From the Discovery Research Laboratories 1, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Wadai 10, Tsukuba, Ibaraki 300-4293, Japan
Received for publication, September 21, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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So far some nuclear receptors for bile acids have
been identified. However, no cell surface receptor for bile acids has
yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile
acids specifically induced receptor internalization, the activation of
extracellular signal-regulated kinase mitogen-activated protein kinase,
the increase of guanosine 5'-O-3-thio-triphosphate binding
in membrane fractions, and intracellular cAMP production in Chinese
hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5
mRNA showed that it was abundantly expressed in
monocytes/macrophages in human and rabbit. Treatment with bile acids
was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by
transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5
originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not
influence those in the original THP-1 cells, suggesting that TGR5 is
implicated in the suppression of macrophage functions by bile acids.
Bile acids are not simply byproducts of cholesterol metabolism but
play essential roles in the absorption of dietary lipids and in the
regulation of bile acid synthesis (1). Farnesoid X receptor and
pregnane X receptor have been recently identified as specific nuclear
receptors for bile acids (2-5). Through the activation of farnesoid X
receptor bile acids repress the expression of cholesterol
7 Reporter Assay--
Expression vectors with human TGR5 cDNA
(pAKKO-TGR5) and rat Gi cDNA (pAKKO-Gi)
were, respectively, constructed by inserting their coding regions into
pAKKO-111H (12). Chinese hamster ovary (CHO) dhfr Internalization--
The expression vector with a fusion protein
of human TGR5 and green fluorescent protein (TGR5-GFP), pAKKO-TGR5-GFP,
was constructed by the insertion of a fused DNA so that the human TGR5-
and GFP-coding regions were connected in tandem. Mock CHO cells seeded
onto chambered coverglasses (Nalgene) were transfected with
pAKKO-TGR5-GFP and cultured overnight. After treatment with 50 µM taurine-conjugated lithocholic acid (TLCA) for 30 min,
the cells were examined under a confocal fluorescence microscope.
Cells Stably Expressing TGR5--
CHO cells expressing human
TGR5 (CHO-TGR5) cells were established by transfecting pAKKO-TGR5 into
CHO dhfr Extracellular Signal-regulated Kinase
Mitogen-activated Protein (MAP) Kinase Activation Assay--
CHO-TGR5
and mock CHO cells were cultured in a medium containing 0.5% dialyzed
fetal bovine serum and then additionally cultured overnight in a medium
containing 0.1% bovine serum albumin. The cells were preincubated with
fresh medium for 3 h and then exposed to TLCA at 2 µM. Western blotting was performed with a PhosphoPlus p44/42 MAP kinase (Thr-202/Tyr-204) antibody kit (Cell Signaling Technology).
Guanosine 5'-O-3-thio-triphosphate (GTP cAMP Production Assay--
CHO-TGR5 cells (2 × 104) were incubated with the samples for 20 min in the
presence of 0.2 mM 3-isobutyl-1-methylxanthine (Sigma). Rabbit adherent alveolar macrophage cells (AMs) (2 × 105 cells) were treated with TLCA (200 µM)
for 4 min in the presence of 1 mM
3-isobutyl-1-methylxanthine. THP-TGR5 or THP-1 cells (1 × 105 cells) were treated with bile acids (50 µM) for 20 min in the presence of 1 mM
3-isobutyl-1-methylxanthine. The amount of cAMP was determined with a
cAMP-Screen System (Applied Biosystems).
Quantitative Reverse Transcription-PCR--
Poly(A)+
RNAs from human tissues and a human blood fraction multiple tissue
cDNA panel were purchased from Clontech.
After a 48-h culture, AMs in the culture plates were washed twice with fresh medium. Total RNAs were extracted from the adherent cells or
rabbit tissues using an Isogen (Nippongene). Random-primed cDNAs
were synthesized and then subjected to quantitative reverse transcription-PCR analysis using an ABI Prism 7700 sequence detector (14).
Phagocytosis and Cytokine Secretion Assays--
AMs were
obtained by the lavage of lungs of female New Zealand White rabbits
weighing 2.5-3.0 kg (Kitayama LABES), purified through gradient
centrifugation with a Ficoll-Paque Plus (Amersham Pharmacia), and then
suspended in Dulbecco's modified Eagle's medium containing 2% fetal
bovine serum, nonessential amino acids, and antibiotics. The viability
of the cells was more than 95% as determined by trypan blue-exclusion
tests. The cells were comprised of more than 90% macrophages as
determined by phagocytic tests and morphological criteria. Rabbit AMs
thereby obtained were cultured overnight and used for experiments.
