Coffee Induces Expression of Glucuronosyltransferases by

GASTROENTEROLOGY 2010;139:1699 –1710
Coffee Induces Expression of Glucuronosyltransferases by the Aryl
Hydrocarbon Receptor and Nrf2 in Liver and Stomach
SANDRA KALTHOFF, URSULA EHMER, NICOLE FREIBERG, MICHAEL P. MANNS, and CHRISTIAN P. STRASSBURG
See editorial on page 1453.
BACKGROUND & AIMS: Coffee is one of the most
widely consumed beverages worldwide. Epidemiologic data
indicate that coffee consumption protects against the progression of chronic liver disease and development of hepatocellular carcinoma and diabetes, but the mechanisms are
not clear. UDP glucuronosyltransferases (UGT1A) are proteins with indirect antioxidant, cytoprotective, and genoprotective capabilities; we examined UGT1A regulation in response to coffee in cultured cells and mice. METHODS:
HepG2 and CaCo2 cells were incubated with regular, metalor paper-filtered, decaffeinated, or instant coffee; green or
black tea; cocoa; or metabolic products of caffeine. The
effects of UGT1A regulation were investigated with reporter
gene assays, immunoblot, TaqMan polymerase chain reaction, mutagenesis, and short interfering (si)RNA analyses.
We also studied the effects of coffee in humanized transgenic mice that express human UGT1A. RESULTS: Incubation of cells with coffee induced transcription of UGT1A1
(5.4-fold), UGT1A3 (5.2-fold), UGT1A4 (4.8-fold), UGT1A7
(6.2-fold), UGT1A8 (5.2-fold), UGT1A9 (3.5-fold), and
UGT1A10 (6.1-fold). Induction was independent of caffeine,
methylxanthines, or the diterpenes cafestol and kahweol.
Mutagenesis and short interfering RNA knockdown studies
showed that UGT1A is regulated by the aryl hydrocarbon
receptor (AhR) and the nuclear factor erythroid-related factor 2 (Nrf2) by cis-acting antioxidant and xenobiotic response elements (ARE/XRE). In transgenic UGT1A mice,
administration of coffee resulted in a 10- and 14-fold induction of UGT1A transcription in liver and stomach, respectively. CONCLUSIONS: UGT1A genes are induced
in vitro and in vivo by coffee, independent of caffeine
content, cafestol, or kahweol. Coffee up-regulates glucuronidation by AhR signaling and Nrf2 binding to the
ARE/XRE. Glucuronidation could mediate the protective and antioxidant effects of coffee.
Keywords: Liver Cancer; Glucuronidation; Coffee.
T
ogether with green tea, coffee is one of the most
widely consumed beverages worldwide. The value of
coffee beans as a commodity is only second to crude oil.
Inhabitants of Western countries consume as much as 3
cups of coffee daily.1 Coffee represents a readily available
and much sought after caffeine delivery system because
of the desired stimulatory effects on its consumers. However, apart from caffeine, coffee contains a plethora of
complex organic compounds.2 Coffee represents a rich
source of phenols, polyphenols, flavanoids, and nonflavanoids, a number of which have been associated with
antioxidant properties. A high proportion of chlorogenic
acid and coffee bean flavanoids survive typical roasting
temperatures of up to 230°C, but roasting also leads to
mutagenic polyaromatic hydrocarbons.2,3 Nevertheless,
epidemiologic and study data suggest that coffee consumption is associated with a decreased risk of a number
of diseases. In 1986, Arnesen et al4 observed lower ␥
glutamyltransferase activities in coffee drinkers in the
Tromso Heart study. This finding has been replicated in
subsequent studies,5 including an analysis of the third
National Health and Nutrition Examination Survey
(NHANES III) in 20056 that showed an inverse correlation of coffee intake and alanine aminotransferase activities. Coffee consumption has been associated with reduced risks of hepatocellular carcinoma (HCC),7 liver
cirrhosis,8,9 and disease progression in chronic hepatitis
C,10 as well as with protective effects in Parkinson’s disease11 and type 2 diabetes,12 and a controversial protection against colorectal cancer.13
To date, the responsible protective mechanisms of coffee remain to be fully elucidated. It has been suggested
that caffeine may be responsible,6 possibly by impaired
transforming growth factor ␤ signaling.14 However, the
role of caffeine alone was questioned by findings in other
studies.5,15,16 Coffee diterpenes (cafestol and kahweol)
have been suggested to induce glutathione-S-transferases
(GSTs) preventing benzo(␣)pyrene genotoxicity,17 and
nuclear factor erythroid-related factor 2 (Nrf2)–mediated
antioxidant action.18
Abbreviations used in this paper: ARE, antioxidant response element;
HCC, hepatocellular carcinoma; Nrf2, nuclear factor erythroid-related
factor 2; PCR, polymerase chain reaction; siRNA, short interfering RNA;
tBHQ, tert-butylhydroquinone; TCDD, 2,3,7,8-tetrachlordibenzo-p-dioxin;
UGT, UDP-glucuronosyltransferase; XRE, xenobiotic response element.
© 2010 by the AGA Institute
0016-5085/$36.00
doi:10.1053/j.gastro.2010.06.048
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Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
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KALTHOFF ET AL
On the basis of these data, we hypothesized that detoxification systems such as the UDP-glucuronosyltransferases (UGTs) may be regulated by coffee and thereby
contribute toward cytoprotection and disease susceptibility. UGTs catalyze the formation of glucuronides from a
broad array of potentially cytotoxic or genotoxic compounds,19 which include human carcinogens and reactive
oxygen species.20 –22 In addition, genetic UGT variants
with reduced catalytic activity have been identified as risk
factors for HCC and other cancers.19,23–25 Regulation of
UGTs by the xenobiotics present in roasted coffee would,
therefore, represent a physiologically plausible mechanism of cytoprotection.
