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Originally published In Press as doi:10.1074/jbc.M606718200 on December 13, 2006

J. Biol. Chem., Vol. 282, Issue 7, 4693-4701, February 16, 2007
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Oxysterol Nuclear Receptor LXRbeta Regulates Cholesterol Homeostasis and Contractile Function in Mouse Uterus*

Kevin Mouzat{ddagger}1, Magali Prod'Homme{ddagger}, David H. Volle{ddagger}2, Benoit Sion§, Pierre Déchelotte, Karine Gauthier||, Jean-Marc Vanacker||, and Jean-Marc A. Lobaccaro, Professor of the Université Blaise Pascal{ddagger}3

From the {ddagger}UMR CNRS 6547, "LXRs, Oxysterols, and Steroidogenic Tissues," and Research Center for Human Nutrition, 63177 Aubière, France, §Laboratoire de Biologie du Développement et de la Reproduction, Université d'Auvergne, 63058 Clermont-Ferrand, France, CHU Clermont-Ferrand, Service d'Anatomie Pathologique, Hôtel Dieu, Boulevard Léon Malfreyt, 63058 Clermont-Ferrand, France, and ||INSERM EMI 0229, 34298 Montpellier, France

Received for publication, July 14, 2006 , and in revised form, December 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The uterus is an organ where lipid distribution plays a critical role for its function. Here we show that nuclear receptor for oxysterols LXRbeta prevents accumulation of cholesteryl esters in mouse myometrium by controlling expression of genes involved in cholesterol efflux and storage (abca1 and abcg1). Upon treatment with an LXR agonist that mimics activation by oxysterols, expression of these target genes was increased in wild-type mice, whereas under basal conditions, lxr{alpha};beta-/- mice exhibited a marked decrease in abcg1 accumulation. This change resulted in a phenotype of cholesteryl ester accumulation. Besides, a defect of contractile activity induced by oxytocin or PGF2{alpha} was observed in mice lacking LXRbeta. These results imply that LXRbeta provides a safety valve to limit cholesteryl ester levels as a basal protective mechanism in the uterus against cholesterol accumulation and is necessary for a correct induction of contractions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The uterus is schematically divided into two distinct zones: endometrium and myometrium. The endometrium, located in the inner part of the organ, is the site of blastocyst implantation, and its epithelium undergoes cyclic radical changes under the control of ovarian sex steroid hormones (1); estrogens are responsible for epithelial cell hyperplasia, whereas progesterone blocks cell proliferation and induces differentiation. The myometrium (2), in the outer part of the uterus, accounts for more than 60% of the whole organ (3) and has a primordial role in uterine function. Whereas muscle quiescence due to high progesterone levels is essential during most of the pregnancy (4), efficient myometrium contractility is fundamental for a normal labor (for a review, see Ref. 5). This switch results from a modification in the plasma ratio of estrogens to progesterone signal that acts as primary event of the parturition. These hormonal changes also induce an increase in the level of endometrial prostaglandins, which play a role in the initiation and maintenance of labor, acting via specific relaxatory or contractile receptors on myometrium initiating contractions (6). Interestingly, it has also been demonstrated that increased production of surfactant protein A by the fetal lung near term causes activation and migration of fetal amniotic fluid macrophages to the maternal uterus, where increased production of interleukin-1beta activates NF-{kappa}B, leading to labor (7, 8). The inversion of the estradiol/progesterone ratio induces the expression of OXTR (oxytocin receptor). When activated by oxytocin, a neuropeptide produced by the pituitary, this receptor has a primordial contractile activity on the myometrium during labor. Besides, lipid distribution in myometrium is modified during pregnancy in humans. Although no change in total phospholipids occurs during pregnancy (9), modifications in membrane fluidity take place. Hence, transfers of omega 3 and omega 6 polyunsaturated fatty acids, essential for normal fetal growth and development, from the mother to the fetus have been suggested (10). Likewise, an increase in local and circulating cholesterol concentrations is observed (11). Although the role of this plasma cholesterol increase is not clear, apart from the anabolic support for the fetus (12), it has been clearly established that this molecule is essential to modulate membrane receptor activity and stability, especially those of OXTR. Indeed, Gimpl and Fahrenholz (13) observed an enrichment of oxytocin receptors in cholesterol-rich plasma membranes in HEK 293 fibroblast stably expressing the human oxytocin receptor. In addition, cholesterol stabilizes the receptor in a high affinity state for agonists and protects it from thermal denaturation (for a review, see Ref. 14). Smith et al. (15) showed that an abnormal increase in the cholesterol content of uterine smooth muscle cells reduces the amplitude of contractions induced by oxytocin in rat. Moreover, cholesterol depletion with methyl-beta-cyclodextrin could increase the contractions of myometrium strips isolated from rat (15) or guinea pig (16).

