A G Protein-coupled Receptor Responsive to Bile Acids

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. 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 (1Russell D.W. Setchell K.D.R. Biochemistry. 1992; 31: 4737-4749Google Scholar). Farnesoid X receptor and pregnane X receptor have been recently identified as specific nuclear receptors for bile acids (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Google Scholar, 3Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Google Scholar, 4Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Google Scholar, 5Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Google Scholar). Through the activation of farnesoid X receptor bile acids repress the expression of cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid synthesis (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Google Scholar,3Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Google Scholar). The activation of pregnane X receptor by bile acids results in both the repression of cholesterol 7α-hydroxylase and the transcriptional induction of cytochrome P450 3a, the bile acid-metabolizing enzyme (4Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Google Scholar,5Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Google Scholar). 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 (6Keane R.M. Gadacz T.R. Munster A.M. Birmingham W. Winchurch R.A. Surgery. 1984; 95: 439-443Google Scholar). At such high concentrations, bile acids are known to exhibit immunosuppressive effects on cell-mediated immunity and macrophage functions (6Keane R.M. Gadacz T.R. Munster A.M. Birmingham W. Winchurch R.A. Surgery. 1984; 95: 439-443Google Scholar, 7Kimmings A.N. van Deventer S.J.H. Obertop H. Rauws E.A.J. Gouma D.J. J. Am. Coll. Surg. 1995; 181: 567-581Google Scholar, 8Drivas G. James O. Wardle N. Br. Med. J. 1976; 26: 1568-1569Google Scholar). The phagocytic capacity of the reticuloendothelial system including Kupffer cells is depressed in cholestasis or obstructive jaundice (8Drivas G. James O. Wardle N. Br. Med. J. 1976; 26: 1568-1569Google Scholar). Cholestatic jaundice frequently causes infectious complications and endotoxemia, which are closely related to elevated serum bile acid levels (7Kimmings A.N. van Deventer S.J.H. Obertop H. Rauws E.A.J. Gouma D.J. J. Am. Coll. Surg. 1995; 181: 567-581Google Scholar, 9Pain J.A. Cahill C.J. Bailey M.E. Br. J. Surg. 1985; 72: 942-945Google Scholar). Furthermore, bile acids including deoxycholic acid (DCA) 1The abbreviations used are: DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LPS, lipopolysaccharide; IL, interleukin; TNFα, tumor necrosis factor α; 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; GTPγS, guanosine 5′-3-O-(thio)triphosphate 1The abbreviations used are: DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; LPS, lipopolysaccharide; IL, interleukin; TNFα, tumor necrosis factor α; 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; GTPγS, guanosine 5′-3-O-(thio)triphosphate 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 α (TNFα) (10Greve J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar, 11Calmus Y. Guechot J. Podevin P. Bonnefis M.T. Giboudeau J. Poupon R. Hepatology. 1992; 16: 719-723Google Scholar). 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. 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 (12Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Google Scholar). 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). 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. CHO cells expressing human TGR5 (CHO-TGR5) cells were established by transfecting pAKKO-TGR5 into CHO dhfr− cells (12Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Google Scholar). 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. 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). Membrane fractions prepared from CHO-TGR5 and mock CHO cells as described elsewhere (13Ohtaki T. Ogi K. Masuda Y. Mitsuoka K. Fujiyoshi Y. Kitada C. Sawada H. Onda H. Fujino M. J. Biol. Chem. 1998; 273: 15464-15473Google Scholar) 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]GTPγS (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]GTPγS bound to the membrane fractions. 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 mm3-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 mm3-isobutyl-1-methylxanthine. The amount of cAMP was determined with a cAMP-Screen System (Applied Biosystems). 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 (14Fujii R. Fukusumi S. Hosoya M. Kawamata Y. Habara Y. Hinuma S. Sekiguchi M. Kitada C. Kurokawa T. Nishimura O. Onda H. Sumino Y. Fujino M. Regul. Pept. 1999; 83: 1-10Google Scholar). 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. TNFα concentrations (which could be neutralized by the anti-TNFα antibody) in the supernatants were measured by bioassay using L929 cells (15Evans T.J. Mol. Biotechnol. 2000; 15: 243-248Google Scholar). 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 (16Fukushima N. Ishii I. Contos J.J.A. Weiner J.A. Chun J. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 507-534Google Scholar), 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 (17Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Google Scholar, 18Hinuma S. Onda H. Fujino M. J. Mol. Med. 1999; 77: 495-504Google Scholar), 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 α 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. 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 (19Kallal L. Benovic J.L. Trends Pharmacol. Sci. 2000; 21: 175-180Google Scholar, 20Xu Y. Zhu K. Hong G. Wu W. Baudhuin L.M. Xiao Y. Damron D.S. Nat. Cell Biol. 2000; 2: 261-267Google Scholar). In the absence of a ligand, TGR5-GFP was typically localized at the plasma membrane (Fig.2 A, left panel) but internalized into the cytoplasm in response to TLCA (Fig.2 A, 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]GTPγS binding to these fractions (Fig.2 B). 