Expression and function of the bile acid receptor TGR5 in Kupffer cells

Kupffer cells are resident macrophages in the liver and play a central role in the hepatic response to injury. Bile acids can impair macrophage function leading to decreased cytokine release. TGR5 is a novel, membrane-bound bile acid receptor, and it has been suggested that the immunosuppressive effect of bile acids can be mediated by TGR5. However, the function of TGR5 in Kupffer cells has not been studied and a direct link between TGR5 and cytokine production in macrophages has not been established. The present study demonstrates that TGR5 is localized in the plasma membrane of isolated Kupffer cells and is responsive to bile acids. Furthermore, bile acids inhibited LPS-induced cytokine expression in Kupffer cells via TGR5-cAMP dependent pathways. TGR5-immunoreactivity in Kupffer cells was increased in rat livers following bile-duct ligation, suggesting that TGR5 may play a protective role in obstructive cholestasis preventing excessive cytokine production thereby reducing liver injury.     

Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1

Bile acids play essential roles in the absorption of dietary lipids and in the regulation of bile acid biosynthesis. Recently, a G protein-coupled receptor, TGR5, was identified as a cell-surface bile acid receptor. In this study, we show that bile acids promote glucagon-like peptide-1 (GLP-1) secretion through TGR5 in a murine enteroendocrine cell line STC-1. In STC-1 cells, bile acids promoted GLP-1 secretion in a dose-dependent manner. As STC-1 cells express TGR5 mRNA, we examined whether bile acids induce GLP-1 secretion through TGR5. RNA interference experiments showed that reduced expression of TGR5 resulted in reduced secretion of GLP-1. Furthermore, transient transfection of STC-1 cells with an expression plasmid containing TGR5 significantly enhanced GLP-1 secretion, indicating that bile acids promote GLP-1 secretion through TGR5 in STC-1 cells. Bile acids induced rapid and dose-dependent elevation of intracellular cAMP levels in STC-1 cells. An adenylate cyclase inhibitor, MDL12330A, significantly suppressed bile acid-promoted GLP-1 secretion, suggesting that bile acids induce GLP-1 secretion via intracellular cAMP production in STC-1 cells.     

The bile acid membrane receptor TGR5: a novel pharmacological target in metabolic,inflammatory and neoplastic disorders

Abstract

TGR5 is the G-protein–coupled bile acid-activated receptor, found in many human and animal tissues. Considering different endocrine and paracrine functions of bile acids, the current review focuses on the role of TGR5 as a novel pharmacological target in the metabolic syndrome and related disorders, such as diabetes, obesity, atherosclerosis, liver diseases and cancer. TGR5 ligands improve insulin sensitivity and glucose homeostasis through the secretion of incretins. The bile acid/TGR5/cAMP signaling pathway increases energy expenditure in brown adipose tissue and skeletal muscle. Activation of TGR5 in macrophages inhibits production of proinflammatory cytokines and attenuates the development of atherosclerosis. This receptor has been detected in many cell types of the liver where it has anti-inflammatory effects, thus reducing liver steatosis and damage. TGR5 also modulates hepatic microcirculation and fluid secretion in the biliary tree. In cell culture models TGR5 has been linked to signaling pathways involved in metabolism, cell survival, proliferation and apoptosis, which suggest a possible role of TGR5 in cancer development. Despite the fact that TGR5 ligands may represent novel drugs for prevention and treatment of different aspects of the metabolic syndrome, clinical studies are awaited with the perspective that they will complete TGR5 biology and identify efficient and safe TGR5 agonists.     

Bile acid induction of cytokine expression by macrophages correlates with repression of hepatic cholesterol 7alpha-hydroxylase

