Bile acid signaling through FXR induces intracellular adhesion molecule-1 expression in mouse liver and human hepatocytes
Pu Qin,*
Lisa A. Borges-Marcucci,*
Mark J. Evans, and
Douglas C. Harnish
Wyeth Research, Cardiovascular and Metabolic Disease Research, Collegeville, Pennsylvania
Submitted 2 February 2005
; accepted in final form 1 April 2005
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ABSTRACT
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Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 35 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-
, VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependent repression of cholesterol 7
-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time- dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-1 promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-
cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events.
inflammation
BILE ACIDS ARE AMPHIPATHIC detergents necessary for the digestion and absorption of fat-soluble nutrients. Bile acids are synthesized in the liver by either a neutral or classic pathway resulting in formation of cholic acid (CA) or by an acidic or alternative pathway leading to the synthesis of chenodeoxycholic acid (CDCA). In humans, the classic biosynthetic pathway predominates via the rate-limiting enzyme cholesterol-7
-hydroxylase (CYP7A1). After their synthesis, bile acids are excreted to the intestine where they facilitate the absorption of dietary lipid and lipid-soluble nutrients. In addition, bile acids are also important in the excretion of cholesterol and toxic metabolites. The biosynthesis of bile acids from cholesterol is the most significant pathway for the elimination of cholesterol from the body.
It has been recently demonstrated that bile acids are the physiological ligands for the farnesoid X receptor (FXR) (24, 38). FXR is expressed mainly in tissues that are exposed to high concentrations of bile acids including the liver and intestine (24, 38). Activated FXR binds to the promoters of its target genes typically via an IR-1 element and enhances the transcription of these genes, including small heterodimer partner (SHP) (11, 33), bile salt export pump (BSEP), and ileal bile acid binding protein (1, 3, 12, 22). The induction of SHP by FXR results in the negative regulation of bile acid synthesis through the regulation of CYP7A1 and sterol 12
-hydroxylase (CYP8B1) (11, 22, 42). Thus FXR is an important regulator for bile acid homeostasis, and inactivation of FXR in mice results in elevated serum bile acids and cholesterol, increased triglyceride levels, increased hepatic cholesterol, and a proatherogenic serum lipoprotein profile (17).
Although the role of bile acids in regulation of bile acid and lipid homeostasis has been well established, its potential role in inflammation has not been defined. There has been a number of reports in the literature suggesting a link between bile acids and inflammation. When accumulated in high concentrations, hydrophobic bile acids can result in an inflammatory phenotype as evidenced during cholestasis (13). The induction of proinflammatory genes such as EGF, transforming growth factor-
1 (TGF-
1), TNF-
, IL-1, and ICAM-1 have been demonstrated in experimental models of cholestasis after bile duct ligation resulting in neutrophil-induced liver injury (14, 30). In addition, before the development of genetically modified mice that are prone to atherosclerosis, cholate supplementation to a high-fat, high-cholesterol diet was necessary to induce consistent aortic lesion formation in C57BL/6 mice (28). This diet has also been shown to induce the expression of a number of inflammatory genes in the liver including serum amyloid A2 (SAA-2), monocyte chemotactic protein, VCAM-1, TNF-
, and regulated on activation normal T cell expressed and secreted (RANTES) (9, 18) with the induction of VCAM-1 specifically being attributed to the presence of cholate (36).
The mechanism by which bile acids could be inducing hepatic and vascular wall inflammation is unclear. The inflammation induced by prolonged exposure to high levels of bile acids could simply be due to its associated cytotoxicity (7). Alternatively, bile acids could elicit their proinflammatory phenotype through activation of kinase pathways including protein kinase C (34), extracellular signal-regulated kinase (31), or c-Jun NH2-terminal kinase (15). In addition, bile acids can stimulate cytokine production in macrophages that can then modulate adjacent hepatocytes (26). In this report, we demonstrate a novel pathway in which bile acids can induce inflammatory gene expression through direct FXR signaling.