Cholesterol and its derivatives are vital nutrients that may also have a major impact on gene expression, and thus their intracellular quantities must be tightly regulated. Among the various transcription factors involved in these regulations, liver X receptor {alpha} (LXR{alpha}, NR1H3) and beta (LXRbeta, NR1H2) play a central role (for a review, see Ref. 17). They belong to a subclass of nuclear receptors that form obligate heterodimers with 9-cis-retinoic acid receptors and are bound to and activated by a class of naturally occurring oxysterols (18, 19). In the absence of ligand, the retinoid X receptor/LXR heterodimer is constitutively linked to specific DNA target sequences and interacts with corepressors, thus blocking transcription initiation (20, 21). The use of LXR-deficient mice (lxr-/-) has also helped to elucidate the role of these nuclear receptors in various physiologic functions (17), and many target genes have been described, such as the ATP-binding cassette transporter A1 (ABCA1) (22–24), ABCG5, and ABCG8 (25), responsible for the cholesterol cellular efflux, and SREBP1c (sterol response element-binding protein 1c) involved in lipid metabolism (26).

In this paper, we demonstrate that LXRbeta functions in the uterus as a sensor to prevent accumulation of cholesteryl esters by coordinately regulating expression of genes encoding proteins involved in cholesterol efflux (ABCA1 and ABCG1). Hence, mice lacking LXRbeta present an abnormal and specific accumulation of cholesteryl esters in uterine myocytes. Besides, these animals show defects in induced contractile activity in uterus.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—lxr knock-out mice (lxr{alpha}-/-, lxrbeta-/-, and lxr{alpha}; beta-/-) and their wild-type controls were maintained on a mixed strain background (C57BL/6:129Sv) and housed in a temperature-controlled room with a 12-h light/dark cycle (27). All experiments were performed on age-matched female mice. For all studies shown, mice were fed ad libitum with water and Global-diet® 2016S from Harlan (Gannat, France) containing 16% protein, 4% fat, and 60% carbohydrates. For all experiments, except for contractile activity assays and the mice used for experiments shown in Fig. 6, animals were treated with a superovulation protocol (intraperitoneal injection of 7 IU of pregnant mare's serum gonadotropin on day 1, 5 IU of human chorionic gonadotropin on day 3) and sacrificed on day 5 at the end of metaestrus. For real time quantitative reverse transcription-PCR (qPCR)4 experiments, mice were gavaged with 45 mg/kg T0901317 (T1317) (Cayman Chemical, Montigny le Bretonneux, France) or vehicle (methyl-cellulose) as previously described (28). For contractile activity assays and the mice used for experiments shown in Fig. 6, estrus was induced with a single injection of 10 µg of estradiol benzoate (Sigma) 18 h before sacrifice. To reduce the effect of stress, the elapsed time between the capture of a mouse and its sacrifice was under 30 s. In some experiments, uteri were longitudinally cut, and the mucosa were gently scraped, as previously described for the intestine (29). Both mucosa and muscular parts were stored in liquid N2 for RNA extraction. All aspects of animal care were approved by the Regional Ethics Committee (authorization CE1-04).

Anatomy and Pathology Analyses—Uteri from 3-month-old mice were collected, fixed, and embedded in paraffin, and 5-µm-thick sections were prepared and stained with hematoxylin/eosin/safran. Lipid staining of each organ collected was performed on 8-µm-thick cryosections with 1,2-propanediol (Sigma) for 1 min and in oil red O (Sigma) for 4 min as described (30). Cross-sectional areas of the various parts of the uteri (circular and longitudinal muscular layers and endometrium) were quantified using Axiovision 4.2 software (Carl Zeiss Vision GmbH, Le Pecq, France).

For semithin sections, chemicals were from Sigma and Agar Scientific (Saclay, France). Uteri were fixed in 1.2% (v/v) glutaraldehyde buffered in 0.07 M sodium cacodylate at pH 7.4 containing 0.05% (w/v) ruthenium red for 1 h at room temperature. Samples were postfixed with 1% (v/v) osmium tetroxide in the same buffer devoid of ruthenium red for 1 h. Organs were then dehydrated in ethanol baths and propylene oxide (three times for 20 min) and embedded in propylene oxide and epon epikote resin (v/v) overnight and in epon twice for 3 h. Resin polymerization was conducted at 60 °C for 72 h. Semithin sections (0.8 µm) were cut with a diamond knife (Leica Ultracut S; Rueil-Malmaison, France), and stained with azure 2 followed by the addition of 1 N NaOH to stop the reaction.

