From the UR 545 INSERM, Département
d'Athérosclérose, Institut Pasteur de Lille and the
Faculté de Pharmacie, Université de Lille II, Lille 59019, France, the § Oncology and Molecular Endocrinology Research
Center, CHUL Research Center and Faculty of Pharmacy, Laval University,
Quebec G1V 4G2, Canada, and ¶ Institute of Experimental
Cardiology, Russian Cardiology Complex, Moscow 121552, Russia
Received for publication, January 22, 2003
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ABSTRACT |
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Peroxisome proliferator-activated
receptor (PPAR) Peroxisome proliferator-activated receptors (PPARs)1
belong to the family of nuclear receptors
that are ligand-activated transcriptional regulators of gene
expression. Upon ligand activation, PPAR Fatty acids and derivatives are natural ligands for PPAR Glucuronides are naturally occurring conjugation metabolites of
xenobiotics and endobiotics (15). Glucuronide conjugation (or
glucuronidation), the process underlying their formation, is considered
detoxifying, because glucuronides usually possess less intrinsic
biological or chemical activity than their parent aglycones and are
rapidly excreted. UDP-glucuronosyltransferase (UGT) enzymes catalyze
the glucuronidation reaction, which consists of the addition of the
glycosyl group from a nucleotide sugar (UDP-glucuronic acid (UDPGA)) to
a wide variety of endogenous and exogenous compounds (15). Endogenous
glucuronidated substances are, for instance, androgens, estrogens,
progestins, bile acids, fatty acids, retinoids, and bilirubin (16).
Numerous exogenous compounds are glucuronidated by UGTs, including
environmental compounds, plant steroids, and important pharmacological
agents (such as nonsteroidal anti-inflammatory drugs, opioids, or
fibrates) (11, 16). More than 60 mammalian UGT isoforms have been
separated into two families and four subfamilies based on their primary amino acid sequence identity (17). In humans, members of the UGT1A
subfamily are encoded by a complex gene, which contains at least 17 exons spanning over 200 kb (18). Located on chromosome 2q37, the UGT1A
gene complex leads to the production of 13 different mRNAs by
alternative splicing; whereas exons 14-17 are shared between all UGT1A
isoforms, the 13 first exons encode the amino-terminal part of each
protein and share between 37 and 90% amino acid sequence identity
(16). The common carboxyl part of UGT1A proteins contains the cofactor
UDPGA binding site, whereas the substrate specificity of each enzyme is
determined by the amino-terminal part of the protein (17).
Belonging to the UGT1A subfamily, UGT1A9 catalyzes the conjugation of
various bulky phenols, N-hydroxyarylamines, steroids, and
several drugs (16, 19). UGT1A9 glucuronidates a variety of endogenous
molecules, such as retinoic acid or thyroid hormones (T3 and T4) and is
mainly involved in the metabolism of catecholestrogens (4-hydroxyestradiol (4OH-E1) and 4-hydroxyestrone
(4OH-E2)) (20, 21). Recent studies revealed that UGT1A9
also catalyzes glucuronidation of the synthetic PPAR activators,
gemfibrozil, fenofibrate, and troglitazone (22-25). Furthermore,
preliminary data indicate that UGT1A9 may conjugate natural PPAR Several studies in both humans and other animals reported that
treatment with PPAR Animal Studies--
Animal studies were performed in compliance
with European Union specifications regarding the use of laboratory
animals. Details of experimental conditions have been described
previously (31). Briefly, male Sv/129 homozygous wild type (+/+)
(n = 6) and PPAR Cell Culture--
Mononuclear cells were isolated from blood of
healthy donors by Ficoll gradient centrifugation and cultured as
described (5, 33). Mature monocyte-derived macrophages were used for
experiments after 10 days of culture. For treatment with PPAR RNA Purification and Reverse Transcription (RT)--
Total RNA
from cells was isolated using Trizol as specified by the supplier
(Invitrogen). RNA was isolated from mouse livers by the acid guanidium
thiocyanate/chloroform method (36). Two micrograms of total RNA from
cultured cells and mouse livers were reverse transcribed using random
hexamer primers and 200 units of Moloney murine leukemia virus reverse
transcriptase (Invitrogen).
Real Time PCR--
Reverse transcribed UGT1A9 and 28 S cDNAs
were quantified by real time PCR on a MX 4000 apparatus (Stratagene)
using the previously described specific primers for UGT1A9 and 28 S
(37, 38). Oligonucleotides for murine UGT1A9 were mUGT1A9F (sense)
5'-GAAGAACATGCATTTTGCTCCT-3' and mUGT1A9R (antisense)
5'-CTGGGCTAAAGAGGTCTGTCATAGTC-3'. PCR amplifications were performed in
a volume of 25 µl containing a 100 nM concentration of
each primer, 4 mM MgCl2, the Brilliant Quantitative PCR Core Reagent Kit mix as recommended by the
manufacturer (Stratagene), and SYBR Green 0.33X (Sigma). The conditions
were 95 °C for 10 min, followed by 40 cycles of 30 s at
95 °C, 30 s at 60 °C, and 30 s at 72 °C. UGT1A9
mRNA levels were subsequently normalized to 28 S mRNA. A
nonparametric Mann-Whitney test was used to analyze for significant
difference between the experimental groups of wild type and PPAR Plasmid Cloning and Site-directed Mutagenesis--
A 1.1-kb
genomic fragment containing a portion of the UGT1A9 promoter (from
nucleotide +53 to Transient Transfection Assays--
For all assays, 40 × 103 COS cells were transfected with 100 ng of the indicated
luciferase reporter plasmids, 50 ng of the pCMV- Electrophoretic Mobility Shift Assays (EMSAs)--
Human PPAR Adenovirus Generation--
The recombinant adenovirus
(Ad)-GFP and Ad-PPAR Microsome Purification and Glucuronidation Assays--
The human
liver microsome preparation, purchased from the Human Cell Culture
Center, Inc. (Laurel, MD) was from a pool of female subjects.