In this study, we demonstrate that treatment of C57BL/6 mice with CA results in the induction of ICAM-1, VCAM-1, TNF-
, and SAA-2. Moreover, in HepG2 cells, ICAM-1 expression was induced by the synthetic FXR agonist GW4064 (25) in a dose- and time-dependent manner. The induction of ICAM-1 by GW4064 could be antagonized by treatment with the FXR antagonist guggulsterone or through inhibition of FXR expression by siRNA. FXR was demonstrated to directly interact with a RARE/FXRE site within the human ICAM-1 promoter. Finally, we demonstrate that the activation of ICAM-1 by liganded FXR could be further enhanced by cotreatment with cytokines suggesting a potential cooperation between the two signaling pathways during inflammatory events.
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MATERIALS AND METHODS
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Mice.
Male C57BL/6 mice (1620 g) (Taconic) were separated into groups of eight. The mice were fed a chow diet or high-fat diet (20% fat, 0.15% cholesterol) supplemented with CA (0.11%) as indicated for up to 5 wk. At the end of the experimental period, the liver was dissected and immediately frozen in liquid nitrogen. Liver total RNA was prepared by using Trizol reagent (GIBCO-BRL) and further purified using RNeasy kit (Qiagen). Gene expression analysis was performed using real-time RT-PCR as described previously (9).
Cell culture.
Primary human hepatocytes cultured in a 24-well plate (Cambrex) were treated immediately on arrival. HepG2 cells were maintained in growth medium (DMEM; GIBCO-BRL) supplemented with heat-inactivated 10% FBS, 1% Glutamax, 1% MEM nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were maintained in 37°C in a 5% CO2 incubator. The cells were seeded in at 3.0 x 105 cells/well in a 12-well dish. The cells were treated with 1100 µM CDCA (Sigma), 11,000 nM GW4064, 5 µM guggulsterone (Steraloids), or 1 µg/ml TNF-
(Roche Applied Science) as indicated. Total RNA samples were harvested using the RNeasy Mini Kit (Qiagen), and real-time RT-PCR was performed as described previously (9).
Western blots.
The HepG2 cell protein extracts were separated on a 415% SDS-PAGE and then transferred to polyvinylidine difluoride membrane. The membrane was blotted using a rabbit anti-ICAM-1 or goat anti-actin antibody (1:1,000 dilution, SC-7891 and SC-1616, respectively, Santa Cruz Biotechnology). Secondary horseradish peroxidase-labeled anti-rabbit and anti-goat antibodies (1:2,500 dilution) were purchased from Amersham and Pierce, respectively. Detection was performed using the enhanced chemiluminescence plus reagent (Amersham Biosciences), and quantitation was performed using the Scion Image software.
FXR siRNA.
FXR siRNA SMART pool and control siRNA were purchased from Dharmacon (sequence information is patented and protected by Dharmacon) and transfected into HepG2 cells using Lipofectamine 2000 (Invitrogen). After an overnight transfection, the cells were treated with vehicle or 1 µM GW4064 for 24 h. Total RNA was isolated, and the levels of SHP, FXR, and ICAM-1 were assayed by real-time RT-PCR.
Promoter studies.
The human ICAM-1 promoter constructs cloned in pGL3-Basic luciferase vector were described previously (4). The point mutations were generated using the QuickChange Mutagenesis Kit from Stratagene. The first three nucleotides in the RARE site were mutated from 5'-GGGTCATCGCCCTGCCA-3' to 5'-ataTCATCGCCCTGCCA-3'. The luciferase promoter construct (0.5 µg/well),
-galactosidase (
-gal) plasmid (0.5 µg/well), FXR full-length cDNA (0.2 µg/ml, Invitrogen), and retinoid X receptor (RXR) full-length cDNA (50 ng/well) were transfected into HepG2 cells overnight using Lipofectamine 2000 reagent (Invitrogen). The cells were treated with vehicle or 1 µM GW4064 for 24 h, and luciferase and
-gal activity was determined.
EMSA.