Analysis of Lipid Content—Lipids were extracted as described (31) and analyzed on high-performance thin layer chromatography plates. Free cholesterol and cholesteryl esters were identified and quantified against standards by densitometry (Sigma Scan Pro; Sigma) as previously described (27).

Real-time PCR—Total RNA was isolated using the Trizol method (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (Promega, Charbonnières, France) and random hexamer primers (Promega) according to the manufacturer's recommendations. The real time PCR was performed on an iCycler (Bio-Rad). Four µl of 1:50 diluted cDNA template were amplified by 0.75 units of HotMaster TaqDNA polymerase (Eppendorf, Brumath, France) using SYBR Green dye to measure duplex DNA formation. Primers are given in Table 1.


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  		TABLE 1
Sequence primers used for qPCR

 
Western Blot Analysis—Protein extracts (30 µg) from whole uterus were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences). Membranes were incubated overnight at 4 °C with primary polyclonal antibodies raised against either ABCA1 (1:500; Novus Biological, Montluçon, France), ER{alpha} (estrogen receptor {alpha}) (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA), ERR{alpha} (estrogen-related receptor {alpha}) (1:200; Santa Cruz Biotechnology), progesterone receptor (1:200; Santa Cruz Biotechnology), noncleaved or cleaved SREBP1c (1:500 and 1:400; Santa Cruz Biotechnology), SCD1 (stearoyl CoA-desaturase 1) (1:200; Santa Cruz Biotechnology), PGF2R (PGF2{alpha} receptor) (1:500; Cayman Chemical), or beta-actin (1:2000; Santa Cruz Biotechnology) followed by a 1-h incubation with a peroxidase-conjugated anti-rabbit or anti-goat IgG (1:10,000 or 1:5000, respectively; Sigma). Peroxidase activity was detected using the Western Light System (PerkinElmer Life Sciences). Protein -fold changes were measured by densitometry of the x-ray films using Quantity One version 4.6.1 software (Bio-Rad).

Measurement of Uterus Contractions in Vitro—Uteri were quickly dissected and carefully cleaned of surrounding fat prior to being suspended in organ baths (50 ml) filled with a Dejalon solution (155 mM NaCl, 5.7 mM KCl, 0.55 mM CaCl2, 6.0 mM NaHCO3, 2.8 mM glucose, pH 7.4), equilibrated with air, and kept at 37 °C as described (32) for measurement of tension. A resting tension (2 g) was applied to the suspended uteri. Contractions were recorded with a force-displacement transducer (MyographESAO® 4, Jeulin, Evreux, France) and analyzed with Sérénis® software (Jeulin). Uteri were incubated with increasing concentrations of synthetic oxytocin (Syntocinon®, Novartis Pharma, Rueil-Malmaison, France) or luprostiol, analogous of PGF2{alpha} (Prosolvin®, Intervet, Angers, France). Results are expressed as a dose-response curve showing the uterine tension minus the basal tension.

Statistical Analysis—For statistical analysis, Student's t test was performed to determine whether there were significant differences between the groups. A p value of 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Loss of LXRbeta Results in Perturbations of Lipid Content in Uterus—No significant difference in the somatic indexes of uteri was observed among the genotypes of the wild type (0.38% ± 0.03, n = 5), the lxr{alpha}-/- mice (0.37% ± 0.04, n = 4), the lxrbeta-/- mice (0.40% ± 0.02, n = 7), and the lxr{alpha};beta-/- mice (0.41% ± 0.01, n = 5) at 3 months of age. Gross examination of uterus sections from lxr-deficient mice did not reveal any perturbation of the structures as assessed by the presence of an apparently normal endometrium, characterized by a monolayer of epithelial cells and a stroma, and the presence of circular and longitudinal layers of smooth muscle in myometrium (Fig. 1A, a–d). The uterus structure remains stable even after 12 months of age in all genotypes (data not shown). Determination of the cross-sectional area of the smooth muscle pointed out no significant variation in the various knock-out mice compared with the wild-type (Table 2). Higher magnification (x400) did not reveal any perturbation of the endometrium structure (data not shown), whereas vacuoles were visible in layers of myometrium from lxrbeta-/- (Fig. 1A, g) and lxr{alpha};beta-/- mice (Fig. 1A, h), localized in the cytoplasm of myocytes (Fig. 1B). Because LXRs are known to have an important role in the regulation of lipid metabolism in various tissues, we examined whether some differences between wild-type and LXR-deficient mice in uterus lipid content were present. Histological analysis using oil red O staining performed on frozen sections pointed to an abnormal accumulation of neutral lipids in vacuoles observed in myometrium of lxrbeta-/- (Fig. 1A, k) and lxr{alpha}; beta-/- (Fig. 1A, l) mice, whereas no difference among the various genotypes was seen in the endometrium (data not shown). This lipid accumulation was visible in LXRbeta-deficient mice as young as 1 month old (data not shown). Since lxr{alpha}-/- mice appeared to have no lipid-rich vacuole (Fig. 1A, j), we concluded that the phenotype was due to the absence of LXRbeta.


