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Potential conflict of interest: Nothing to report.
The farnesoid X receptor (FXR) is a nuclear receptor that plays key roles in hepatoprotection by maintaining the homeostasis of liver metabolism. FXR null mice display strong hepatic inflammation and develop spontaneous liver tumors. In this report, we demonstrate that FXR is a negative modulator of nuclear factor κB (NF-κB)–mediated hepatic inflammation. Activation of FXR by its agonist ligands inhibited the expression of inflammatory mediators in response to NF-κB activation in both HepG2 cells and primary hepatocytes cultured in vitro. In vivo, compared with wild-type controls, FXR−/− mice displayed elevated messenger RNA (mRNA) levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interferon-inducible protein 10, and interferon-γ in response to lipopolysaccharide (LPS). Examination of FXR−/− livers showed massive necroses and inflammation after treatment with LPS at a dose that does not induce significant liver damage or inflammation in wild-type mice. Moreover, transfection of a constitutively active FXR expression construct repressed the iNOS, COX-2, interferon-inducible protein 10 and interferon-γ mRNA levels induced by LPS administration. FXR activation had no negative effects on NF-κB-activated antiapoptotic genes, suggesting that FXR selectively inhibits the NF-κB-mediated hepatic inflammatory response but maintains or even enhances the cell survival response. On the other hand, NF-κB activation suppressed FXR-mediated gene expression both in vitro and in vivo, indicating a negative crosstalk between the FXR and NF-κB signaling pathways. Our findings reveal that FXR is a negative mediator of hepatic inflammation, which may contribute to the critical roles of FXR in hepatoprotection and suppression of hepatocarcinogenesis. (HEPATOLOGY 2008;48:1632–1643.)
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Inflammatory responses play important roles in pathological conditions of the liver in both humans and experimental animals.1, 2 Therefore, the precise control of inflammation is essential for the prevention of chronic inflammatory disorders, as well as for inhibiting the exacerbation or progression of diseases, including many types of cancers.3 Hepatocellular carcinoma (HCC) often occurs as a secondary condition to chronic hepatitis and it is a prototypical inflammation-associated cancer.4 Recently, several reports have linked the nuclear factor κB (NF-κB) signaling pathway to HCC, thereby providing insight into the molecular mechanism by which inflammation affects HCC development.5
NF-κB has received considerable attention as a key regulator of inflammation, because activated NF-κB is frequently detected in various inflammatory diseases and tumors.4 The activation of NF-κB, which down-regulates the transcriptional activity of multiple steroid/nuclear receptors,6 is one of the critical cellular responses to acute infections and inflammations.3, 7 One of the pivotal functions of NF-κB is rapid activation in response to lipopolysacchride (LPS) or proinflammatory cytokines, which is an evolutionally conserved defensive mechanism against infections. Recent animal studies provide strong, direct genetic evidence that the classical, IKK-dependent NF-κB activation pathway is a crucial mediator of tumor promotion.4, 8 The classic NF-κB consists of a p65 (RelA) and p50 heterodimer that is activated in response to various stimuli, including LPS, tumor necrosis factor (TNF-α), double-stranded RNA, and ultraviolet radiation. Functional crosstalk between NF-κB and several other nuclear receptors—such as the nuclear steroid and xenobiotic receptor, estrogen receptor, and androgen receptor—has been reported and suggested to have different physiological significance in xenobiotic or lipid metabolisms and inflammation.2, 6
Nuclear receptors are ligand-activated transcription factors that have central roles in nearly every aspect of development and adult physiology,9 and several nuclear receptors play important roles in regulating inflammatory responses.10 For example, peroxisome proliferator-activated receptors (PPARs) and liver X receptors (LXRs) have been reported to be molecular links between lipid metabolism and inflammatory responses.11, 12 Recently, it was shown that the nuclear steroid and xenobiotic receptor SXR and NF-κB mutually suppress each other.6
Another nuclear receptor, farnesoid X receptor (FXR), is a bile acid receptor that is essential for bile acid homeostasis, as well as for normal lipid and glucose metabolism.13–15 FXR binds as a heterodimer with retinoid X receptor (RXR) and coordinates the expression of genes involved in bile acid production, efflux, and influx, as well as detoxification in the liver, which suggests that a key function of FXR is for the maintenance of bile acid homeostasis and reduction of bile acid toxicity.16 We recently showed that FXR−/− mice displayed prominent liver injury and inflammation and developed spontaneous liver tumors as they aged.17 A similar finding indicated that the expression of inflammatory genes in the liver was elevated in FXR null mice.14 We hypothesized that FXR may directly modulate liver inflammation.
