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Originally published In Press as doi:10.1074/jbc.M608993200 on December 11, 2006

J. Biol. Chem., Vol. 282, Issue 7, 4393-4399, February 16, 2007
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Interleukin-1 Receptor Antagonist Induction as an Additional Mechanism for Liver Receptor Homolog-1 to Negatively Regulate the Hepatic Acute Phase Response*

Nicolas Venteclef and Philippe Delerive1

From the Cardiovascular and Urogenital Center of Excellence for Drug Discovery, GlaxoSmithKline, 25 Avenue du Quebec, 91951 Les Ulis, France

Received for publication, September 21, 2006 , and in revised form, November 29, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor believed to play a key role in bile acid metabolism, cholesterol homeostasis, and intestinal cell crypt renewal. LRH-1 has recently been reported to negatively regulate the hepatic acute phase response by antagonizing, at least in part, the CCAAT/enhancer-binding protein signaling pathway. Here we have shown, using adenovirus-mediated LRH-1 overexpression and gene-silencing experiments, that the interleukin-1 receptor antagonist (IL-1RA) gene is a novel LRH-1 target gene in hepatic cells. Promoter mapping and chromatin immunoprecipitation experiments revealed that LRH-1 regulates IL-1RA gene expression under inflammatory conditions at the transcriptional level via the binding to an LRH-1 response element. Interestingly, IL-1RA induction by an intraperitoneal injection of lipopolysaccharide is significantly lower in LRH-1 heterozygous compared with wild-type mice, demonstrating the contribution of LRH-1 in IL-1RA gene regulation. Finally, RNA interference experiments indicate that LRH-1 blocks the hepatic acute phase response by, at least in part, inducing IL-1RA expression. Taken together, these results lead to the identification of IL-1RA as a novel LRH-1 target gene and demonstrate the existence of multiple mechanisms contributing to the overall anti-inflammatory properties of LRH-1 in hepatic cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The liver receptor homolog-1 (LRH-12; NR5A2) is a member of the nuclear receptor superfamily (1). Nuclear receptors have central roles in nearly every aspect of development and adult physiology. LRH-1 is the mammalian homolog of the Drosophila Fushi Tarazu F1 receptor (FTZ-F1; NR5A3) and, similar to FTZ-F1, binds its cognate target sequence (5'-(Py)CAAGG(Py)-C(Pu)-3') as a monomer (2). It is highly expressed in the ovary, liver, intestine, and pancreas (2–4). Several groups have recently shown by x-ray crystallography that the human LRH-1 ligand binding pocket can bind various phospholipids, including phosphoinositides and phosphatidylethanolamine, linking phospholipid metabolism to gene transcription (5–8). LRH-1 is involved in the regulation of the expression of transcription factors implicated in embryonic development, such as Oct4 and the hepatic nuclear factors HNF-3beta, HNF4{alpha}, and HNF1{alpha} (9, 10). Recently, LRH-1 was shown to regulate estrogen production through the control of aromatase (CYP19) gene transcription in ovarian and adipose tissue (10, 11) and adiponectin in adipocytes (12). Furthermore, LRH-1 has recently been reported to be involved in intestinal crypt cell renewal by coactivating beta-catenin on the cyclin D1 promoter (13). In addition, LRH-1 is believed to be a key player in cholesterol homeostasis (1). LRH-1 is known to play a pivotal role in the transcriptional regulation of CYP7A1, the rate-limiting enzyme of the bile acid biosynthetic pathway (3) and CYP8B1, the oxysterol 12{alpha}-hydroxylase required for cholic acid production (14). Moreover, LRH-1 has been reported to regulate the expression of APO A1 (15), ABCG5/ABCG8 (16), CETP (17), SR-B1 (4), and the carboxyl ester lipase (18), thereby implicating this receptor in high density lipoprotein remodeling and cholesterol transport.