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  		TABLE 2
Surface analysis of the cross-sectional area of the uteri Quantifications (mean ± S.E.) represent the relative surface of each part of the uterus: endometrium and circular (CL) and longitudinal (LL) layers. The number of analyzed cross-sections is indicated in parentheses.

 
Semithin sections (0.8 µm) of osmium tetroxide-fixed uteri were performed to precisely determine the localization of these vacuoles. Azure 2 dye, which stains lipids in yellow, showed that these vacuoles were localized in the cytoplasm of myocytes (Fig. 1B) and did not result of an infiltration of adipose tissue within the smooth muscle, as also suggested by the absence of any significant increase levels of adipocyte marker mRNA, such as the fatty acid-binding protein (aP2) and PPAR{alpha} and -{gamma} (peroxisomal proliferator-activated receptors {alpha} and {gamma}) (see Fig. 4A).


Figure 1
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  		FIGURE 1.
LXRbeta-deficient mice present an abnormal accumulation of neutral lipids in uterine myocytes only. A, histological examination of uteri from wild-type (WT), LXR{alpha}-, and/or beta-deficient mice. a–h, hematoxylin/eosine/safran staining at two magnifications. Squared portions indicate the magnified view; i–l, oil red O staining; bars indicate the different sizes (200 or 20 µm). Black arrowheads, epithelium. Black arrows, cytoplasmic vacuoles in myocytes. LL, longitudinal muscular layer; CL, circular muscular layer; E, endometrium. B, azure blue 2B staining of semithin sections from the muscular layer. Lipids are stained in yellow. Dashed line, plasma membrane of a myocyte. Bar, 5 µm. C, oil red O staining was performed on frozen sections from wild-type and lxr{alpha};beta-/- mice of 3 months of age as described under "Experimental Procedures." Bar, 20 µm.

 
In order to determine whether this lipid accumulation was generalized to various muscles, oil red O staining was performed on frozen slides of three different types of muscle: intestine (duodenum), heart, and rough muscle (quadriceps) of 3-month-old wild-type and lxr{alpha};beta-/- females (Fig. 1C). None of the tested tissues were stained positively, except uterine smooth muscle, which was used as a control. These data led us to suggest the existence of tissue-specific mechanisms by which LXRbeta regulates lipid homeostasis in uterine smooth muscle.

LXRbeta Null Mice Have Elevated Uterus Cholesteryl Esters—To determine the nature of lipids accumulated in the uterus, thin layer chromatography analyses were performed on whole lipid extracts from uteri of 3- and 12-month old mice. Although LXR-mediated triacylglycerol accumulation had already been reported in vascular smooth muscle cells (33), biochemical analysis revealed that only the fraction containing cholesteryl esters was significantly increased after normalizing to uterus weight at 3 months (23.5- and 37.2-fold in lxrbeta-/- and lxr{alpha};beta-/- mice, compared with wild-type mice; p < 0.0005) and 12 months of age (27.6- and 66.5-fold in lxrbeta-/- and lxr{alpha};beta-/- mice, compared with wild-type mice; p < 0.0005) (Fig. 2). lxr{alpha}-/- mice presented the same low amount of cholesteryl esters as the wild-type mice. Together, these data suggest that the increase in the oil red O staining observed in the lxrbeta-/- and lxr{alpha}; beta-/- uterus was due to the accumulation of cholesteryl esters. Whatever the age considered, cholesteryl ester concentration was significantly higher in lxr{alpha};beta-/- uterus than in lxrbeta-/-. This could suggest a mechanism of a slight redundancy between the two isoforms, where LXR{alpha} could partially reverse the drastic phenotype induced by absence of LXRbeta. No significant differences in free cholesterol (Fig. 2, white bars), triacylglycerol, and phospholipid contents (data not shown) were observed among the genotypes.

Both LXR{alpha} and LXRbeta Are Expressed in the Various Compartments of the Uterus—The results described above suggested that the LXR-dependent changes observed in the cholesteryl esters content were primarily due to LXRbeta. We wondered whether this specificity in the LXR isoform was due to the exclusive expression of LXRbeta in the tissue. The presence of LXR{alpha} and LXRbeta mRNA was checked by qPCR on whole uterus as well as mucosa and muscular parts. As shown in Fig. 3, lxr{alpha} and lxrbeta were detected in myometrium and endometrium parts of the uterus. In the myometrium, both isoform mRNAs were in a comparable amount compared with whole uterus. In order to confirm that we had an enrichment of the muscular part, OXTR mRNA was amplified, and the highest accumulation was obtained in the myometrium fraction, as expected.