After pretreatment with bile acids (100 µM) for 16 h, AMs were incubated with heat-inactivated yeast cells in the presence
of fresh rabbit serum for 40 min, and then the AMs containing yeast
cells were counted under a microscope. In the assay for cytokine
secretion, AMs were preincubated with bile acids for 1 h and then
treated with 1 ng/ml LPS (Escherichia coli O111:B4, Wako) in
the presence of bile acids for 12 h. THP-TGR5 or THP-1 cells were
treated as in AMs with the exception of LPS concentration at 50 ng/ml.
TNF Identification of a Specific GPCR for Bile Acids--
In searching
for GPCRs in the GenBankTM data base, we found a human
genomic DNA sequence (AC021016) coding for a novel GPCR. Based on this
sequence, we isolated a cDNA encoding the GPCR, designated as TGR5,
from human spleen cDNAs. We subsequently isolated TGR5 cDNAs in
various species. Human TGR5 shared 86, 90, 82, and 83% amino acid
identity, respectively, with that in bovine, rabbit, rat, and mouse
(Fig. 1). Among the known
GPCRs, TGR5 shared at most 30, 29, 26, and 25% amino acid identity
with EDG6, EDG8, EDG1, and EDG7 (16), respectively. We thus began
studies to identify ligands for TGR5 as an orphan GPCR. Although we
previously reported a strategy to identify ligands for orphan GPCRs by
detecting signal transduction (17, 18), in this study we employed a new
method. We co-transfected a reporter gene (cAMP-responsive element
fused to luciferase gene (pCRE-Luc, Clontech)) and
expression vectors of human TGR5 and rat G protein
Although TGR5 was suggested to be a GPCR based on its sequence (Fig.
1), we expressed TGR5-GFP in CHO cells and then examined its
subcellular localization (19, 20). In the absence of a ligand, TGR5-GFP
was typically localized at the plasma membrane (Fig.
2A, left panel) but
internalized into the cytoplasm in response to TLCA (Fig.
2A, right panel). To confirm further that the
interaction of TLCA and TGR5 occurred in the plasma membrane, we
prepared membrane fractions from CHO-TGR5 and examined
[35S]GTP Tissue Distribution of TGR5 mRNA--
In our preliminary
experiments, the expression levels of TGR5 mRNA were high in human,
bovine, and rabbit but very low in rat and mouse (data not shown). We
therefore analyzed its tissue distribution in human and rabbit by
reverse transcription-PCR. High levels of TGR5 mRNA were detected
in human placenta and spleen, whereas moderate levels were found in
various other tissues including lung and fetal liver (Fig.
4A). In fractionated human
leukocytes, TGR5 mRNA was detected mainly in the resting
CD14+ monocytes (Fig. 4B). Among rabbit tissues,
the highest level of TGR5 mRNA was detected in the spleen (Fig.
4C). We also detected a high level of TGR5 mRNA in AMs,
indicating that at least one of the major cells expressing TGR5 is a
monocyte/macrophage. We therefore used rabbit AMs in the following
experiments.
Analyses for Role of TGR5 in Macrophages--
An increase of
intracellular cAMP reportedly results in the suppression of
LPS-stimulated cytokine production in macrophages (23). In addition,
CD14 has been shown to function as the LPS receptor (24). Because, as
demonstrated above, bile acids were supposed to affect macrophage
functions via TGR5, we examined this point. TLCA was found to increase
cAMP production in AMs (Fig.
5A). TLCA, glycolithocholic
acid, and LCA all significantly suppressed phagocytic activity in AMs
(Fig. 5B). Furthermore, TLCA greatly reduced the induction
of cytokine mRNAs (i.e. TNF We have isolated a novel GPCR, TGR5, on the basis of sequence
information of the databases. TGR5 was found to be identical to
hGPCR19, which has been very recently reported by another group (25).
However, the ligands and functions of this receptor have been
unidentified. In this paper, we have demonstrated that TGR5 functions
as a cell surface receptor responsive to bile acids as agonists.
