The G-Protein-coupled bile acid receptor, Gpbar1 (TGR5), negatively regulates hepatic inflammatory response through antagonizing nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) in mice†‡§
fax: 626-256-8704; and to: Wendong Huang, Ph.D., Department of Gene Regulation and Drug Discovery, Beckman Research Institute, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010. E-mail: firstname.lastname@example.org; fax: 626-256-8704
Department of Gene Regulation and Drug Discovery, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA
School of Medicine, Henan University, Kaifeng 475001, P. R. China
Potential conflicts of interest: Nothing to report.
This work was supported by the City of Hope Gastrointestinal Cancer Program (Gastrointestinal Cancer Research Pilot Fund; to Y.-D.W.) and the National Cancer Institute (1R01CA139158-01A2; to W.H.).
Gpbar1 (TGR5), a membrane-bound bile acid receptor, is well known for its roles in regulation of energy homeostasis and glucose metabolism. TGR5 also displays strong attenuation of macrophage reactivity in vitro, but the physiological roles of TGR5 in inflammatory response, and its mechanism, is unknown. Here, we demonstrate that TGR5 is a negative modulator of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB)-mediated inflammation. TGR5 activation suppresses the phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), the translocation of p65, NF-κB DNA-binding activity, and its transcription activity. Furthermore, TGR5 activation enhances the interaction of IκBα and β-arrestin2. Suppression of NF-κB transcription activity and its target gene expression by TGR5 agonist are specifically abolished by the expression of anti-β-arrestin2 small interfering RNA. These results show that TGR5 suppresses the NF-κB pathway by mediation of the interaction between IκBα and β-arrestin2. In a lipopolysaccharide (LPS)-induced inflammation model, TGR5−/− mice show more severe liver necroses and inflammation, compared with wild-type (WT) mice. Activation of TGR5 by its agonist ligand inhibits the expression of inflammatory mediators in response to NF-κB activation induced by LPS in WT, but not TGR5−/−, mouse liver. Conclusion: These findings identify TGR5 as a negative mediator of inflammation that may serve as an attractive therapeutic tool for immune and inflammatory liver diseases. (HEPATOLOGY 2011;)
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Chronic inflammation is increasingly recognized as an important component of tumorigenesis and metabolic diseases.1, 2 For example, hepatocellular carcinoma (HCC) is a prototypical inflammation-associated cancer that often occurs secondary to chronic hepatitis. Moreover, chronic inflammation is an important mediator of insulin resistance and type 2 diabetes in obese individuals.2 Thus, the precise control of inflammation is essential for the prevention of chronic inflammatory disorders, including many types of cancers and metabolic disorders.3, 4
Nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) has received considerable attention as a key regulator of immunity, inflammation, and carcinogenesis.1, 5 The classic NF-κB consists of a p65 (RelA) and p50 heterodimer sequestered in the cytoplasm of unstimulated cells by its assembly with the inhibitor, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα). Upon stimulation with proinflammatory ligands, such as lipopolysaccharide (LPS) and tumor necrosis factor (TNF), IκBα is phosphorylated by IκB kinase (IKK) and is then subjected to ubiquitination and proteasome-mediated degradation, which results in the nuclear translocation of NF-κB and the activation of its target genes.6 Under normal conditions, NF-κB activation is transient and tightly controlled. Conversely, chronic activation of NF-κB signaling is frequently detected in numerous human inflammatory and autoimmune diseases, cancers, and diabetes.7, 8 Mounting evidence supports the notion that constitutive NF-κB activation is fundamental to the pathobiology of these human diseases.9 Therefore, defining new therapeutic targets that antagonize NF-κB signaling is crucial for further understanding the regulation of this pathway and the development of novel therapeutic strategies to inhibit prolonged activation of this pathway in these human diseases.