EMSA studies were performed using the digoxigenin (DIG) gel shift kit from Roche Applied Biosciences. The probes used were 1) wild-type (WT) probe, 5'-CCCCCGGGGTCATCGCCCTGCCACCGCCGC-3'; 2) mutated (MUT) probe: 5'-CCCCCGataTCATCGCCCTGCCACCGCCGC-3'; 3) IR-1 probe: 5'-GAAACTGAGGGTCAGTGACCCAAGTGAAG-3'; and 4) control probe provided by the manufacturer. The putative binding sites are underlined, and the mutated nucleotides are in lowercase letters. WT, MUT, and IR-1 probes were labeled with DIG and purified with mini-quick spin oligo columns (Roche). FXR and RXR-
proteins were in vitro transcribed and translated from FXR and RXR-
cDNA using the TnT quick-coupled transcription/translation systems (Promega). FXR and RXR-
proteins were preincubated with competitors (antibodies or unlabeled probes) for 3 min at room temperature and then mixed with DIG-labeled probes. The reactions were incubated at room temperature for 25 min and then separated on a 10% acrylamide gel. The nucleotides were transferred to a nylon membrane and detected using an anti-DIG antibody according to the manufacturers protocol. For the supershift experiment, FXR and RXR antibodies (SC-13063 and SC-553, respectively, Santa Cruz Biotechnology) were used at a 1:1,000 dilution.
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RESULTS
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CA induces inflammatory gene expression.
Previous studies (9, 18) have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat diet containing CA. Studies were undertaken to determine the involvement of cholate in mediating these inflammatory gene inductions. C57BL/6 mice were fed a chow, high-fat diet supplemented with 0.5% cholate or high-fat diet alone for 5 wk. Induction of the inflammatory genes ICAM-1, VCAM-1, SAA-2, and TNF-
mRNA occurred in mice fed a high-fat diet containing CA but not in mice fed a high-fat diet without CA(Fig. 1). This suggests that the induction of these genes is dependent on CA.

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Fig. 1. Hepatic inflammatory gene expression is induced by cholate. C57BL/6 mice were fed with a chow diet, high-fat diet (HF) with 0.5% cholate (CA) or high-fat diet without CA for 5 wk. The hepatic mRNA levels of ICAM-1, VCAM-1, TNF- , and serum amyloid A2 (SAA-2) were examined by real-time RT-PCR and normalized to GAPDH. Values are reported as means ± SE with the mean expression level in mice fed the chow diet defined as 1. *P < 0.05 vs. mice on a chow diet.
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To further prove the role of CA in the induction of these inflammatory genes and to eliminate the potential confounding toxic metabolic effects associated with 5-wk exposure to CA (7), C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of CA (0.011%) for 5 days. Real-time RT-PCR analysis demonstrated a dose-dependent induction in the hepatic mRNA levels of ICAM-1, VCAM-1, TNF-
, and SAA-2 with maximal induction observed at the 0.3% dose for TNF-
(2-fold) and at 1.0% for ICAM-1 (3.1-fold), VCAM-1 (2.2-fold), and SAA-2 (28-fold; Fig. 2). As positive controls, the induction of SHP and repression of CYP7A1 by CA were observed as previously reported (19). These data support the premise that bile acids such as CA can induce inflammatory gene expression in the liver.
FXR is required for the induction of ICAM-1 in HepG2 cells.