Material and Methods
Cell Culture Experiments
Hepatoma (HepG2), colon carcinoma (CaCo2),
and esophagus carcinoma (KYSE70) cells were grown in
RPMI 1640 (HepG2 and KYSE70) or Dulbecco’s modified
Eagle’s medium with nonessential amino acids (CaCo2)
supplemented with 10% fetal bovine serum.
Standardized Preparation of Coffee, Cocoa,
and Tea
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Stock solutions were prepared to represent the concentration and preparation mode of commonly used beverages: regular, decaffeinated, filtered, boiled, and instant coffee. For preparation of coffee and decaffeinated coffee, 150
mL of water (Aqua Irrigation Solution; DeltaSelect,
Dreieich, Germany) was boiled in a beaker and cooled for 10
seconds. Six grams of ground coffee powder (Jacobs Krönung/Jacobs Krönung decaffeinated; Kraft Foods, Bremen,
Germany) was added, incubated for 1 minute, and subsequently filtered through a paper coffee filter (Mellita, Minden, Germany). The undiluted filtrate was used for mice
treatment, and diluted (12% coffee ⫹ 88% medium) filtrate
was used for cell culture treatment. Two grams of Jacobs
Krönung instant coffee (Kraft Foods) was solubilized in 150
mL of boiled water. For boiled coffee, 150 mL of water was
boiled, cooled for 10 seconds, and 6 g of Jacobs Krönung
coffee powder was added and incubated for 1 minute. The
mixture was filtered through a metal sieve. Six grams of
cocoa (Krüger, Bergisch Gladbach, Germany) was diluted in
150 mL of boiled water. For green tea, 150 mL of water were
boiled and cooled for 2 minutes and 1 tea bag (green tea
Meßmer, Seevetal, Germany) was incubated for 3 minutes.
For preparation of black tea, 1 tea bag (black tea; Thiele Tee,
Emden, Germany) was incubated in 150 mL of boiled water
for 3 minutes. To show concentration dependency 1%, 4%,
8%, and 12% coffee stock solutions were used for luciferase
experiments (data not shown). Induction was highest with
12%, which was used subsequently. As a control for filtered
coffee 150 mL of water was boiled, cooled for 10 seconds,
and paper filtered. For all other beverages water served as a
negative control. Caffeine and theophylline concentrations
were determined by enzyme-linked immunoabsorbent assay
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Table 1. Chemical Composition of the Tested Beverages
Coffee
Decaffeinated coffee
Instant coffee
Boiled coffee
Cocoa
Green tea
Black tea
Caffeine
(mg/cup, 150 mL)
Theophylline
(mg/cup, 150 mL)
95.8 ⫾ 0.8
3 ⫾ 0.3
93.4 ⫾ 1.8
128 ⫾ 3.6
14.5 ⫾ 1
30.6 ⫾ 0.8
37 ⫾ 1.6
6.9 ⫾ 0.5
0.6 ⫾ 0.1
5.2 ⫾ 0.4
6.7 ⫾ 1
10.75 ⫾ 0.5
2 ⫾ 0.2
3.4 ⫾ 0.3
Data are mean ⫾ SD.
(Neogen, Lexington, KY; Table 1). The antioxidative capacities indicating polyphenol content were determined by an
OxiSelect Oxygen Radical Antioxidant Capacity Activity Assay (Cell BioLabs, San Diego, CA).
Methylxanthines and Coffee Lipids Cafestol
and Kahweol
One cup of coffee following the above protocol
contains approximately 100 mg of caffeine, which are metabolized to 70 mg of paraxanthine, 9 mg of theobromine,
and 9 mg of theophylline.26 Accordingly, these methylxanthine amounts (Sigma-Aldrich, Traufkirchen, Germany)
were used per 150 mL of medium. Cafestol and kahweol
(Sigma-Aldrich) were solubilized in dimethyl sulfoxide and
diluted in medium to concentrations of 5, 30, and 56
␮mol/L.
RNA Isolation and Reverse Transcription
Polymerase Chain Reaction
HepG2, CaCo2, and KYSE70 cells were treated
with coffee, tea, or cocoa solutions for 24 hours. Total
RNA was isolated with TRIzol (Invitrogen, Karlsruhe,
Germany). To isolate RNA from mouse organs, 100 mg
of frozen tissue was homogenized in TRIzol. RNA (5 ␮g)
was used for cDNA synthesis with the use of oligo(dT)primed Superscript III reverse transcriptase (Invitrogen).
Quantitative Real-Time Polymerase Chain
Reaction
By TaqMan, 0.2 ␮g of cDNA were analyzed (ABI
Prism 7000 sequence detection system; Applied Biosystems, Foster City, CA) with the use of UGT1A isoformspecific primers and probes (Supplementary Table 1).
Ct-values were normalized against ␤-actin.
Generation of Luciferase Reporter Gene
Constructs
A 1000-base pair (bp) DNA fragment of the
UGT1A1 5=-upstream region was amplified by polymerase
chain reaction (PCR) from 2 healthy blood donors exhibiting the A(TA)6TAA or the A(TA)7TAA (UGT1A1*28)
variant, respectively. The UGT1A3 258-bp DNA fragment
and the UGT1A4 513-bp fragment were cloned from
genomic DNA.27,28 UGT1A7 5= upstream DNA fragments
were amplified by PCR from 2 healthy blood donors
exhibiting the ⫺57G or the ⫺57T variant, respectively.29
A 500-bp (UGT1A8, UGT1A10) and a 530-bp (UGT1A9)
DNA fragment of each UGT1A 5= upstream sequence was
amplified by PCR from a healthy blood donor (all primers are shown in Supplementary Table 2). The PCR fragments were directionally inserted (XhoI and NheI) into
pGL3 basic (Promega, Mannheim, Germany). Mutagenesis of putative xenobiotic response element (XRE) and
antioxidant response element (ARE) binding sites was
performed by primer extension with the use of primers
specified in Supplementary Table 3. All inserts were sequenced in full.