Although nuclear receptors for bile acids have been reported, we
believe this is the first report on the identification of a GPCR
responsive to bile acids. We have found that the primary structures and
responsiveness to bile acids are highly conserved in TGR5 among human,
bovine, rabbit, rat, and mouse, suggesting that TGR5 has some important
physiological functions. We tried to demonstrate a direct binding of
[3H]TLCA to the membrane fractions of CHO-TGR5 but failed
because [3H]TLCA showed high nonspecific binding to
various substrates and cell membrane fractions (data not shown). We
think that synthetic compounds with high affinity to TGR5 will be
required to demonstrate the direct binding of a ligand to TGR5 in
future studies. However, instead of that, we demonstrated that TGR5
functions as a cell surface receptor responsive to bile acids on the
basis of several lines of evidence. By visualization using a fusion
protein of TGR5 and GFP, we found that the fusion protein was
apparently localized at the membrane of CHO cells, and bile acids
induced the internalization of the fusion protein from the cell
membrane to the cytoplasm. Furthermore, we demonstrated that
[35S]GTP Some lipid mediators reportedly have not only nuclear receptors but
also cell surface receptors (26). However, our research indicates that
the nuclear and cell surface bile acid receptors possess distinctive
functions. For example, taurine- or glycine-conjugated forms of bile
acids showed agonistic activity on TGR5. However, they are reportedly
inactive to the nuclear receptors in the absence of a specific
transporter, even though bile acids usually exist as conjugated forms.
In addition, the effective doses of the bile acids were lower for TGR5
than for the nuclear receptors (i.e. EC50 > 10 µM). Finally, the tissue distribution of TGR5 mRNA
differed from those of the nuclear receptors; high levels of TGR5
mRNA were detected in the placenta, spleen, and
monocytes/macrophages, whereas the nuclear receptors are mainly
expressed in the liver, kidney, and intestine (2-5).
Although immunosuppressive effects of bile acids have been reported
(6-11), the precise mechanisms have remained unclear. The phagocytic
capacity of the macrophages including Kupffer cells is depressed in
cholestasis or obstructive jaundice (8). Furthermore, bile acids
including DCA and CDCA have been reported to suppress LPS-induced
production of cytokines in macrophages, including IL-1, IL-6, and
TNF Although our results suggest that TGR5 plays a role in the regulation
of macrophage functions by bile acids, we do not rule out the
possibility that TGR5 has other unknown important functions, because
TGR5 mRNA is widely distributed not only in lymphoid tissues but
also in other tissues. Our findings that TGR5 is responsive to bile
acids will give an important clue in revealing the physiological functions of TGR5 in future studies.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase, the rate-limiting enzyme in bile acid synthesis (2,
3). The activation of pregnane X receptor by bile acids results in both
the repression of cholesterol 7
-hydroxylase and the transcriptional
induction of cytochrome P450 3a, the bile acid-metabolizing enzyme (4,
5). However, no cell surface receptor for bile acids has yet been
identified. In hepatobiliary diseases including obstructive jaundice,
viral hepatitis, and primary biliary cirrhosis, the mean serum
concentration of bile acids exceeds 100 µM (range,
70-400 µM), whereas normally this remains below 10 µM (6). At such high concentrations, bile acids are known
to exhibit immunosuppressive effects on cell-mediated immunity and
macrophage functions (6-8). The phagocytic capacity of the
reticuloendothelial system including Kupffer cells is depressed in
cholestasis or obstructive jaundice (8). Cholestatic jaundice frequently causes infectious complications and endotoxemia,
which are closely related to elevated serum bile acid levels (7, 9).
Furthermore, bile acids including deoxycholic acid
(DCA)1 and chenodeoxycholic
acid (CDCA) have been demonstrated to have inhibitory activities on the
lipopolysaccharide (LPS)-induced production of cytokines in
macrophages, including interleukin (IL)-1, IL-6, and tumor necrosis
factor
(TNF
) (10, 11). However, the precise mechanisms involved
have remained unclear. Here we show that a novel G
protein-coupled receptor (GPCR), TGR5, is responsive to bile acids and
discuss the possibility that bile acids suppress macrophage functions
via TGR5.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells
stably transfected with only pAKKO-111H (mock CHO cells) were cultured
in a medium and used as host cells. TGR5, luciferase, and
Gi were transiently expressed in the host cells by
co-transfection using a LipofectAMINE 2000 (Invitrogen). After culture
overnight, the cells were incubated with test compounds for 4 h.
