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Endocrinology Vol. 143, No. 7 2559-2570
Copyright © 2002 by The Endocrine Society

RECEPTORS

Estrogen Receptor {alpha} Inhibits IL-1ß Induction of Gene Expression in the Mouse Liver

Mark J. Evans, Kehdih Lai, Lucinda J. Shaw, Douglas C. Harnish and Christopher C. Chadwick

Wyeth Research, Collegeville, Pennsylvania 19426

Address all correspondence and requests for reprints to: Dr. Mark Evans, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426. E-mail: . evansm{at}wyeth.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogens have been suggested to modulate several inflammatory processes. Here, we show that IL-1ß treatment induced the expression of approximately 75 genes in the liver of ovariectomized mice. 17{alpha}-Ethinyl estradiol (EE) pretreatment reduced the IL-1ß induction of approximately one third of these genes. Estrogen receptor {alpha} (ER{alpha}) was required for this inhibitory activity, because EE inhibition of IL-1ß-stimulated gene expression occurred in ERß knockout mice, but not in ER{alpha} knockout mice. EE treatment induced expression of 40 genes, including the transcriptional repressor short heterodimer partner and prostaglandin D synthase, known modulators of nuclear factor-{kappa}B signaling. However, the ER agonists genistein and raloxifene both inhibited IL-1ß gene induction without stimulating the expression of prostaglandin D synthase, short heterodimer partner, or other ER-inducible genes, indicating that induction of gene expression was not required for ER inhibition of IL-1ß signaling. Finally, the ability of EE to repress IL-1ß gene induction varied among tissues. For example, EE inhibited IL-1ß induction of lipopolysaccharide-induced c-x-c chemokine (LIX) in the liver, but not in the spleen or lung. The degree of EE repression did not correlate with ER expression. cAMP response element binding protein-binding protein (CBP)/p300 levels also varied between tissues. Together, these results are consistent with a model of in vivo ER interference with IL-1ß signaling through a coactivator-based mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEPSIS IS THE 11th leading cause of death in the United States and is a major cause of mortality in the intensive care unit, with mortality rates of 40–60% (1). The initial manifestation of sepsis is extensive inflammation, primarily due to increased TNF{alpha}, IL-1, and IL-6 levels (2) resulting from bacterial products such as lipopolysaccharides (LPS). Animal models of sepsis have suggested that anticytokine therapy could provide benefit in sepsis (3). However, several randomized clinical trials of antiinflammatory therapies, including the use of naturally occurring antagonists or blocking antibodies directed against TNF{alpha} or IL-1ß, have not shown any improvement in survival from sepsis (1), perhaps due to the redundant nature of cytokine signaling pathways.

In humans, gender has recently been shown to be a significant predictor of survival from sepsis, with a hospital mortality rate of 70% for male patients, but only 26% for female patients in a prospective study (4). Surprisingly, although the women studied in this trial were postmenopausal, their plasma levels of 17ß-estradiol were in the high normal physiological range (250 pg/ml) and were significantly higher than those in the male patients, possibly due to inhibited degradation of estradiol by the liver due to sepsis. Similarly, female mice are better able than male mice to tolerate sepsis after cecal ligation and puncture (5). Male rats also develop greater uveitis than female rats after injection of LPS (6). Further, ovariectomized females develop greater uveitis than intact females, and administration of 17ß-estradiol to ovariectomized females reduces the development of uveitis. Estrogen thus may be a significant component for the gender effects seen in sepsis.

In vitro, estrogen has been demonstrated to interfere with cytokine signaling in several systems. One of the best characterized systems is estrogen inhibition of IL-1ß induction of IL-6 expression, which occurs in both rodent and human bone marrow-derived cell lines (7). This decrease in IL-6 expression occurs through estrogen receptor (ER) inhibition of both nuclear factor-{kappa}B (NF-{kappa}B) and NF-IL6 activation of the IL-6 promoter (8, 9). The DNA binding domain of the ER is not required for inhibition of IL-6 promoter activity (10), but the carboxyl-terminal activation function-2 (AF2) domain of ER is necessary for this inhibition (11). Similarly, ER can inhibit TNF-{alpha} induction of gene expression (12) in an AF2 domain-dependent manner (13). Further studies of ER inhibition of NF-{kappa}B activity have suggested multiple potential mechanisms, including direct interaction between ER and NF-{kappa}B (9, 10), inhibition of NF-{kappa}B binding to its cognate DNA recognition site (14), or stabilization of I{kappa}B{alpha} (15). Overexpression of the coactivator cAMP response element binding protein-binding protein (CBP) in HepG2 cells (16) or the related coactivator p300 in smooth muscle cells (17) reduces the inhibitory effects of ER on NF-{kappa}B activation of gene expression. Coactivator competition between ER and NF-{kappa}B may thus be the predominant mechanism in cultured cells with limiting amounts of coactivators, whereas in other cell types with higher levels of coactivator expression different mechanisms may mediate ER inhibition of gene induction by cytokines. Which, if any, of these mechanisms occur in vivo with physiological levels of ER, coactivators, and cytokine signaling pathway components is unclear.