Luciferase Assays
Dual luciferase assays (Dual-Reporter Assay; Promega) with HepG2, CaCo2, or KYSE70 cells were performed
as previously described.27 One day after transfection, cells
were treated with coffee, cocoa, or tea for 48 hours. All
experiments were performed in triplicate in at least 3–10
independent experiments. Results were analyzed with the
use of Microsoft Excel software (Microsoft, Redman, WA)
and show fold induction of luciferase activity. Error bars
represent standard deviations. Statistical analysis was performed with the Student’s t test. Differences were considered significant when P values were below .05.
Transfection of Short Hairpin RNA
For short interfering RNA (siRNA) experiments,
100 pmol of siRNA (MWG Biotech, Ebersberg, Germany)
against Nrf2 (AAGAGUAUGAGCUGGAAAAACTT), AhR
(AAGCGGCAUAGAGACCGACUUTT), or nonsilencing
control (UAAUGUAUUGGAACGCAUATT) was transfected in 1 mL of OPTI-MEM (Invitrogen) into KYSE70
cells seeded into 12-well plates with the use of lipofectamine 2000 (Invitrogen). The final concentration of
siRNA was 100 nmol/L. Reporter gene constructs were
transfected 6 hours later with the use of lipofectin, and
cells were treated with coffee, cocoa, or tea on the next
day for a further 48 hours. Knockdown efficiency of
siRNAs was determined by Western blot analysis (described below). For complete UGT1A knockdown
KYSE70, CaCo2, and HepG2 cells were treated with 100
nmol/L siRNA (Applied Biosystems) against UGT1A exon
2 (GGAUCAAUGGUCUCAGAAAtt). Cells were counted
24 and 48 hours after treatment.
Hydrogen Peroxide Production
Hydrogen peroxide concentrations in supernatants of treated cells were determined with the use of the
H2O2 detection kit (Assay Designs, Ann Arbor, MI).
Western Blot Analysis
KYSE70 cells were treated with coffee (12% of stock
solution), decaffeinated coffee (12% of stock solution),
2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD; 5 nmol/L), or
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tert-butylhydroquinone (tBHQ; 100 ␮m). Total cell lysate
(20 ␮g) was boiled for 10 minutes in Laemmli sample buffer
(2% sodium dodecyl sulfate, 62.5 mmol/L Tris-HCl, pH 6.8,
10% glycerol, and 0.001% bromphenol blue) and separated
by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis before electrotransfer onto a nitrocellulose membrane. Incubation with primary antibodies (anti-AhR, sc8087, anti-Nrf2, sc-722, anti-UGT1A1, and sc-27415; Santa
Cruz Biotechnology Inc, Santa Cruz, CA) was carried out in
10% dry milk. After incubation with secondary antibodies
(Millipore, Schwalbach, Germany), protein was visualized by
chemiluminescence (Pierce, Rockford, IL) on X-ray film.
Staining with ␤-actin antibody (sc-69879) was used as
loading control. UGT1A protein expression in mice
tissue was performed as described before30 (antibodies:
UGT1A1: sc-27415, Santa Cruz Biotechnology Inc;
UGT1A3: H00054659-M02, Abnova, Walnut, CA;
UGT1A4: AV46829, Sigma-Aldrich; UGT1A6: 458416,
BD Biosciences, San Jose, CA).
Humanized UGT1A Transgenic Mice
To study in vivo UGT1A regulation by coffee, a
humanized transgenic mouse model was used. Recently,
a UGT1A transgenic mouse model containing the complete human UGT1A locus was described.31 In this study
a transgenic mouse model was used that contained the
human first exons of UGT1A isoforms UGT1A1, UGT1A3,
UGT1A4, UGT1A5, UGT1A6, UGT1A7, and UGT1A9 and
the respective promoter regions that were based on BAC
clone RP11-943B10 (imaGenes GmbH, Berlin, Germany).
DNA was sequenced by capillary electrophoresis in an
ABI Prism 300 automated sequencer (Applied Biosystems) to exclude polymorphisms. In the 7 founder animals the human transgene was confirmed by DNA analysis. A mouse line carrying 6 gene copies was selected
(determined by real-time quantitative PCR) with 10 ng of
purified genomic DNA in an ABI Prism 7000 Sequence
Detection System (Applied Biosystems) with the use of
qPCR MasterMix (Euro Gentec, Cologne, Germany). Fluorescent in situ hybridization confirmed transgene insertion into a single region of the genome. Six male and 6
female 8-week-old transgenic UGT1A mice were treated
with undiluted coffee (for preparation see above) as their
drinking water for 3 days. Each control pool contained 4
mice given normal drinking water. The animal treatment
was approved by the Animal Ethics Committee of the
state Lower Saxony, Germany.