Microsomal proteins were purified from HEK 293 cells stably expressing
UGT1A9, THP1 cells treated or not with PPAR activators, or wild type or
PPAR UGT1A9 Catalyzes Glucuronidation of Fibrates and
Troglitazone--
Recent studies have indicated that UGT1A9 catalyzes
glucuronidation of gemfibrozil, fenofibric acid, and troglitazone,
suggesting that this enzyme is involved in the metabolism of synthetic
PPAR activators (22-25). To further characterize the role of UGT1A9 in
fibrate and glitazone glucuronide conjugation, microsomes from HEK 293 cells stably expressing UGT1A9 or from human liver (as positive
control) were incubated with gemfibrozil, fenofibric acid, clofibric
acid, ciprofibric acid, troglitazone, pioglitazone, and rosiglitazone
in the presence of radiolabeled cofactor UDPGA (Table
I). Whereas all fibrates were
glucuronidated by microsomes from UGT1A9-HEK 293 cells and human liver,
only a low level of troglitazone glucuronide formation was detected,
and pioglitazone and rosiglitazone were not glucuronidated in either
microsome preparation (Table I). UGT1A9 glucuronidated all fibrates
tested with a similar efficacy (between 259 and 343 pmol·min PPAR
To investigate whether PPAR
In differentiated murine adipocyte 3T3-L1 cells, which express PPAR
Monocyte-derived macrophages, which express both PPAR PPAR PPAR PPAR
Next, the wild type and mutated DR1 sites were cloned in multiple
copies upstream of the luciferase reporter gene driven by the
heterologous thymidine kinase promoter (TKpGL3), and cotransfection experiments were performed (Fig. 6).
Luciferase activity was increased upon cotransfection of the plasmid
containing three copies of the wild type DR1 with the PPAR PPAR In the present study, we identify UGT1A9 as a novel PPAR As previously reported (22-25), UGT1A9 catalyzes the glucuronide
conjugation of gemfibrozil, fenofibric acid, and troglitazone; however,
the present study also evidences the role of this enzyme in
glucuronidation of ciprofibric and clofibric acids. In contrast, glucuronidation of rosiglitazone and pioglitazone was not detected with
both microsomal preparations from UGT1A9-expressing HEK 293 cells and
human liver. This absence of glucuronidation is consistent with
previous observations indicating that only traces of pioglitazone and
rosiglitazone glucuronides are detected in human urine, whereas their
major metabolites correspond to sulfate conjugates (46, 47). The
present study, consistent with previous reports (24), indicates that
gemfibrozil is glucuronidated with a 2-3-fold higher activity than
other fibrates by human liver microsomes. This observation may explain,
at least partly, the very short plasma half-life of this compound when
compared with other fibrates (48). However, UGT1A9 glucuronidates all
fibrates with similar rates, and this difference of plasma half-life
may involve other UGT enzymes, such as UGT1A1, UGT2B7, or UGT2B15,
which also conjugate both gemfibrozil and fenofibrate (24).
UGT1A9 catalyzes glucuronidation of many drugs and exogenous compounds.
Thus UGT1A9 induction by PPAR Since UGT1A9 catalyzes the glucuronidation of fibrates and
troglitazone, its induction by PPARs suggests that synthetic agonists of nuclear receptors may regulate their own metabolism through up-regulation of UGT enzymes. Interestingly, this enzyme also glucuronidates polycyclic aromatic hydrocarbons, such as
6-hydroxychrysene or 4-methylumbelliferone (55), and polycyclic
aromatic hydrocarbons induce UGT1A9 and UGT1A6 expression in Caco2
cells (55). Whereas the molecular mechanisms of this transcriptional
regulation of polycyclic aromatic hydrocarbon-inducible UGTs have not
been clearly established, this may occur through the aryl hydrocarbon
receptor (55). Very recent observations indicate that UGT1A9 also
catalyzes the glucuronidation of arachidonic and linoleic acid
metabolites, such as 5-HETE, 12-HETE, 15-HETE, or 13-HODE.2
Some of these compounds, such as 15-HETE and 13-HODE, are endogenous PPAR Increased UGT1A9 expression is accompanied by an enhanced
catecholestrogen glucuronidation activity in PPAR agonist-treated THP-1
cells. Catecholestrogens such as 4OH-E1 and 4OH-E2 are formed from
estrogen through a hydroxylation process catalyzed by CYP1A1, CYP1A2,
and CYP1B1 (60) and are major endogenous substrates for UGT1A9 (21).