Estrogens bind to two distinct nuclear receptors, ER{alpha} and ERß (18). These two receptors have a nearly identical DNA binding domain and can both activate transcription through binding to identical ER response elements (19, 20, 21). ER{alpha} and ERß also have identical core AF2 domain sequences (22) and can bind a common set of coactivators, including SRC-1, SRC-2, and SRC-3 (23, 24). However, although ER{alpha} and ERß bind 17ß-estradiol with comparable affinity (25), they share only 60% homology in the ligand binding domain and differ entirely in the AF1 domain. These differences may be responsible for emerging distinctions between ER{alpha} and ERß, including the opposite effects of these receptors on AP1 site activity (26). The liver expresses predominantly ER{alpha} (25) with low levels of ERß (27). Although the liver contains predominantly hepatocytes, sinusoidal endothelial cells and Kupffer cells also express ER (28). The distribution of ER{alpha} and ERß among these cell types has not yet been determined. Both hepatocytes and Kupffer cells contribute to the liver response to LPS. For example, LPS induction of macrophage inflammatory protein-2 (MIP-1ß), RANTES (regulated upon activation, normal T cell expressed and secreted), and monocyte chemoattractant protein-5 (MCP-5) is reduced in Kupffer cell-depleted mice, whereas LPS induction of inducing protein-10, KC, MIP-2, and MCP-1 is not altered (29). Endothelial cells, particularly endothelial cells at sites of injury, predominantly express ERß (30), suggesting that either receptor could modulate inflammation in the liver.

Recently, we (31) demonstrated that estrogen can inhibit high fat diet-induced inflammation in the mouse liver through an ER-dependent mechanism. Although likely to include cytokine signaling networks, the pathway for diet-induced inflammation in this model has not yet been determined, nor is it clear that the protective effects of estrogen in this model occur through direct estrogen activity within the liver as opposed to estrogen activity in other organs such as the digestive tract. As IL-1 administration can mimic many aspects of sepsis (32), here we have used an IL-1ß-dependent short-term model of mouse liver inflammation more likely to mimic clinical sepsis inflammation to examine the roles of estrogen, ER{alpha}, and ERß in inhibition of cytokine signaling in the liver. We demonstrate that gene induction by ER is not required for estrogen’s protective effects, and that the inhibitory activity of estrogen on a given gene differs between tissues. Together, these findings suggest that the proposed coactivator sharing mechanism for ER inhibition of NF-{kappa}B activity may occur in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Ovariectomized C57BL/6 mice (16–20 g) were purchased from Taconic Farms (Lexington, KY). ER{alpha} knockout mice (33) were bred onto a C57BL/6 strain background, or ERß knockout mice on a 129 strain background were generated and ovariectomized internally. No ERß mRNA can be detected in several tissues analyzed in these ERßKO mice. All mice were fed a casein-based diet to reduce phytoestrogen exposure. For some studies, 0.3% cholic acid (Sigma, St. Louis, MO) was added to powered casein diet. After 5–7 d of recovery, the mice were treated by daily sc injections of vehicle (90% corn oil/10% ethanol) or vehicle containing compounds. On the fifth day of treatment, the mice received an ip injection of PBS containing 20 µg/kg IL-1ß 1 h after receiving the sc injection. One hour later, the mice were euthanized, and total liver, spleen, or lung RNA was prepared using TRIzol reagent (Life Technologies, Inc., Grand Island, NY). For all studies, groups consisted of six to eight animals, with all animals treated in accord with accepted standards of care as specified by the Wyeth animal care committee.