Results
UGT1A Gene Regulation by Coffee
Significant induction of luciferase activity by regular as well as decaffeinated coffee was observed. Regular
coffee induced UGT1A1 (5.4-fold), UGT1A3 (5.2-fold),
UGT1A4 (4.8-fold), UGT1A7 (6.2-fold), UGT1A8 (5.2-fold),
UGT1A9 (3.5-fold), and UGT1A10 (6.1-fold) in KYSE70
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Figure 1. (A) Induction of different UGT1A reporter gene constructs by coffee in luciferase assays (KYSE70 cells). Different UGT1A–5=-upstream regions
were significantly induced by regular and decaffeinated coffee in comparison to empty vector control (basic). UGT1A1, UGT1A7, and UGT1A10 coffee
induction in HepG2 (B) and CaCo2 (C) cells. (D) Western blot of UGT1A1 protein induction by regular and decaffeinated coffee in HepG2, CaCo2, and
KYSE70 cells. Human liver microsomes (HLMs) were used as positive control. (E) Hydrogen peroxide concentration in supernatants of treated (48 hours)
KYSE70 cells. (F) Antioxidative capacity of tested beverages determined by enzyme-linked immunoabsorbent assay.
cells (Figure 1A). In HepG2 and CaCo2 cells induced
luciferase activity was observed for UGT1A1, UGT1A7,
and UGT1A10 reporter gene constructs with regular coffee, decaffeinated coffee, and instant coffee (Figure 1B
and C). This indicates that UGT1A induction did not
require caffeine. In HepG2, CaCo2, and KYSE70 cells
treated with regular and decaffeinated coffee, an increase
of UGT1A1 protein was observed, which did not depend
on caffeine (Figure 1D). Coffee treatment did not lead to
significant hydrogen peroxide generation (Figure 1E), nor
did coffee exhibit the highest antioxidant activity (Figure
1F) compared with the other beverages used.
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Regulation of UGT1A1, UGT1A7, and
UGT1A10 by Different Coffee Preparations,
Cocoa, Green Tea, and Black Tea
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coa, green tea, and black tea (KYSE70 cells) were performed.
The UGT1A1 1000-bp wild-type reporter gene construct was
induced by coffee, decaffeinated coffee, and instant coffee to
a comparable extent (ca 5.5-fold; Figure 2A). UGT1A1 induction was weaker with boiled coffee (4.1-fold) and green
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Induction experiments with regular filtered coffee,
decaffeinated filter coffee, instant coffee, boiled coffee, co-
UGT1A REGULATION BY COFFEE
Figure 2. Induction of UGT1A1 (A), UGT1A7 (B), and UGT1A10 reporter gene constructs (C) in KYSE70 cells. Luciferase activity with the UGT1A7
⫺57G (UGT1A7*12) and UGT1A1 A(TA)7(TAA) (UGT1A1*28) variants was compared with wild type. Significant differences are relative to the basic
vector. In single nucleotide polymorphism constructs significance was compared with wild type. (D) Luciferase activity of the UGT1A1, UGT1A7, and
UGT1A10 reporter gene constructs in KYSE70 cells after treatment with caffeine (100 mg/150 mL) and its primary dimethylxanthine metabolites
paraxanthine (70 mg/150 mL), theobromine (9 mg/150 mL), and theophylline (9 mg/150 mL). Similar results for Caco2 and HepG2 cells are not
shown. (E) Treatment of KYSE70 cells with the coffee lipids cafestol and kahweol (C⫹K) (each 5, 30, or 56 ␮mol/L).
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tea (4.4-fold) and insignificant with cocoa and black tea. A
construct containing UGT1A1*28 [Gilbert’s syndrome;
A(TA)6TAA⬎A(TA)7TAA] did not significantly reduce induction compared with wild-type UGT1A1. However, there
was a trend toward reduction for coffee (5.4-fold ⬎ 4.2fold), decaffeinated coffee (5.4-fold ⬎ 4.6-fold), and green
tea (4.4-fold ⬎ 3.5-fold).
The UGT1A7 wild-type construct was significantly induced by coffee (6.2-fold), decaffeinated coffee (5.9-fold),
instant coffee (6.8-fold), boiled coffee (9-fold), and green
tea (4.8-fold; Figure 2B). Cocoa and black tea failed to
significantly induce UGT1A7. A construct containing the
UGT1A7*12 polymorphism (⫺57 T⬎G) led to significant reduction of coffee (3.3-fold), decaffeinated coffee
(2.7-fold), and instant coffee-mediated induction (3.5fold) relative to wild type.
To assess the effect of UGT1A expression on cell proliferation, siRNA knockdown of UGT1A1 exon 2 was
performed, which completely abrogates all UGT1A transcripts. In KYSE70, HepG2, and CaCo2 cells this had no
effect on cell proliferation, indicating that modulation of
UGT1A expression does not affect cell proliferation rates
(data not shown).
In the absence of known promoter variants of UGT1A10
only the wild-type sequence was studied. There was significant up-regulation by coffee (6.1-fold), decaffeinated
coffee (6-fold), instant coffee (9.8-fold), boiled coffee (9.5fold), cocoa (11.2-fold), green tea (5.7-fold), and black tea
(3-fold; Figure 2C). In combination, UGT1A induction
depends on the beverage preparation but not on caffeine.
Black tea showed only weak inducibility; green tea
showed a moderate induction of UGT1A1, UGT1A7, and
UGT1A10.
Inducibility Is Independent of
Methylxanthines, Cafestol, and Kahweol
To clarify the role of caffeine or its metabolites,
KYSE70, HepG2, and Caco2 cells were treated with caffeine
(100 mg/150 mL), paraxanthine (70 mg/150 mL), theobromine (9 mg/150 mL), and theophylline (9 mg/150 mL).26 In
KYSE70 cells, luciferase activity with UGT1A1 and UGT1A10
was not significantly induced (Figure 2D), and UGT1A7
induction by paraxanthine (2-fold) was low.
Induction experiments were then expanded to cafestol
and kahweol present in non–paper-filtered coffee. Because
in Figure 2B and C induction was higher with boiled (metalfiltered) coffee, this may be an effect of cafestol and kahweol. However, there was no significant up-regulation of
UGT1A7 and UGT1A10 by 30 ␮mol/L cafestol and kahweol
(3.4/3.7-fold) compared with the control (basic) vector (Figure 2E). Treatment with 5 ␮mol/L cafestol and kahweol
even led to reduced UGT1A7 and UGT1A10 expression,
which was not observed for UGT1A1 but is unlikely to affect
overall UGT1A inducibility.