Luciferase activity was measured with a PicaGene LT.2.0 (Toyo Ink).
cells (12). THP-1 cells expressing human TGR5
(THP-TGR5) cells were established by transfecting pcDNA 3.1 (Invitrogen) inserted with human TGR5 cDNA and selecting
neomycin-resistant cells.
S) Binding
Assay--
Membrane fractions prepared from CHO-TGR5 and mock CHO
cells as described elsewhere (13) were suspended at 500 µg/ml in a
binding buffer (pH 7.4) containing 50 mM Tris, 150 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 30 µM GDP, and 0.05% CHAPS. The
membrane fractions (196 µl) were mixed with TLCA (2 µl of dimethyl
sulfoxide solution) and 100 nM [35S]GTP
S
(Amersham Biosciences) (2 µl). After incubation at 25 °C for 60 min, the reaction mixtures were diluted with 1.8 ml of a chilled
washing buffer, which was a modified binding buffer without GDP, and
then filtered through nitrocellulose filters (Schleicher & Schuell).
The filters were washed with 1.8 ml of the washing buffer, dried, and
subjected to a liquid scintillation counter to measure
[35S]GTP
S bound to the membrane fractions.
concentrations (which could be neutralized by the anti-TNF
antibody) in the supernatants were measured by bioassay using L929
cells (15).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit
Gi (to reduce the basal level of luciferase) into CHO
cells. We then screened more than 1,000 compounds by measuring
luciferase activities induced in response to intracellular cAMP
production and detected specific increases due to bile acids
including TLCA, lithocholic acid (LCA), DCA, and CDCA at 25 µM. In addition, we confirmed that TGR5 derived from not
only human but also the other all species examined responded similarly
to these compounds in this assay (data not shown), suggesting that TGR5
functions as a receptor for bile acids commonly in mammals.
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Fig. 1.
Amino acid sequences of human, bovine,
rabbit, rat, and mouse TGR5. Residues identical in at least two
sequences are boxed. The predicted seven-transmembrane
domains (TM1-7) are indicated in bars above the
sequences (27). The nucleotide and amino acid
sequence data for human, bovine, rabbit, rat, and mouse TGR5 cDNAs
appear in the DDBJ/EMBL/GenBankTM data base with accession
numbers AB089307, AB089306, AB089309, AB089310, and AB089308,
respectively.
S binding to these fractions (Fig.
2B). Significant levels of the binding were detected at 1 µM TLCA. They reached 4-5 times the basal level at
10-100 µM TLCA in a dose-dependent manner. However, such increases in [35S]GTP
S binding were not
detected in the membrane fractions of mock CHO cells. Extracellular
signal-regulated kinase MAP kinase is reportedly activated in the
signal transduction of GPCRs (20, 21). Treatment with TLCA rapidly
increased extracellular signal-regulated kinase MAP kinase activity in
CHO-TGR5 cells but not in mock CHO cells (Fig. 2C). In
addition we found that TLCA, LCA, DCA, CDCA, and cholic acid (CA)
dose-dependently induced the production of cAMP in CHO-TGR5
cells (Fig. 3A) at the median
effective concentrations (EC50) of 0.33, 0.53, 1.01, 4.43, and 7.72 µM, respectively. These bile acids did not
induce the production of cAMP in mock CHO cells (data not shown). We
examined various cholesterol metabolites and related compounds in cAMP
production in CHO-TGR5 cells (Fig. 3B). The agonistic
activities seen appeared to increase in accordance with hydrophobicity
and not only free forms but also taurine and glycine conjugates
were active. Ursodeoxycholic acid and cholesterol were only slightly
active, but pregnandione showed significant activity. These results
suggest that the hydroxy groups as well as the 5
-cholanic acid
structure are important for the ligands to exhibit agonistic activity
on TGR5.
(E)-([tetrahydrotetramethylnaphthalenyl]propyl)benzoic acid (TTNPB), rifampicin, and 22(R)-hydroxysterol,
which are potent agonists for farnesoid X receptor, pregnane X
receptor, and liver X receptor, respectively (3, 4, 22), showed little
activity to TGR5. When we compared stable CHO cell lines expressing
various receptors, TLCA induced a response to TGR5 but not to EDG6,
EDG7, or EDG8 (data not shown). Altogether, our results unequivocally demonstrate that TGR5 functions as a specific cell surface receptor for
bile acids.
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Fig. 2.
TGR5 as a specific cell surface receptor for
bile acids. A, internalization of TGR5 induced by TLCA.