Microarray analysis
For microarray studies, polyadenylated [poly(A)] RNA was purified using the polyATtract system (Promega Corp., Madison, WI). Double-stranded cDNA was synthesized from 1 µg poly(A) RNA using the SuperScript System (Life Technologies, Inc.). Briefly, 1 µg poly(A) RNA was mixed with 100 pmol oligonucleotide GGCCATGGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24 in 20 µl water, annealed at 70 C for 10 min, and quick-chilled. Buffer, dithiothreitol, and dNTP mix were then added and incubated at 37 C for 2 min. SuperScript II reverse transcriptase was added, and the 37 C incubation was continued for 60 min. Second-strand synthesis was performed by adding reaction buffer, deoxy-NTPs (200 µM), DNA ligase (10 U), DNA polymerase (40 U), ribonuclease H (2 U), and water (to a final volume of 150 µl), and the reaction was incubated for 2 h at 16 C. This was followed by addition of 10 U T4 DNA polymerase and incubation at 16 C for 5 min. The cDNA was purified by phenol chloroform extraction, precipitated, and transcribed in vitro using T7 RNA polymerase. The cRNA was purified by RNeasy column (QIAGEN, Chatsworth, CA) and fragmented by incubation in 40 mM Tris (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate buffer at 94 C for 35 min. Fifteen micrograms of fragmented RNA were hybridized sequentially to Mu11KsubA and Mu11KsubB GeneChips (Affymetrix, Santa Clara, CA) at 45 C as recommended by the manufacturer. The hybridized chips were washed as recommended by the manufacturer, and scanned and analyzed using GeneChip 3.1 software (Affymetrix). Expression levels in animals receiving the vehicle pretreatment plus IL-1ß or 17{alpha}-ethinyl estradiol (EE) pretreatment plus IL-1ß were determined using expression levels in animals receiving vehicle pretreatment and PBS treatment as the reference. Hybridization intensities were normalized using global scaling to a target intensity of 2500. The 5'/3' ratio for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ranged from 0.93–1.08, and that for ß-actin ranged from 0.67–0.81. Fold change values were determined from the GeneChip 3.1 software. Two independent experiments with separate sets of animals were performed. Changes were considered reproducible only if the same direction change occurred with magnitude greater than 2-fold in each experiment.

Real-time PCR
Selected regulated genes identified with the gene chip were verified by real-time RT-PCR using an ABI PRISM 7700 Sequence Detection System according to the manufacturer’s protocol (PE Applied Biosystems, Foster City, CA). TaqMan primers and probes were made for each gene of interest. The data were analyzed using Sequence Detector version 1.7 software (PE Applied Biosystems) and were normalized to GAPDH using the PE Applied Biosystems primer set. Statistical significance was determined by ANOVA.

Cell transfections
HepG2 cells were maintained in growth medium at 37 C in a 5% CO2 incubator. The cells were seeded in growth medium [phenol red-free DMEM (Life Technologies, Inc.) supplemented with heat-inactivated 10% FBS, 1% Glutamax, 1% MEM nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin] at 2.5 x 105 cells/well in a 12-well dish (Falcon) before transfection. The cells were transfected by the calcium phosphate coprecipitation method using an NF-{kappa}B luciferase reporter, a control ß-galactosidase reporter, and either pcDNA3-ER{alpha} or ERß expression vectors as described previously (16). Luciferase activity was determined by a chemiluminescent method using a Luciferase Assay System (Promega Corp.). ß-Galactosidase activity was determined using Galacto-Light (Tropix, Inc., Bedford, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To determine whether estrogen can alter IL-1ß signaling in vivo, ovariectomized C57BL/6 mice were treated with vehicle or EE for 5 d. On the fifth day, the mice received an ip injection of IL-1ß. One hour later, the livers were removed, and RNA was isolated and analyzed for expression levels of approximately 11,000 genes (Table 1Go). Ovariectomized animals that received vehicle treatment and no IL-1ß were used as a reference group for basal RNA expression (this level of expression was defined as 1.0 for each gene). Seventy-five known genes were induced at least 2-fold by IL-1ß treatment of ovariectomized mice. Many of these genes are known targets of IL-1ß regulation, including numerous chemokines, cytokines, acute phase proteins, and adhesion molecules. An additional prominent class of genes induced by IL-1ß include numerous transcription factors, particularly members of the NF-{kappa}B transcription factor family (NFKB1, relB, NFKB2, and relA) and the AP1 family (jun and junB). IL-1ß treatment also reduced the expression of five genes, including the transcription cofactors hairy and enhancer of split 1 and short heterodimer partner (SHP).


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  		Table 1. IL-1ß regulation of gene expression in the mouse liver

 
IL-1ß induction of many genes was reproducibly altered by estrogen treatment (Table 1Go), as graphically depicted in Fig. 1Go. Genes induced by IL-1ß are in the upper right quadrant, and genes inhibited by IL-1ß are in the lower left quadrant. The majority of genes lie close to the diagonal line, indicating that IL-1ß induction of these genes was not influenced by EE. However, many genes showed altered expression in animals receiving EE treatment. For example, transglutaminase showed higher induction in EE- and IL-1ß-treated animals (8.0-fold) than in vehicle- and IL-1ß-treated animals (2.2-fold). This is consistent with the known induction of transglutaminase expression by both estrogen (34) and increased NF-{kappa}B activity (35). In contrast, approximately one third of genes had decreased expression in animals treated with EE and IL-1ß compared with animals treated with vehicle and IL-1ß. For genes such as fnk and JAK-binding protein (JAB), RNA levels in the EE- plus IL-1ß-treated animals were reduced completely to the basal expression levels. However, for most genes, such as LPS-induced c-x-c chemokine (LIX) and bcl-3, RNA expression levels in the EE- and IL-1ß-treated animals were reduced about 50% from RNA levels in the vehicle- and IL-1ß-treated animals. Finally, the reduction in SHP mRNA levels by IL-1ß was also significantly attenuated in animals treated with EE.