To further elucidate the role of bile acids in the induction of hepatic inflammatory genes, studies were performed in primary human hepatocytes and the human hepatic cell line HepG2. Primary human hepatocytes were treated with 100 µM CDCA or the synthetic FXR agonist GW4064 (1 µM) for 6 and 24 h, and the mRNA levels were determined by real-time RT-PCR. Both SHP and ICAM-1 were induced by CDCA and GW4064 treatment at both time points (Fig. 3). In contrast, SAA-2 mRNA levels were only slightly induced (1.4-fold) by GW4064, whereas VCAM-1 mRNA levels were not regulated at all (data not shown). To further study the regulation of ICAM-1 by FXR, HepG2 cells were treated with increasing concentrations of CDCA or GW4064 for 24 h. ICAM-1 mRNA levels were dose dependently increased by both CDCA and GW4064 treatment with the maximal fold induction of 2.7 and 2.5 at 100 µM CDCA and 100 nM GW4064, respectively (Fig. 4A). Similar results were also obtained in the human hepatocyte cell line, Hep3B (data not shown). As expected, SHP mRNA was upregulated, whereas CYP7A1 mRNA was downregulated by CDCA and GW4064 in a dose-dependent manner consistent with previous observations (16). Time-course studies demonstrated a rapid induction of ICAM-1 and SHP mRNA within 2 h by GW4064 treatment and persisting for at least 24 h (Fig. 4B), which resulted in the induction of ICAM-1 protein levels (Fig. 4C). The ability of the selective FXR agonist GW4064 to induce ICAM-1 expression supports the involvement of FXR signaling in mediating this effect.

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Fig. 3. Chenodeoxycholate (CDCA) and GW4064 (GW) treatments induce the expression of SHP and ICAM-1 in primary human hepatocytes. Primary human hepatocytes were treated with 100 µM CDCA or 1 µM GW for 6 and 24 h. Total RNA samples were harvested. The mRNA levels of SHP and ICAM-1 were examined by real-time RT-PCR and normalized to GAPDH. This experiment was performed once in triplicate. Fold inductions by CDCA and GW treatment were calculated and graphed (means + SE are shown).
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Fig. 4. Farnesoid X receptor (FXR) is involved in the induction of ICAM-1 in HepG2 Cells. A: HepG2 cells were treated with increasing concentrations of CDCA or GW for 24 h, and the mRNA levels of SHP, CYP7A1, and ICAM-1 were examined by real-time RT-PCR and normalized to GAPDH. B: HepG2 cells were treated with 1 µM GW between 2 and 24 h. The mRNA levels of SHP and ICAM-1 were examined by real-time RT-PCR and normalized to GAPDH. Values are reported as means ± SE for each group from at least 3 experiments done in duplicate. *P < 0.05 vs. vehicle (Veh)-treated cells. C: HepG2 cells were treated with 100 µM CDCA or 1 µM GW for 24 h, and the protein levels of ICAM-1 and actin were determined by Western blot analysis. A representative blot is shown as well as the quantitation from 3 separate experiments. The levels of ICAM-1 proteins were quantified by Scion Image software and normalized to the levels of actin protein. Fold inductions by CDCA and GW treatment were calculated and graphed (means ± SE is shown)
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To further confirm the role of FXR in the induction of ICAM-1, HepG2 cells were treated with 100 nM GW4064 alone or cotreated with 5 µM of the FXR-selective antagonist guggulsterone (Fig. 5A) (5, 35). The GW4064-mediated elevation of SHP and ICAM-1 mRNA was effectively inhibited by guggulsterone coadministration. In addition, transfection of FXR siRNA resulted in a 50% suppression of endogenous FXR mRNA levels, which led to an
50% decrease in the GW4064-mediated induction of SHP mRNA levels (from a 60% increase to a 30% increase over vehicle-treated cells, Fig. 5B). A similar decrease in the GW4064-mediated induction in ICAM-1 expression was also observed following FXR siRNA transfection (from a 130% increase to a 60% increase over vehicle-treated cells, Fig. 5B). These data further support a role for FXR in mediating ICAM-1 expression in HepG2 cells.

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Fig. 5. The induction of ICAM-1 in HepG2 cells is dependent on FXR. A: HepG2 cells were treated with 100 nM GW or 100 nM GW with 5 µM guggulsteron (GW + Gug) for 24 h. The mRNA levels of SHP and ICAM-1 were determined by real-time RT-PCR and normalized to GAPDH. B: HepG2 cells were transfected with FXR SMART pool (Dharmacon) at a final concentration of 200 nM or control siRNA for overnight and then treated with Veh or 1 µM GW for 24 h. The mRNA levels of FXR, SHP, and ICAM-1 were determined by real-time RT-PCR and normalized to GAPDH. Values are reported as means ± SE for each group from at least 3 experiments done in triplicate. *P < 0.05 vs. Veh-treated cells.