Recently, we have been shown that LRH-1 is a negative regulator of the hepatic acute phase response (APR) (19). Ectopic expression of LRH-1 using adenovirus resulted in the inhibition of IL-1beta- and IL-6-mediated induction of acute phase gene expression, such as haptoglobin (HP), serum amyloid A, and C-reactive protein in cultured hepatocytes. In addition, LRH-1 partial deficiency led to an exacerbated inflammatory response in vitro and in vivo, indicating that LRH-1 is a physiological modulator of hepatic APR. Moreover, molecular studies revealed that LRH-1 negatively interferes with the development of the APR by, at least in part, antagonizing C/EBP transcriptional activity (19).

To better understand the role of LRH-1 in the control of APR, we compared LRH-1-mediated APR gene regulation in HepG2 cells after a short or prolonged cytokine exposure. We found that the interleukin-1 receptor antagonist (IL-1RA) gene is directly regulated by LRH-1 in vivo and in vitro. Furthermore, we provide some evidence that IL-1RA induction by LRH-1 contributes to the overall anti-inflammatory properties of LRH-1 during the APR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—HepG2 cells (American Type Culture Collection, Manassas, VA) were maintained in basic Eagle’s medium (BME) supplemented with 2 mM glutamine, 1% non-essential amino acids, and 10% (v/v) fetal calf serum in an atmosphere of 5% CO2 at 37 °C. HepG2 cells stably expressing short hairpin (sh)RNA targeting LRH-1 expression or LacZ as control were previously described (19).

Plasmids—The plasmid pSG5-LRH-1 has been previously described (15). The pSG5 plasmid was purchased from Stratagene (La Jolla, CA). The human IL-1RA promoter construct (-1150 to +1) was obtained by PCR amplification using human genomic DNA (Clontech) as the template. The resulting PCR product was inserted as a KpnI/HindIII fragment into pGL3 basic vector (Promega) yielding IL-1RA-Luc. The mutation of the LRH-1 binding site within the human IL-1RA promoter was obtained by site-directed mutagenesis (Stratagene, La Jolla, CA) using the following oligonucleotides: 5'-TTCCAAAAAGAGAAACCCTTTCTGTTGTCACTTTCA-3' and 5'-TGAAAGTGACAACAGAAAGGGTTTCTCTTTTTGGAA-3'. All constructs were verified by DNA sequence analysis.

Transient Transfection Assays—HepG2 cells, plated in 24-well plates at 50–60% confluence in BME supplemented with 10% fetal calf serum, were transiently transfected with reporter and expression plasmids using FuGENE 6 reagent (Roche Molecular Biochemical, Indianapolis, IN) as indicated in the figure legends. The pSEAP2 expression plasmid (Clontech) was co-transfected to assess transfection efficiency. 48 h post-transfection, cells were collected and assayed for luciferase and alkaline phosphatase activities. All experiments were repeated at least three times.

RNA Interference—Twenty-one RNA nucleotides directed against human IL-1RA (GenBankTM accession number NM_173842 [GenBank] ) and the non-silencing control small interfering RNA (siRNA) were obtained from Qiagen (Valencia, CA). HepG2 cells (40% confluence) were transfected with siRNAs by using Hi-perfect (Qiagen) following the manufacturer’s instruction. Twenty-four hours post-transfection, the cells were refed with fresh medium for the indicated time period.

RNA Analysis—Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer’s instructions. Total RNA was treated with DNaseI (Ambion, Inc., Austin, TX) at 37 °C for 30 min followed by inactivation at 75 °C for 5 min. Real time quantitative PCR (RT-QPCR) assays were performed using an Applied Biosystems 7900 sequence detector. Total RNA (1 µg) was reverse-transcribed with random hexamers using the TaqMan reverse transcription reagent kit (Applied Biosystems) following the manufacturer’s protocol. Gene expression levels were determined by Sybr green assays as described previously (15). Cyclophilin transcript was used as an internal control to normalize the variations for RNA amounts. Gene expression levels are expressed relative to cyclophilin mRNA levels. All of the results presented are expressed as mean ± S.E. All of the primers used in this study are available upon request.