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  		Figure 1. Effects of EE on IL-1ß induction of gene expression in mice. The IL-1ß fold induction of gene expression in animals receiving EE treatment (y-axis) is plotted vs. the IL-1ß fold induction in animals receiving vehicle treatment (x-axis) for all genes listed in Table 1Go. Genes with IL-1ß induction unaffected by EE treatment would lie along the dotted diagonal line. Genes above the diagonal line are stimulated by EE treatment, genes below the diagonal line are inhibited by EE treatment. The positions of the genes fnk, JAB, LIX, bcl-3, SHP, and transglutaminase are indicated by open circles.

 
The reduced level of IL-1ß-induced gene expression could be due to either of two processes. Firstly, EE treatment might alter the basal expression of a gene but not have any effect on the ability of IL-1ß to induce that gene. In this case, the fold induction of the gene would be expected to be the same in animals treated with either vehicle or EE. Alternatively, EE might have no effect on basal expression but, rather, might specifically inhibit IL-1ß induction of gene expression. In this case the fold induction of the gene should be reduced in EE-treated animals compared with vehicle-treated animals. To distinguish between these two mechanisms, the IL-1ß fold regulation of fnk, JAB, LIX, bcl-3, and SHP was determined in animals treated with vehicle or EE (Fig. 2AGo). These genes were chosen to cover a range of inductions by IL-1ß (bcl-3 > JAB > LIX > fnk) or repression by IL-1ß (SHP). The fold induction of fnk, JAB, and LIX was significantly reduced, and EE did not alter the basal expression of these three genes (not shown). In contrast, the magnitude of IL-1ß regulation of bcl-3 and SHP was the same in animals receiving vehicle or EE (Fig. 2AGo). The regulation of these genes by EE was due to changes in basal expression, with the basal expression of bcl-3 reduced by EE, and the basal expression of SHP increased by EE (Fig. 2BGo). The IL-1ß induction of bcl-3 was 6.2-fold in vehicle-treated animals and 5.2-fold in EE-treated animals. The IL-1ß regulation of SHP was 0.14-fold in vehicle-treated animals and 0.15-fold in EE-treated animals. IL-1ß regulation of these genes was unaffected by EE treatment. Thus, estrogen can influence in vivo IL-1ß signaling both by reducing the basal expression of IL-1ß target genes and by reducing the ability of IL-1ß to induce gene expression.



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  		Figure 2. Regulation of basal and IL-1ß-induced gene expression by EE. A, Ovariectomized C57BL/6 mice were treated sc with vehicle or 100 µg/kg·d EE for 5 d. On the fifth day, animals received vehicle or 20 µg/kg IL-1ß by ip injection. One hour later, liver total RNA was prepared and analyzed by real-time PCR for fnk, JAB, LIX, bcl-3, and SHP expression, with normalization for GAPDH expression. The values reported are the IL-1ß fold change for each gene in either vehicle-treated animals (black bars) or EE-treated animals (gray bars). *, P < 0.05 for comparison of EE plus IL-1ß induction vs. vehicle plus IL-1ß induction. B, The expression levels (mean ± SEM) of bcl-3 and SHP were determined for livers from ovariectomized C57BL/6 mice treated with vehicle (gray bars), vehicle plus IL-1ß (black bars), EE (gray hatched bars), or EE plus IL-1ß (black hatched bars). The expression values were normalized for GAPDH, with expression in vehicle-treated animals defined as 1.0. The numerical fold change induced by IL-1ß treatment is shown for animals treated with either vehicle or EE. *, P < 0.01 for comparison of EE to vehicle groups. **, P < 0.01 for comparison of EE plus IL-1ß to vehicle plus IL-1ß.

 
Estrogens can regulate gene expression through numerous mechanisms. Many of these processes are mediated by ER{alpha} or ERß, although estrogens can also regulate physiological processes through antioxidant mechanisms (36). To determine whether ER activity was required for EE regulation of IL-1ß gene induction, mice were treated with ER agonists, including EE, 17ß-estradiol, genistein, and raloxifene, as well as the ER antagonist ICI182780 (Fig. 3Go). The agonists all decreased the expression of fnk, JAB, LIX, and bcl-3, with EE and 17ß-estradiol having the greatest activity, genistein having intermediate activity, and raloxifene having the weakest activity. In contrast, ICI182780 did not inhibit IL-1ß induction of any of these genes. To confirm the necessity of ER for these regulations and to determine which receptor subtype was required, wild-type mice, ER{alpha} knockout mice, and ERß knockout mice were treated with vehicle or EE followed by a 1-h IL-1ß treatment (Fig. 4Go). EE had no effect on the expression of fnk, JAB, LIX, bcl-3, or SHP in the ER{alpha} knockout mice. In contrast, the regulation of these genes was similar in the ERß knockout mice and the wild-type mice. Together, these results indicate that estrogen regulation of IL-1ß signaling in the liver was primarily mediated through agonist activation of ER{alpha}.