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]GTPγS 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 (20Xu Y. Zhu K. Hong G. Wu W. Baudhuin L.M. Xiao Y. Damron D.S. Nat. Cell Biol. 2000; 2: 261-267Google Scholar, 21English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Google Scholar). Treatment with TLCA rapidly increased extracellular signal-regulated kinase MAP kinase activity in CHO-TGR5 cells but not in mock CHO cells (Fig. 2 C). 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. 3 A) 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. 3 B). 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 5β-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 (3Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Google Scholar, 4Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Google Scholar, 22Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Google Scholar), 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.Figure 3Promotion 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.View Large Image Figure ViewerDownload (PPT) 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.4 A). In fractionated human leukocytes, TGR5 mRNA was detected mainly in the resting CD14+ monocytes (Fig. 4 B). Among rabbit tissues, the highest level of TGR5 mRNA was detected in the spleen (Fig.4 C). 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. An increase of intracellular cAMP reportedly results in the suppression of LPS-stimulated cytokine production in macrophages (23Yoshimura T. Kurita C. Nagao T. Usami E. Nakao T. Watanabe S. Kobayashi J. Yamazaki F. Tanaka H. Inagaki N. Nagai H. Pharmacology. 1997; 54: 144-152Google Scholar). In addition, CD14 has been shown to function as the LPS receptor (24Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Google Scholar). 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.5 A). TLCA, glycolithocholic acid, and LCA all significantly suppressed phagocytic activity in AMs (Fig. 5 B). Furthermore, TLCA greatly reduced the induction of cytokine mRNAs (i.e. TNFα, IL-1α, IL-1β, IL-6, and IL-8) in AMs stimulated with LPS (Fig. 5 C). Finally, LPS-induced TNFα secretion was significantly reduced with LCA, DCA, and CDCA and their taurine- or glycine-conjugated forms (Fig.5 D). 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.6 A). LPS-stimulated TNFα 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, Band C). Notably, the relative inhibitory activities of bile acids on TNFα secretion from THP-TGR5 cells almost paralleled those seen in rabbit AMs.Figure 6Immunosuppression 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 TNFα secretion in THP-TGR5 cells by bile acids. C, effect of bile acids on LPS-induced TNFα secretion in THP-1 cells. THP-TGR5 or THP-1 cells were treated as in Fig. 5 D 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'st test). T, taurine-conjugated; F, free.View Large Image Figure ViewerDownload (PPT) 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 (25Takeda S. Kadowaki S. Haga T. Takaesu H. Mitaku S. FEBS Lett. 2002; 520: 97-101Google Scholar). 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]GTPγS binding were specifically induced in the membrane fractions prepared from CHO-TGR5 by TLCA. Because the replacement of GDP and GTPγS 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]GTPγS 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 Gαs but not to Gαq or Gαi. Some lipid mediators reportedly have not only nuclear receptors but also cell surface receptors (26Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Google Scholar). 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 (2Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Google Scholar, 3Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Google Scholar, 4Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Google Scholar, 5Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Google Scholar). Although immunosuppressive effects of bile acids have been reported (6Keane R.M. Gadacz T.R. Munster A.M. Birmingham W. Winchurch R.A. Surgery. 1984; 95: 439-443Google Scholar, 7Kimmings A.N. van Deventer S.J.H. Obertop H. Rauws E.A.J. Gouma D.J. J. Am. Coll. Surg. 1995; 181: 567-581Google Scholar, 8Drivas G. James O. Wardle N. Br. Med. J. 1976; 26: 1568-1569Google Scholar, 9Pain J.A. Cahill C.J. Bailey M.E. Br. J. Surg. 1985; 72: 942-945Google Scholar, 10Greve J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar, 11Calmus Y. Guechot J. Podevin P. Bonnefis M.T. Giboudeau J. Poupon R. Hepatology. 1992; 16: 719-723Google Scholar), the precise mechanisms have remained unclear. The phagocytic capacity of the macrophages including Kupffer cells is depressed in cholestasis or obstructive jaundice (8Drivas G. James O. Wardle N. Br. Med. J. 1976; 26: 1568-1569Google Scholar). 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 TNFα (10Greve J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar, 11Calmus Y. Guechot J. Podevin P. Bonnefis M.T. Giboudeau J. Poupon R. Hepatology. 1992; 16: 719-723Google Scholar). 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 (6Keane R.M. Gadacz T.R. Munster A.M. Birmingham W. Winchurch R.A. Surgery. 1984; 95: 439-443Google Scholar). Taken together, it is unlikely that the immunosuppressive functions of bile acids are the results of damage to cell membrane. Greve et al. (10Greve J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar) demonstrate that bile acids such as DCA and CDCA inhibit LPS-induced TNFα secretion in human lymphocytes (10Greve J.W. Gouma D.J. Buurman W.A. Hepatology. 1989; 10: 454-458Google Scholar). 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 (23Yoshimura T. Kurita C. Nagao T. Usami E. Nakao T. Watanabe S. Kobayashi J. Yamazaki F. Tanaka H. Inagaki N. Nagai H. Pharmacology. 1997; 54: 144-152Google Scholar). We showed here that TGR5 was abundantly expressed in monocytes/macrophages and that bile acids including LCA, DCA, and CDCA inhibited LPS-stimulated TNFα secretion in rabbit AMs. In addition, these bile acids clearly suppressed LPS-stimulated TNFα 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. We thank Drs. Y. Sumino, O. Nishimura, and H. Onda for helpful discussions and Dr. H. Komatsu and A. Katano for collaboration.