A G Protein-coupled Receptor Responsive to Bile Acids*

Yuji Kawamata, Ryo Fujii, Masaki Hosoya, Masataka Harada, Hiromi Yoshida, Masanori Miwa, Shoji Fukusumi, Yugo Habata, Takashi Itoh, Yasushi Shintani, Shuji HinumaDagger, Yukio Fujisawa, and Masahiko Fujino

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 7alpha -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 7alpha -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 alpha  (TNFalpha ) (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

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- 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).

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- 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.

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 (GTPgamma 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]GTPgamma 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]GTPgamma S bound to the membrane fractions.

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. TNFalpha concentrations (which could be neutralized by the anti-TNFalpha antibody) in the supernatants were measured by bioassay using L929 cells (15).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  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.

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]GTPgamma 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]GTPgamma 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 5beta -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]GTPgamma S binding to membrane fractions of CHO-TGR5. Binding of [35S]GTPgamma S to TGR5-CHO cell () and mock CHO cell (open circle ) membrane fractions was determined in the binding buffer containing 30 µM GDP and the indicated concentrations of TCLA. The increase in [35S]GTPgamma 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.

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.


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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.

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. TNFalpha , IL-1alpha , IL-1beta , IL-6, and IL-8) in AMs stimulated with LPS (Fig. 5C). Finally, LPS-induced TNFalpha 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 TNFalpha 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 TNFalpha 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 TNFalpha 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 TNFalpha secretion in THP-TGR5 cells by bile acids. C, effect of bile acids on LPS-induced TNFalpha 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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]GTPgamma S binding were specifically induced in the membrane fractions prepared from CHO-TGR5 by TLCA. Because the replacement of GDP and GTPgamma 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]GTPgamma 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 Galpha s but not to Galpha q or Galpha i.

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 TNFalpha (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 TNFalpha 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 TNFalpha secretion in rabbit AMs. In addition, these bile acids clearly suppressed LPS-stimulated TNFalpha 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.

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.

    ACKNOWLEDGEMENTS

We thank Drs. Y. Sumino, O. Nishimura, and H. Onda for helpful discussions and Dr. H. Komatsu and A. Katano for collaboration.

    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.

Dagger 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

    ABBREVIATIONS

The abbreviations used are: DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LPS, lipopolysaccharide; IL, interleukin; TNFalpha , tumor necrosis factor alpha ; 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; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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