We report a novel role for FXR in the control of liver inflammation—that of antagonizing the NF-κB signaling pathway. Our results identify FXR as a potential regulator of hepatic inflammation and suggest that FXR ligands may be used to treat liver inflammatory diseases and prevent hepatocarcinogenesis. On the other hand, activated NF-κB repressed the FXR signaling pathway, suggesting negative crosstalk between the FXR and NF-κB signaling pathways.
Chenodeoxycholic acid, LPS (from Escherichia coli 0111:B4) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were purchased from Sigma Chemical (St. Louis, MO). GW4064 and 6α-ethylchenodeoxycholic acid (6ECDCA) were provided by Dr. Barry M. Forman. TNF-α was purchased from R&D Systems, Inc. The phFXR, phRXR expression vectors, and FXR-dependent reporter (EcRE-LUC) were created in our laboratory. The p65 expression vector and the phRL-TK vector were kindly provided by Xufeng Chen and Akio Kruoda (both City of Hope, Duarte, CA), respectively. Estrogen receptor-α reporter plasmid and ERα expression plasmid were provided by Dr. Barry M. Forman. The NF-κB–dependent reporter (NF-κBx3-LUC) was kindly provided by Dr. Peter Tontonoz (UCLA, Los Angeles, CA) and Dr. Bruce Blumberg (UCLA, Los Angeles, CA).
Cell Culture and Transient Transfection.
Human hepatoblastoma cells (HepG2) were seeded into 6-well plates (1×106 cells/well) and grown in complete culture medium [high-glucose Dulbecco's modified Eagle's medium (with L-glutamine) supplied with 10% (vol/vol) inactivated fetal calf serum and 1% (vol/vol) antibiotics–antimycotics] as described.18 The following day, cells were treated with GW4064 (2 μM) or 6ECDCA (3 μM). Eighteen hours after treatment, the cells were treated with TPA (50 nM), LPS (1 μg/mL), or TNF-α (10 ng/mL) and then collected for RNA isolation after a 6-hour incubation.
Transient transfection of HepG2 cells was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Twenty-four hours after transfection, cells were pretreated with GW4064 (2μM) or 6ECDCA (3μM) for 18 hours, unless stated otherwise. Then cells were treated with or without LPS or TPA. Following a 6-hour incubation, cells were harvested and the luciferase activity was determined using a dual-luciferase reporter assay system in accordance with the manufacturer's instructions (Promega, Madison, WI). Luciferase activities were normalized via cotransfection of the control thymidine kinase-driven Renilla luciferase plasmid, phRL-TK. Data are expressed as relative fold activation to that of nonstimulated (−) sets.
Primary Mouse Hepatocyte Culture.
Primary hepatocytes from 8-week-old mice were prepared as described.19–21 Cells were treated with GW4064 (2μM) and 6ECDCA (3μM). Eighteen hours after treatment, cells were treated with LPS (20 μg/mL), TPA (150 nM), or TNF-α (10 ng/mL) and then collected for RNA isolation after a 6-hour incubation.
RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction.
Total RNA isolation from HepG2 cells, primary mouse hepatocytes, and mouse livers and quantitative real-time polymerase chain reaction (PCR) were performed as described.17 Amplification of β-actin was used as an internal reference. Primers sequences are available on request.