In combination, induction of UGT1A1, UGT1A7 and
UGT1A10 by coffee is not mediated by caffeine, its dim-
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ethylxanthine metabolites, or the coffee lipids cafestol
and kahweol.
Identification of Responsible DNA Binding
Motifs in the UGT1A1-, UGT1A7-, and
UGT1A10-5=–Upstream Regions
Previous data suggest an effect of coffee on Nrf2
signaling. In addition, xenobiotics generated by roasting are
likely to activate AhR signaling. This may proceed by ARE
and XRE DNA binding motifs; thus, the 5= upstream sequence of UGT1A1, UGT1A7, and UGT1A10 was analyzed
(Figure 3A). All identified putative XRE and ARE DNA
binding motifs were mutagenized individually in the
UGT1A1 1000-bp construct to examine their function. Mutagenesis of XRE-102, XRE-586, XRE-706, or ARE-96 led to
a significant reduction of regular coffee and decaffeinated
coffee–mediated induction of UGT1A1 (Figure 3B). XRE586 and XRE-706 are therefore responsible for the higher
inducibility of the UGT1A1 1000-bp construct.
In the UGT1A7 promoter, 1 XRE and 2 ARE binding
sites were identified (Figure 3A). Mutagenesis of XRE-101
or ARE-143 significantly reduced induction (Figure 3C).
However, mutagenesis of ARE-187 led to a lower decrease
of UGT1A7 inducibility, which was not significant with
the use of decaffeinated coffee.
In the UGT1A10 promoter, 4 XRE binding sites and 1
ARE binding site were identified (Figure 3A). Mutagenesis of XRE-176 and XRE-256 did not affect UGT1A10
induction (Figure 3D). However, when XRE-101, XRE136, or ARE-149 was mutagenized, inducibility was significantly reduced.
In summary, mutagenesis indicates XRE- and ARE-mediated regulation of UGT1A transcription in response to
coffee.
siRNA Mediated Knockdown of AhR and
Nrf2 Affects Inducibility of UGT1A1,
UGT1A7, and UGT1A10 by Coffee and
Decaffeinated Coffee
A direct involvement of AhR and Nrf2 was examined by siRNA experiments. After treatment with the AhR
ligand TCDD or the Nrf2 activator tBHQ (for 24 and 48
hours), AhR and Nrf2 protein amounts were increased in
KYSE70 cells (Figure 4A), which was reduced or absent in
the presence of AhR/Nrf2 siRNA treatment (Western
blots). AhR as well as Nrf2 siRNA abolished regular and
decaffeinated coffee–mediated induction of UGT1A1,
UGT1A7, and UGT1A10 (Figure 4B–D). These data suggest that both AhR and Nrf2 are involved in UGT1A
induction by coffee.
UGT1A mRNA Induction by Coffee in
HepG2, CaCo2, and KYSE70 Cells
UGT1A mRNA level induction was examined by
TaqMan PCR. In HepG2, CaCo2, and KYSE70 cells
treated with either regular or decaffeinated coffee
UGT1A1 mRNA was most strongly induced (HepG2,
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Figure 3. Identification of putative DNA binding motifs (A) and their mutagenesis in the UGT1A1 (B), UGT1A7 (C), and UGT1A10 (D) genes. Both
XRE and ARE elements participate in the transcriptional regulation of UGT1A genes in response to coffee. Significance was determined in
comparison to wild-type (nonmutagenized) constructs.
18-fold; CaCo2, 29-fold; KYSE70, 20-fold; Figure 5). In
HepG2 cells inducibility was additionally observed for
UGT1A3 (11.8-fold), UGT1A4 (5.9-fold), and UGT1A6
(5.8-fold; Figure 5A) RNAs. Again, decaffeinated coffee
showed no differences in inducibility. UGT1A8 mRNA
was not detectable in HepG2 cells. In CaCo2 cells, inducibility was detected for UGT1A3 (7.7-fold), UGT1A4
(10-fold), UGT1A6 (8.7-fold), UGT1A7 (8.8-fold), and
UGT1A8 (8.1-fold) mRNAs (Figure 5B). In KYSE70 cells,
UGT1A3 (7-fold), UGT1A4 (7.2-fold), UGT1A6 (5.8-fold),
UGT1A7 (4-fold), UGT1A8 (4.1-fold), and UGT1A10
(6.6-fold) mRNAs were induced (Figure 5C). The KYSE70
cells were additionally treated with AhR, Nrf2, and control siRNAs to show the dependence of coffee-mediated
inducibility on AhR and Nrf2. SiRNA treatment completely abolished UGT1A induction (Figure 5C).
As additional control experiments we examined whether
coffee (and not only synthetic inducers as shown in Figure
4) directly increases AhR and Nrf2 protein levels. HepG2,
CaCo2, and KYSE70 cell lysates after 48 hours of treatment
with coffee showed up-regulation in all 3 cell lines (Figure
5D and E).
In combination, coffee regulates human UGT1A
genes by AhR and Nrf2 signaling at the transcriptional
level.
In Vivo Regulation of UGT1A mRNA
Transcription by Coffee
In humanized transgenic UGT1A mice orally exposed to coffee for 3 days UGT1A induction in different
hepato-gastrointestinal organs was detectable by TaqMan PCR. Figure 6A–F shows examples of UGT1A
mRNA as well as protein induction by coffee. In the liver,
UGT1A1 expression is highly up-regulated (10-fold upregulation in female liver) in comparison to controls (3
pools of untreated UGT1A transgenic mice; Figure 6A).
UGT1A3 and UGT1A6 mRNAs are up-regulated only in
the colon of male mice (Figure 6B and C). UGT1A mRNA
induction was highest in the stomach, in which UGT1A1
(11-fold), UGT1A4, and UGT1A6 (14-fold) were up-regulated in male and female humanized UGT1A transgenic
mice (Figure 6D–F). These data provide direct evidence of
significant coffee-mediated in vivo regulation of human
UGT1A expression.