The left panel shows CHO cells expressing TGR5-GFP. The
right panel shows CHO cells expressing TGR5-GFP after
treatment with TLCA (50 µM) for 30 min. Bars
indicate 4 µm. B, TLCA-induced [35S]GTP S
binding to membrane fractions of CHO-TGR5. Binding of
[35S]GTP
S to TGR5-CHO cell (
) and mock CHO
cell (
) membrane fractions was determined in the binding buffer
containing 30 µM GDP and the indicated concentrations of
TCLA. The increase in [35S]GTP
S binding was indicated
as ratios of total binding to basal binding. Data represent the
mean ± S.E. in three independent experiments of triplicate
assays. C, extracellular signal-regulated kinase MAP kinase
activation in CHO-TGR5 cells by TLCA. CHO-TGR5 or mock CHO cells were
subjected to Western blot analysis after treatment with TLCA (2 µM) for the indicated periods.
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Fig. 3.
Promotion of cAMP production in CHO-TGR5
cells by bile acids. A, dose-responsive analyses for
cAMP production induced by bile acids. The inset shows the
chemical structure of major bile acids. B, comparison of
cAMP production stimulatory activities in bile acids and in related
compounds. CHO-TGR5 cells were treated with the indicated compounds at
2 µM. T, taurine-conjugated;
G, glycine-conjugated; F, free. Data represent
the mean values ± S.E. (n = 3) of percentages in
cAMP production in LCA at 10 µM. UDCA,
ursodeoxycholic acid; TTNPB,
(E)-([tetrahydrotetramethylnaphthalenyl]propyl)benzoic
acid.
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Fig. 4.
Distribution of TGR5 mRNA.
A, expression of TGR5 mRNA in human tissues.
B, expression of TGR5 mRNA in fractionated human
leukocytes. C, tissue distribution of TGR5 mRNA in
rabbit tissues. Poly(A)+ or total RNA preparations were
subjected to quantitative reverse transcription-PCR using a ABI Prism
7700 sequence detector. Each column represents the mean
value in duplicate determinations.
, IL-1
, IL-1
, IL-6,
and IL-8) in AMs stimulated with LPS (Fig. 5C). Finally,
LPS-induced TNF
secretion was significantly reduced with LCA, DCA,
and CDCA and their taurine- or glycine-conjugated forms (Fig.
5D). These relative inhibitory activities mostly agreed with
the cAMP production stimulatory activities observed on CHO-TGR5 cells.
To determine whether the effects of these bile acids were exhibited
through TGR5, we established a stable human monocytic cell line
expressing TGR5 by transfecting an expression vector of human TGR5 into
THP-1 cells. The original THP-1 cells expressed little TGR5 mRNA.
TLCA, LCA, and DCA significantly induced cAMP production in THP-TGR5
cells, whereas TLCA did not do so in THP-1 cells (Fig.
6A). LPS-stimulated TNF
secretion was markedly reduced by bile acids including TLCA, LCA, DCA,
and CDCA in THP-TGR5 cells but not in THP-1 cells (Fig. 6, B
and C). Notably, the relative inhibitory activities of bile
acids on TNF
secretion from THP-TGR5 cells almost paralleled those
seen in rabbit AMs.
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Fig. 5.
Immunosuppression by bile acids in rabbit
AMs. A, increase in cAMP production in AMs by TLCA.
B, suppression of phagocytosis in AMs by bile acids.
C, suppression of cytokine mRNA expression in AMs by
TLCA. After pretreatment with TLCA (50 µM) for 1 h,
AMs were incubated with LPS (1 ng/ml) for 2 h in the presence or
absence of TLCA (50 µM). D, suppression of
LPS-induced TNF secretion from AMs by bile acids. After treatment
with bile acids, AMs were incubated with LPS (1 ng/ml) for 12 h in
the presence or absence of bile acids. Data represent the mean
values ± S.E. (n = 3). **, p < 0.01, compared with control (Student's t test).
T, taurine-conjugated; G,
glycine-conjugated; F, free.
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Fig. 6.
Immunosuppression by bile acids via TGR5 in
THP-1 cells expressing TGR5. A, increase in cAMP
production in THP-TGR5 or THP-1 cells by bile acids. B,
suppression of LPS-induced TNF secretion in THP-TGR5 cells by bile
acids. C, effect of bile acids on LPS-induced TNF
secretion in THP-1 cells. THP-TGR5 or THP-1 cells were treated as in
Fig. 5D with the exception of LPS concentration at 50 ng/ml.