The glucuronidation of catecholestrogens is a potentially important
catabolic pathway for the elimination of these genotoxic steroid
metabolites from a given tissue and the prevention of cell damages they
can induce (61, 62). Indeed, the hydroperoxide-dependent
oxidation of catecholestrogens to quinones and the
NADPH-dependent reduction of the quinones back to
hydroquinones, yield semiquinone free radical intermediates and
superoxide radicals (63). The generation of free radicals has been
postulated to mediate DNA damage, leading to tumor development (61-63). Thus, by enhancing catecholestrogen glucuronidation catalyzed by UGT1A9, PPAR activators may contribute to protect cells against the
potential carcinogenic formation of free radicals.
UGT1A9 expression and glucuronidation activity are induced following
PPAR In conclusion, the present study demonstrates that PPAR and
are ligand-activated transcription factors
belonging to the nuclear receptor family. PPAR
mediates the
hypolipidemic action of the fibrates, whereas PPAR
is a receptor for
the antidiabetic glitazones. In the present study, the
UDP-glucuronosyltransferase (UGT) 1A9 enzyme is identified as a PPAR
and PPAR
target gene. UGTs catalyze the glucuronidation reaction,
which is a major pathway in the catabolism and elimination of numerous
endo- and xenobiotics. Among the UGT1A family enzymes, UGT1A9
metabolizes endogenous compounds, including catecholestrogens, and
xenobiotics, such as fibrates and to a lesser extent troglitazone.
Treatment of human hepatocytes and macrophages and murine adipocytes
with activators of PPAR
or PPAR
resulted in an enhanced UGT1A9
expression and activity. In addition, disruption of the PPAR
gene in
mice completely abolished the PPAR
agonist-induced UGT1A9 mRNA
and activity levels. A PPAR response element was identified in the
promoter of UGT1A9 at positions
719 to
706 bp by transient
transfection and electromobility shift assays. Considering the role of
UGT1A9 in catecholestrogen metabolism, PPAR
and PPAR
activation
may contribute to the protection against genotoxic catecholestrogens by
stimulating their inactivation in glucuronide derivatives. Furthermore,
since UGT1A9 is involved in the catabolism of fibrates, these results
suggest that PPAR
and PPAR
may control the intracellular level of
active fibrates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and PPAR
regulate gene
transcription by dimerizing with the retinoid X receptor (RXR) and
binding to PPAR response elements (PPREs) within the regulatory regions
of target genes (1). These PPREs usually consist of a direct repeat of
the hexanucleotide AGGTCA sequence separated by one or two nucleotides
(DR1 or DR2) (1). Furthermore, both PPAR
and PPAR
negatively
interfere with proinflammatory transcription factors pathways by a
mechanism termed transrepression (2). PPAR
is highly expressed in
tissues such as liver, muscle, kidney, and heart, where it stimulates
the
-oxidative degradation of fatty acids (3). PPAR
is
predominantly expressed in adipose tissue, where it promotes adipocyte
differentiation and lipid storage (4). Both PPAR
and PPAR
are
expressed in cells of the immune system, where they regulate the
inflammatory response, influence cell proliferation and apoptosis, and
modulate lipid homeostasis (5, 6).
and
PPAR
. Natural eicosanoids derived from arachidonic acid via the
lipoxygenase pathway, such as 8-hydroxytetraenoic acid (8-HETE), 15-HETE, and leukotriene B4, as well as oxidized phospholipids activate
PPAR
(7-9). PPAR
is a receptor for eicosanoid metabolites formed
via the cyclooxygenase (15d-prostaglandin J2) and lipoxygenase pathways
(15-HETE, 9-hydroxyoctadecadienoic acid (9-HODE), and 13-HODE) (10).
Synthetic agonists of PPARs are used in the treatment of metabolic
diseases, such as dyslipidemia and type 2 diabetes. The antidiabetic
glitazones (e.g. troglitazone, rosiglitazone, and
pioglitazone), which are insulin sensitizers, are high affinity ligands
for PPAR
(7). The hypolipidemic fibrates (gemfibrozil, bezafibrate,
ciprofibrate, and fenofibrate) are PPAR
ligands (7). Both natural
and synthetic ligands of PPARs share similar metabolic pathways, since
eicosanoids, fibrates, and troglitazone are excreted as glucuronide
conjugates in humans (11-14).
and
PPAR
agonists, such as 15-HETE and
13-HODE.2 Human UGT1A9 is
expressed in various tissues, including liver, intestine, kidney,
mammary gland, and ovary (21).
activators results in enhanced glucuronidation activity and UGT expression (26-29). In the Helsinki Heart Study population, gemfibrozil treatment resulted in a significant elevation of plasma androstane-3
,17
-diol glucuronide levels (26). In rats,
but not in mice, fenofibrate increases bilirubin glucuronidation in
liver microsomes (27, 28). In the same line, clofibrate induces the
bilirubin-conjugating UGT1A1 protein in microsomes from rat liver (29).
In rodents, clofibrate induces the glucuronidation of an antithrombotic
thioxyloside (LF 4.0212), which is catalyzed by UGT1A9 in humans (30).
Based on these observations, we investigated in the present study
whether UGT1A9 expression is regulated by PPAR
and PPAR
agonists.
Our results demonstrate that UGT1A9 is a positively regulated PPAR
and PPAR
target gene. This increased expression is observed in human
hepatocytes and macrophages as well as in mouse 3T3-L1 adipocytes.