Lipopolysaccharide (LPS) Challenge—Experimental protocols were approved by the GlaxoSmithKline Institutional Animal Care and Use Committee. Female wild-type and LRH-1+/– mice (9) (n = 8/group) received an intraperitoneal injection of a LPS solution (40 µg/mice) or saline. 2.5 h post-injection, the animals were sacrificed and blood was recovered for serum preparation and the liver was quickly removed, frozen in liquid nitrogen, and used for RNA extraction.

Enzyme-linked Immunosorbent Assays (ELISAs)—Acute phase protein levels were measured by ELISA assays (IL-1-RA, RayBiotech, Inc.; HP, ICL).

Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed using the EpiQuick ChIP kit (Epigentek, P-2002) following the manufacturer’s instructions. LRH-1 and RNA polymerase 2 recruitments to the IL-1RA promoter were determined using a monoclonal anti-FLAG or a polyclonal anti-RNA polymerase 2 antibody, respectively. IL-1RA promoter occupancy was then assessed by PCR amplification of its proximal region using the following oligonucleotides: IL-1RA-ChIP-F (5'-CAAGCTAGCGTGACACAACAGGAAGGG-3') and IL-1RAChIP-R (5'-ATAAAGCTTGTGACTGCAAGCAAGGCAGTGG-3'). PCR reactions were analyzed by electrophoresis on a 1% agarose gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We recently established LRH-1 as a physiological modulator of the acute phase response in vitro and in vivo (19). LRH-1 antagonizes cytokine-mediated gene induction in hepatocytes by, at least in part, negatively interfering with the C/EBP signaling pathway. To get a better understanding of the role of LRH-1 in the control of this APR, HepG2 cells were infected for 24 h with an adenovirus encoding LRH-1 or green fluorescent protein (GFP) (as a control) and subsequently stimulated for 6 or 24 h with a combination of both IL-1beta and IL-6, the main effectors of the APR (20). At the end of the treatment period, total RNA were prepared and positive and negative acute phase gene expressions were monitored by RT-QPCR. As a control, ectopic expression of LRH-1 (m.o.i. = 10) in HepG2 cells resulted in a robust induction of both ApoA1 (3.0-fold ± 0.12) and SR-B1 (2.66-fold ± 0.10) mRNA levels, two well defined LRH-1 target genes (data not shown). The combination of IL-1beta and IL-6 strongly increased HP, serum amyloid A, PAI-1, FBG, and IL-1RA gene expression levels in a time-dependent manner, whereas it significantly diminished transferrin gene expression (Fig. 1) in agreement with previous reports (21, 22). In unstimulated cells, LRH-1 overexpression did not affect acute phase gene expression. However, LRH-1 significantly inhibited the cytokine-elicited inflammatory response, and this gene repression occurred in a time-dependent manner (Fig. 1, A, B, D, and E). Interestingly, PAI-1 levels were not affected by LRH-1 overexpression after 6 or 24 h of cytokine stimulation (Fig. 1C). Finally, LRH-1 surprisingly potentiated cytokine-mediated interleukin-1 receptor antagonist gene expression only after 24 h of cytokine treatment (Fig. 1F). Two forms of IL-1RA protein have been reported in mice and humans, a secreted (sIL-1RA) and intracellular form (icIL-1RA) resulting from the activity of two different promoters and differential splicing (23). However, hepatic cells only express the secreted form (24). Because IL-1RA is a well known anti-inflammatory acute phase protein (24, 25) and because LRH-1 seems to positively regulate this gene, we next focused our attention on the regulation of IL-1RA by LRH-1 and its biological relevance.


Figure 1
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  		FIGURE 1.
LRH-1 inhibits cytokine-mediated APR gene expression in a time-dependent fashion. HepG2 cells (A–F) cultured in 24-well plates were infected for 24 h with Ad-LRH-1 or Ad-GFP (m.o.i. = 10). Cells were then stimulated with the combination of IL-1beta (10 ng/ml) and IL-6 (10 ng/ml), or vehicle (phosphate-buffered saline 0.1% bovine serum albumin) for 6 and 24 h. At the end of the treatment, total RNA were extracted and gene expression levels were monitored by RT-QPCR.