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Mapping the FXR-responsive element in the human ICAM-1 promoter.
To determine the mechanism of FXR regulation on ICAM-1, a series of promoter deletion constructs was created and tested in HepG2 cotransfection assays. As shown in Fig. 6, GW4064 treatment resulted in a threefold induction in luciferase activity with the ICAM-1 promoter construct containing sequences from 1127 to +18. Deletion of promoter sequences from 1127 to 309 did not affect the GW4064-mediated induction in luciferase activity. However, deletion from 309 to 244 resulted in a complete loss of GW4064-mediated induction. The sequence between 309 and 244 contains a RARE (reviewed in Ref. 37) and mutation of this site within the context of the 309 to +18 promoter region (see Fig. 7A) also resulted in the loss of the FXR-mediated induction.
This RARE site appears to contain an imbedded FXRE site (Fig. 7A). To determine whether FXR directly binds to this element, EMSA assays were performed. In vitro transcribed/translated FXR and RXR-
proteins were incubated with labeled WT RARE probe from the ICAM-1 promoter, consensus FXRE probe (IR-1 element from BSEP), or the mutated RARE probe (MUT; Fig. 7, B and C). A shifted band is observed when both FXR and RXR-
proteins are present and only with the WT and IR-1 probe but not with the MUT probe. The shifted band can be competed off by excess amounts of unlabeled WT or IR-1 probe (Fig. 7C), whereas excess amounts of unlabeled MUT probe or a control nucleotide probe (unrelated sequences) does not compete off the shifted band. To further confirm that FXR and RXR-
proteins bind to this element, FXR and RXR-
proteins were incubated with labeled WT probe and antibodies against the NH2-terminal of FXR or RXR-
(Fig. 7D). Both antibodies greatly reduced the intensity of the shifted band, suggesting that the FXR-RXR heterodimer can bind to this RARE/FXRE site to mediate the induction of ICAM-1 transcription directly.
Coordinative induction of ICAM-1 expression by FXR agonist and proinflammatory cytokine.
Because cytokine signaling can regulate genes involved in bile acid synthesis (6, 10) and is linked with inflammatory gene expression, we wanted to determine whether there was any cooperation between bile acids and cytokines in influencing ICAM-1 expression. From previous experiments (4), TNF-
was shown to modulate ICAM-1 expression through the NF-
B element just downstream from the newly identified FXRE, and it is likely that these two signaling pathways may coordinately impact the ICAM-1 promoter. HepG2 cells were treated with TNF-
and GW4064 for 24 h, and the mRNA levels of ICAM-1 and FXR were examined by real-time RT-PCR (Fig. 8). TNF-
and GW4064 treatment elevated ICAM-1 mRNA levels to a similar extent (2- to 3-fold) in HepG2 cells without affecting FXR mRNA levels. When given in combination, an additive effect on ICAM-1 induction was observed. These data suggest that the FXR-dependent induction of ICAM-1 in hepatocytes can be further enhanced by additional proinflammatory signals such as TNF-
.
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DISCUSSION
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It has been well established that high doses of bile acids can induce inflammation in the liver and cause liver damage in disease conditions such as primary biliary cirrhosis, cholestasis, and primary sclerosing cholangitis (8). In this study, we determined that bile acids alone, in the absence of high fat and high cholesterol, could mediate the induction of a number of inflammatory genes in the mouse liver. In addition, we identified a novel role for FXR being involved in this activity, specifically in mediating the induction of ICAM-1 in human hepatocytes. This induction of inflammatory gene expression is likely to initiate or contribute to bile acid-induced inflammation.