Induction of UGT1A9 gene expression is accompanied by an increased
glucuronidation activity of catecholestrogens and fibrates. This
positive regulation of UGT1A9 expression occurs at the transcriptional
level by binding of PPAR
and PPAR
to a DR1 response element
located at
719 to
706 bp in the promoter region of the UGT1A9 gene.
Furthermore, the role of PPAR
in the fibrate-dependent
induction of UGT1A9 is established by the absence of fibrate response
in PPAR
-null mice.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
null (
/
) (n = 6) mice (32) (kind gift of Dr. F. Gonzalez, NCI, National Institutes of
Health, Bethesda, MD) were fed for 17 days with standard mouse chow
diet containing 0.2% (w/w) fenofibrate or not. At the end of the
treatment period, the animals were fasted for 4 h and sacrificed,
and livers were removed immediately, weighed, rinsed in 0.9% (w/v)
NaCl, frozen in liquid nitrogen, and stored at
80 °C until RNA or
microsome preparation.
and
PPAR
agonists, medium was changed to medium without serum but
supplemented with 1% nutridoma HU (Roche Molecular Biochemicals).
Human monocytic THP-1 cells (ATCC, Manassas, VA) were maintained in
RPMI 1640 medium (Invitrogen) containing 10% fetal calf serum
(FCS) and differentiated for 72 h with 100 nM phorbol
12-myristate 13-acetate. Differentiated cells were incubated for
24 h in the presence or absence of the indicated PPAR activators.
Human primary hepatocytes were isolated as described previously (34)
and incubated for the indicated times in dexamethasone-free Williams'
E medium containing fenofibric acid (250 µM). Mouse
3T3-L1 preadipocyte cells were grown in Dulbecco's modified Eagle's
medium (Invitrogen) containing 10% FCS and differentiated in
adipocytes according to the method of Bernlohr et al. (35).
Two-day postconfluent cells were switched to differentiation medium
(Dulbecco's modified Eagle's medium, 10% FCS, 10 µg/ml insulin, 1 µM dexamethasone, and 0.5 mM
3-methyl-1-isobutylxanthine) for 10 days. Cells were then cultured in
postdifferentiation medium (Dulbecco's modified Eagle's medium, 10%
FCS, and insulin) with or without rosiglitazone (1 µM).
Isolation and stable expression of the human UGT1A9 cDNA in human
embryonic kidney (HEK) 293 cells has been previously described
(21).
null mice.
1115) was amplified by PCR, using forward
5'-CTAGCAGAAGCTTCAGTAGGTGGGAGAAATACCAGCAC-3' and reverse
5'-CTAGCAGCTCGAGGAAAGACCGTCTCTTACTGGCAAG-3' primers into which
XhoI and HindIII sites were introduced,
respectively. The PCR amplification was performed in a final reaction
volume of 50 µl under the following conditions: denaturation at
96 °C for 3 min, 35 cycles of 15 s at 94 °C, 40 s at
60 °C, and 15 s at 72 °C, followed by a final extension at
72 °C for 7 min. The PCR product was cloned into the XhoI
and HindIII restriction sites of the pGL3 vector to generate
the A9prom construct. Mutations were introduced in the DR1 site using
the QuikChange site-directed mutagenesis kit (Stratagene) and the
A9promDR1mt
(5'-TGACATCACCTCTGATTTCAAGGAGTGCTCAG-3') oligonucleotide (nucleotides in boldface type are the mutated bases). The A9DR1-TKpGL3 and A9DR1mt-TKpGL3 plasmids were obtained by
cloning three copies of the corresponding annealed oligonucleotides in
the thymidine kinase promoter-driven luciferase reporter (TKpGL3) vector.
-galactosidase
expression vector, and with or without 20 ng of the pSG5-mRXR
,
pSG5-hPPAR
or pSG5-hPPAR
expression vectors, as indicated. All
samples were complemented with pBS-SK+ plasmid (Stratagene) to an
identical amount of 500 ng/well. COS cells were transfected using the
ExGen reagent (Euromedex) for 3 h at 37 °C and subsequently
incubated overnight with Dulbecco's modified Eagle's medium, 0.2%
FCS and then treated for 24 h with either Me2SO
(vehicle), Wy14643 (50 µM), or rosiglitazone (1 µM), as indicated.
and PPAR
and mouse RXR
proteins were synthesized in
vitro using the TNT Quick Coupled Transcription/Translation System
(Promega, Madison, WI). Double-stranded oligonucleotides were
end-labeled with [
-32P]ATP using T4-polynucleotide
kinase. For EMSA using in vitro produced proteins, PPAR
,
PPAR
, and/or RXR proteins were incubated for 15 min at room
temperature in a total volume of 20 µl containing 2.5 µg of
poly(dI-dC) and 1 µg of herring sperm DNA in binding buffer as
previously described (39). The radiolabeled probes (A9DR1wt
(5'-GACATCACCTCTGACCTCAAGGAG-3'), A9DR1mt5'
(5'-GACATAAAATCTGACCTCAAGGAG-3'), and A9DR1mt3'
(5'-GACATCACCTGTAAAATCAAGGAG-3'), where underlined nucleotides represent response element half-sites and
bases in boldface type are mutated) were added, and the binding reaction was incubated for a further 15 min at room temperature. The
protein complexes were resolved by 4% nondenaturing polyacrylamide gel
electrophoresis in 0.25× Tris-borate-EDTA (TBE) at room temperature. For supershift experiments, anti-PPAR
or anti-PPAR
antibodies (0.2 µg) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated for 20 min in the binding buffer before the addition of
in vitro produced proteins as described above. For
competition experiments, the indicated excess quantities of unlabeled
oligonucleotides were added to the binding reaction just before the
labeled probes.
were obtained by homologous
recombination in Escherichia coli (40) after insertion of
the cDNAs into the pAdCMV2 vector (Q.BIOgene, Illkirch, France).