 


Figure 2
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  		FIGURE 2.
LRH-1 overexpression potentiates cytokine-mediated IL-1RA up-regulation in a dose-dependent manner. A and B, HepG2 cells cultured in 24-well plates were infected for 24 h with Ad-LRH-1 or Ad-GFP at various m.o.i. (1, 5, and 10). Cells were then stimulated with a combination of IL-1beta (10 ng/ml) and IL-6 (10 ng/ml) or vehicle (phosphate-buffered saline 0.1% bovine serum albumin) for 24 h. At the end of the treatment, total RNA were extracted and IL-1RA and HP gene expressions were monitored by RT-QPCR. C, ELISA assay analysis of IL-1RA in the culture medium of HepG2 cells infected with Ad-LRH-1 or Ad-GFP (m.o.i. = 10) and stimulated for 24 h. D, Western blot analysis of HP in the culture medium of HepG2 cells infected with Ad-LRH-1 or Ad-GFP (m.o.i. = 10) and stimulated for 24 h.

 
LRH-1-mediated IL-1RA gene induction occurred in a dose-dependent manner (Fig. 2A). Again, in the absence of cytokine stimulation, LRH-1 was not able to up-regulate IL-1RA expression. This dose-dependent IL-1RA gene activation was correlated with a strong dose-dependent HP gene suppression after 24 h of IL-1beta/IL-6 treatment (Fig. 2B) in agreement with our previous results (Fig. 1A) (19). Interestingly, HepG2 cells secreted very low levels of IL-1RA, which were strongly stimulated in response to both IL-6 and IL-1beta mixture (Fig. 2C), as measured by ELISA assay. This effect was significantly potentiated by LRH-1 overexpression, whereas HP secretion was completely abrogated, as demonstrated by Western blot analysis (Fig. 2D). Taken together, these results indicate that LRH-1 regulates IL-1RA gene expression and protein levels in IL-1beta/IL-6-stimulated HepG2 cells.


Figure 3
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  		FIGURE 3.
Cytokine-mediated IL-1RA expression is reduced in LRH-1-deficient HepG2 cells. shLRH-1 or shLacZ HepG2 cells were stimulated with the combination of IL-1beta (10 ng/ml) and IL-6 (10 ng/ml) or vehicle (phosphate-buffered saline 0.1% bovine serum albumin) for 24 h. At the end of the treatment period, HP and IL-1RA gene expression levels were measured by RTQPCR (*, p < 0.05 shLacZ versus shLRH-1).

 
To gain further insight into the role of LRH-1 in the control of IL-1RA gene expression, gene-silencing experiments were carried out. IL-1RA mRNA levels were quantified in HepG2 cells stably expressing a shRNA targeting LRH-1 expression or a shRNA targeting LacZ as a control. As expected, IL-1beta/IL-6 treatment led to a robust (6-fold) induction of IL-1RA gene expression in shLacZ cells. Interestingly, this gene up-regulation was significantly less pronounced in LRH-1-deficient cells (2-fold), suggesting that LRH-1 is involved in cytokine-mediated IL-1RA gene expression. Of note, IL-1RA expression under resting conditions was not different in shLacZ compared with shLRH-1 HepG2 cells, indicating that LRH-1 does not contribute to IL-1RA basal gene expression (Fig. 3A). As a control, the cytokine-mediated inflammatory response was exacerbated in LRH-1-deficient HepG2 cells as illustrated by HP mRNA quantification in line with our previous report (19). Collectively, these data suggest that LRH-1 participates in cytokine-mediated but not basal IL-1RA gene expression in HepG2 cells.

