme | Relative Vmax (pmol/min·mg protein) | Km (μm) | Glucuronidation efficiency (L·min−1·mg−1·10−6) | Ref. no. |
|---|---|---|---|---|---|
| 3α-Diol | UGT2B7 | 46.3 | 0.5 | 92.6 | This work |
| UGT2B15 | 125.4 | 2.2 | 57.0 | 18 | |
| UGT2B17 | 70.8 | 1.1 | 64.4 | 17, 31 | |
| ADT | UGT2B7 | 97.0 | 9.7 | 10.0 | 30 |
| UGT2B17 | 21.7 | 0.6 | 36.2 | 17, 31 | |
| DHT | UGT2B15 | 10.4 | 2.8 | 3.7 | 18 |
| UGT2B17 | 63.1 | 0.7 | 90.1 | 17 |
| Substrate | Enzyme | Relative Vmax (pmol/min·mg protein) | Km (μm) | Glucuronidation efficiency (L·min−1·mg−1·10−6) | Ref. no. |
|---|---|---|---|---|---|
| 3α-Diol | UGT2B7 | 46.3 | 0.5 | 92.6 | This work |
| UGT2B15 | 125.4 | 2.2 | 57.0 | 18 | |
| UGT2B17 | 70.8 | 1.1 | 64.4 | 17, 31 | |
| ADT | UGT2B7 | 97.0 | 9.7 | 10.0 | 30 |
| UGT2B17 | 21.7 | 0.6 | 36.2 | 17, 31 | |
| DHT | UGT2B15 | 10.4 | 2.8 | 3.7 | 18 |
| UGT2B17 | 63.1 | 0.7 | 90.1 | 17 |
The value of UGT2B7 apparent Km on 3α-diol was determined using microsome preparation from HK293 stably expressing UGT2B7, as described in Materials and Methods. Result represents the mean of two independent experiments. Other Km values were determined as reported by the references.
Tissue distribution of the human UGT2B transcripts
Due to the high homology among the members of the UGT2B subfamily, a specific RT-PCR strategy was used to determine the tissue distribution of these isoenzymes. The oligonucleotides used for the RT-PCR were specific for UGT2B7 and UGT2B10 and were designed not to amplify other UGT2B sequences known to date. We demonstrated the expression of UGT2B7 and UGT2B10 in the liver, kidney, mammary gland, lung, small intestine, and LNCaP cell line (Fig. 3). UGT2B10 was also expressed in testis, placenta, spleen, stomach, and T47-D and ZR-75–1 breast cancer cell lines. Adipose and adrenal do not express UGT2B7 or UGT2B10. Moreover, UGT2B7 transcript was absent in placenta, testis, prostate, and breast cancer cell lines. Oligonucleotides specific for the GAPDH transcript were used as a positive control for the RT-PCR reaction and yielded a product of appropriate length in each RNA sample.
A, Tissue distribution of UGT2B7 and UGT2B10 transcripts. Total RNA from various tissues were analyzed by RT-PCR analysis, using oligonucleotides specific for UGT2B7 and UGT2B10 transcripts. One tenth of each UGT2B7 and UGT2B10 PCR product was separated on a 1.0% agarose gel. The specificity of PCR product was confirmed by direct sequencing. GAPDH was used as a positive control for RT-PCR reactions. B, Comparison of UGT2B7 and UGT2B10 transcript expressions with those of other human UGT2B isoforms.
Discussion
The present report describes the relative enzymatic properties and transcript and protein stabilities of the human UGT2B enzymes as well as an exhaustive tissue distribution of the UGT2B7 and UGT2B10 transcripts. Previous cloning and characterization of UGT2B proteins suggested that these enzymes have overlapping substrate specificities. However, no study clearly demonstrated the relative importance of each UGT2B isoenzyme to the steroid metabolism occurring in extrahepatic tissues. In the present study, all UGT2B enzymes have been stably expressed in the same cell line model, human embryonal kidney (HK293) cells, which is devoid of steroid transferase activity. Assays were conducted under identical conditions and were normalized with levels of UGT2B protein. Therefore, it was possible to evaluate accurately the glucuronidation efficiency of each UGT isoenzyme for the steroid hormones and to clearly identify the isoforms implicated in C18 and C19 steroid catabolism.