Viral stocks were then created as previously described (41). Viral
titers were determined by plaque assay on HEK 293 cells and defined as
plaque-forming units/ml. 0.8 × 106 HepG2 cells were
infected at an input multiplicity of 100 virus particles/cell by adding
virus stocks directly to the culture medium. After 3 h of
infection, cells were subsequently incubated for 24 h with either
Me2SO (vehicle) or Wy14643 (50 µM).
-null mouse livers as previously described (21). Enzyme assays
were performed as reported (21). Briefly, microsomes were incubated
with 25 µM [14C]UDP-glucuronic acid, 2 mM unlabeled UDPGA, and 200 µM substrates in
a final volume of 100 µl of glucuronidation assay buffer (21). Assays
were performed at 37 °C for 30 min for fibrates, 1 h for 4OH-E1 and 4OH-E2, or 4 h for glitazones
and were terminated by adding 100 µl of methanol. 100 µl of
glucuronidation assays were applied onto a TLC plate (Merck) and
migrated using a toluene/methanol/acetic acid (7:3:1) mix. The extent
of substrate glucuronidation was analyzed and quantified by
PhosphorImager analysis (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1·mg of protein
1 for
gemfibrozil and ciprofibric acid, respectively), whereas gemfibrozil
was glucuronidated 2-fold more by human liver microsomes, compared with
other fibrates (Table I). Interestingly, troglitazone was
glucuronidated with a 10-fold lower activity than fibrates by both
UGT1A9 and human liver microsomes, whereas pioglitazone and
rosiglitazone were not glucuronidated in human liver. These results
demonstrate that UGT1A9 is able to convert fibrates and, to a lesser
extent, troglitazone into their glucuronide derivatives.
In vitro glucuronidation of fibrates and glitazones by microsomes
isolated from human liver and HEK 293 cells expressing UGT1A9
and PPAR
Activators Induce UGT1A9 Expression--
Since
UGT1A9 is expressed in various tissues (21, 42, 43), the regulation of
its gene expression by ligand-activated PPAR
and PPAR
was studied
in different cell types. Primary human hepatocytes, which express
PPAR
, were treated with fenofibric acid (250 µM) for
6, 12, and 24 h, and the level of UGT1A9 mRNA expression was
determined by real time RT-PCR. An increase in the concentration of
UGT1A9 mRNA was observed in fenofibric acid-treated compared with
vehicle-treated cells (Fig.
1a). This effect occurred in a
time-dependent manner, with a 4.2-fold increase of UGT1A9 transcript levels after 24 h of treatment.
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Fig. 1.
PPAR induces human
UGT1A9 mRNA in hepatocytes and HepG2 cells, and
PPAR
stimulates murine UGT1A9 expression in
3T3-L1 adipocytes. a, primary human hepatocytes were
treated with Me2SO (vehicle) or fenofibric acid (250 µM) for 6, 12, or 24 h. b, HepG2 cells
were infected with Ad-PPAR
or Ad-GFP for 3 h and subsequently
stimulated for 24 h with vehicle or Wy14643 (50 µM).
c, 3T3-L1 adipocytes were treated or not with rosiglitazone
(1 µM) for 24 h. Human and murine UGT1A9 RNA levels
were measured by real time RT-PCR. Values are expressed as means ± S.D. (n = 3), relative to the control set as
1.
induces UGT1A9 expression, human
hepatoma HepG2 cells were infected with an adenovirus coding for
PPAR
(Fig. 1b). Wy14643 treatment induced a 2-fold higher UGT1A9 expression in noninfected as well as in control Ad-GFP-infected cells. Infection with the PPAR
-adenovirus produced a more than 3-fold increase in UGT1A9 expression when compared with noninfected or
Ad-GFP-infected cells, and the addition of Wy14643 resulted in 4-fold
higher UGT1A9 mRNA levels (Fig. 1b). These results
demonstrate that PPAR
overexpression positively regulates the
expression of UGT1A9.
at high levels (44, 45), rosiglitazone treatment resulted in 3-fold
higher expression of UGT1A9 compared with vehicle-treated cells (Fig.
1c).
and PPAR
(5), were treated with increasing concentrations of the PPAR
agonist
Wy14643 or the PPAR
activator rosiglitazone (Fig. 2a). Both treatments resulted
in an increased UGT1A9 expression. In monocyte-derived macrophage THP-1
cells that also express both nuclear receptors (5), treatment with
Wy14643 resulted in a 2-fold induction of UGT1A9 mRNA, whereas
rosiglitazone provoked a 2.4-fold accumulation of this transcript (Fig.
2b). Taken together, these results demonstrate that
activation of PPAR
or PPAR
induces the UGT1A9 gene expression in
different human and mouse cell types.
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Fig. 2.