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  		Figure 3. Regulation of IL-1ß gene induction by ER ligands. Ovariectomized mice were treated sc for 5 d with vehicle, 100 µg/kg·d EE, 1 mg/kg·d 17ß-estradiol (1 E2), 10 mg/kg·d 17ß-estradiol (10 E2), 30 mg/kg·d genistein, 5 mg/kg·d raloxifene, or 10 mg/kg·d ICI182780 (ICI). On d 5 the mice received ip injections of vehicle (gray bars) or 20 µg/kg IL-1ß (black bars). One hour later, liver total RNA was prepared. Gene expression in each animal was quantified by real-time PCR, with normalization for GAPDH expression. All values are reported relative to the mean expression in vehicle-treated animals, defined as 1.0. *, P < 0.05 for comparison to vehicle- plus IL-1ß-treated animals.

 


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  		Figure 4. ER dependence of EE regulation of IL-1ß gene induction. Ovariectomized C57BL/6 wild-type mice (WT), C57Bl/6 ER{alpha}-/- (ER{alpha}KO) mice, or 129 ERß-/- (ERßKO) mice were treated sc for 5 d with vehicle (black bars) or 10 µg/kg·d EE (gray bars), followed by a 1-h treatment with 20 µg/kg IL-1ß. A control group of animals from each strain was treated with vehicle for 5 d, followed by a 1-h treatment with PBS. Liver expression levels of fnk, JAB, LIX, bcl-3, and SHP (mean ± SEM) were quantified for each individual animal by real-time PCR, with the mean expression in the control animals receiving vehicle and PBS defined as 1.0. *, P < 0.05 for comparison of IL-1ß induction of each gene between EE-treated animals and vehicle-treated animals.

 
SHP functions as a corepressor for many transcription factors, suggesting that EE induction of basal SHP levels could inhibit gene induction by IL-1ß. Feeding mice a diet enriched in cholic acid induces the expression of SHP (37, 38, 39), which, in turn, inhibits the transcription of cholesterol 7{alpha}-hydroxylase (CYP7A1), the rate-limiting enzyme in the bile acid synthetic pathway. As expected, feeding cholic acid to ovariectomized mice induced SHP expression and nearly completely repressed the expression of CYP7A1 (Fig. 5Go). Although IL-1ß decreased SHP mRNA levels in the animals fed cholate, there was no corresponding increase in CYP7A1 expression, presumably due to a longer than 1 h half-life for the SHP protein. IL-1ß induction of fnk, JAB, LIX, and bcl-3 was similar in animals receiving either the control or cholic acid-enriched diets. This inability of bile acid induction of SHP to inhibit IL-1ß induction of gene expression is consistent with the ability of genistein and raloxifene to inhibit IL-1ß induction of gene expression without increasing SHP expression (Fig. 3Go). Thus, induction of SHP is not sufficient for inhibition of IL-1ß signaling and does not appear to be involved in EE regulation of IL-1ß signaling.



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  		Figure 5. IL-1ß induction of gene expression in mice receiving a cholate diet. Ovariectomized mice were fed either a standard casein diet or a casein diet supplemented with 0.3% cholic acid and treated with vehicle for 5 d. On d 5, 1 h after the animals received either vehicle (gray bars) or IL-1ß (black bars) treatment, liver RNA was prepared and analyzed by real-time PCR for expression levels of SHP, cholesterol 7{alpha}-hydroxylase (CYP7A1), fnk, JAB, LIX, and bcl-3.

 
Although induction of SHP was not necessary for ER{alpha} repression of IL-1ß signaling, EE pretreatment might have induced the expression of other genes responsible for the inhibition of IL-1ß activity. Analysis of the microarray data indicated that EE pretreatment increased the expression of 38 known genes by at least 2-fold (Table 2Go). Many of the identified genes are known targets for estrogen regulation, including inositol-1-phosphate synthase (40), intestinal trefoil factor (41), creatine kinase (42), and signal transducer and activator of transcription-5A (43). Interestingly, prostaglandin D2 synthase expression was increased dramatically by EE pretreatment. In cultured cells, overexpression of prostaglandin D synthase inhibits NF-{kappa}B activity by blocking the activity of I {kappa} kinase (44). However, genistein did not stimulate the expression of prostaglandin D synthase (Fig. 6Go), but did inhibit IL-1ß induction of fnk, JAB, LIX, and bcl-3. Raloxifene weakly induced prostaglandin D synthase expression, but it was less efficacious than genistein for inhibiting IL-1ß induction of fnk, JAB, LIX, and bcl-3. Thus, there was no correlation between the induction of prostaglandin D synthase and the repression of IL-1ß induction of gene expression. Genistein and raloxifene also did not stimulate the expression of several other genes induced by EE, including inositol-1-phosphate synthase, intestinal trefoil factor, apolipoprotein A IV, and C1qB (Fig. 6Go). Induction of gene expression does not therefore appear to be a necessary component for ER{alpha} inhibition of IL-1ß gene induction.