Figure 2
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  		FIGURE 2.
Uteri from LXR-deficient mice accumulate cholesteryl esters at 3 and 12 months of age. Analysis of free cholesterol (open bars) and cholesteryl ester (black bars) concentrations were determined as described under "Experimental Procedures" from whole uteri. Histograms are indicated as means ± S.E. (n = 5), except for lxr{alpha};beta-/- at 12 months (n = 8). *, p < 0.05; ***, p < 0.005. WT, wild type.

 


Figure 3
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  		FIGURE 3.
LXR{alpha} and LXRbeta are expressed in the myometrium and endometrium. RNAs were prepared from whole uteri and from epithelial and muscular parts of the uterus as described under "Experimental Procedures" to determine the levels of mRNA expression of LXR{alpha}, LXRbeta, and OXTR. Quantification (mean ± S.E.) was done by qPCR (n = 4–6). Results obtained in the whole uteri are indicated as 1.

 
LXRs Regulate Cholesterol Efflux and Fatty Acid Metabolism in the Uterus in Vivo—To explore the underlying molecular mechanisms that might account for the cholesteryl ester accumulation in LXRbeta-deficient mice, gene expression was examined by qPCR from whole uteri of wild-type and lxr{alpha}; beta-/- animals gavaged with the potent synthetic LXR agonist T1317 (Fig. 4A). In both genotypes, basal and T1317-induced levels of genes encoding SRBI (scavenger receptor BI) involved in cell cholesterol entry, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (red) and synthase (syn), responsible for de novo cholesterol synthesis, and ACAT1 and -2 (acyl-coenzyme A:cholesterol acyltransferase 1 and 2, respectively), implicated in cholesterol esterification, were unchanged. In contrast, expression of abca1 and abcg1, encoding two cholesterol efflux transporters, showed an LXR-dependant regulation. T1317 treatment induced an increase of abca1 and abcg1 accumulation in uteri from wild-type mice (2.7- and 5.2-fold increase, respectively; p < 0.01). No induction of the LXR target genes was seen in lxr-deficient mice. As expected, a higher accumulation of ABCA1 was observed in the T1317-treated wild-type mice (5-fold compared with the vehicle-treated animals; p < 0.01) (Fig. 6B).


Figure 4
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  		FIGURE 4.
LXRs regulate genes involved in lipid homeostasis in uterus. A, genes involved in cholesterol entry and efflux and adipocyte markers. B, genes involved in fatty acid synthesis. Transcripts were quantified by qPCR analysis (mean ± S.E.). n = 5–8. *, p < 0.05 versus vehicle-gavaged wild-type mice; **, p < 0.01 versus vehicle-gavaged wild-type mice; ***, p < 0.005 versus vehicle-gavaged wild-type mice.

 
Not surprisingly, transcripts of the low density lipoprotein receptor ldlr (low density lipoprotein receptor) was significantly lower in lxr{alpha};beta-/- mice, since this gene is known to be regulated by the intracellular concentration of oxysterols through a negative regulation loop (34). Interestingly, although the basal level of abca1 was unchanged, basal accumulation of abcg1 was significantly lower in lxr{alpha};beta-/- females (66% less than the wild-type mice; p < 0.05) (Fig. 4A). It is assumed that this decrease could be considered as the primum movens of the phenotype, since the intracellular cholesterol increase cannot induce ABCA1 and ABCG1 transporters, leading to its sequestration and accumulation in myocytes. It could thus be suspected that LXRs regulate cholesterol efflux in uterus myocytes.

In addition, RNA accumulation of known target genes of LXRs involved in fatty acid metabolism was studied (Fig. 4B). T1317 treatment induced the accumulation of srebp1c (31.3-fold, p < 0.005) and lpl (lipoprotein lipase) (2.6-fold, p < 0.01) in wild-type mice, encoding SREBP1c and LPL, respectively. Quite surprisingly, the level of the noncleaved form of SREBP1c did not appear to be different among the genotypes, whatever the treatment (Fig. 6B). Likewise, no variation of the cleaved form was observed in the same samples (Fig. 6B). Interestingly, the gene encoding the fatty acid synthase fas, which has been shown to be an LXR target gene, is basally less expressed in lxr{alpha};beta-/- females compared with the wild type and not induced by T1317 in the wild-type mice. scd1 and scd2, encoding stearoyl CoA-desaturase 1 and 2, show a higher accumulation in the uteri from T1317-treated wild-type mice (4.0- and 1.7-fold, respectively; p < 0.05). A not significant higher protein level of SCD1 (3-fold) was observed only in the wild-type mice after the T1317 treatment (Fig. 6B), whereas SCD2 was undetectable in all of the samples (data not shown). These results suggested that, as already described in other tissues, LXRs might be involved in the regulation of fatty acid homeostasis in the uterus.