To elucidate the molecular mechanism by which LRH-1 controls IL-1RA gene expression, a 1-kb fragment of the secreted IL-1RA gene promoter (26) was inserted upstream from the luciferase reporter gene. This promoter construct was active in HepG2 cells, as previously demonstrated by several groups (24, 27). IL-1beta/IL-6 treatment for 24 h led to a 6.5-fold activation of this promoter construct (Fig. 4A). Co-transfection of the LRH-1 expression vector enhanced this cytokine-induced promoter activity in line with our gene expression data (Figs. 1F and 2A). However, in the absence of stimulation, LRH-1 overexpression significantly increased the basal promoter activity (3.5-fold) in contrast with the previous results (Figs. 1F and 2A). Bioinformatic analysis of the human IL-1RA promoter revealed the presence of a potential LRH-1 binding site located roughly 120 bp upstream from the transcription initiation site. To demonstrate that this site is a functional LRH-1 binding site, a site-directed mutagenesis experiment was performed. Mutation of this putative LRH-1 binding site did not affect IL-1RA basal promoter activity as well as its induction by IL-1beta/IL-6 (Fig. 4A). By contrast, this mutated reporter construct lost its responsiveness to LRH-1 overexpression in the presence or absence of cytokine stimulation (Fig. 4A), demonstrating that this site is functionally required for LRH-1 to mediate IL-1RA gene regulation. Electrophoretic mobility shift assays performed using in vitro translated LRH-1 protein revealed that LRH-1 is, in fact, able to bind to this response element in vitro (data not shown). To unequivocally demonstrate that LRH-1 binds to this IL-1RA promoter region in vivo, ChIP assays were performed in HepG2 cells (Fig. 4B). HepG2 cells were infected with the adenovirus encoding a human FLAG-tagged LRH-1 or Ad-GFP for 24 h and subsequently treated with the interleukin mixture for 3 h. After the formaldehyde cross-linking, LRH-1 and the RNA polymerase 2 recruitment to the IL-1RA promoter was determined using monoclonal anti-FLAG and anti-polymerase 2 antibodies, respectively. In the absence of stimulation, we were unable to detect RNA polymerase 2 recruitment to the promoter, which is consistent with the weak IL-1RA gene expression. As expected, cytokine treatment for 3 h induced the association of the RNA polymerase 2 to the promoter. This specific recruitment was dramatically increased by LRH-1 overexpression (Fig. 4B). Interestingly, LRH-1 by itself is unable to trigger RNA polymerase 2 binding to the promoter, in agreement with the gene expression data (Figs. 1 and 2). As a matter of fact, LRH-1 was found to be associated to this promoter region only in cytokine-stimulated HepG2 cells, demonstrating the specific binding to this region in vivo. Taken together, these data indicate that LRH-1 regulates IL-1RA gene transcription via the binding to a response element located in the proximal promoter under inflammatory conditions.


Figure 4
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  		FIGURE 4.
LRH-1 regulates IL-1RA expression at the transcriptional level via the binding to an LRH-1 response element. A, HepG2 cells were transfected with the wild-type human IL-1RA or a LRH-1 response element-mutated construct (100 ng) and pSG5-LRH-1 (200 ng) or empty vector (pSG5). 24 h post-transfection, cells were stimulated with a combination of IL-6 (10 ng/ml) and IL-1beta (10 ng/ml) or vehicle (phosphate-buffered saline 0.1% bovine serum albumin) for 24 h. Thereafter, luciferase activity was determined. B, HepG2 cells infected with Ad-LRH-1 or Ad-GFP (m.o.i. = 10), stimulated with a combination of IL-1beta (10 ng/ml) and IL-6 (10 ng/ml) or vehicle (phosphate-buffered saline 0.1% bovine serum albumin) for 3 h, were subjected to ChIP assay using a FLAG (Sigma M2) or RNA polymerase 2 antibody. In vivo IL-1RA promoter occupancy was assessed by PCR amplification of the proximal promoter region. mut, mutant.

 


Figure 5
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  		FIGURE 5.
Reduced IL-1RA expression upon LPS challenge in LRH-1 heterozygous mice. Wild-type and LRH-1+/– mice (n = 8/group) received an intraperitoneal injection of a LPS solution (40 µg/animal) or saline. 2.5 h post-injection, animals were sacrificed, and plasma acute phase proteins were measured by ELISA assays (C and D). Liver gene expressions were also assessed by RT-QPCR (A and B). (*, p < 0.05, wild-type (WT) + LPS versus heterozygous + LPS).