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  		Table 2. EE induction of gene expression in the mouse liver

 


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  		Figure 6. Induction of gene expression by ER ligands. Liver expression of prostaglandin D synthase, inositol-1-phosphate synthase (IPS), intestinal trefoil factor (ITF), apolipoprotein A-IV (apoA IV), and complement protein C1qB was determined for the mice treated with 100 µg/kg·d EE, 30 mg/kg·d genistein, 5 mg/kg·d raloxifene, or 10 mg/kg·d ICI182780 (ICI) as described in Fig. 4Go. On d 5 the mice received ip injections of vehicle (gray bars) or 20 µg/kg IL-1ß (black bars). Values are the mean ± SEM, with mean expression in animals treated with vehicle plus PBS defined as 1.0. *, P < 0.05 for comparison to the vehicle- plus PBS-treated animals.

 
The ability of EE to inhibit IL-1ß induction of only a subset of genes suggests that gene-specific factors regulate this inhibition. Genes that show EE inhibition of IL-1ß induction and genes that lack EE inhibition of IL-1ß induction share common primary transcription factors such as NF-{kappa}B. However, each individual promoter has an overall unique suite of transcription factors, probably recruiting a distinct set of coactivators. Nuclear receptor interaction with coactivators has been suggested to play an important role in the pathway cross-talk seen in transfected cultured cells (16). In this model, in vivo ER inhibition of IL-1ß induction of gene expression would be expected to differ between different tissues, as the levels of coactivators and corepressors varies between tissues. We therefore examined the ability of EE to inhibit IL-1ß gene inductions in liver, spleen, and lung. Liver expressed almost exclusively ER{alpha}, spleen expressed lower levels of ER{alpha}, and lung expressed low levels of ER{alpha} and high levels of ERß mRNA (Fig. 7AGo). IL-1ß induced the expression of fnk, JAB, LIX, and bcl-3 in all three organs, although the magnitude of the induction varied significantly. In the liver, EE pretreatment inhibited IL-1ß induction of these genes. In the spleen, EE pretreatment inhibited the induction of fnk and JAB, but did not inhibit the induction of LIX or bcl-3. In the lung, EE treatment did not inhibit the induction of any of these genes (Fig. 7BGo). This was not due to a general inability of ERß to block IL-1ß signaling, because cotransfection of either an ER{alpha} or an ERß expression plasmid along with an NF-{kappa}B-driven reporter plasmid into HepG2 cells resulted in an agonist-dependent inhibition of NF-{kappa}B induction of gene expression (Fig. 8Go). Thus, ERß has the intrinsic ability to inhibit IL-1ß signaling. The tissue specificity of EE inhibition of IL-1ß inductions combined with the lack of requirement for EE induction of gene expression agrees with a model for in vivo ER regulation of IL-1ß signaling through coactivator interactions. CBP and p300 mRNA expression levels were several-fold higher in spleen and lung than in liver (Fig. 7BGo). Limiting amounts of CBP in liver might allow ER to inhibit the induction of genes such as LIX, whereas the higher levels of CBP expression in the spleen and lung might overcome this repression.



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  		Figure 7. Regulation of IL-1ß induction of gene expression by EE in the spleen and lung. A, Ovariectomized C57BL/6 mice were treated sc with vehicle for 5 d. Total RNA was prepared from liver, spleen, and lung and analyzed for ER{alpha} and ERß expression by real-time PCR. Values are the mean ± SEM after normalization for GAPDH levels. B, Ovariectomized C57BL/6 mice were treated sc for 5 d with vehicle (open and black bars) or 10 µg/kg·d EE (gray bars). On d 5, the mice received an ip injection of PBS (open bars) or 20 µg/kg IL-1ß (black and gray bars). After 1 h, total RNA was prepared from liver, spleen, and lung. The expression levels of fnk, JAB, LIX, bcl-3, CBP, and p300 were determined for each tissue by real-time PCR. The expression levels in the mice that received vehicle and IL-1ß treatment (black bars) or EE and IL-1ß treatment (gray bars) are reported relative to those in mice that received vehicle and PBS treatment (open bars), with the mean value for this group defined as 1.0 for each gene in each tissue. *, P < 0.05 for comparison of EE plus IL-1ß to vehicle plus IL-1ß.