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Figure 4. (A) Western blot confirming knockdown of AhR (top) and Nrf2 (bottom) by specific siRNAs at 24 and 48 hours in KYSE70 cells induced
with either TCDD or tBHQ 6 hours after knockdown. siRNA-mediated knockdown of AhR and Nrf2 (KYSE70 cells) abolished both regular and
decaffeinated coffee–mediated luciferase induction of UGT1A1 (B), UGT1A7 (C), and UGT1A10 (D). Significance was determined in relation to
constructs treated with control siRNA.
Discussion
Coffee consumption has been associated with protection against inflammation, fibrosis, and neoplastic disease, spanning conditions, which include liver fibrosis,
chronic hepatitis C, Parkinson’s disease, and HCC. An
attractive mechanistic hypothesis is the induction of
cytoprotective and genoprotective antioxidant mechanisms.32,33 As a roasted plant-derived product, coffee contains a plethora of potentially active ingredients that are
potential inducers for oxidative and phase II metabolism.27,34 The present study performed in vitro and in
vivo provides direct evidence that coffee is a potent inducer of the human UGT1A genes.
Glucuronidation exerts protective effects by detoxifying and eliminating oxidative intermediates and reactive
oxygen species generated by phase I metabolism. Our
study shows a surprisingly substantial induction of the
entire family of UGT1A genes in reporter gene experiments, at the transcriptional level and at the protein level.
This contrasts with earlier findings that failed to identify
coffee-mediated UGT induction.35 In that study, a lower
coffee concentration used in vitro with CaCo2 cells did
not induce UGT1A1 and UGT1A6 mRNAs. In our study
a higher concentration of 12% (extracted from 6 g of
coffee powder with 150 mL of water) was used that
up-regulated mRNA not only in CaCo2, HepG2, and
KYSE70 cells but also in vivo in mice, possibly indicating
that the experimental outline of Okamura et al35 was not
capable of detecting UGT1A inducibility. Because of the
variability of common coffee preparations, we developed
standard preparations, including metal- and paper-filtered coffee, decaffeinated and instant coffee, as well as
green and black tea and cocoa, to re-create everyday
practices of beverage preparation.1 Paper-filtered coffee is
less rich in cafestol and kahweol. Decaffeinated coffee
lacks caffeine, and tea was included because it contains
caffeine but is not processed by roasting. The different
preparations analyzed in this study led to various degrees
UGT1A REGULATION BY COFFEE
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November 2010
Figure 5. TaqMan PCR quantification of UGT1A mRNA induction by regular and decaffeinated coffee in HepG2 (A), CaCo2 (B), and KYSE70 cells
(C). Significance was determined in relation to control-treated cells. In all cell lines UGT1A1 mRNA induction was highest. (C) KYSE70 cells were
additionally treated with AhR, Nrf2, and control siRNAs, abolishing inducibility of all UGT1A genes. Significance was determined in relation to cells
treated with control siRNA. Increase of AhR (D) and Nrf2 (E) protein in lysates of coffee-treated HepG2, CaCo2, and KYSE70 cells detected by
Western blot.
of UGT1A induction (Figure 2A–C). However, apart from
black tea, all used preparations were observed to significantly induce UGT1A gene transcription, indicating that
compounds other than caffeine and coffee diterpenes are
likely to be responsible for UGT1A up-regulation. We
were able to exclude caffeine and methylxanthines, as
well as the coffee diterpenes cafestol and kahweol as main
responsible inducers of UGT1A transcription (Figure 2D
and E).
These findings are of interest because they expand
other hypotheses. Caffeine increases intracellular cyclic
adenosine monophosphate and has been shown to inhibit the synthesis of connective tissue growth factor by
inducing proteasomal degradation of Sma- and Madrelated proteins and thereby disrupting transforming
growth factor ␤ signaling.36 The cytochrome P450 (CYP)
1A2-generated primary caffeine metabolite paraxanthine
is capable of exerting this effect14 but was found to be
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GASTROENTEROLOGY Vol. 139, No. 5
BASIC–
ALIMENTARY TRACT
Figure 6. Induction of UGT1A mRNA (TaqMan PCR relative to actin) and protein (Western blot) in humanized UGT1A transgenic mice after 3 days
of oral coffee intake. (A–F) Examples of UGT1A expression in different organs of the hepato-gastrointestinal tract are shown. Significance levels were
determined between treated and untreated animals.
inactive toward inducing UGT1A transcription. Despite
epidemiologic evidence that associated caffeine with reduced aminotransferase activities,6 some studies suggest
that protective effects of coffee may not be related to
caffeine alone,5,15,16 which is supported by this study. In
a recent study, caffeine consumption was linked to reduced liver fibrosis.8 However, caffeine sources other
than coffee showed only a modest effect,8 suggesting that
there may be additional mechanisms related to other
coffee-derived constituents. Other well-studied ingredients in coffee are the diterpenes cafestol and kahweol.
Both have been suggested to be anticarcinogenic by inhibiting aflatoxin B DNA binding,37 promoting glutathione-S-transferase induction and protection against aromatic hydrocarbon genotoxicity,17,38 and by protecting
against tetrahydrocarbon hepatotoxicity in mice.39 However, cafestol and kahweol were not observed to play a
role in the transcriptional regulation of UGT1A expression. Cafestol and kahweol were previously shown in
mice to regulate drug metabolizing enzymes by way of
the Nrf2 pathway.40 We, therefore, demonstrated by mutagenesis of ARE and XRE DNA binding motifs and
siRNA knockdown that UGT1A regulation by coffee proceeds by cis-acting Nrf2/ARE in addition to AhR/XRE
elements (Figures 3–5). This cafestol and kahweol independent transcriptional regulation was coordinated such
that siRNA knockdown of either Nrf2 or AhR was capable of completely abolishing coffee-mediated inducibility.
