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• W2077671696 abstract "The uterus is an organ where lipid distribution plays a critical role for its function. Here we show that nuclear receptor for oxysterols LXRβ 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, lxrα;β-/- 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 PGF2α was observed in mice lacking LXRβ. These results imply that LXRβ 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. The uterus is an organ where lipid distribution plays a critical role for its function. Here we show that nuclear receptor for oxysterols LXRβ 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, lxrα;β-/- 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 PGF2α was observed in mice lacking LXRβ. These results imply that LXRβ 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. 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.Dockery P. Rogers A.W. Baillieres Clin. Obstet. Gynaecol. 1989; 3: 227-248Abstract Full Text PDF PubMed Scopus (29) Google Scholar); estrogens are responsible for epithelial cell hyperplasia, whereas progesterone blocks cell proliferation and induces differentiation. The myometrium (2.Berto A.G. Sampaio L.O. Franco C.R. Cesar Jr., R.M. Michelacci Y.M. Biochim. Biophys. Acta. 2003; 1619: 98-112Crossref PubMed Scopus (42) Google Scholar), in the outer part of the uterus, accounts for more than 60% of the whole organ (3.McCormack S.A. Glasser S.R. Endocrinology. 1980; 106: 1634-1649Crossref PubMed Scopus (159) Google Scholar) and has a primordial role in uterine function. Whereas muscle quiescence due to high progesterone levels is essential during most of the pregnancy (4.Di Renzo G.C. Mattei A. Gojnic M. Gerli S. Curr. Opin. Obstet. Gynecol. 2005; 17: 598-600Crossref PubMed Scopus (59) Google Scholar), efficient myometrium contractility is fundamental for a normal labor (for a review, see Ref. 5.Huszar G. Roberts J.M. Am. J. Obstet. Gynecol. 1982; 142: 225-237Abstract Full Text PDF PubMed Scopus (73) Google Scholar). 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.Myatt L. Lye S.J. Prostaglandins Leukot. Essent. Fatty Acids. 2004; 70: 137-148Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). 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-1β activates NF-κB, leading to labor (7.Condon J.C. Jeyasuria P. Faust J.M. Mendelson C.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4978-4983Crossref PubMed Scopus (265) Google Scholar, 8.Mendelson C. Condon J. J. Steroid Biochem. Mol. Biol. 2005; 93: 113-119Crossref PubMed Scopus (68) Google Scholar). 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.Pulkkinen M.O. Nyman S. Hamalainen M.M. Mattinen J. Gynecol. Obstet. Invest. 1998; 46: 220-224Crossref PubMed Scopus (13) Google Scholar), 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.Holman R.T. Johnson S.B. Ogburn P.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4835-4839Crossref PubMed Scopus (182) Google Scholar). Likewise, an increase in local and circulating cholesterol concentrations is observed (11.Potter J. Nestel P.J. Clin. Chim. Acta. 1978; 87: 57-61Crossref PubMed Scopus (4) Google Scholar). Although the role of this plasma cholesterol increase is not clear, apart from the anabolic support for the fetus (12.Brizzi P. Tonolo G. Esposito F. Puddu L. Dessole S. Maioli M. Milia S. Am. J. Obstet. Gynecol. 1999; 181: 430-434Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), 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.Gimpl G. Fahrenholz F. Eur. J. Biochem. 2000; 267: 2483-2497Crossref PubMed Scopus (82) Google Scholar) 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.Gimpl G. Fahrenholz F. Physiol. Rev. 2001; 81: 629-683Crossref PubMed Scopus (2285) Google Scholar). Smith et al. (15.Smith R.D. Babiychuk E.B. Noble K. Draeger A. Wray S. Am. J. Physiol. 2005; 288: C982-C988Crossref PubMed Scopus (90) Google Scholar) 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-β-cyclodextrin could increase the contractions of myometrium strips isolated from rat (15.Smith R.D. Babiychuk E.B. Noble K. Draeger A. Wray S. Am. J. Physiol. 2005; 288: C982-C988Crossref PubMed Scopus (90) Google Scholar) or guinea pig (16.Buxton I.L. Vittori J.C. Proc. West Pharmacol. Soc. 2005; 48: 126-128PubMed Google Scholar). 