The bile acid receptor, Gpbar1 (TGR5), is a regulator of energy homeostasis,10 bile acid homeostasis,11 as well as glucose metabolism.12 TGR5 is a member of the G-protein-coupled receptor (GPCR) family, which contains seven transmembrane domains and transduces extracellular signals through heterotrimeric G proteins. Recent in vitro studies, using macrophages and Kupffer cells from wild-type (WT) animals, suggested that TGR5 may be involved in the suppression of macrophage and Kupffer cell functions in response to bile acid treatment.13, 14 The physiological role of TGR5 in inflammatory response, and the mechanism by which TGR5 has its immunoregulation function, is still unclear.
In this article, using a specific TGR5 agonist, we identify TGR5 as a negative regulator of NF-κB-mediated inflammation in a β-arrestin2-dependent manner, and demonstrate that TGR5 ligands have utility in reducing LPS-induced inflammation in the liver. These findings suggest TGR5 is a potential target for therapeutic intervention in inflammatory liver diseases.
Male mice fighting are able to cause liver inflammation and liver injury.15, 16 Therefore, in this study, we used only female mice. Eight-week-old WT (C57BL/6J) and TGR5−/− female mice (on C57BL/6J background; Merck Research Laboratories, Kenilworth, NJ)17 were maintained in a pathogen-free animal facility under a standard 12-hour light-dark cycle. Mice were fed a diet containing 10 mg of 23(S)-mCDCA/kg diet or standard rodent chow for 3 days. After that, mice were fasted overnight and then injected intraperitoneally (i.p.) with a single dose of LPS (20 mg/kg) or phosphate-buffered saline (PBS), followed by feeding water ad libitum. Six hours after the injection, mice were killed, and the liver was removed for further analysis. All procedures followed National Institutes of Health (Bethesda, MD) guidelines for the care and use of laboratory animals.
Reagents and Plasmids.
Reagents and plasmids are outlined in the Supporting Information.
Cell Culture and Transient Transfection.
Cell culture and transient transfection are outlined in Supporting Information.
Isolation of Mouse Bone-Marrow–Derived Macrophages and Kupffer Cells.
Mouse bone-marrow–derived macrophages were derived according to previously published methods.18 Mouse Kupffer cells were prepared as described previously.13 Cells were pretreated with 23(S)-mCDCA (10 μM). Eighteen hours after treatment, the cells were treated with LPS (1 ng/mL), then collected for RNA isolation after a 4-hour incubation.
Analysis of Alanine Aminotransferase and Aspartate Transaminase and Histology.
Analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the staining for liver sections, is described in the Supporting Information.
RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction.
Total RNA isolation from cells and tissues and quantitative real-time polymerase chain reaction (qRT-PCR) are described in the Supporting Information.
Enzyme-Linked Immunosorbent Assay.
The method for enzyme-linked immunosorbent assay (ELISA) is described in the Supporting Information.
Protein Extract Preparation and Immunoblot Analysis.
Protein extract preparation and immunoblotting analysis are described in the Supporting Information.
Electrophoretic Mobility-Shift Assay.
HepG2 cells or mouse macrophages were transfected with p65 expression plasmid or control plasmid with or without cotransfection of TGR5 plasmid. After 24-hour transfection, cells were treated with 10 μM of 23(S)-mCDCA or dimethyl sulfoxide (DMSO) (control) for 24 hours. Finally, nuclear proteins were extracted for electrophoretic mobility-shift assay (EMSA). EMSA assays were performed as previously described.19 The following oligonucleotide was used for the EMSA assay: NF-κB-binding site; 5‘- tcgagggctggggattccccat-3’.
The plasmids, pFLAG-IκBα, pHA-β-arrestin2, and mTGR5, were cotransfected into HEK293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cell treatment and immunoprecipitation for cells and liver tissue were performed as described in the Supporting Information.
β-arrestin2 Small Interfering RNA.
β-arrestin2 siRNA and control small interfering (si)RNA were purchased from Santa Cruz Biotechnology (Santa Cruz, California) and transfected into HepG2 cells using siRNA transfection reagent (Santa Cruz Biotechnology). Cell treatment is described in the Supporting Information.