Our study initiated from the observation of the increased expression of proinflammatory genes in the liver of C57BL/6 mice fed an atherogenic diet (20% fat, 0.15% cholesterol, and 0.5% cholate) for 5 wk (9). We demonstrate here that CA alone can mediate the induction of at least a subset of these proinflammatory genes such as VCAM-1, ICAM-1, TNF-
, and SAA-2. This is supported by a previous study (36) in which the induction of VCAM-1 as well as other proinflammatory genes such as the chemokines scya6 and scya9 were attributed solely to the cholate component of the atherogenic diet. In that study, SAA-2 expression was also demonstrated to be induced by either the cholesterol or cholate component of the diet. Therefore, it appears that bile acids themselves are inducers of proinflammatory gene expression in the liver.
Through a series of in vitro experiments including agonist, antagonist, and siRNA, we provide strong evidence that bile acids induce the expression of ICAM-1 through an FXR-dependent mechanism. Using promoter mapping and EMSA assays, we showed that FXR and RXR bind to a RARE/FXRE site within the promoter of ICAM-1. Mutations in both the upstream half-site (Fig. 6) as well as the downstream half-site (data not shown) of this RARE/FXRE site in ICAM-1 promoter abolished the induction in luciferase activity by GW4064. This suggested that FXR likely functions as a heterodimer with RXR and was supported by the EMSA experiments. Interestingly, the EMSA experiments showed that the amount of binding of FXR-RXR to the ICAM-1 promoter did not change following GW4064 or CDCA treatment (data not shown). Thus the induction in the transcription of ICAM-1 may be a result of ligand-dependent displacement of corepressors with coactivators associated with the FXR-RXR heterodimer but not due to the amount of FXR-RXR associated with the response element.
ICAM-1 can be induced by proinflammatory cytokines such as TNF-
, INF-
, and IL-1
in endothelial cells as well as hepatocytes (21, 27, 32, 41). When HepG2 cells were cotreated with TNF-
and GW4064, an additive effect in the induction of ICAM-1 was observed, suggesting that the FXR- and cytokine-signaling pathways can cooperate and may impact inflammatory events associated with elevated levels of bile acids. This may occur during cholestasis where both elevated levels of cytokines and bile acids are observed. The neutrophil-induced liver injury in the bile duct-ligated mouse model of cholestasis is reduced in both ICAM-1- and CD18-deficient mice (13, 14). Therefore, the potential arises that the neutrophil-induced liver injury resulting from elevated bile acid levels is mediated in part due to the activation of ICAM-1 expression by FXR. On the other hand, FXR activation can also have beneficial effects in this model through induction of bile salt exporters that facilitate the clearance of bile acids during cholestasis, thereby reducing the toxic and potentially proinflammatory effect of bile acids (20).
The FXR-dependent induction of ICAM-1 may also translate into the vasculature where FXR expression has recently been demonstrated (2). The induction of ICAM-1 is known to contribute to the leukocyte-induced inflammation in vascular tissue that can eventually lead to the formation of atheromatus lesions. The contribution of FXR in atherosclerotic lesion development has not yet been studied. However, studies by Paigens laboratory (28) demonstrating the requirement of cholate for the development of consistent aortic lesions in the C57BL/6 strain of mice support a potential link between bile acid signaling and vascular wall inflammation. This may be relevant in settings of a high-cholesterol diet because cholesterol is converted to bile acids in the liver. Mice on a high-cholesterol diet do have increased bile acid secretion and serum bile acid levels (20, 29, 39, 40), which may directly impact the vasculature.
In summary, we demonstrate that bile acid signaling can result in hepatic inflammatory gene expression in vivo and have identified a novel role for FXR in mediating ICAM-1 gene expression. Moreover, bile acid and cytokine signaling can result in an additive enhancement in ICAM-1 expression. The consequence of this proinflammatory mechanism in vivo is currently being investigated but may play a role in diseases associated with elevated levels of bile acids such as cholestatic diseases or atherosclerosis.
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FOOTNOTES
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Address for reprint requests and other correspondence: D. C. Harnish, Wyeth Research. Cardiovascular & Metabolic Disease Research, N2236, 500 Arcola Rd, Collegeville, PA 19426 (E-mail: harnisd{at}wyeth.com)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* P. Qin and L. A. Borges-Marcucci contributed equally to this work. 
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