PPAR and
PPAR
activators increase UGT1A9 mRNA in
human primary and THP-1 macrophages. a, primary human
macrophages were treated with Wy14643 (25, 50, or 75 µM)
or rosiglitazone (0.5, 1, or 2 µM) for 24 h.
b, differentiated THP-1 macrophages were treated for 24 h with Me2SO (Vehicle), Wy14643 (50 µM), or rosiglitazone (1 µM). UGT1A9
mRNA levels were measured by real time RT-PCR, and expressed
relative to control set as 1. Values are means ± S.D.
(n = 3).
and PPAR
Activators Induce UGT1A9 Activity in Human
THP-1 Cells--
To determine whether the induction of UGT1A9
expression is accompanied by changes in its activity, THP-1 cells, in
which UGT1A9 is induced both by PPAR
and PPAR
activators, were
treated with Wy14643 (50 µM) or rosiglitazone (1 µM) for 36 h and analyzed for their glucuronidation
efficiency using 4OH-E1, 4OH-E2, gemfibrozil, fenofibric acid, or rosiglitazone as substrate (Fig.
3). Glucuronidation of rosiglitazone by
microsomes purified from THP-1 cells was detected but was too low to be
quantifiable (data not shown). However, treatment with Wy14643 or
rosiglitazone provoked an increased glucuronidation of all other
substrates tested. As such, Wy14643 induced 4OH-E2,
4OH-E1, gemfibrozil, and fenofibric acid glucuronidation 2-, 1.7- 2.2-, and 1.9-fold, respectively, whereas rosiglitazone induced glucuronidation of these substrates by 2.1-, 1.9-, 2.2-, and
1.6-fold, respectively (Fig. 3). These results clearly demonstrate that
both PPAR
and PPAR
agonists induce the glucuronidation activity
of UGT1A9 in THP-1 cells.
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Fig. 3.
PPAR and
PPAR
activators increase glucuronidation
activity in THP-1 macrophages. Microsomes purified from PPAR
agonist- or vehicle-treated THP-1 macrophages were incubated with
[14C]UDPGA and 4-hydroxyestradiol, 4-hydroxyestrone,
gemfibrozil, or fenofibric acid (200 µM) as indicated
under "Materials and Methods." Radiolabeled glucuronide derivatives
were subsequently analyzed by thin layer chromatography and quantified
by PhosphorImager analysis. Values represent the means ± S.D.
(n = 3).
Gene Disruption Abolishes Fibrate Induction of Murine
UGT1A9--
The effect of PPAR
gene disruption on the induction of
UGT1A9 mRNA and activity levels by fenofibrate was investigated in mice (Fig. 4). In wild-type mice,
fenofibrate treatment provoked a 2-fold increase in UGT1A9 mRNA
levels as compared with control animals (Fig. 4a). By
contrast, fenofibrate failed to induce UGT1A9 gene expression in
PPAR
-null mice. Moreover, glucuronidation assays using liver
microsomes revealed that fenofibrate treatment increases the levels of
4OH-E2, 4OH-E1, gemfibrozil, and fenofibric acid glucuronidation activity in wild type but not in PPAR
-null mice
(Fig. 4, b-e). These results demonstrate that PPAR
is
required for the induction of UGT1A9 expression and activity by
fenofibrate.
View larger version (21K):
[in a new window]
Fig. 4.
PPAR is required for
the induction of UGT1A9 gene expression by fibrates. Wild type
(+/+) and PPAR
null (
/
) mice were treated with 0.2% (w/w)
fenofibrate mixed in chow diet (
) or chow diet (
) for 17 days
(a). Total RNA was extracted from livers and analyzed for
mUGT1A9 expression by real time RT-PCR. Values are expressed as
means ± S.D. (n = 6), relative to the control set
as 1. Statistically significant differences between untreated and
fenofibrate-treated mice are indicated by asterisks
(Mann-Whitney test; ***, p < 0.001). b-e,
microsomes were incubated with radiolabeled UDPGA (25 µM), unlabeled UDPGA (2 mM), and
4-hydroxyestradiol (b), 4-hydroxyestrone (c),
gemfibrozil (d), or fenofibric acid (e) (200 µM) as indicated under "Materials and Methods."
Radiolabeled glucuronide derivatives were subsequently analyzed by thin
layer chromatography and quantified by PhosphorImager analyses. Values
represent means ± S.D. (n = 3).
and PPAR
Activate the UGT1A9 Gene
Promoter--
To decipher the molecular mechanisms of UGT1A9 induction
by PPAR activators, a 1.1-kb fragment of the human UGT1A9 gene promoter was cloned in front of the pGL3-luciferase reporter gene (Fig. 5a). Cotransfection of this
plasmid in COS cells in the presence or absence of murine RXR
, human
PPAR
, or human PPAR
and subsequent incubation with Wy14643 or
rosiglitazone resulted in an increased activity of the UGT1A9 promoter
(Fig. 5, b and c). The UGT1A9 promoter contains a
DR1 sequence at positions
719 to
706. To test whether this site
could mediate the induction by PPAR
and PPAR
, mutations were
introduced in the context of the 1.1-kb UGT1A9 promoter construct (Fig.
5a). Mutation of this DR1 site abolished the induction of
UGT1A9 promoter activity by ligand-activated PPAR
and PPAR
(Fig.
5, b and c).
View larger version (23K):
[in a new window]
Fig. 5.