In this experiment normalization of the glucuronidation rates was performed using the EL-93 antibody, a specific anti-UGT2B antibody that was used to quantify the expression of exogenous UGT2B enzymes in HK293 stably transfected with UGT2B4, UGT2B7, UGT2B15, and UGT2B17. This antibody has been extensively used for determination of exogenous UGT2B expression in stable cell lines, with both human and monkey isoforms (18, 19, 20, 31, 35, 36, 37, 38), immunochemistry on monkey tissues (Barier, O., C. Girard, L. Berger, M. El Alfy, A. Bélanger, and D. W. Hum, unpublished results), and, finally, comparison of the protein expression levels of human and monkey isoforms (18, 19, 39, 40). As the recognition of each UGT2B isoform is the basis of this work, it is important to note that the affinities of the EL-93 antibody for the different isoforms were tested few years ago. Indeed, we tested the affinity of the antibody against UGT2B17 and UGT2B15 fusion proteins. We demonstrated that the recognition of these two fusion proteins was identical for both anti-UGT EL-93 antibody and anti-T7-Tag antibody using several antiimmune serum dilutions and several enzyme concentrations. Furthermore, the use of anti-His-Tag antibody demonstrated similar results with the UGT2B15 and UGT2B17 fusion proteins (our unpublished results). With these results, we concluded that the affinity of the anti-UGT2B polyclonal EL-93 antibody is not significantly different for the human UGT2B isoforms. These results are not surprising, because the human UGT2B enzymes show 77–97% homology in their amino acid sequences, and this polyclonal antibody recognizes several epitopes.
The EL-93 antibody was first used to quantify the UGT2B17 protein expression level after DHT and epidermal growth factor (EGF) treatment in LNCaP cells (41). It demonstrated that DHT in combination with EGF decreased protein expression by 73%. The quantification of the protein correlates exactly with the 75% decrease observed in glucuronidation of DHT, which is conjugated by UGT2B17 (41). This experiment is a good example of the linear relationship between glucuronide formation and the amount of protein determined by Western blotting. The EL-93 was also used in other studies to normalize UGT2B activity with the protein expression level. Indeed, the apparent Vmax of UGT2B15(D85) and UGT2B15(Y85) were normalized for the amount of each protein expressed, and this led to a 2-fold higher glucuronidation rate for the UGT2B15(Y85) allele (18). Moreover, the EL-93 antibody was recently used to normalize the UGT2B7(H268) and UGT2B7(Y268) glucuronidation rates for the 3′-azido-3′-deoxythimidine substrate (39). The combination of these results demonstrates that the EL-93 antibody is the most appropriate tool to quantify and compare the expression of the UGT2B isoforms.
Quantification of the UGT2B protein expression level underlined the importance of glucuronidation activity normalization when comparing the activities of the different isoforms, as the expressions of UGT2B4, UGT2B15, and UGT2B17 were, respectively, 90%, 70%, and 50% lower than that of UGT2B7. Substrate specificity and relative kinetic analyses indicated that the latter isoform has the broadest substrate specificity, conjugating androgens, estrogens, and catechol estrogens. UGT2B7 is the only UGT2B isoenzyme demonstrating reactivity toward estradiol. Moreover, the glucuronidation rates for estriol and catechol estrogens, normalized by Western blot, were clearly higher than that of any other UGT2B enzymes. The importance of UGT2B7 as an estrogen-conjugating enzyme is reinforced by its expression of UGT2B7 in mammary gland. Surprisingly, the UGT2B7 transcript was not detected in T47-D and ZR-75–1 breast cancer cell lines. This absence may contribute to maintain higher levels of potent estrogens, stimulating the growth of these two estrogen-dependent cell lines. In addition to its C18 conjugating activity, UGT2B7 also had a high glucuronidation velocity toward the 5α-reduced androgen metabolites ADT and 3α-Diol, whereas very low activity was observed for Testo and DHT. The expression of UGT2B7 in liver and several steroid target tissues probably contributes to the circulating level of ADT-glucuronide (ADT-G), the major androgen metabolite found in the circulation (7). However, UGT2B7 could not be detected in the prostate, which has been reported to contain high levels of ADT-G (17, 42). UGT2B17, which glucuronidates ADT at a 5-fold lower rate than UGT2B7, is the only known enzyme expressed in the prostate able to conjugate ADT to its glucuronide derivative (17). The Vmax/Km ratio indicated that UGT2B17 was more efficient for the glucuronidation of this substrate than any other UGT2B isoforms. Moreover, UGT2B17 showed high glucuronidation activity for Testo and DHT, as previously observed (17). Interestingly, our data also showed the high lability of the protein, suggesting that a change in the expression of the mRNA would rapidly affect protein expression. The major difference between UGT2B15 and UGT2B17 resides in the glucuronidation of ADT, which is catalyzed by UGT2B17, but not by UGT2B15. Testo, which was previously found to be glucuronidated only by UGT2B17 (29), was also conjugated by UGT2B7 and UGT2B15.