Eight-week-old mice, unless stated otherwise, were used in this work. The wild-type and FXR−/− mice (a gift from Dr. Frank Gonzalez at the National Institutes of Health)22 were maintained in a pathogen-free animal facility under a standard 12:12-hour light/dark cycle. Mice were fed standard rodent chow and water ad libitum. Eight-week-old female wild-type and FXR−/− mice were fasted overnight and then injected intraperitoneally 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 blood and livers were removed for further analysis. All procedures followed National Institutes of Health guidelines for the care and use of laboratory animals.
Adenovirus that expressed VP16 (the transactivation domain of herpes simplex virus) alone (Ad-VP16) or murine FXRα2 fused to VP16 (FXRα2-VP16; constitutively active) was provided by Dr. Peter A. Edwards (University of California, Los Angeles, CA). Adenovirus was amplified in HEK293 cells and purified with an Adeno-X Virus Purification Kit (Clontech Laboratories, Inc, Mountain View, CA). Wild-type or FXR−/− mice were injected in the tail vein with 1 × 109 plaque-forming units per mouse of either Ad-VP16 or FXRα2-VP16 (four mice per group). After 7 days, mice were fasted overnight and then injected intraperitoneally with a single dose of LPS (30 mg/kg) or PBS. After 6 hours, mice were killed and livers were removed for further analysis.
Analysis of Alanine Aminotransferase Activity and Liver Histology.
Alanine aminotransferase activity analysis, hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining were performed as described.17
The nuclear extracts of mouse liver and HepG2 cells were prepared as reported by Najima et al.23 and Bontemps et al.,24 respectively. Immunoblot analysis was performed as described.18 The samples were blotted using p65 (Cell Signaling) or Lamin B1 (Santa Cruz Biotechnology) antibodies.
Nuclear extracts of HepG2 cells were prepared as described in the Immunoblot Analysis section above. Electrophoretic mobility-shift assays (gel-shift assays) were performed as described.25, 26 The following oligonucleotide was used for the gel-shift assay: NF-κB-binding site; 5′- tcgagggctggggattccccat-3′.
All data represent at least three independent experiments and are expressed as the mean ± SD. The Student t test was used to calculate P values. A P value less than 0.05 was considered significant.
FXR−/− Mouse Primary Hepatocytes Are Sensitive to Activation of NF-κB.
Because some nuclear receptors such as pregnane X receptor and LXR regulate the inflammatory response by repressing NF-κB signaling, we hypothesized that FXR−/− mice are more sensitive than wild-type mice to inflammation mediated by NF-κB. We first compared the messenger RNA (mRNA) levels of proinflammatory genes in primary hepatocytes from wild-type and FXR−/− mice after activating the NF-κB pathway with TPA, tumor necrosis factor-α (TNF-α) or LPS. Primary FXR−/− hepatocytes that were treated with TPA expressed higher levels of TNF-α and cyclooxygenase-2 (COX-2) mRNA than did untreated primary FXR−/− hepatocytes. This induction was considerably reduced in wild-type primary hepatocytes (Fig. 1A). TPA-treated primary hepatocytes from FXR−/− mice expressed higher levels of interleukin (IL)-6 mRNA than nontreated FXR−/− hepatocytes, but this change in expression was not observed in wild-type controls (Fig. 1A). We also compared the expression of proinflammatory genes in primary hepatocytes from FXR−/− and wild-type mice after treatment with TNF-α (Fig. 1B) and LPS (Fig. 1C). Induction of hepatic inducible nitric oxide synthase (iNOS), IL-1α, and IL-6 expression in response to TNF-α or LPS was significantly higher in FXR−/− primary hepatocytes than in wild-type hepatocytes. These results suggest that FXR−/− primary hepatocytes are more sensitive than wild-type hepatocytes to NF-κB activation.