Nrf2-mediated regulation in response to coffee is in
agreement with the findings in another study18 that additionally postulated an inhibitory effect of coffee on
CYP activity. This inhibitory effect may in fact be related
to the simultaneous activation of UGT1A genes as a
means of antioxidant action. Our data therefore suggest
that Nrf2- and AhR-dependent activation of glucuronidation proceeds independent of cafestol and kahweol, as
well as caffeine or its methylxanthine metabolites. Glucuronidation may therefore represent a protective mechanism activated by coffee and coordinately controlled by
Nrf2/ARE and AhR/XRE signaling.
Genetic variants of UGT1A genes that alter catalytic
activity are present in whites at frequencies between 1%
and 40%.19 Case control studies have discussed these
variants as risk factors for cancer development, including
HCC (reviewed in Strassburg et al19). In particular
UGT1A7, which catalyzes the detoxification of hydroxylated benzo(␣)pyrenes30 has been linked to HCC. The
induction of UGT1A7 and other UGT1As by coffee would
thus represent a plausible explanation for cancer protection. In this study 2 variants were studied: UGT1A1*28
(Gilbert’s syndrome, reviewed in Strassburg41) and
UGT1A7*12.29 Both are characterized by 70% reduced
gene transcription compared with wild-type (UGT1A1*1
and UGT1A7*1) sequence. Our analysis showed that only
in UGT1A7*12 (Figure 2B) was inducibility significantly
impaired, which strengthens the hypothesis of UGT1A7
UGT1A REGULATION BY COFFEE
1709
polymorphisms as cancer risk factors.19 This does not
appear to be an effect of altered cell proliferation by the
modulation of UGT1A transcription because a complete
knockdown of all UGT1A transcripts did not alter cell
proliferation in different cell lines (data not shown).
To show that oral intake of coffee was indeed able to
regulate UGT1A expression in the digestive tract that establishes direct contact to coffee but also in the liver, which
would require induction through the blood stream, we
treated humanized transgenic UGT1A mice with coffee. In
vivo induction of human UGT1A gene transcription was
observed in the gastrointestinal tract as well as in the liver,
indicating that coffee can regulate glucuronidation in various organs on direct and indirect contact (Figure 6).
In conclusion, this study adds a new mechanistic hypothesis for the protective action of coffee mediated by
UGT1A gene activation capable of conferring indirect
antioxidant activity, cytoprotection, and genoprotection.
UGT1A regulation is affected by coffee consumption,
which is coordinately mediated by the Nrf2/ARE and the
AhR/XRE signaling pathways. It does not depend on
caffeine or coffee diterpenes. Although the responsible
compound (or compounds) for this regulation has not
yet been identified, our study indicates that pharmacologic intervention with caffeine-free coffee extracts may
represent an attractive future strategy of chemoprotection in risk groups for HCC, chronic hepatitis, or tumor
development.
Supplementary Material
Note: To access the supplementary material
accompanying this article, visit the online version of
Gastroenterology at www.gastrojournal.org, and at doi:
10.1053/j.gastro.2010.06.048.
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Received December 30, 2009. Accepted June 10, 2010.
Reprint requests
Address requests for reprints to: Christian P. Strassburg, MD,
Department of Gastroenterology, Hepatology and Endocrinology,
Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover,
Germany. e-mail: [email protected]; fax: (49)
511-532-4896.
Conflicts of interest
The authors disclose no conflicts.
Funding
This work was supported by the Deutsche
Forschungsgemeinschaft SFB/TRR77 project A4 (to C.P.S.).
November 2010
UGT1A REGULATION BY COFFEE
Supplementary Table 1. Primers and Probes for TaqMan