The kinetic analyses revealed that UGT2B15 had the highest 3α-Diol-conjugating activity, which was 1.8- and 2.7-fold the activities of UGT2B17 and UGT2B7, respectively. These data suggest that in addition to UGT2B7, UGT2B15 is strongly implicated in the formation of 3α-Diol-G in extrahepatic tissues. Indeed, UGT2B15 is expressed in adipose tissue, where expression of UGT2B7 and UGT2B17 were not detected. Therefore, UGT2B15 may have a role in 3α-Diol glucuronidation in this tissue, which is in agreement with the increased circulating 3α-Diol-G levels found in obese men while ADT-G levels remain unchanged (43).
Normalization of glucuronidation activity with protein expression level showed the low activity of UGT2B4 on steroids as previously observed (19, 24, 44). This widely expressed enzyme (19) conjugates a wide variety of substrates, including androgens and catechol estrogens, and therefore, UGT2B4 may be a cell type-specific steroid-conjugating enzyme, complementing other UGT2B enzymes in the glucuronidation of androgens, estrogens, and catechol estrogens. However, despite the fact that UGT2B4 is mainly active on bile acid (hyodeoxycholic acid) (19, 44, 45), it remains obvious that this isoform has the lowest steroid-conjugating activity in the UGT2B subfamily.
Northern blot analysis performed on stable cells demonstrated variable expression levels of the UGT2B4, UGT2B7, UGT2B15, and UGT2B17 transcripts in HK293 cells. UGT2B15 and UGT2B17 were expressed almost equally in these stable cells, approximately 2-fold higher than UGT2B7, whereas UGT2B4 showed only half the mRNA expression compared with UGT2B7. Comparison of the mRNA/protein ratio showed a 3.7- to 6.2-fold higher ratio for UGT2B4, UGT2B15, and UGT2B17 than for UGT2B7.
Previous observations demonstrated a decrease in UGT2B17 glucuronidation velocity after 1 h of cycloheximide treatment and a half-life of 3 h, suggesting a rapid turnover of the protein (17, 41). However, it was recently demonstrated that a decrease in UGT2B20 activity could be observed without changes in protein expression (38). Our results indicate that inhibition of translation induced by cycloheximide treatment caused a decrease in UGT2B17 protein levels after 1 h, whereas other UGT2B tested were stable for more than 12 h. These results are in agreement with those previously reported (17). Indeed, a decrease in DHT glucuronidation was observed after treatment of LNCaP cells with cycloheximide (46). This partial lost of glucuronidation activity can be related to UGT2B17, which has a very short half-life of only 3 h, whereas the UGT2B15 protein was stable for at least 24 h (41). Thus, the 50% of the residual DHT-conjugating activity observed after 24 h of cycloheximide treatment in the LNCaP cell line cannot be attributed to UGT2B17. Based on these results, it seems that the endogenous UGT2B enzymes expressed in LNCaP cells have different levels of stability and that the more stable UGT2B isoforms, such as UGT2B15, are implicated in the formation of DHT glucuronide in this cell line. Antibodies that can recognize specifically each UGT2B members could be useful tools to establish the identity of the UGT responsible for this residual activity in LNCaP cells. Moreover, specific antibodies could contribute to the understanding of the regulation of UGT2B enzymes by visualizing directly the variations in protein expression of each isoform. Such a demonstration of changes in protein expression has been reported previously, where rat UGT1 protein expression variations were followed with antibodies specific for each isoform (47). However, in our case, homology between UGT2B subfamily members makes it difficult to obtain antibodies specific for each UGT2B isoform. Several strategies, including immunization of specific peptides, were used to obtain such monoclonal antibodies. However, no significant results were demonstrated with these antibodies, and thus, the EL-93 antibody remains the best tool to investigate human UGT2B protein expression.