Inhibition of NF-κB–Regulated Proinflammatory Genes by FXR Agonists.
Our previous work has indicated that the effects of FXR agonists in HepG2 cells are through FXR.18 To investigate whether activation of FXR has any effect on the NF-κB pathway, we tested the influence of FXR agonists on the expression of TNF-α, COX-2, and iNOS in HepG2 cells. Cells that were pretreated with the FXR agonists GW4064 and 6ECDCA showed significantly less TPA-induced expression of TNF-α mRNA than did non-pretreated cells (Fig. 2). A similar inhibition of expression of COX-2 and iNOS by 6ECDCA was observed in response to stimulation with LPS or TNF-α, respectively (Fig. 2). To confirm that these effects were mediated by FXR, we also tested the influence of FXR agonists on the expression of proinflammatory gene response to NF-κB activation in primary hepatocytes from wild-type and FXR−/− mice. Inhibition of LPS-induced iNOS expression by the FXR agonists GW4064 and 6ECDCA was preserved in primary hepatocytes from wild-type mice, but was abolished in FXR−/− hepatocytes (Fig. 3). GW4064 and 6ECDCA also repressed the TNF-α-induced expression of the NF-κB target gene monocyte chemoattractant protein-1 in wild-type hepatocytes, but not in FXR−/− hepatocytes (Fig. 3). Our results indicate that FXR activation represses the expression of NF-κB–regulated genes in HepG2 cells and mouse primary hepatocytes. Similar phenomenon on FXR activation by its synthetic ligands (GW4064 and 6ECDCA) down-regulating iNOS and COX-2 was observed by Li et al.27 in vascular smooth muscle cells.
Activation of FXR Antagonizes NF-κB Signaling.
Because FXR agonists such as GW4064 and 6ECDCA inhibited the expression of NF-κB target genes, we next tested whether FXR agonists inhibited NF-κB activity at the level of gene transcription. We cotransfected HepG2 cells with an NF-κB reporter plasmid and the control plasmid phRL-TK and assessed the effects of GW4064 on the regulation of NF-κB reporter activity. Treatment with known NF-κB pathway activators such as TPA and LPS resulted in six-fold and two-fold greater NF-κB reporter activity, respectively (Fig. 4A,B). NF-κB activity induced by TPA or LPS was inhibited by GW4064 treatment. Transfection of these cells with FXR/RXR inhibited NF-κB activity in the absence of ligand, suggesting that FXR may suppress NF-κB activity without addition of exogenous ligand due to the fact that HepG2 cells may synthesize bile acid to activate FXR as described.28 However, addition of GW4064 further enhanced this repression (Fig. 4A,B). Furthermore, to eliminate the possibility that the compounds were affecting other pathways, we used p65 overexpression to activate the NF-κB reporter.6 Overexpression of p65 significantly activated the NF-κB reporter (Fig. 4C). NF-κB activity was inhibited by GW4064 in the presence of FXR/RXR, but GW4064 treatment had no significant effect on NF-κB activity in the absence of the FXR/RXR expression vectors. The observed inhibition was proportional to the amounts of FXR/RXR vectors (Fig. 4C). The results were confirmed via transfections of a shorter incubation (1 hour) with GW4064 and FXR expression alone (Supporting Fig. 1A-D). The results of FXR expression alone suggest that FXR alone (without RXR) may suppress NF-κB activity (Supporting Fig. 1B-D). To evaluate the specificity of effects of FXR on NF-κB report activity, a negative control transfection was also performed using estrogen receptor-α reporter plasmid (Supporting Fig. 1E). These results indicate that activation of FXR can antagonize NF-κB activity at the level of gene transcription.
Anti-inflammatory Activity of FXR In Vivo.