 


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  		Figure 8. In vitro inhibition of IL-1ß signaling by ER{alpha} and ERß. HepG2 cells were transfected by electroporation with an NF-{kappa}B-driven luciferase reporter plasmid, a ß-galactosidase control reporter plasmid, and either an human ER{alpha} or ERß expression plasmid. Four hours later the cells were treated with vehicle (gray bars), 10 nM 17ß-estradiol (black bars), or 10 nM 17ß-estradiol and 1 µM ICI182780 (hatched bars). The following day, the cells were activated by the addition of 100 U/ml IL-1ß. After 6 h, cell extracts were prepared and analyzed for luciferase expression. Values are the mean ± SEM from triplicate determinations after normalization for ß-galactosidase activity. The mean luciferase expression in cells receiving vehicle treatment was defined as 1.0. *, P < 0.01 compared with vehicl- treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To begin to understand the in vivo mechanisms for interaction between estrogen activity and inflammation, we first examined the effect of estrogen on a simplified system, focusing on gene induction in the mouse liver 1 h after a single injection of IL-1ß. Approximately 75 known genes were induced at least 2-fold by IL-1ß. Many of these genes have previously been identified as IL-1ß targets. For example, several cytokines, such as MCP-1 (Scya2), MIP-2 (Scyb2), inducing protein-10 (Scyb10), MIG (Scyb9), intercrine (Scya7), KC (Gro1), LIX, (Scyb5), MIP-1{gamma} (Scya9), and IL-1ß itself were all induced. Similarly, the adhesion molecules E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1 were all induced by IL-1ß. Additionally, the expression of many transcription factors was increased, predominantly members of the NF-{kappa}B family, including p105 (Nfkb1), p100 (Nfkb2), p65 (relA), and relB. Additionally, IL-1ß induced the expression of c-jun, E74-like factor 3 (ELF3), CCAAT/enhancer binding protein-{delta} (C/EBP{delta}), and NF-E2-related factor 2 (NRF2). Many of these transcription factor genes have been previously shown to be targets of IL-1ß induction, including c-jun in the murine macrophage J774.1 cell line (45) and C/EBP{delta} in chondrocytes (46). In fact, IL-1ß induction of cyclooxygenase-2 and secreted type II phosphatase A2 is completely dependent upon C/EBP{delta} and does not require NF-{kappa}B (46). Similarly, the induction of heme oxygenase-1 by several stimuli has been shown to depend upon the transcription factor NRF-2 (47). Thus, the induction of heme oxygenase-1 seen in the liver here may have been a secondary effect due to IL-1ß induction of NRF-2. Further studies in NRF-2 knockout mice (48) could begin to delineate these in vivo pathways. Finally, although not commonly identified as decreasing gene expression, IL-1ß treatment strongly decreased the expression of five genes, including the transcription cofactors hairy and enhancer of split 1 and SHP.

Pretreatment with EE inhibited IL-1ß induction of many of these genes. There was no correlation between the degree of induction by IL-1ß and EE inhibition, as EE repressed the induction of ELF-3 (induced 23-fold), bcl-3 (induced 12-fold), LIX (induced 5-fold), JAB, and fnk (both induced 3-fold). Additionally, there was no overall gene family pattern to regulation by EE. For example, of the cytokines induced by IL-1ß, EE treatment inhibited the induction of MIG and LIX, but not the remaining members of this family. Similarly, EE treatment inhibited IL-1ß induction of p105 and p100, but not that of p65 or relB. NF-{kappa}B is actually a family of transcription factors composed of various heterodimers, such as p65/p50, and different target genes can have different sensitivity to the distinct heterodimers (49, 50). One possibility is that genes such as MIG and LIX are more dependent upon cellular levels of p105 (p50) or p100 (p52). Alternatively, activation of these genes may be more dependent on ELF3, which was very strongly inhibited by EE pretreatment.

For mechanistic studies of the effects of EE on IL-1ß induction of gene expression, we focused on the genes LIX, bcl-3, fnk, and JAB to cover a range of IL-1ß inductions. These results showed that EE inhibition of IL-1ß signaling is mediated by ER{alpha}, as regulation is lost in ER{alpha} knockout mice, but not in ERß knockout mice. However, the inhibition of IL-1ß signaling by ER{alpha} does not require classical ER induction of gene expression. Pretreatment with genistein or raloxifene blocked IL-1ß gene induction. ICI182780 blocked this activity of genistein and raloxifene, indicating that these compounds also acted through ER{alpha} (not shown). However, neither genistein nor raloxifene stimulated the expression of several marker genes, including prostaglandin D synthase, intestinal trefoil factor, inositol-1-phosphate synthase, apolipoprotein A IV, and complement component C1qB. Finally, EE induction of the corepressor SHP did not mediate the inhibition of IL-1ß signaling, as induction of SHP by feeding a cholate-containing diet did not inhibit IL-1ß induction. Together these results indicate that in vivo estrogen inhibition of IL-1ß activity occurs through a nonclassical mechanism.