Figure 5
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  		FIGURE 5.
Uteri from mice lacking LXRbeta present a defect of induced contractions. Contractile activity was assessed on uteri from estrogen-treated wild-type and lxr-deficient mice. Main amplitude contraction was recorded in response to synthetic oxytocin or PGF2{alpha} analog luprostiol. **, p < 0.01 versus wild-type mice; #, p < 0.05 versus lxr{alpha}-/- mice; ##, p < 0.01 versus lxr{alpha}-/- mice. Each point represents the mean ± S.E. n = 6–9 animals.

 
Uteri of LXRbeta-deficient Mice Present Contraction Defects— Since myometrium is the major actor during labor, we investigated whether the cholesteryl ester accumulation could modify uterine contractile activity. Assays were thus performed on uteri from the various genotypes in order to measure the response of the muscle to either oxytocin or luprostiol, a PGF2{alpha} analog. Uterus from wild-type and lxr{alpha}-/- females presented a similar increase of contraction amplitudes when oxytocin (Fig. 5A) or luprostiol (Fig. 5B) were added in increasing concentrations in the media. Although uteri from lxrbeta-/- and lxr{alpha};beta-/- mice presented no difference compared with the wild-type mice in the basal contractions (Fig. 5), the organs were less responsive to higher concentrations of oxytocin and PGF2{alpha} analog (p < 0.01 compared with the wild-type and lxr{alpha}-/- mice, n = 6–9).

Interestingly, the measured efficient doses of oxytocin and luprostiol to induce the maximal amplitude of contraction were identical in all genotypes (5 x 10-3 units/ml and 3 x 10-4 mg/ml for oxytocin and luprostiol, respectively). Although these data suggested that the amount of receptors was not affected, we analyzed by qPCR the levels of OXTR and PGF2R. As shown in Fig. 6A, no basal variation was observed between the wild-type and lxr{alpha};beta-/- mice; besides, T1317 gavage had no significant effect on OXTR and PGF2R RNA levels, as well as on PGF2R protein accumulation (Fig. 6B). Likewise, because mice were synchronized by the intraperitoneal injection of E2, we hypothesized that the estradiol-dependent activities, which regulate the expression of various genes, such as OXTR, could have been altered in the engineered mice. qPCR analyses showed that contraction impairment was not due to a decrease in mRNA or protein levels of ER{alpha} and progesterone receptor and ERR{alpha} (Fig. 6, A and B). Altogether, these data suggested that the phenotype observed in mice lacking LXRbeta was probably more due to a muscular defect than to a drastic steroid hormone signaling defect, as pointed out by the significant decrease of sm22{alpha} (transgelin-encoding gene) encoding transgelin, a calponin that is expressed exclusively in smooth muscle-containing tissues of adult animals (65% decrease in the lxr{alpha};beta-/- mice compared with the wild-type mice; p < 0.05). sm22{alpha} is one of the earliest markers of differentiated smooth muscle cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we detail the discovery of LXRbeta as an important regulator of cholesterol homeostasis in the uterus through its ability to modulate transcription of genes encoding proteins that regulate cholesterol efflux (ABCA1 and ABCG1) and fatty acid synthesis (LPL, SREBP-1c, and SCD1/2). Besides, the uteri of mice lacking LXRbeta present an abnormal capacity to contract under oxytocin or PGF2{alpha} signals. LXRbeta appears thus to provide a cholesterol safety valve that operates as in other tissues as a sterol sensor and thereby maintains the concentration of free cholesterol below toxic levels. In mice lacking LXRbeta, LXR{alpha} does not present a totally redundant function.

LXRbeta Regulates Cholesterol Efflux within the Myocytes—The ability of LXRs to control muscular lipid metabolism is reaching a high interest state. Studies (35, 36) showed that LXR activation can promote triglyceride accumulation in the presence of high glucose concentration in skeletal muscle cells, via the induction of the expression of lipogenic enzymes, such as SREBP1c (26), FAS (fatty acid synthase) (37), and SCD1 (38). In parallel, LXRs have been described as regulators of cholesterol efflux from the rough muscle by increasing the efflux of intracellular cholesterol to extracellular acceptors, such as high density lipoprotein (39). Despite these data, little was known about the role of these nuclear receptors in smooth muscle. Davies et al. (33) pointed out that T1317 could induce triacylglycerol accumulation in human vascular smooth muscle cells by activating FAS, SREBP-1c, and SCD-1. We found that mice lacking LXRbeta presented a high accumulation of cholesteryl esters in longitudinal layers as well as circular layers of myometrium. Since abcg1 is a target gene of LXRs in the uterus, as shown by the induction of the mRNA accumulation after the gavage of wild-type mice with T1317, and because its basal level was significantly lower in mice lacking LXRbeta, we hypothesized that LXRbeta is a central sensor of cholesterol status of the uterine myocytes. Moreover, this role seems to be specific for the uterus, since the other muscles from lxrbeta-/- mice tested so far (heart, duodenum, and quadriceps) did not show such a profile of oil red O staining. Analysis of the molecular mechanisms leading to such specificity would be of interest in order to screen whether expression of cofactors within these organs could in part explain the phenotype.