Yang et al.17 reported that FXR−/− livers from 9- to 12-month-old mice displayed prominent liver injury and inflammation although there were no obvious tumors. We tested expression of some proinflammatory NF-κB target genes in wild-type and FXR−/− livers from 12-month-old mice. TNF-α, IL-1α, and IL-2 were up-regulated in FXR−/− livers compared with wild-type livers (Fig. 5A). To further address whether FXR may modulate inflammatory gene expression in vivo, we compared the induction of inflammatory genes by LPS in wild-type and FXR−/− mice (n = 6). Induction of hepatic iNOS, COX-2, interferon-inducible protein 10 (IP-10), and interferon-γ (IFN-γ) expression in response to LPS was significantly greater in FXR−/− mice compared with wild-type (Fig. 5B). The difference in expression of iNOS and IFN-γ between FXR−/− and wild-type mice was considerably greater than the difference in expression of COX-2 and interferon-inducible protein 10, suggesting that certain inflammatory genes are more sensitive to the loss of FXR signaling in vivo.
The levels of alanine transaminase, a marker of liver damage, were also significantly increased by treatment with LPS in FXR−/− mice compared with wild-type mice (Fig. 5C). Examination of liver pathology revealed that massive necroses and inflammation were present in FXR−/− mice, but not in wild-type controls after injection of LPS (Fig. 5D). The liver injury induced by LPS was further confirmed by TUNEL assays. Considerable TUNEL-positive staining was detected in the livers of FXR−/− mice, but not in wild-type mice, after administration of LPS (Fig. 5E). The results suggest that FXR−/− mice are more sensitive to inflammatory stimuli.
To better understand the physiological role of hepatic FXR in the suppression of inflammation, we injected wild-type mice with adenovirus that expressed VP16 (Ad-VP16), the transactivation domain of herpes simplex virus, or murine FXRα2 fused to VP16 (Ad-FXRα2-VP16). FXR-VP16 is constitutively active in the absence of FXR ligands.15 Hepatic expression of FXRα2-VP16 led to the induction of the FXR target genes small heterodimer partner and bile salt export pump (Fig. 5F). In contrast, hepatic expression of FXRα2-VP16 suppressed the LPS-induced expression of interferon-inducible protein 10, interferon-γ, iNOS, and COX-2 (Fig. 5F). The results were confirmed by hepatic expression of FXRα2-VP16 in FXR−/− mice (Fig. 5G). Collectively, these results demonstrate that FXR is a negative regulator of the hepatic inflammation in vivo.
FXR Activation Suppressed NF-κB Transcriptional Activity by Decreasing the Binding Between NF-κB and DNA Sequences.
The nuclear p65 levels in mouse livers and HepG2 cells were shown in Fig. 6A -C. It can be seen that LPS administration increased nuclear p65 levels in both wild-type and FXR−/− mice. However, there was no difference of nuclear p65 levels between wild-type and FXR−/− mice after LPS administration. Hepatic expression of FXRα2-VP16 did not reduce nuclear p65 induced by LPS (Fig. 6B). Similarly, FXR activation did not change the levels of nuclear p65 induced by p65 overexpression in HepG2 cells (Fig. 6C). Both results in vitro and in vivo indicate that FXR activation did not change the translocation of p65. The binding of NF-κB to DNA sequences was then tested via electrophoretic mobility-shift assay using nuclear extracts from HepG2 cells. FXR activation clearly reduced the binding activity of NF-κB to DNA sequences induced by p65 overexpression (Fig. 6D). These results suggest that FXR activation may suppress NF-κB transcriptional activity by decreasing the binding between NF-κB and DNA sequences.
Activation of FXR Dose Not Suppress the Expression of NF-κB Antiapoptosis Target Genes.