Structural analysis of the ER ligand binding domain suggested the presence of multiple conformations. The position of helix 12 in AF2 has been shown to depend critically on the identity of the bound ligand (51). In the ER{alpha}/17ß-estradiol complex, helix 12 is located in a full agonist position favorable for interaction with coactivators. In contrast, in the ER{alpha}/raloxifene or ER/tamoxifen structure, the position of helix 12 is dramatically shifted into a full antagonist conformation (52, 53). In the ERß/genistein structure, helix 12 is also displaced into a quasi antagonist position (51). If ER{alpha} behaves similarly, these structural results would correlate with our in vivo data. The two compounds, genistein and raloxifene, that do not position helix 12 in the agonist position also did not induce gene expression in vivo. Further, these results suggest that the in vivo inhibition of IL-1ß activity by ER{alpha} did not depend upon helix 12 conformation. Peptide inhibition studies have shown that multiple distinct sites on ER can interact with coactivators (54). Thus, structural changes induced in common by EE, raloxifene, and genistein in alternative regions of ER, such as AF1, that have not been included in the crystal structures obtained to date may be important for the in vivo inhibitory activity of ER.

Although some studies have suggested a direct binding interaction between ER and NF-{kappa}B, recent studies suggest a more indirect interaction (reviewed in Ref. 55), either by competition for limiting amounts of coactivators or through formation of a trimeric complex of ER/coactivator/p65. Tissue expression of coactivators can vary significantly (56), and some coactivators, including the peroxisomal proliferator-activated receptor-{gamma} coactivator-1 and the androgen receptor coactivator FHL2, are expressed in only select tissues (57, 58). If coactivators were involved in ER inhibition of IL-1ß signaling, then genes showing inhibition in the liver, such as JAB, fnk, LIX, and bcl-3, might not show inhibition in other tissues with a different suite of coactivators, as we found to be the case. EE inhibited IL-1ß induction of fnk and JAB in the liver and spleen, but not in the lung. Alternatively, EE inhibited LIX and bcl-3 induction in the liver, but not in the spleen or lung. These results do not seem to depend on expression levels of either ER{alpha} or ERß, because when ER{alpha} or ERß are expressed in the same in vitro context by transfection they are both able to similarly mediate the inhibition of IL-1ß induction of reporter gene expression.

These differences in EE inhibition of IL-1ß activity in different tissues could be due to increases or decreases in coactivator levels. One candidate coactivator is CBP/p300, which is recruited to nuclear receptors primarily through the AF1 domain. In either cultured smooth muscle cells or HepG2 cells, overexpression of CBP can reduce ER inhibition of IL-1ß induction of reporter gene expression (16, 17). P300 expression is higher in rat spleen than liver (56), in agreement with our finding of higher levels of CBP and p300 in mouse spleen and lung than liver. Because CBP/p300 is an important coactivator for both ER{alpha} and NF-{kappa}B, then limiting amounts in the liver might permit ER inhibition of IL-1ß signaling, whereas higher amounts in the spleen might be adequate for saturating both ER{alpha} and NF-{kappa}B. Alternatively, if the trimeric complex model occurs in vivo, than the basis for liver-specific ER inhibition of IL-1ß gene induction would depend upon a coactivator with higher expression levels in the liver than in the lung or spleen. Further studies with knockout animals will be required to delineate which mechanism occurs in the intact animal.

No matter which mechanism is operative, ER{alpha} clearly has the potential to function as a specific inhibitor of inflammatory pathways. Clinical use of hormone replacement therapy in postmenopausal women has proven to have many benefits with few adverse effects, in contrast to prolonged use of glucocorticoid steroids for suppression of inflammatory conditions, which can result in significant bone loss and fractures (59). Development of ER ligands with the selective ability to inhibit inflammatory signaling pathways in multiple tissues may be useful both in acute settings such as sepsis as well as in chronic conditions that are known to have a significant inflammatory component, such as atherosclerosis.


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  		Table 1A. Continued

 

   Footnotes
 
Abbreviations: AF2, Activation function-2; CBP, cAMP response element binding protein-binding protein; C/EBP{delta}, CCAAT/enhancer binding protein-{delta}; EE, 17{alpha}-ethinyl estradiol; ELF3, E74-like factor-3; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JAB, JAK-binding protein; LIX, LPS-induced c-x-c chemokine; LPS, lipopolysaccharide; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NRF2, NF-E2-related factor 2; NF-{kappa}B, nuclear factor-{kappa}B; poly(A), polyadenylated; SHP, short heterodimer partner.

Received January 18, 2002.

Accepted for publication March 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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