All data represent at least three independent experiments and are expressed as the mean ± standard deviation. The Student's t-test was used to calculate P values, unless stated otherwise. For multiple comparisons between groups, a two-way analysis of variance (ANOVA), followed by Bonferroni's post-hoc test, was performed. A P value less than 0.05 was considered significant.
TGR5−/− Mouse Macrophages, Primary Kupffer Cells and Hepatic Tissue Display Elevated Expression of NF-κB-Regulated Proinflammatory Genes.
TGR5 is expressed in macrophages, primary Kupffer cells, and livers.13, 14, 20 It is not expressed in hepatocytes. In this work, we found that, compared with WT controls, macrophages, primary Kupffer cells, and livers from TGR5−/− mice had elevated messenger RNA (mRNA) levels of some proinflammatory NF-κB target genes (Fig. 1A). These elevated genes include inducible nitric oxide synthase (iNOS), interferon-inducible protein-10 (IP-10), and interleukin (IL)-1α in TGR5−/− mouse macrophages; monocyte chemoattractant protein-1 (MCP-1), interferon gamma (IFN-γ), iNOS, and IP-10 in TGR5−/− mouse primary Kupffer cells and IL-1β and IFN-γ in TGR5−/− mouse livers, respectively. Protein levels of IL-1β and IFN-γ in TGR5−/− mouse livers were also elevated, compared with WT controls (Supporting Fig. 1A). These results suggest that TGR5 may be a negative modulator of hepatic inflammation.
TGR5−/− Mouse Macrophages, Primary Kupffer Cells, and Livers Are Sensitive to Activation of NF-κB.
If TGR5 is a suppressor of NF-κB-mediated inflammation, TGR5−/− mice should be more sensitive than WT mice to inflammation mediated by NF-κB. We compared the mRNA levels of proinflammatory genes in macrophages and primary Kupffer cells from WT and TGR5−/− mice after activating the NF-κB pathway with a known NF-κB pathway activator, LPS. LPS-treated TGR5−/− macrophages and primary Kupffer cells expressed higher mRNA levels of NF-κB target genes than did untreated TGR5−/− macrophages and primary Kupffer cells (MCP-1 and IFN-γ in macrophages and MCP-1, iNOS, and IP-10 in primary Kupffer cells; see Fig. 1B). This induction was considerably reduced in WT macrophages and primary Kupffer cells. We then compared the expression of proinflammatory genes in livers from both TGR5−/− and WT mice after treatment with LPS. Induction of MCP-1, IP-10, IFN-γ, and iNOS in response to LPS was significantly greater in TGR5−/− mice than WT mice (Fig. 1B) (protein levels of some proinflammatory genes in mouse livers were measured using ELISA; see Supporting Fig. 1B). The levels of some inflammatory serum markers in TGR5−/− mice were also found significantly higher than that in WT mice after treatment with LPS (Fig. 1C). Those results suggest that certain inflammatory genes are more sensitive to LPS induction in the absence of TGR5 signaling in vivo.
Levels of ALT and AST, two markers of liver injury, were also significantly increased by treatment with LPS in TGR5−/− mice, compared with WT mice (Fig. 1C). We next examined liver pathology, and found that massive inflammation was present in TGR5−/− mice, but not WT mice, after administration of LPS (Fig. 1D). We then performed F4/80 immunohistochemistry staining on liver samples to determine Kupffer cell infiltration. F4/80 is a mature tissue-macrophage marker. The population of F4/80-positive cells in TGR5−/− mouse liver was higher than WT, even without LPS treatment (Fig. 1E). After LPS administration, the numbers of F4/80-positive cells in WT and TGR5−/− mouse livers were increased with 47% and 57%, respectively. Results indicated that LPS increased Kupffer cell infiltration more significantly in TGR5−/− mouse liver. It has been well reported that LPS induces hepatocyte apoptosis in vitro and in vivo.21-24 Liver injury induced by LPS was further confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Compared with WT controls, TUNEL-positive percentage was enhanced in the livers of TGR5−/− mice after injection of LPS (Fig. 1F). Together, both in vitro and in vivo results suggest that macrophages, primary Kupffer cells, and livers from TGR5−/− mice are more sensitive to LPS-induced inflammation.