 
To demonstrate the relevance of the IL-1RA gene regulation by LRH-1 in vivo, the APR was studied in wild-type and heterozygous LRH-1 mice during an LPS challenge, as previously described (19). Briefly, female mice (n = 8/group) received an intraperitoneal injection of a LPS solution (40 µg/animal) or saline. 2.5 h post-injection, animals were sacrificed, and IL-1RA and HP mRNA and protein levels were measured in the liver and in the plasma, respectively. As a control, we verified that LRH-1 gene expression was not affected by the LPS injection (data not shown). The LPS-induced inflammatory response was exacerbated in the LRH-1 heterozygous mice as illustrated by a more pronounced HP gene and protein induction (Fig. 5, B and D) in line with our previous report (19). As expected, LPS injection in wild-type mice resulted in a sharp induction of IL-1RA gene expression (>350-fold) as measured by RT-QPCR (Fig. 5A). By contrast, IL-1RA expression was significantly less induced in LRH-1 heterozygous animals (150-fold compared with 350-fold in wild-type mice) in line with our results obtained in LRH-1-deficient HepG2 cells (Fig. 3). Similar results were obtained at the protein level by ELISA assay (Fig. 5C). This result indicates that LRH-1 is involved in IL-1RA gene regulation in vivo.

Because IL-1RA and HP (or other APR genes such as serum amyloid A and FBG) genes are inversely regulated by LRH-1 in hepatic cells and because IL-1RA is a well described anti-inflammatory protein known to efficiently modulate the inflammatory response in vivo (28–30), it is tempting to speculate that LRH-1 negatively regulates the APR by, at least in part, inducing IL-1RA expression. To support this hypothesis, IL-1RA gene-silencing experiments were carried out in HepG2 cells. Transfection of HepG2 cells with siRNA targeting IL-1RA resulted in a strong inhibition of IL-1RA expression at both mRNA and protein levels (Fig. 6, A and B). To determine whether IL-1RA induction by LRH-1 contributes to its anti-inflammatory properties in hepatic cells, HP gene expression levels were measured in HepG2 cells transfected with siRNA targeting IL-1RA or a non-silencing control and subsequently infected with Ad-LRH-1 (m.o.i. = 10) or Ad-GFP for 24 h. Thereafter, the cells were stimulated in the presence or absence of the cytokine combination for 6 or 24 h. After 6 h of cytokine stimulation, HP expression was up-regulated as expected. This transcriptional activation was not affected by IL-1RA gene knockdown. Moreover, ectopic expression of LRH-1 significantly reduced cytokine-stimulated HP expression, and this inhibitory effect was similar when the IL-1RA gene was inhibited. As previously observed (Fig. 1A), 24-h IL-1beta/IL-6 treatment led, in the control cells, to a more robust induction of HP expression (compared with the 6 h time point) that was completely abolished by LRH-1 overexpression (Fig. 6). Interestingly, this LRH-1-mediated HP gene suppression was significantly less marked in HepG2 cells transfected with siRNA targeting IL-1RA expression (Fig. 6), suggesting that IL-1RA gene induction contributes to the negative regulation of the cytokine-elicited APR by LRH-1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
LRH-1 has been shown to be an important regulator of cholesterol homeostasis and to be involved in intestinal cell crypt renewal (1, 13). In addition, we have recently reported its functional implication in the control of the acute phase response (19). By studying APR gene regulation in HepG2 cells after a short or prolonged cytokine exposure, we first demonstrated that LRH-1 negatively regulates the APR in a time-dependent manner. Indeed, LRH-1-mediated gene suppression was much more pronounced in cells stimulated for 24 h with IL-1beta and IL-6 (Fig. 1). Interestingly, PAI-1 gene expression was not affected by LRH-1 overexpression, suggesting that LRH-1 selectively regulates cytokine-stimulated gene expression in hepatic cells. Similar observations were made with other nuclear receptors controlling the inflammatory response. For instance, glucocorticoids and LXR agonists antagonize NF{kappa}B transcriptional activation (31, 32). However, they inhibit NF{kappa}B-driven transcription in a promoter-specific manner (33). Moreover, these transcriptional cross-talks are often dependent on the nature of the inflammatory stimulus (31, 32). This is consistent with the concept that every nuclear receptor may differentially impact the development of the inflammatory response. These results strongly suggest that LRH-1 likely regulates a specific subset of APR genes. Transcriptome analysis using various inflammatory stimuli will be required to precisely identify this LRH-1-specific subset.