In addition to its roles in regulating proinflammatory genes, another major function of NF-κB is to regulate many antiapoptotic genes, including members of Bcl-2 family such as Bcl-xL and Bfl-1/A1,29 as well as the cellular inhibitors of apoptosis, cIAP1 and cIAP2, TRAF1, TRAF2, and GADD45β.30, 31 To determine the effects of FXR activation on the antiapoptotic genes that are activated by NF-κB, we measured the mRNA levels of NF-κB–activated antiapoptotic genes after FXR agonist treatment. Neither GW4064 nor 6ECDCA suppressed the expression of Bfl-1, GADD45β, and cIAP1 induced by TNF-α in mouse primary hepatocytes (Fig. 7A). Similarly, hepatic expression of FXRα2-VP16 did not suppress the LPS-induced expression of TRAF1, TRAF2, cIAP2, and Bfl-1 mRNA in vivo (Fig. 7B). The results were confirmed by hepatic expression of FXRα2-VP16 in FXR−/− mice (Fig. 7C). These results suggest that FXR activation selectively inhibited NF-κB target genes for hepatic inflammation but not antiapoptotic genes.
Suppression of FXR Signaling by NF-κB Activation.
Because nuclear receptor signaling pathways are repressed in the inflammatory response,2, 6, 32 we investigated whether NF-κB activators inhibited the expression of FXR-mediated target genes in vitro and in vivo. The results show that NF-κB activation suppressed the expressions of FXR and its target genes small heterodimer partner and bile salt export pump (Supporting Fig. 2), which was consistent with the report of Kim et al.33 In addition, NF-κB activation induced by TNF-α repressed FXR activation induced by its ligand GW4064 in hepatocytes of wild-type mice (Supporting Fig. 2B). To further investigate the effects of activation of NF-κB on the transcriptional activity of FXR, we cotransfected HepG2 cells with an FXR reporter (EcRE-LUC) and FXR/RXR expression plasmids. Treatment with GW4064 dramatically induced FXR reporter activity in the presence of FXR/RXR (Fig. 8). TPA and LPS strongly repressed GW4064-induced FXR reporter activity (Fig. 8A,B). Repression of FXR reporter activity by TPA was more prominent than repression induced by LPS, possibly due to the additional inhibition of TPA on expression of FXR and RXR mRNA (Supporting Fig. 2A). The transactivation of NF-κB by overexpression of p65 also repressed GW4064-induced FXR reporter activity in a dose-dependent manner (Fig. 8C), suggesting that activation of NF-κB reciprocally antagonizes FXR activity.
The known functions of FXR in the liver have recently expanded rapidly from initial roles in regulating liver metabolism to also participating in liver regeneration and hepatocarcinogenesis.17, 34 The novel roles of FXR in promoting liver regeneration and protecting against hepatocarcinogenesis are consistent with FXR's previous roles in defending against bile acid toxicity. In contrast to its well-established mechanism in regulating bile acid homeostasis, little is known about how FXR functions in liver regeneration and carcinogenesis. Our results suggest that one potential role for FXR in protecting against hepatocarcinogenesis is by modulating NF-κB–mediated hepatic inflammatory responses. FXR activation strongly suppresses the activity of NF-κB in cell culture experiments in vitro. This is further supported by both primary hepatocyte and animal studies in vivo. However, FXR does not suppress the NF-κB–activated antiapoptotic genes. This differential effect of FXR is consistent with its key role as a hepatocyte protector. There are several implications of modulation of NF-κB by FXR in liver function. First, hydrophobic bile acids induce hepatic inflammation35 and bile acids such as deoxycholic acid can activate NF-κB by increasing the binding of NF-κB to DNA36, 37 during pathological conditions of the liver, such as cholestasis.35 Therefore, FXR may decrease bile acid–induced hepatoxicity through its anti-inflammatory function. Second, gallbladder mucosal inflammation is an important event in cholesterol gallstone disease, and the gallbladder epithelia of FXR−/− mice under lithogenic conditions showed increased inflammation compared with wild-type mice.38 Our findings may be directly related to Moschetta's studies demonstrating that activation of FXR by GW4064 prevents the development of cholesterol gallstone disease in a mouse model.38 Third, NF-κB–mediated hepatic inflammation may contribute to liver insulin resistance, and FXR−/− mice exhibit insulin resistance.15, 39 Our results may provide an explanation for the increased insulin resistance in FXR−/− mice. Finally, hepatic inflammation is closely linked to hepatocarcinogenesis,40, 41 and FXR−/− mice also display intense liver inflammation prior to developing spontaneous liver tumors. The mutual suppression between FXR and NF-κB may be an important mechanism for preventing tumorigenesis.