In the studies reported herein, we show that two complementary experimental models: inbred strains of mice (i.e. C57BL/6 and C3H/HeJ), and a differentiated line of rat hepatoma cells (i.e. L35 cells), require the activation of cytokines by monocyte/macrophages to display bile acid negative feedback repression of cholesterol 7alpha-hydroxylase (CYP7A1). Feeding a bile acid-containing atherogenic diet for 3 weeks to C57BL/6 mice led to a 70% reduction in the expression of hepatic CYP7A1 mRNA, whereas no reduction was observed in C3H/HeJ mice. The strain-specific response to repression of CYP7A1 paralleled the activation of hepatic cytokine expression. Studies using cultured THP-1 monocyte/macrophages showed that the hydrophobic bile acid chenodeoxycholate, a well established potent repressor of CYP7A1, induced the expression of mRNAs encoding interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFalpha). In contrast, the hydrophilic bile acid ursodeoxycholate, which does not repress CYP7A1, did not induce cytokine mRNA expression by THP-1 cells. Chenodeoxycholate activation of cytokines by THP-1 cells was blocked by the peroxisome proliferator-activated receptor gamma agonist rosiglitazone. The expression of cytokines (e.g. IL-1 and TNFalpha) by THP-1 cells paralleled with the ability of these cells to produce conditioned medium that when added to rat L35 hepatoma cells, repressed CYP7A1. Moreover, rosiglitazone, which blocks cytokine activation by macrophages, also blocked the repression of CYP7A1 normally exhibited by C57BL/6 mice fed the bile acid-containing atherogenic diet. The combined data indicate that the activation of cytokines may mediate CYP7A1 repression caused by feeding mice an atherogenic diet containing bile acids.     

Thematic Review Series: Bile Acids: Bile acids as regulatory molecules

In the past, bile acids were considered to be just detergent molecules derived from cholesterol in the liver. They were known to be important for the solubilization of cholesterol in the gallbladder and for stimulating the absorption of cholesterol, fat-soluble vitamins, and lipids from the intestines. However, during the last two decades, it has been discovered that bile acids are regulatory molecules. Bile acids have been discovered to activate specific nuclear receptors (farnesoid X receptor, preganane X receptor, and vitamin D receptor), G protein coupled receptor TGR5 (TGR5), and cell signaling pathways (c-jun N-terminal kinase 1/2, AKT, and ERK 1/2) in cells in the liver and gastrointestinal tract. Activation of nuclear receptors and cell signaling pathways alter the expression of numerous genes encoding enzyme/proteins involved in the regulation of bile acid, glucose, fatty acid, lipoprotein synthesis, metabolism, transport, and energy metabolism. They also play a role in the regulation of serum triglyceride levels in humans and rodents. Bile acids appear to function as nutrient signaling molecules primarily during the feed/fast cycle as there is a flux of these molecules returning from the intestines to the liver following a meal. In this review, we will summarize the current knowledge of how bile acids regulate hepatic lipid and glucose metabolism through the activation of specific nuclear receptors and cell signaling pathways.     

Activation of the bile acid receptor TGR5 enhances LPS-induced inflammatory responses in a human monocytic cell line

Abstract

Introduction: Bile acids are recognized as signaling molecules, mediating their effects both through the cell surface receptor TGR5 and the nuclear receptor FXR. After a meal, approximately 95% of the bile acids are transported from terminal ileum and back to the liver via the portal vein, resulting in postprandial elevations of bile acids in blood. During the digestion of fat, components from the microbiota, including LPS, are thought to reach the circulation where it may lead to inflammatory responses after binding TLR4 immune cells. Both LPS and bile acids are present in blood after a high-fat meal; we therefore wanted to study consequences of a possible interplay between TGR5 and TLR4 in human monocytes. Methods: The monocytic cell line U937 stably transfected with the NF-κB reporter plasmid 3x-κB-luc was used as a model system to study the effects of TGR5 and TLR4. Activation of MAP kinases was studied to reveal functional consequences of triggering TGR5 in U937 cells. Effects of TGR5 and TLR4 activation were monitored using NF-κB luciferase assay and by quantification of the pro-inflammatory cytokines IL-6 and IL-8 using ELISA. Results: In this study, results show that triggering TGR5 with the specific agonist betulinic acid (BA), and the bile acids CDCA or DCA, activated both the main MAP kinases ERK1/2, p38 and JNK, and the NF-κB signaling pathway. We further demonstrated that co-triggering of TLR4 and TGR5 enhanced the activation of NF-κB and the release of inflammatory cytokines in a synergistic manner compared to triggering of TLR4 alone. Conclusions: Thus, two different and simultaneous events associated with the digestive process coordinately affect the function of human monocytes and contribute to enhanced inflammation. Because elevated levels of circulatory LPS may contribute to the development of insulin resistance, the results from this study suggest that bile acids through the activation of TGR5 may have a role in the development of insulin resistance as well.     