PCR
Gene
UGT1Aall
UGT1A1
UGT1A3
UGT1A4
UGT1A5
UGT1A6
UGT1A7
UGT1A8
UGT1A9
UGT1A10
Actin human
Actin mouse
Primer and probes
Forward: GCTATGGCAATTGCTGATGCTTT
Reverse: CGATGGTCGGGTTCCAGTGTA
Probe: FAM-AAAATCCCTCAGACAGTCCT-MGB
Forward: GAATCAACTGCCTTCACCAAAAT
Reverse: AGAGAAAACCACAATTCCATGTTCT
Probe: FAM-CTATCCCAGGAATTTGAA-MGB
Forward: CAGAAGTATGGCAATGTTGAACAATA
Reverse: GCCTCATTATGTAGTAGCTCCACACA
Probe: FAM-TCTTTGGTCTATCATAGGTC-MGB
Forward: TTTTTCTGCCCCTTATGCAAGT
Reverse: ACAGCCACACGGATGCATAG
Probe: FAM-TCAGAGAGAGGTGTCAGTGGTGGATCTTGTTAMRA
Forward: CCATTTCATGGACCCAGGAC
Reverse: AGAAGATGTTCTGTTTCAAAGAACGA
Probe: FAM-AATTTGATCGCCTTTTGCTGGGTCACATAMRA
Forward: CTTCATTGGAGGTATCAACTGTAAGAA
Reverse: AAGAGAAAACCACAATTCCATGTTC
Probe: FAM-AGGAAAGACTTGTCTCAGGAATTTGAAGCCTAMRA
Forward: GAGGATCAGGACCGGGAGTT
Reverse: GAAAATGCACTTCGCAATGGT
Probe: VIC-TGGTTTTTGCCGATGCT-MGB
Forward: GGAGGATCTGGACCGGGAA Reverse:
TGGATGAACTCAGAAATAGAGAAAACAA Probe:
FAM-TGGATTTCGCCGATGCTCAATGG-TAMRA
Forward: AAACCCGTGATGCCCAAC
Reverse: GGCTTCAAATTCCATAGGCAAC
Probe: FAM-TGATCTTCATTGGTGGTATCAACTGCCATCTAMRA
Forward: ACCTCGTACACTCTGGAAGATCAGA
Reverse: GAACTCATTAATAGAGAAAATATACTTTGTGCC
Probe: FAM-AATTCATGGTTTTCGCCCATGCTCA-TAMRA
Forward: TGCCGACAGGATGCAGAAG
Reverse: GCCGATCCACACGGAGTACT
Probe: FAM-AGATCAAGATCATTGCTCCTCCTGAGCGCTAMRA
Forward: ACGGCCAGGTCATCACTATTG
Reverse: CAAGAAGGAAGGCTGGAAAAG
Probe: FAM-CAACGAGCGGTTCCGATGCCC-MGB
1710.e1
Supplementary Table 2. Primers for Cloning UGT1ALuciferase Constructs
Primer
1A1 ⫺1000 Nhe fwd
1A1 pre ATG Xho rev
1A3 ⫺258 Nhe fwd
1A3 pre ATG Xho rev
1A4 ⫺513 bp Nhe fwd
1A4 pre ATG Xho rev
1A7 ⫺530 NheI fwd
1A7 rev XhoI
1A8 ⫺500 Nhe fwd
1A8 Xho rev
1A9 ⫺530 Nhe F
1A9 pre ATG Xho R
1A10 ⫺500 Nhe F
1A10 pre ATG Xho R
Sequence
GGT TGG CTA GCG CTG AGC CCT GAG
TGG CTG AGG
TTT AAC TCG AGG GCG CCT TTG CTC CTG
CCA G
TCT AGC TAG CAC TTG GAT GTT CCC CAG
AG
GTG GCT CGA GCT CAG CAG AAG ACA CG
TTT AAG CTA GCC CTG AAC ACT CTC TGT
TT
TTT AAC TCG AGC TCA GCA GAA GCC
ACC G
TTT AAG CTA GCT CCC AGC TAC TGA GGC
TGA GGC AGG
AAA TTC TCG AGC AGC AGA GAA CTT CAG
CCC AGA GCC
GCC AGC TAG CGC TGG GAA GTC GGT
GCT AAG G
TTT AAC TCG AGA GAG AAC TGC AGC
CCG AGC C
AAA TTG CTA GCA ATA TGT ATG CAT TGC
AGA G
AAA TTC TCG AGC AGC AGA GAA CTG
CAG CTG
GAG ATG CTA GCG AGC CCC AGT TTC
TTG CCA GTT G
TTT AAC TCG AGA GAG AAC TGC AGC
CCG AGC C
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KALTHOFF ET AL
GASTROENTEROLOGY Vol. 139, No. 5
Supplementary Table 3. Primers for Site-Directed
Mutagenesis of Binding Elements
Primer
Sequence
1A1 XRE ⫺102 mut fwd
1A1 XRE ⫺102 mut rev
1A1 XRE ⫺586 mut fwd
1A1 XRE ⫺586 mut rev
1A1 XRE ⫺706 mut fwd
1A1 XRE ⫺706 mut rev
1A1 ARE ⫺96 mut fwd
1A1 ARE ⫺96 mut rev
1A7 XRE ⫺100 mut fwd
1A7 XRE ⫺100 mut rev
1A7 ARE ⫺143 mut fwd
1A7 ARE ⫺146 mut rev
1A7 ARE ⫺187 mut fwd
1A7 ARE ⫺187 mut rev
1A10
1A10
1A10
1A10
1A10
1A10
1A10
1A10
1A10
1A10
XRE
XRE
XRE
XRE
XRE
XRE
XRE
XRE
ARE
ARE
⫺101
⫺101
⫺136
⫺136
⫺176
⫺176
⫺256
⫺256
⫺149
⫺149
mut
mut
mut
mut
mut
mut
mut
mut
mut
mut
fwd
rev
fwd
rev
fwd
rev
fwd
rev
fwd
rev
GCT TTT TAT AGT AAT TAA ACA CAG TC
GAC TGT GTT TAA TTA CTA TAA AAA GC
GGC TCA CCT CAT GGC AAT TAC TCG
TGT GG
CCA CAC GAG TAA TTG CCA TGA GGT
GAG CC
CTC TAC CCC AGA ATT ACC CCC ACC
CC
GGG GTG GGG GTA ATT CTG GGG TAG
G
TAT AGT CAC GTG AAA TTT AAA AAC
ATT AAC
GTT AAT GTT TTT AAA TTT CAC GTG
ACT ATA
ATG AAT AAG TAA TTT GCT TCT TTT
GAG GGC
GCC CTC AAA AGA AGC AAA TTA CTT
ATT CAT
TAT GAG TAA AAA ATT TAA AGT GAA
TGT GA
TCA CAT TCA CTT TAA ATT TTT TAC
TCA TA
CAT ATA AGC AAA ATT TAA AGC AAA
GGC TA
TAG CCT TTG CTT TAA ATT TTG CTT
ATA TG
GGATAAATAAAATTCCTCTATTGGGGTC
GACCCCAATAGAGGAATTTTATTTATCC
ATCATTGGCAGTGAAAATTATTTTTT
AAAAAATAATTTTCACTGCCAATGAT
CAGCAAATGATACAAATTTGTTATCGTTC
GAACGATAACAAATTTGTATCATTTGCTG
CTAAACTCACTTGCAATATACTCTCCCTC
GAGGGAGAGTATATTGCAAGTGAGTTTAG
TATGAGTAAAAAATTTAAAGTGAGTGTGA
TCACACTCACTTTAAATTTTTTACTCATA