Our results indicate that FXR does not affect p65 nuclear translocation. However, FXR may suppress p65 transactivity by decreasing its DNA binding activity. Previous reports have shown that another nuclear receptor, glucocorticoid receptor (GR), antagonizes the action of NF-κB through direct physical protein–protein interactions.42–44 Recently, Pascual et al.45 reported that sumoylation of PPAR-γ is a potential mechanism involved in the inhibition of NF-κB by PPAR-γ ligands. The sumoylation-dependent pathway is also involved in the regulation of proinflammatory genes by LXR.46 Whether this is a common mechanism for FXR remains to be tested.
On the other hand, infections and inflammatory responses have long been observed to suppress hepatic gene expression.2, 6 NF-κB is the central transcriptional regulator of the hepatic inflammatory responses, and it may provide a link between inflammation and the suppression of nuclear receptor signaling in metabolic function by antagonizing the activities of a number of nuclear receptors, including GR, the aryl hydrocarbon receptor, and SXR.6, 44, 47 We can now expand this list to include FXR. We observed two effects of suppression of FXR signaling by NF-κB activation. First, we observed decreased expression of FXR as shown by the marked reduction of FXR and RXR mRNA after injection of LPS into mice. This is consistent with the report by Kim et al.33 Second, we observed direct antagonizing FXR transactivity by NF-κB activation as shown by the suppression of FXR target genes by TNF-α, which was not associated with changes in the expression of FXR and RXR mRNA (Supporting Fig. 2B). This was further confirmed by the observation that LPS (which did not alter FXR or RXR mRNA levels in HepG2 cells), TPA or overexpression of p65 dramatically repressed FXR reporter activity. Gu et al.2 reported that the p65 subunit of NF-κB directly interacts with the DNA-binding domain of RXRα and may prevent its binding to the consensus DNA sequences. Because RXR is a dimerization partner of FXR, it is possible that NF-κB suppresses FXR activity by reducing the number of FXR/RXR complexes.
We noted that activation of FXR repressed specific sets of NF-κB target genes, but not all the target genes in response to the NF-κB activators that we used in this study (TPA, LPS, and TNF-α). This phenomenon has also been observed for other nuclear receptors. Ogawa et al.48 demonstrated that a cohort of genes was sensitive to GR-mediated repression when induced by LPS but was GR resistant when induced by polyinosinic:polycytidylic acid. Similar results were obtained in LXR- and PPAR-γ–mediated repression for inflammatory genes.46 One possibility is that the transrepression programs that are mediated by nuclear receptors are regulated in a signal-specific manner. In addition, the transrepression pathways themselves may be subject to further regulation and can be overridden by specific signals in a gene-specific manner.46 It will be interesting to define the mechanism by which FXR activation inhibits NF-κB in a gene-specific manner.
In conclusion, our results reveal that FXR is a negative mediator of liver inflammation and that there is reciprocal suppression between FXR and NF-κB signaling pathways. These findings support the role of FXR as a central hepatoprotector, and suggest that FXR agonist ligands offer possible therapies to prevent and treat hepatitis, liver fibrosis, and hepatocarcinogenesis.
We thank Keely Walker for proofreading the manuscript; Xufeng Chen, Akio Kruoda, Peter Tontonoz, and Bruce Blumberg for plasmids; and Peter A. Edwards for adenovirus that expressed VP16 alone (Ad-VP16) or murine FXRα2 fused to VP16.