Bile acids induce adhesion molecule expression in endothelial cells through activation of reactive oxygen species, NF-kappaB, and p38

Bile acids are synthesized in the liver, stored in gallbladder, and secreted into the intestine to aid in the absorption of lipid-soluble nutrients. In addition, bile acids also actively participate in regulation of gene expression through their ability to act as ligands for the nuclear receptor farnesoid X receptor or by activating kinase signaling pathways. Under cholestatic conditions, elevated levels of bile acids in the liver induce hepatic inflammation, and because bile acid levels are also elevated in the circulation, they might also induce vascular inflammation. To test this hypothesis, primary human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells were treated with bile acids, and the expression of ICAM-1, VCAM-1, and E-selectin were monitored. The three major bile acids found in the circulation, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, all strongly induced both the mRNA and protein expression of ICAM-1 and VCAM-1. To delineate the mechanism, the experiments were conducted in the presence of various kinase inhibitors. The results demonstrate that the bile acid-mediated induction of adhesion molecule expression occurs by stimulation of NF-kappaB and p38 MAPK signaling pathways through the elevation in reactive oxygen species. The bile acid-induced cell surface expression of ICAM-1 and VCAM-1 was sufficient to result in the increased adhesion of THP-1 monocytes to the HUVEC, suggesting that elevated levels of bile acids in the circulation may cause endothelium dysfunction and contribute to the initiation of early events associated with vascular lesion formation.     

Identification of a G protein-coupled receptor specifically responsive to beta-alanine

We isolated a cDNA encoding an orphan G protein-coupled receptor, TGR7, which has been recently reported to correspond to MrgD. To search for ligands for TGR7, we screened a series of small molecule compounds by detecting the Ca2+ influx in Chinese hamster ovary cells expressing TGR7. Through this screening, we found that beta-alanine at micromolar doses specifically evoked Ca2+ influx in cells expressing human, rat, or mouse TGR7. A structural analogue, gamma-aminobutyric acid, weakly stimulated cells expressing human or rat TGR7, but another analogue, glycine, did not. In addition, beta-alanine decreased forskolin-stimulated cAMP production in cells expressing TGR7, suggesting that TGR7 couples with G proteins Gq and Gi. In guanosine 5'-O-3-thiotriphosphate binding assays conducted using a membrane fraction of cells expressing TGR7, beta-alanine specifically increased the binding of guanosine 5'-O-3-thiotriphosphate. When a fusion protein composed of TGR7 and green fluorescent protein was expressed in cells, it localized at the plasma membrane but internalized into the cytoplasm after treatment with beta-alanine. In addition, we found that beta-[3H]alanine more efficiently bound to TGR7-expressing cells than to control cells. From these results, we concluded that TGR7 functioned as a specific membrane receptor for beta-alanine. Quantitative PCR analysis revealed that TGR7 mRNA was predominantly expressed in the dorsal root ganglia in rats. By in situ hybridization and immunostaining, we confirmed that TGR7 mRNA was co-expressed in the small diameter neurons with P2X3 and VR1, both in rat and monkey dorsal root ganglia. Our results suggest that TGR7 participates in the modulation of neuropathic pain.     