Figure 6
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  		FIGURE 6.
The decrease in the amplitude of contraction is not linked to oxytocin or PGF2{alpha} receptor expression or estrogen signaling pathway defects but to a muscular defect. A, RNAs were extracted from uteri of estrogen-treated wild-type and lxr-deficient mice. Transcripts of genes encoding hormone receptors were quantified by real time PCR analysis (mean ± S.E.). n = 5–8. B, representative Western blots of proteins extracted from uteri of estrogen-treated wild-type and lxr-deficient mice. n = 5. ER{alpha}, estrogen receptor {alpha}; ERR{alpha}, estrogen-related receptor alpha; PR, progesterone receptor; nc-or c-SREBP-1c, noncleaved or cleaved sterol regulatory binding protein-1c, respectively.

 
Moreover, as in other tissues, lipogenic genes are also regulated by LXRs. Whether srebp1c, lpl, and scd1/2 are specific LXRbeta target genes in the uterus has not been determined yet. The status of SCD1 and SCD2 is of particular interest. Indeed, these enzymes are responsible for the {Delta}9-cis desaturation of stearoyl- and palmitoyl-CoA, producing oleoyl- and palmitoleoyl-CoA. Oleoyl-CoA is the substrate for cholesterol acyltransferase and enables more esterification of cholesterol. However, no significant basal change of these enzymes was observed in the lxr{alpha};beta-/- mice. Besides, no clear defect in triglyceride concentration was detected in the uterus although fas presented a lower basal level in lxr{alpha};beta-/- mice.

LXRbeta Deficiency Leads to Uterus Contraction Defect—The decreased total amplitude of contractions did not seem to be due to a loss of muscle mass, as pointed out by the cross-sectional area analysis in the LXRbeta-deficient mice and the somatic index, but rather to a muscular defect as suggested by the decrease of the sm22{alpha} transcript. Besides, although a direct role of LXRbeta in the control of the contractile activity of the uterus could not be ruled out, several lines of evidence suggest that cholesterol could modulate contractile activity of various smooth muscles. Pharmacological depletion of cholesterol by methyl-beta-cyclodextrin abolished induced contractions of ureter and portal vein (40) and arteries (41) in rats. Conversely, Smith et al. (15) showed that cholesterol inhibited uterus contraction of pregnant rat by destabilizing caveolae. Altogether, these data prove that cholesterol concentration has dramatic effects on smooth muscle contraction and that in the uterus, the cholesterol amount is negatively correlated to its ability to contract. Consistently, our results show that LXRbeta-deficient mice, which presented an increased cholesteryl ester concentration, exhibited a lower capacity to contract under stimulation with oxytocin and PGF2{alpha} analog.

It is interesting to note that although lxrbeta-/- and lxr{alpha};beta-/- mice apparently deliver successfully pups at term, LXRbeta-deficient females usually show various signs of fetal resorption in the uterine horns. In some cases, 3–9-month-old lxrbeta-/- and lxr{alpha};beta-/- females develop hind leg paralysis a few days or weeks after delivery. When necropsy of these females is done, nonexpulsed pups could be observed in uterine horns (Fig. 7A). In rare cases, the female died giving birth (Fig. 7B). Hence, it could be hypothesized that the LXR-signaling pathway is one of the actors responsible for lipid change in plasma membrane of uterus myocytes at the term of pregnancy, thus initiating the ability of uterus to properly deliver the pups. Besides, it has been pointed out that obese and overweight women often have dysfunctional labor. Obesity is related to many complications associated with pregnancy (e.g. gestational diabetes mellitus, tromboembolic problems, and hypertensive disorders such as preeclampsia or eclampsia) (42). It has been shown that even with an uncomplicated pregnancy, there was a need for more oxytocin infusion to induce labor in overweight and obese women than in normal weight (42). Moreover, prepregnancy body mass index (43, 44) and increase in its category (45) during pregnancy have been found to be high risk factors for caesarean delivery at term of pregnancy for failure to progress in the labor. However, the molecular mechanisms by which obesity and overweight lead to difficult labor remain unknown so far. Nevertheless, the lxrbeta-/- females could be considered as the first engineered mice presenting an abnormal contraction of uterus that could be used to understand how disequilibrium in the lipid diet could interfere with a normal parturition in humans. Screening of new specific targets of LXRbeta would thus be helpful to study side effects of the overweight on labor.