TGR5 Suppresses the Expression of NF-κB-Mediated Proinflammatory Genes Induced by LPS In Vitro and In Vivo.
We then tested whether ligand-activated TGR5 could inhibit these proinflammatory NF-κB target genes induced by LPS. 23(S)-mCDCA is a new synthetic, highly selective TGR5 agonist.25 We synthesized 23(S)-mCDCA and confirmed its TGR5-specific activity by observing the expected dose-dependent increase in cyclic adenosine monophosphate (cAMP) in WT macrophages, but not TGR5−/− macrophages, and by activation of a CRE-reporter construct only in TGR5-expressing cells (data not shown). Measurements of ALT, AST, and alkaline phosphatase (ALP) and histological staining indicated that administering 23(S)-mCDCA to mice did not induce toxic effects (data not shown). 23(S)-mCDCA treatment repressed LPS-induced IP-10 and IL-6 expression in WT macrophages, but not TGR5−/− macrophages (Fig. 2A). A similar inhibition of mRNA levels of iNOS, MCP-1, cyclooxygenase-2 (COX-2), and IL-6 by the TGR5 agonist was observed in response to stimulation with LPS in WT Kupffer cells, but not TGR5−/− Kupffer cells (Fig. 2A). Kawamata et al.14 and Keitel et al.13 observed a similar down-regulating TNF-α, IL-6, IL-1α, and IL-1β in rabbit macrophages and rat Kupffer cells upon treatment with bile acids. Furthermore, we examined the effects of the TGR5 agonist on the NF-κB pathway in vivo. Livers from WT mice that were pretreated with the TGR5 agonist, 23(S)-mCDCA, showed significantly less LPS-induced expression of IP-10, MCP-1, iNOS, and IFN-γ mRNA than did nonpretreated livers. This inhibition was abolished, or considerably reduced, in TGR5−/− mouse groups (Fig. 2B). Collectively, these results demonstrate that TGR5 plays a protective role against LPS-induced inflammation in vitro and in vivo.
Activation of TGR5 Antagonizes NF-κB Signaling.
We next tested whether TGR5 agonist inhibited NF-κB activity at the levels of gene transcription. We cotransfected HepG2 cells with a NF-κB reporter plasmid and the control plasmid, phRL-TK, and assessed the effects of the TGR5 ligand on the regulation of NF-κB reporter activity. Treatment with known NF-κB pathway activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or LPS resulted in 5.1- and 1.8-fold higher NF-κB reporter activity, respectively (Fig. 3A). NF-κB activity induced by TPA or LPS was suppressed by 23(S)-mCDCA treatment. Transfection of these cells with TGR5 inhibited NF-κB activity in the absence of ligand, suggesting that TGR5 may suppress NF-κB activity without the addition of exogenous ligand, possibly resulting from the fact that GPCRs have constitutive activity.26, 27 Addition of 23(S)-mCDCA further enhanced this repression (Fig. 3A). Furthermore, to eliminate the possibility that the compounds were affecting other pathways, we used p65 overexpression to activate the NF-κB reporter. Overexpression of p65 significantly activated the NF-κB reporter (Fig. 3B,C). NF-κB activity was inhibited by 23(S)-mCDCA in a ligand dose-dependent manner in the absence of the TGR5 expression vector (Fig. 3B). The expression of endogenous TGR5 in HepG2 cells was detected, and endogenous TGR5 function in HepG2 cells was determined by measuring cAMP levels (Supporting Fig. 2). Therefore, the TGR5 ligand suppressing NF-κB activity in the absence of TGR5 overexpression may be through activating endogenous TGR5 in HepG2 cells. Compared with that in the absence of the TGR5 expression vector, TGR5 overexpression enhanced the suppression of NF-κB activity by the TGR5 agonist (Fig. 3C). Moreover, the observed inhibition of NF-κB activity in response to activated TGR5 was proportional to the amounts of TGR5 vector. Inhibition of NF-κB transactivity by TGR5 activation was also confirmed in mouse macrophages (Fig. 3D). These results indicate that activation of TGR5 can antagonize NF-κB activity at the level of gene transcription.