Interferon-gamma-induced apoptosis and activation of THP-1 macrophages

Apoptotic macrophages are frequently observed in human atherosclerotic lesions, and are considered to be involved in plaque instability in atherosclerosis. However, the molecular mechanism that promotes programmed cell death of macrophages in atherosclerosis remains to be elucidated. In this study, we investigated the effects of interferon-gamma (IFN-gamma), a cytokine secreted by activated T helper 1 (Th1) lymphocytes, on apoptotic cell death of THP-1 macrophages. Further we studied whether these apoptotic macrophages could be simultaneously activated in vitro and subsequently overgenerate monocyte chemoattractant protein-1 (MCP-1). When THP-1 macrophages were cultured with various concentrations of IFN-gamma, DNA synthesis was significantly decreased. IFN-gamma was found significantly to induce apoptotic cell death in THP-1 macrophages. RNase protection assay revealed that IFN-gamma up-regulated the mRNA levels of two pro-apoptotic molecules, tumor necrosis factor-alpha receptor 1 (TNFR1) and caspase-8, in THP-1 cells. Furthermore, TNF-alpha antibodies were found completely to neutralize the IFN-gamma-induced inhibition in DNA synthesis as well as apoptotic cell death in macrophages. IFN-gamma was found to activate these macrophages to stimulate MCP-1 production. The results suggest that IFN-gamma not only exerted apoptotic effects on macrophages, but also activated them and subsequently overgenerated MCP-1, and was thus involved in the development and progression of atherosclerosis.     

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

Suppression of interleukin-1 beta and LDL scavenger receptor expression in macrophages by a selective protein kinase C inhibitor.

A human monocytic cell line, THP-1, stimulated with 40 nM phorbol myristate acetate (PMA), differentiated to macrophage-like cells, and exhibited increased expression and release of interleukin-1 beta and expression of acetylated low density lipoprotein (ac-LDL) receptors. A selective inhibitor, MDL 29,152 (4-propyl-5-(4-quinolinyl)-2(3H)-oxazolone) was used to show that this induction required activation of protein kinase C. MDL 29,152 acts in the catalytic domain of protein kinase C and is at least 200-fold selective for protein kinase C over cAMP-dependent protein kinase in THP-1 cells. MDL 29,152 (50 microM) reduced levels of interleukin-1 beta mRNA in PMA-stimulated cells by 76% and eliminated detectable interleukin-1 beta in the media. Flow cytometric analysis showed that 48 h after THP-1 activation, approximately 50% of the cells expressed ac-LDL receptors, while in the presence of 100 microM MDL 29,152, less than 5% of the cells expressed receptors. The relationship between THP-1 differentiation and protein kinase C activation was determined by following the expression of the cell surface antigen MO-1. Expression of MO-1 antigen increases as monocytes differentiate to macrophages. After 48 h of phorbol activation, 90% of the THP-1 population was MO-1-positive; less than 16% of the population was MO-1-positive when 100 microM MDL 29,152 was present. By dual analysis, it was found that within the differentiated, MO-1-positive population, only approximately 50% of the cells also expressed ac-LDL receptors. Based on these findings, we conclude that protein kinase C promotes processes important in THP-1 activation and differentiation to macrophage-like cells including interleukin-1 beta expression and secretion, ac-LDL receptor and MO-1 expression.     

Viral activation of macrophages through TLR-dependent and -independent pathways

Induction of cytokine production is important for activation of an efficient host defense response. Macrophages constitute an important source of cytokines. In this study we have investigated the virus-cell interactions triggering induction of cytokine expression in macrophages during viral infections. We found that viral entry and viral gene products produced inside the cell are responsible for activation of induction pathways leading to IFN-alphabeta expression, indicating that virus-cell interactions on the cell surface are not enough. Moreover, by the use of cell lines expressing dominant negative versions of TLR-associated adaptor proteins we demonstrate that Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta is dispensable for all virus-induced cytokine expression examined. However, a cell line expressing dominant negative MyD88 revealed the existence of distinct induction pathways because virus-induced expression of RANTES and TNF-alpha was totally blocked in this cell line whereas IFN-alphabeta expression was much less affected in the absence of signaling via MyD88. In support of this, we also found that inhibitory CpG motifs, which block TLR9 signaling inhibited early HSV-2-induced TNF-alpha and RANTES expression dramatically whereas IFN-alphabeta induction was only slightly affected. This suggests that virus activates macrophages through distinct pathways, of which some are dependent on TLRs signaling through MyD88, whereas others seem to be independent of TLR signaling. Finally we demonstrate that IFN-alphabeta induction in HSV-2-infected macrophages requires a functional dsRNA-activated protein kinase molecule because cells expressing a dsRNA-dependent protein kinase version unable to bind dsRNA do not express IFN-alphabeta on infection.     