Data represent the mean values ± S.E. (n = 3).
**, p < 0.01, compared with control (Student's
t test). T, taurine-conjugated; F,
free.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S binding were specifically induced in the
membrane fractions prepared from CHO-TGR5 by TLCA. Because the
replacement of GDP and GTP
S is specifically induced in G proteins
coupling to GPCRs, our results indicate that TGR5 is specifically
activated by TLCA. Taken together with the results of internalization
and [35S]GTP
S binding, TGR5 is thought to be directly
responsive to bile acids. The treatment of bile acids specifically
induced the activation of extracellular signal-regulated kinase MAP
kinase and intracellular cAMP production in CHO cells expressing TGR5. However, we could not detect any apparent change in intracellular Ca2+ in CHO cells expressing TGR5, suggesting that TGR5
couples to G
s but not to G
q or
G
i.
(10, 11). One possible explanation for the immunosuppression is
that bile acids might give damage to cell membranes. However, we
confirmed that cell viabilities were more than 90% even after the
treatment of rabbit AMs with bile acids up to 200 µM. It
has been reported that cell viabilities of lymphocytes are not affected
by the incubation with 250 µM DCA, CDCA, and
ursodeoxycholic acid (6). Taken together, it is unlikely that the
immunosuppressive functions of bile acids are the results of damage to
cell membrane. Greve et al. (10) demonstrate that bile acids
such as DCA and CDCA inhibit LPS-induced TNF
secretion in human
lymphocytes (10). They have demonstrated that these bile acids do not
inactivate endotoxin directly, as measured in a chromogenic Limulus
test, indicating that the effect of bile acids is not a result of
direct interaction between bile acids and LPS. In our experiments, bile
acids induced cAMP production in rabbit AMs and THP-TGR5 cells. It has
been known that an increase of intracellular cAMP results in the
suppression of LPS-stimulated cytokine production in macrophages (23).
We showed here that TGR5 was abundantly expressed in
monocytes/macrophages and that bile acids including LCA, DCA, and CDCA
inhibited LPS-stimulated TNF
secretion in rabbit AMs. In addition,
these bile acids clearly suppressed LPS-stimulated TNF
secretion in
THP-TGR5 cells but not in parental THP-1 cells. These results suggest
that the suppression of macrophage functions by bile acids is at least
partly mediated via TGR5 through an increase of cAMP production.
However, we could not directly demonstrate that the suppression of
macrophage functions was mediated via TGR5 by means of loss-of-function
experiments. To confirm the physiological functions of TGR5, we tried
to design small interfering RNA for TGR5 to knock out the TGR5
functions, but we failed to obtain effective small interfering RNAs
because TGR5 is encoded by a GC-rich sequence so that it was very
difficult to design proper small interfering RNAs. We actually designed five different small interfering RNAs, but all of them were ineffective to suppress the expression of TGR5. We think that to solve this issue
synthetic antagonists with high affinity will be necessary.
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ACKNOWLEDGEMENTS |
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We thank Drs. Y. Sumino, O. Nishimura, and H. Onda for helpful discussions and Dr. H. Komatsu and A. Katano for collaboration.
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FOOTNOTES |
---|
* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB089307, AB089306, AB089309, AB089310, and AB089308, respectively.
To whom correspondence should be addressed. Tel.: 81-298-64-5035;
Fax: 81-298-64-5000; E-mail: Hinuma_Shuji@takeda.co.jp.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M209706200
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ABBREVIATIONS |
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The abbreviations used are:
DCA, deoxycholic
acid;
CDCA, chenodeoxycholic acid;
LPS, lipopolysaccharide;
IL, interleukin;
TNF, tumor necrosis factor
;
GPCR, G protein-coupled
receptor;
CHO cells, Chinese hamster ovary cells;
TGR5-GFP, a fusion
protein of human TGR5 and green fluorescent protein;
TLCA, taurine-conjugated lithocholic acid;
CHO-TGR5 cells, CHO cells
expressing human TGR5;
THP-TGR5 cells, THP-1 cells expressing human
TGR5;
MAP kinase, mitogen-activated protein kinase;
AMs, adherent
alveolar macrophage cells;
LCA, lithocholic acid;
CA, cholic acid;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid;
GTP
S, guanosine 5'-3-O-(thio)triphosphate.
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