The binding of NF-κB to its response elements was then examined via EMSA using nuclear extracts from HepG2 cells. TGR5 activation dramatically reduced the binding activity of NF-κB to DNA sequences induced by p65 overexpression (Fig. 3E). Results in HepG2 cells were also confirmed in mouse macrophages (Fig. 3F). These results suggest that TGR5 activation may suppress NF-κB transcriptional activity by decreasing the binding of NF-κB to its response elements.
TGR5 Inhibits Phosphorylation of IκBα and Nuclear Translocation of p65.
IκBα phosphorylation and nuclear p65 levels in HepG2 cells are shown in Fig. 4A. TGR5 activation by 23(S)-mCDCA dramatically suppressed the level of phosphorylated IκBα induced by TNF-α and almost completely abolished the nuclear translocation of p65 induced by p65 overexpression. These results were further confirmed in livers from WT and TGR5−/− mice (Fig. 4B). Increase of IκBα phosphorylation levels in response to LPS was greater in TGR5−/− mice than WT mice (Fig. 4B). In response to TGR5 ligand treatment, the increase of LPS-induced IκBα phosphorylation was completely abolished in WT mouse livers, whereas an approximately 40% decrease was observed in TGR5−/− mouse livers. TGR5 agonist administration inhibited LPS-induced nuclear p65 levels in WT mice, but not TGR5−/− mice (Fig. 4B). These results suggest that TGR5 activation inhibits both IκBα phosphorylation and p65 nuclear translocation, which may contribute to antagonize the transactivity of NF-κB.
TGR5 Antagonizes NF-κB Pathway in a β-Arrestin2-Dependent Manner.
The ubiquitously expressed protein, β-arrestin2, is a multifunctional signaling molecule. It was originally identified as a negative regulator of GPCR signaling.28 It has been demonstrated that β-arrestin2 is able to bind to NF-κB inhibitor IκBα in the cytoplasm to inhibit NF-κB activity.29, 30 In this work, we further investigated the function of β-arrestin2 in NF-κB signaling. HEK293 cells cotransfected with TGR5, β-arrestin2, and IκBα were challenged with TGR5 agonist 23(S)-mCDCA, then the cell extracts were subjected to immunoprecipitantion. TGR5 activation enhanced β-arrestin2 interaction with IκBα (Fig. 5A). These results were also confirmed in mouse livers. TGR5 ligand administration increased β-arrestin2 interaction with IκBα in WT, but not in TGR5−/− mouse livers (Fig. 5B). Knockdown of β-arrestin2 abolished the inhibition of TGR5 activation on NF-κB transactivity and its target gene expression induced by p65 overexpression (Fig. 5C-E). These results indicate that TGR5 inhibits NF-κB in a β-arrestin2-dependent manner.
GPCRs comprise the largest protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal-transduction pathways through agonist binding to an orthosteric binding site. GPCRs regulate cell migration, proliferation, differentiation, and survival and play a major role in the development and progression of many diseases, such as inflammatory diseases and cancer.31, 32 Many GPCRs induce NF-κB activation,33, 34 whereas only a few GPCRs inhibit NF-κB-mediated inflammation.35 Two GPCRs, the A2A and A2B adenosine receptors, suppress the NF-κB pathway in a specific gene- and cell-type–dependent manner.35-37 Activation of β2-adrenergic receptor, a subtype of GPCRs, inhibits NF-κB activity by means of β-arrestin interaction with IκBα.29 Our data show that TGR5 is a potential suppressor of NF-κB-dependent inflammatory response. TGR5 activation is able to enhance β-arrestin2 interaction with IκBα. TGR5 antagonizing NF-κB signaling was abolished by the expression of anti-β-arrestin2 siRNA (Fig. 5). These results suggest that TGR5 inhibits NF-κB in a β-arrestin2-dependent manner, and the inhibition of NF-κB-mediated inflammation by some GPCRs could share the same mechanism. It is interesting to study the mechanism of the TGR5-dependent β-arrestin2–IκBα interaction. We ruled out the possibility that TGR5 may interact with β-arrestin2. We also ruled out another possibility that activation of TGR5 may reduce the interaction of casein kinase II and β-arrestin2 and thus enhance the β-arrestin2–IκBα interaction.29 It will be interesting to continue the study in future work.