G蛋白偶联胆汁酸受体1促进胃癌细胞的增殖、迁移和侵袭的实验研究

目的:探讨G蛋白偶联胆汁酸受体1(G-protein coupled bile acid receptor 1,GPBAR1/TGR5)对胃癌细胞增殖、迁移和侵袭的影响。方法:免疫组织化学染色方法(Immunohistochemistry,IHC)检测胃癌及癌旁组织芯片中TGR5表达情况;qRT-PCR及Western blot检测胃癌细胞系中TGR5表达水平;小干扰RNA处理AGS、MKN-45胃癌细胞后构建TGR5敲减细胞系,慢病毒载体转染胃癌SGC-7901细胞构建TGR5过表达细胞系;CCK-8实验、平板克隆形成实验、裸鼠皮下移植瘤实验检测TGR5对细胞增殖的影响;流式细胞仪检测TGR5对细胞周期及凋亡的影响;Tanswell实验检测TGR5对胃癌细胞迁移及侵袭的影响;Western blot检测上皮间充质转化(Epithelial-mesenchymal transition,EMT)相关分子β-连环蛋白(β-catenin)、锌脂蛋白转录因子(Snail)、E盒结合锌指蛋白(Zinc finger E-box binding homeobox 1,ZEB)1在AGS、MKN-45及SGC-7901胃癌细胞中的表达。结果:TGR5在胃癌及癌旁组织中均有表达,胃癌组织TGR5高表达率(41.0%)显著高于癌旁组织(9.5%),伴肠化生癌旁组织TGR5高表达率(50%)显著高于不伴肠化生的癌旁组织(0%),胃癌组织TGR5表达与肿瘤大小相关。TGR5在正常人胃上皮永生化细胞株GES-1及各胃癌细胞系中均有表达。TGR5表达敲低的AGS和MKN-45细胞增殖能力减弱、凋亡率显著升高、侵袭和迁移能力显著降低。过表达TGR5的SGC-7901细胞增殖能力增强、克隆形成能力提高、凋亡率明显减低、侵袭和迁移能力显著升高。此外,TGR5过表达显著上调了间质细胞标志物β-catenin、Snail、ZEB1的表达水平。结论:TGR5能够增强胃癌细胞增殖及迁移能力,并抑制细胞凋亡。TGR5可能通过EMT途径介导胃癌细胞转移。     

Troglitazone but not conjugated linoleic acid reduces gene expression and activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophages

Gene expression and activity of matrix-metalloproteinases (MMP)-2 and -9 in macrophages are reduced through peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent inhibition of NF-kappaB. Since conjugated linoleic acids (CLAs) are PPARgamma ligands and known to inhibit NF-kappaB via PPARgamma, we studied whether CLA isomers are capable of reducing gene expression and gelatinolytic activity of MMP-2 and -9 in PMA-differentiated THP-1 macrophages, which has not yet been investigated. Incubation of PMA-differentiated THP-1 cells with either c9t11-CLA, t10c12-CLA or linoleic acid (LA), as a reference fatty acid, resulted in a significant incorporation of the respective fatty acids into total cell lipids relative to control cells (P<.05). Treatment of PMA-differentiated THP-1 cells with 10 and 20 mumol/L troglitazone but not with 10 or 100 mumol/L c9t11-CLA, t10c12-CLA or LA reduced relative mRNA concentrations and activity of MMP-2 and MMP-9 compared to control cells (P<.05). DNA-binding activity of NF-kappaB and PPARgamma and mRNA expression of the NF-kappaB target gene cPLA(2) were not influenced by treatment with CLA. In contrast, treatment of PMA-differentiated THP-1 cells with troglitazone significantly increased transactivation of PPARgamma and decreased DNA-binding activity of NF-kappaB and relative mRNA concentration of cPLA(2) relative to control cells (P<.05). In conclusion, the present study revealed that CLA isomers, in contrast to troglitazone, did not reduce gene expression and activity of MMP-2 and -9 in PMA-differentiated THP-1 macrophages, which is probably explained by the observation that CLA isomers neither activated PPARgamma nor reduced DNA-binding activity of NF-kappaB. This suggests that CLA isomers are ineffective in MMP-associated extracellular matrix degradation which is thought to contribute to the progression and rupture of advanced atherosclerotic plaques.