Figure 6
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  		FIGURE 6.
LRH-1 negatively interferes with the cytokine-elicited inflammatory response by, at least in part, inducing IL-1RA expression. A, HepG2 cells were transfected with a siRNA-targeting IL-1RA gene or a control siRNA (20 nM). 24 h post-transfection, HepG2 cells were stimulated by a combination of IL-1beta (10 ng/ml) and IL-6 (10 ng/ml) or vehicle (phosphate-buffered saline 0.1% bovine serum albumin). IL-1RA mRNA levels were measured by RT-QPCR. B, quantification of IL-1RA in the culture medium was performed by ELISA assay. C, HepG2 cells were transfected with a siRNA-targeting IL-1RA and a control siRNA (20 nM each) and infected with an Ad-LRH-1 or Ad-GFP (m.o.i. = 10). 24 h post-infection, HepG2 cells were then stimulated with a combination of IL-1beta (10 ng/ml) and IL-6 (10 ng/ml) or vehicle for 6 and 24 h. After stimulation, HP mRNA levels were measured by RT-QPCR. (*, p < 0.05,). CTL, control; RNAi, RNA interference.

 
IL-1RA gene was identified in this study as a novel LRH-1 target gene under inflammatory conditions both in vitro and in vivo. In fact, LRH-1 was found to potentiate cytokine-induced IL-1RA expression in hepatic cells (Figs. 1, 2, and 5). Furthermore, gene-silencing experiments indicated that LRH-1 is a physiological modulator of IL-1RA expression under inflammatory conditions (Fig. 3A). Promoter studies and ChIP assays revealed that LRH-1 regulates IL-1RA expression at the transcriptional level via binding to a LRH-1-response element located 120 bp upstream from the transcriptional start site (Fig. 4). This IL-1RA gene induction was significantly impaired in LRH-1 heterozygous mice subjected to an LPS challenge, demonstrating the in vivo relevance of this mechanism (Fig. 5). PPAR{alpha} (peroxisome proliferator-activated receptor {alpha}) was recently reported to regulate IL-1RA expression in chondrocytes by a similar mechanism (27). Indeed, PPAR{alpha}-mediated IL-1RA gene promoter activation required the activation of both NF{kappa}B and C/EBP transcription factors (27). Whether LRH-1 functionally cooperates with both NF{kappa}B and C/EBP signaling pathways is still to be determined and will require further experiments.

IL-1RA was originally described as an IL-1 inhibitory activity in the urine of patients with fever (see Ref. 25 for review). This naturally occurring anti-inflammatory protein competitively blocks the binding of IL-1{alpha} and IL-1beta to type I and type II IL-1 receptors but does not transduce a signal (34). Two forms of IL-1RA protein have been reported in mice and humans, a secreted (sIL-1RA) and intracellular form (icIL-1RA) resulting from the activity of two different promoters and differential splicing (23). IL-1RA knock-out mice have been shown to develop chronic inflammatory arthropathy (28), to display a hypersensitivity to bacterial LPS (35) and to an arterial inflammatory state (30), and finally to present an exaggerated inflammatory response associated with an increased mortality during the APR (29). Furthermore, IL-1RA has been shown to be highly produced by the liver during the APR and therefore is considered as a positive acute phase protein (24, 36). Interestingly, hepatic cells only produce the secreted form (24). In addition to its crucial role in regulating IL-1 signaling in various inflammatory states, IL-1RA deficiency has been associated with major metabolic dysfunctions (37, 38). Isoda et al. (37) reports a liver enlargement with intrahepatic lipid deposits under high fat diet in IL-1RA knock-out mice. This is mainly because of an impaired bile acid metabolism resulting in an increase in very low density lipoprotein and low density lipoprotein-cholesterol and a decrease in high density lipoprotein-cholesterol (37). IL-1RA may be an important molecular link between lipid metabolism and inflammation. The role of IL-1RA in bile acid biosynthesis via small heterodimer partner regulation is intriguing, because LRH-1 has also been shown to play a key role in CYP7A1 and CYP8B1 gene regulation (1). Whether LRH-1 regulates bile acid production via an IL-1RA-dependent mechanism will have to be investigated. Nevertheless, by controlling IL-1RA hepatic and systemic levels (Fig. 6) and by affecting cholesterol homeostasis, LRH-1 may impact the development of chronic inflammatory diseases such as atherosclerosis.