We noted that TGR5 activation repressed specific sets of NF-κB target genes, but not all the target genes, in response to LPS in vitro and in vivo. This phenomenon has also been observed for bile acid nuclear receptor farnesoid X receptor.38 It will be interesting to define the mechanism by which TGR5 activation inhibits NF-κB in a gene-specific manner.
NF-κB is the central transcriptional regulator of inflammatory and immune responses.39 Constitutive NF-κB activation has been implicated in the malignant progression of numerous human inflammatory diseases, metabolic diseases, cancers, and diabetes.40 Inhibiting the aberrant activation of NF-κB signaling can slow down or stop these disease processes.41, 42 In this study, our analysis results of inflammatory gene expression revealed that TGR5 has anti-inflammatory properties in the mouse liver. Our data show that TGR5 activation prevents the phosphorylation of IκBα, nuclear translocation of p65, and NF-κB DNA-binding activity.
Activation of NF-κB in Kupffer cells promotes liver cancer development through IL-6 and liver-inflammatory responses.43 Blockage of NF-κB by deletion of IKKβ in Kupffer cells, in addition to hepatocytes, strongly inhibited diethylnitrosamine-induced HCC development.43 Thus, the suppression of NF-κB might be a therapeutical strategy for treating liver cancer, because the loss of NF-κB in Kupffer cells might suppress cancer. TGR5 is highly expressed in Kupffer cells of the liver.13, 14 In this study, we demonstrated that TGR5 activation is able to strongly suppress NF-κB-induced inflammation in vitro and in vivo, which suggests that TGR5 may be a desirable therapeutic target for liver cancer treatment.
It has been reported that TGR5 could be a potential target for the treatment of diabesity and associated metabolic disorders.10, 12, 44, 45 For example, Watanabe et al. reported that TGR5 activation by bile acids induces energy expenditure in muscle and brown adipose tissue.10 Thomas et al. found that TGR5 activation improves glucose tolerance and insulin sensitivity in fat-fed mice.12 These diseases, such as obesity, insulin resistance, and type 2 diabetes, are also closely associated with chronic inflammation, characterized by abnormal cytokine production, increased acute-phase reactants, and activation of a network of inflammatory signaling pathways.4, 46, 47 Inhibition of NF-κB-related inflammation is able to improve glucose metabolism in vivo.48, 49 Here, our data show that TGR5 is a negative modulator of NF-κB-mediated inflammation. Therefore, there is a potential link between anti-inflammation and treatment of obesity and diabetes through TGR5. TGR5 may be an attractive therapeutic target for metabolic disorders through not only regulation of energy and glucose homeostasis, but also suppression of NF-κB signaling.
In conclusion, our results reveal that TGR5 is a negative regulator of NF-κB-mediated hepatic inflammation, and indicate that TGR5 ligands have utility in anti-inflammation. These findings suggest that TGR5 is a potential target for anti-inflammatory drug design, and its agonist ligands offer possible therapies to prevent and treat inflammatory liver diseases.
The authors thank Dr. Galya Vassileva in Merck Research Laboratories and Merck Research Laboratories for TGR5−/− mice, Dr. Peter Tontonoz, Dr. Bruce Blumberg, Xufeng Chen, Akio Kruoda, and Dr. Gang Pei for plasmids, and Sofia Loera for conducting the immunohistochemical staining in the Anatomic Pathology Core Facility of City of Hope.