PPAR and
PPAR
induce UGT1A9 promoter activity via a
PPRE located between
719 and
706 bp. a, schematic
representation of the human UGT1A9 gene promoter; 1.1 kb of the UGT1A9
promoter was cloned in front of the luciferase reporter gene, and the
potential PPAR response element was mutated by site-directed
mutagenesis. Mutated bases are underlined, and
arrows represent response element half-sites. b
and c, COS cells were transfected with the indicated human
UGT1A9 promoter-driven luciferase (Luc) reporter plasmids
(100 ng) in the absence or presence of pSG5-mRXR
, pSG5-hPPAR
(b), or pSG5-hPPAR
2 (c) (20 ng) and a
cytomegalovirus-driven
-galactosidase expression plasmid
(pCMV-
-galactosidase; 50 ng). Cells were subsequently treated or not
with Wy14643 (50 µM) or rosiglitazone (1 µM) for 24 h. Values are expressed as -fold
induction of the controls (pGL3) set at 1, normalized to internal
-galactosidase activity as described under "Materials and
Methods." Values represent the means ± S.D.
or
PPAR
plasmids. This activity was further enhanced by the addition of
their respective ligands (Fig. 6b). In contrast, no change
in activity was observed when either the empty TKpGL3 vector or the
TKpGL3 vector containing three copies of the mutated DR1 were
transfected. Taken together, these data indicate that the UGT1A9
promoter DR1 is a positive cis-acting element by which
ligand-activated PPAR
and PPAR
induce human UGT1A9 promoter
activity.
View larger version (26K):
[in a new window]
Fig. 6.
PPAR and
PPAR
activate the DR1-719 response
element. a, three copies of the wild type or mutated
PPREs were cloned upstream of the thymidine kinase (TK)
minimal promoter-driven luciferase reporter (TKpGL3). b and
c, COS cells were transfected with the indicated plasmids
(100 ng), pCMV-
-galactosidase (50 ng), and in the absence or
presence of pSG5-mRXR
, pSG5-hPPAR
(b), or
pSG5-hPPAR
2 (c) (20 ng). Cells were subsequently treated
or not with Wy14643 (50 µM) or rosiglitazone (1 µM) for 24 h. Values are expressed as -fold
induction of the controls (pGL3) set at 1, normalized to internal
-galactosidase activity as described under "Materials and
Methods." Values represent the means ± S.D.
and PPAR
Bind the PPRE within the UGT1A9 Gene
Promoter--
To determine whether human PPAR
or PPAR
bind the
PPRE in the UGT1A9 promoter, EMSAs were performed using this response
element as radiolabeled probe (Fig. 7).
As expected, in the absence of RXR, neither PPAR
nor PPAR
bound
the probe (Fig. 7, a and b, lane
2). In the presence of RXR, both PPAR
and PPAR
bound
the DR1 site (Fig. 7, a and b, lane
4). Furthermore, these complexes were supershifted by
anti-PPAR
or anti-PPAR
antibodies (lane 5).
Whereas both PPAR
/RXR and PPAR
/RXR heterodimers bound to the wild
type probe, no protein-DNA complex was observed when using the mutated
A9DR1mt5' and A9DR1mt3' probes (Fig. 7, a and b,
lanes 9 and 13). For competition
experiments, increasing amounts (1-, 10-, 50-, and 100-fold excess) of
unlabeled oligonucleotides encompassing either a consensus DR1 site
(DR1cons.) or the A9DR1wt, A9DR1mt5', or A9DR1mt3' sites
were added to binding reactions containing either PPAR
or PPAR
in
the presence of RXR (Fig. 7, c and d). Both
PPAR
and PPAR
binding on the A9DR1wt was strongly competed by the
DR1 consensus site and to a lower extent by the UGT1A9 DR1 itself (Fig.
7, c and d). By contrast, the mutated A9DR1
oligonucleotides did not efficiently compete for PPAR
or PPAR
binding to the DR1. Taken together, these data demonstrate that PPAR
and PPAR
bind to the UGT1A9 PPRE site at positions
719 to
706.
View larger version (57K):
[in a new window]
Fig. 7.
PPAR and
PPAR
bind to the PPRE in the UGT1A9
promoter. a and b, EMSAs were performed with
end-labeled wild type or mutated A9DR1 probes in the presence of
unprogrammed reticulocyte lysate, RXR, PPAR
(a), PPAR
(b), and both RXR and PPAR
or PPAR
as indicated.
Supershift experiments were carried out using anti-PPAR
or
anti-PPAR
antibodies (0.2 µg). c and d,
competition EMSA on radiolabeled A9DR1 probe were performed by adding a
1-, 10-, 50-, or 100-fold molar excess of the indicated cold consensus
DR1 (DR1 cons.), A9DR1wt, A9DR1mt5', or A9DR1mt3'
oligonucleotides in EMSA with unprogrammed reticulocyte lysate, RXR,
and PPAR
or PPAR
.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
PPAR
target gene. The induction of UGT1A9 mRNA by PPAR
or PPAR
agonists was observed in human hepatocytes and macrophages, human THP-1 and HepG2 cell lines, and murine 3T3-L1 adipocytes. The
identification of a PPRE in the UGT1A9 gene provides the first evidence
for a direct regulation of a human UGT enzyme by PPARs.