Figure 7
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  		FIGURE 7.
In rare cases, nondelivered pups are found blocked in the uterus horn or in the vagina of LXRbeta-deficient mice. A, left, necropsy of a 6-month-old lxrbeta-/- mouse presenting an abdominal distension and suffering of hind leg paralysis 2 weeks after delivery. Right, necropsy of a 6-month-old lxr{alpha};beta-/- mouse. The right uterine horn was opened. Dashed line, nonexpulsed fetus; asterisk, placenta; arrowheads, ovary; H, uterine horn; C, cervix; bar, 0.5 cm. B, lxr{alpha};beta-/- female dead during parturition. Note the pup blocked in the cervix/vagina.

 
LXR{alpha} Does Not Have Redundant Functions in the Uterus—It has been generally admitted that LXRbeta is ubiquitously expressed, whereas LXR{alpha} expression is limited to tissues where lipid metabolism is high. Actually, it looks as if very few tissues do not express LXRbeta (46). So far, few physiologic functions have been associated with LXRbeta in vivo, since, conversely to lxr{alpha}-/- mice, lxrbeta-/- mice do not present clear cut phenotypes. Until now, only two functions have been reported for LXRbeta in mice. Komuves et al. (47) were the first to report an alteration in the LXRbeta-deficient mice, which presented an abnormal differentiation of the epidermis, whereas LXR{alpha}-deficient mice appeared normal. The authors showed that only LXRbeta was present in the affected tissue, suggesting that this phenotype could develop because of the lack of LXR{alpha} expression and thus the absence of any possible redundancy. In the testis, we5 and others (48, 49) described that LXRbeta was important for the regulation of the cholesterol metabolism in Sertoli cells. Conversely to epidermis, LXR{alpha} is expressed in the Sertoli cells.5 The present data point out a combined phenotype of cholesteryl ester accumulation and lower contraction capacity due to the lack of LXRbeta while LXR{alpha} is expressed. This fact has to be paralleled with the phenotype observed in the lxr{alpha}-/- mice fed a high amount of cholesterol, where the presence of LXRbeta could not rescue the regulation of cyp7a1 (50). Hence, although both receptors could bind the same DNA sequence in vitro (17), LXR{alpha} and LXRbeta can differentially regulate gene expression, and thus in the uterus they clearly do not have overlapping roles. These data strongly support the existence of specific molecular mechanisms leading to LXRbeta transactivation, such as specific promoter sequences or specific cofactors for each isoform. Such elements remain to be discovered.


   FOOTNOTES
 
* This work was supported by the Centre National de la Recherche Scientifique, the Université Blaise Pascal, Fondation pour la Recherche Médicale Grant INE2000-407031/1, and the Fondation BNP-Paribas. 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 Recipient of a doctoral fellowship from the Ministère de l'Education Nationale de Recherche et de la Technologie. Back

2 Present address: Institute of Genetics and Molecular and Cellular Biology, 67404 Illkirch Cedex, France. Back

3 To whom correspondence should be addressed: UMR CNRS-Université Blaise Pascal 6547 and Research Center for Human Nutrition, 24 Ave. des Landais, 63177 Aubière Cedex, France. Tel.: 33-473-40-74-16; Fax: 33-473-40-70-42; E-mail: j-marc.lobaccaro{at}univ-bpclermont.fr.

4 The abbreviations used are: qPCR, real time quantitative reverse transcription-PCR; T1317, LXR agonist T0901317. Back

5 D. H. Volle, K. Mouzat, R. Duggavathi, B. Siddeek, P. Déchelotte, B. Sion, G. Veyssière, M. Benahmed, and J. M. A. Lobaccaro, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank J. P. Saru, S. Monceau, C. Puchol, and S. Plantade for excellent technical assistance; Dr. G. Prensier (UMR CNRS 6023) for help in the semithin section analysis; Dr. D. Gallot (Obstetric and Gynecology Department, CHU Clermont-Ferrand) for obtaining the synthetic hormones and for helpful discussions; Drs. G. Veyssière, S. Baron, F. Caira, and J. Henry-Berger (UMR CNRS 6547) for critically reading the manuscript; Dr. D. J. Mangelsdorf (Howard Hughes Medical Institute, Dallas, TX) for providing the mice; and members of the Chester laboratory for assistance in animal dissections.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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