In the present report the rapid protein turnover of UGT2B17 was confirmed by glucuronidation assays, which showed a decrease in activity after 1 h of treatment. Therefore, the high UGT2B17 mRNA/protein ratio can be explained by the labile nature of the protein; however, the 3.9- and 6.2-fold mRNA/protein ratios for UGT2B4 and UGT2B15 could not be attributed to protein instability, because cycloheximide treatment revealed that both proteins were stable for more than 12 h. The discrepancies between transcript and protein levels observed with HK293-UGT stable cells could be accounted for by a lower UGT2B4 and UGT2B15 translation efficiency compared with UGT2B7 in these cells. Comparison of UGT2B17 protein stability with the highly homologous UGT2B15, which was stable for at least 12 h, suggests that minor changes in primary structure can have deleterious effects on protein stability (41). To ensure that a decrease in immunoreactive complexes could not be caused by a decrease in transcription, the total RNA of stable cell lines was extracted after each treatment to perform Northern blot analysis.
UGT2B transcript stability was also assessed with actinomycin D, demonstrating the high degree of stability of the UGT2B mRNAs. Indeed, only UGT2B4 transcript expression was diminished by 50% after 12 h of treatment, whereas no expression change was observed for the UGT2B7, UGT2B15, and UGT2B17 transcripts. These results are supported by the regulation experiments performed in cell lines that express endogenous UGT2B enzymes. Indeed, it was demonstrated that an incubation period of at least 6 days is required to observe a significant variation in glucuronidation rates and transcript expression levels after treatment with DHT (41, 46), EGF (41), interleukins (ILs) (48), and fibroblastic growth factor (49). Moreover, results obtained with the UGT2B17 promoter region demonstrated that the regulation after IL-1α treatment occurs at the transcription level (48), where a 90% inhibition of transcription after IL-1α treatment was observed with a 3-kb portion of the UGT2B17 promoter. These results demonstrate that the glucuronidation activity observed after 6 days of IL-1α treatment cannot be related to de novo mRNA synthesis or UGT2B17 protein stability, and thus, the UGT2B17 transcript must be very stable. Taken together, these results demonstrate that UGT transcript and protein are stable, with the exception of the UGT2B17 protein, and that a differential translation efficiency is observed among UGT isoforms. Therefore, in human target cells, posttranscriptional events, such as protein turnover, might play a role in the maintenance of different UGT protein levels.
In conclusion, our data indicate that three of four UGT2B isolated to date, namely UGT2B7, UGT2B15, and UGT2B17, conjugate Testo, DHT, and 3α-Diol. However, ADT, the major androgen metabolite, is glucuronidated only by UGT2B7 and UGT2B17. Glucuronidation efficiency demonstrated that UGT2B7 is the major isoform implicated in 3α-Diol glucuronidation. Moreover, high glucuronidation rates were observed in estrogens and catechol estrogens, which is concordant with the expression of UGT2B7 in mammary gland. Although UGT2B17 shows better Testo- and DHT-conjugating activities, glucuronidation of C19 steroids by UGT2B15 remains important, because it is the only isoform expressed in adipose tissue. Our results demonstrated that UGT2B4 had low glucuronidation rates toward all classes of steroid molecules; thus, it might reside in the complement of other UGT2B isoenzymes in specific steroid target cells. This study clearly demonstrates that despite overlapping substrate specificities, each UGT2B enzyme that is expressed in a tissue-specific manner has a different function and provides new insights in the physiological relevance of each UGT2B isoform in steroid target tissues.
We thank Dr. Pei Min Rong and Pierre-Michel Simard for technical assistance, and Dr. Thomas R. Tephly for providing the UGT2B7-HK293 cell line.
Author notes
These authors contributed equally.
Recipient of a scholarship from the Fonds de la Recherche en Santé du Québec.
Recipient of a scholarship from the Medical Research Council of Canada.