Several concurring mechanisms may explain the anti-inflammatory activities of LRH-1 during the APR in vitro and in vivo. We previously demonstrated that LRH-1 antagonizes cytokine-mediated gene expression by, at least in part, inhibiting the C/EBP signaling pathway (19). In addition, Mueller et al. (39) has recently reported the involvement of LRH-1 in extra-adrenal glucocorticoid synthesis in the intestine, with likely consequences for immune homeostasis. Here, we have identified a novel mechanism by which LRH-1 may exert those effects in hepatic cells (Fig. 7). Kinetic studies indicated that LRH-1 blocks cytokine-induced APR gene expression more efficiently after 24 h than 6 h of stimulation (Fig. 1), raising the possibility that an additional mechanism may be required to fully inhibit this inflammatory response. The identification of IL-1RA (a well known anti-inflammatory protein) as a novel LRH-1 target gene led us to speculate that LRH-1 may regulate the APR by inducing this gene. Gene-silencing experiments revealed that IL-1RA induction contributes to the overall anti-inflammatory properties of LRH-1 after 24 h but not after 6 h of cytokine stimulation (Fig. 6). This result strongly suggests that LRH-1 negatively regulates this APR by first inhibiting C/EBP transcriptional activity and thereafter by inducing IL-1RA expression leading to IL-1 signaling blockade (Fig. 7). The relative contribution of these different mechanisms on the development of acute or chronic inflammatory responses remains to be determined.


Figure 7
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  		FIGURE 7.
Model of control of the hepatic APR by LRH-1. SAA, serum amyloid A; C/EBPbeta, CCAAT/enhancer-binding protein beta; FBG, fibrinogen beta.

 
In conclusion, our results lead to the identification of IL-1RA as a novel LRH-1 target gene and demonstrate the existence of multiple mechanisms contributing to the overall anti-inflammatory properties of LRH-1 in hepatic cells.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: GlaxoSmithKline R & D, Cardiovascular and Urogenital Center of Excellence for Drug Discovery, 25 Ave. du Québec, 91951 Les Ulis, France. Tel.: 33-169296081; Fax: 33-169074892; E-mail: pxd14884{at}gsk.com.

2 The abbreviations used are: LRH-1, liver receptor homolog-1; APR, acute phase response; HP, haptoglobin; IL-1RA, interleukin-1 receptor antagonist; siRNA, small interfering RNA; RT-QPCR, real time quantitative PCR; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; ChIP, chromatin immunoprecipitation; m.o.i., multiplicity of infection; sh, short hairpin; C/EBP, CCAAT/enhancer-binding protein; Ad, adenovirus. Back


   ACKNOWLEDGMENTS
 
We thank Jason Smith for technical contribution, Stéphane Huet (GlaxoSmithKline (GSK) Cardiovascular and Urogenital Center of Excellence for Drug Discovery) and Bryan Goodwin (GSK Molecular Discovery Research (MDR)) for critical reading of the manuscript, John Bisi (GSK MDR) for providing FLAG-tagged Ad-LRH-1, and Beverly H. Koller and Anne Latour (Department of Medicine, University of Chapel Hill, Chapel Hill, NC) for LRH-1 heterozygous mice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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