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Oncology and Molecular Endocrinology Research Center (D.T., J.-S.C., É.L., D.W.H., A.B.) and Medical Research Council Group in Molecular Endocrinology (A.B.), CHUL Research Center, Laval University, Québec, Canada G1V 4G2.
Address all correspondence and requests for reprints to: Dr. Alain Bélanger, Oncology and Molecular Endocrinology Research Center, CHUL Research Center, 2705 boulevard Laurier, Québec, Canada G1V 4G2. E-mail: alain.belanger{at}crchul.ulaval.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,17ß-diol more
efficiently than any other human UGTB isoform. UGT2B15 and UGT2B17
showed similar glucuronidation activity for androstane-3,17ß-diol
(30% lower than that of UGT2B7), whereas UGT2B17 demonstrated the
highest activity for androsterone, testosterone, and
dihydrotestosterone. UGT2B4 demonstrates reactivity toward 5-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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-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-3,17ß-diol
(3-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 3-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 3-Diol, estriol, and
4hydroxyestrone (4-OH-E1) (19),
whereas UGT2B7, UGT2B15, and UGT2B17 catalyze the conjugation of the
17ß-hydroxy position of DHT, testosterone (Testo), and 3-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 5-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 3-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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-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 x
10-3 pmol) were
radiolabeled with the random primed DNA labeling kit (Roche Molecular Biochemicals) in the presence of
[-32P]dCTP. The radiolabeled cDNAs were
pooled and used as probe. The Northern blot was prehybridized in 40%
formamide, 5 x Denhardt’s solution, 5 x 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 x 106
cpm/ml cDNA probe in the same solution for 16 h at 42 C. The blot
was washed twice in 0.5 x 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 x 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
3-diol was performed by incubating 15 µg microsomal protein from
HK293 cells stably expressing UGT2B7 with from 0.1–1.0
µM 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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.
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). 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).
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-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 3-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
3-diol than 2B15 and 2B17 (Table 2).
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). 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 5-reduced androgen
metabolites ADT and 3-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
3-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 3-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 3-Diol
glucuronidation in this tissue, which is in agreement with the
increased circulating 3-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-1 treatment occurs at the transcription
level (48), where a 90% inhibition of transcription after
IL-1 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-1 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 3-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 3-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.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally.
3 Recipient of a scholarship from the Fonds de la Recherche en
Santé du Québec.
4 Recipient of a scholarship from the Medical Research Council of
Canada.
Received August 11, 2000.
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 S. A. Gestl, M. D. Green, D. A. Shearer, E. Frauenhoffer, T. R. Tephly, and J. Weisz Expression of UGT2B7, a UDP-Glucuronosyltransferase Implicated in the Metabolism of 4-Hydroxyestrone and All-Trans Retinoic Acid, in Normal Human Breast Parenchyma and in Invasive and in Situ Breast Cancers Am. J. Pathol., April 1, 2002; 160(4): 1467 - 1479. [Abstract] [Full Text] [PDF]
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