and PPAR
agonists may stimulate the
catabolism of these substances. Drug or xenobiotic metabolizing enzymes
play central roles in the biotransformation, metabolism, and/or
detoxification of foreign compounds that are introduced in the human
body. Glucuronide conjugates are among the major types of naturally
occurring metabolites of xenobiotics (49). Whereas the nuclear
receptors pregnane-X receptor and constitutive androstane receptor play
essential roles in the control of drug-metabolizing enzyme expression,
PPARs are also important xenobiotic sensors, at least in rodents
(50-52). Thus, PPAR
regulates, in rats, the expression of various
cytochrome P450s (CYPs) such as CYP1A2, -2A1, -2B1, -2B2, and -3A4, as
well as conjugating enzymes such as glutathione
S-transferase, GSTA1 and GSTM2 (50). Furthermore, in mice
PPAR
positively regulates the expression of mdr2
(or P-glycoprotein, Pgp, Abcb4), a major transporter of glucuronide derivatives localized in the canalicular membrane of
hepatocytes (53, 54). These data indicate that PPARs are involved in
the control of cellular metabolic processes, and the identification of
UGT1A9 as a PPAR target gene demonstrates a direct role of these
nuclear receptors as regulators of the metabolism of endogenous and
exogenous compounds in humans.
and PPAR
ligands (7). This suggests that not only synthetic but also natural activators of PPARs may control the expression of
enzymes involved in their inactivation pathways. Identification of UGTs
as target genes of receptors activated by their substrates suggests
that drug-conjugating enzymes may control intracellular levels of
receptor ligands. Furthermore, metabolites of the cyclooxygenase and
lipoxygenase pathways, such as 12- and 15-HETE and 13-HODE are highly
synthesized in smooth muscle cells and/or endothelial cells of the
atherosclerotic plaque (56-58). The release of these proinflammatory
molecules stimulates the inflammatory response and activates monocyte
adhesion and migration in the vascular subendothelium (56-59). PPAR
and PPAR
are highly expressed in cells of the arterial wall, where
they control the expression of numerous genes involved in the
inflammatory response, macrophage differentiation, apoptosis, and
cholesterol efflux (10). Thus, the PPAR-dependent induction
of HETEs and 13-HODE glucuronidation catalyzed by UGT1A9 in macrophages
may potentially contribute to the anti-inflammatory effect of PPAR
and PPAR
ligands.
activator treatment of various cell types, including mouse
adipocyte 3T3-L1 cells. Interestingly, there is accumulating evidence
that PPAR
ligands inhibit estrogen biosynthesis in breast adipose
tissue (64). Local estrogen biosynthesis is catalyzed by the P450
aromatase (CYP19), and recent data have established that ligands for
PPAR
and RXR inhibit aromatase expression in human breast adipose
tissue (65). Whereas this inhibition was shown to be independent of DNA
binding, it is believed that PPAR
agonists may have useful
therapeutic benefits in the management of breast cancer by reducing
local estrogen biosynthesis. The data presented here demonstrate that
PPAR
not only decreases estrogen biosynthesis but could also locally
induce their catabolism to catecholestrogen glucuronides. Thus, the
UGT1A9-induced expression may be part of a complex process by which
PPAR
activators decrease estrogen levels in adipose tissue.
and PPAR
activators enhance UGT1A9 expression and activity. However, whereas all
clinically used fibrates are glucuronidated, the only glitazone
conjugated by this enzyme (i.e. troglitazone) is no longer
used in clinic (66), and pioglitazone and rosiglitazone are not
glucuronidated. These observations suggest that treatment with agonists
of both receptors may affect the transcriptional activity of PPAR
,
whereas PPAR
-dependent pathways may not be affected. In
addition, molecules that activate both PPAR
and PPAR
are actually
under development, and examination of their glucuronidation by UGT1A9
may be useful to understand their pharmacokinetic properties,
which may influence their hypolipidemic and hypoglycemic activities.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Bruno Derudas for technical assistance. Pr. B. Van Vlijmen (Department of Cardiology, Leiden University, Leiden, The Netherlands) is acknowledged for providing the pAd vector.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Fondation Lefoulon-Delalande, Institut de France (to O. B.), European Community Grant ERBFMBICT983214 (to I. P. T.), Fonds Européens de Developpement Régional, Conseil Régional Région Nord/Pas-de-Calais Genopole Project 01360124, grants from the Leducq Foundation (to B. S. and J. C. F.), Canadian Institutes of Health Research Grants MOP-44032 and MSH-46809, and Fonds de la Recherche en Santé du Québec Grant 13408-166 (to C. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed: Unité
INSERM 545, Institut Pasteur de Lille, 1 rue du Pr. Calmette, BP 245, 59019 Lille, France. Tel.: 33-3-20-87-73-87; Fax: 33-3-20-87-71-98; E-mail: bart.staels@pasteur-lille.fr.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M300749200
2 A. Bélanger, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; PPRE, PPAR response elements; HETE, hydroxytetraenoic acid; HODE, hydroxyoctadecadienoic acid; UGT, UDP-glucuronosyltransferase; UDPGA, UDP-glucuronic acid; RT, reverse transcription; 4OH-E1, 4-hydroxyestradiol; 4OH-E2, 4-hydroxyestrone; FCS, fetal calf serum; EMSA, electrophoretic mobility shift assay; HEK, human embryonic kidney; CYP, cytochrome P450.
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