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 α (LXRα, NR1H3) and β (LXRβ, NR1H2) play a central role (for a review, see Ref. 17.Beaven S.W. Tontonoz P. Annu. Rev. Med. 2006; 57: 313-329Crossref PubMed Scopus (192) Google Scholar). 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.Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1445) Google Scholar, 19.Janowski B.A. Grogan M.J. Jones S.A. Wisely G.B. Kliewer S.A. Corey E.J. Mangelsdorf D.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 266-271Crossref PubMed Scopus (781) Google Scholar). 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.Hu X. Li S. Wu J. Xia C. Lala D.S. Mol. Endocrinol. 2003; 17: 1019-1026Crossref PubMed Scopus (112) Google Scholar, 21.Wagner B.L. Valledor A.F. Shao G. Daige C.L. Bischoff E.D. Petrowski M. Jepsen K. Baek S.H. Heyman R.A. Rosenfeld M.G. Schulman I.G. Glass C.K. Mol. Cell. Biol. 2003; 23: 5780-5789Crossref PubMed Scopus (190) Google Scholar). The use of LXR-deficient mice (lxr-/-) has also helped to elucidate the role of these nuclear receptors in various physiologic functions (17.Beaven S.W. Tontonoz P. Annu. Rev. Med. 2006; 57: 313-329Crossref PubMed Scopus (192) Google Scholar), and many target genes have been described, such as the ATP-binding cassette transporter A1 (ABCA1) (22.Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12097-12102Crossref PubMed Scopus (834) Google Scholar, 23.Venkateswaran A. Repa J.J. Lobaccaro J.M. Bronson A. Mangelsdorf D.J. Edwards P.A. J. Biol. Chem. 2000; 275: 14700-14707Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 24.Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (847) Google Scholar), ABCG5, and ABCG8 (25.Repa J.J. Berge K.E. Pomajzl C. Richardson J.A. Hobbs H. Mangelsdorf D.J. J. Biol. Chem. 2002; 277: 18793-18800Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar), responsible for the cholesterol cellular efflux, and SREBP1c (sterol response element-binding protein 1c) involved in lipid metabolism (26.Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1408) Google Scholar). In this paper, we demonstrate that LXRβ 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 LXRβ present an abnormal and specific accumulation of cholesteryl esters in uterine myocytes. Besides, these animals show defects in induced contractile activity in uterus. Animals−lxr knock-out mice (lxrα-/-, lxrβ-/-, and lxrα; β-/-) 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.Cummins C.L. Volle D.H. Zhang Y. McDonald J.G. Sion B. Lefrancois Martinez A.M. Caira F. Veyssiere G. Mangelsdorf D.J. Lobaccaro J.M.A. J. Clin. Invest. 2006; 116: 1902-1912Crossref PubMed Scopus (139) Google Scholar). 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) 4The abbreviations used are: qPCR, real time quantitative reverse transcription-PCR; T1317, LXR agonist T0901317. experiments, mice were gavaged with 45 mg/kg T0901317 (T1317) (Cayman Chemical, Montigny le Bretonneux, France) or vehicle (methyl-cellulose) as previously described (28.Volle D.H. Repa J.J. Mazur A. Cummins C.L. Val P. Henry-Berger J. Caira F. Veyssiere G. Mangelsdorf D.J. Lobaccaro J.M.A. Mol. Endocrinol. 2004; 18: 888-898Crossref PubMed Scopus (42) Google Scholar). 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.Repa J.J. Turley S.D. Lobaccaro J.M.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1146) Google Scholar). 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.Frenoux J.-M. Vernet P. Volle D.H. Britan A. Saez F. Kocer A. Henry-Berger J. Mangelsdorf D.J. Lobaccaro J.M. Drevet J.R. J. Mol. Endocrinol. 2004; 33: 361-375Crossref PubMed Scopus (44) Google Scholar). 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.Grizard G. Sion B. Bauchart D. Boucher D. J. Chromatogr. B Biomed. Sci. Appl. 2000; 740: 101-107Crossref PubMed Scopus (48) Google Scholar) 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.Cummins C.L. Volle D.H. Zhang Y. McDonald J.G. Sion B. Lefrancois Martinez A.M. Caira F. Veyssiere G. Mangelsdorf D.J. Lobaccaro J.M.A. J. Clin. Invest. 2006; 116: 1902-1912Crossref PubMed Scopus (139) Google Scholar). 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, Charbonnie`res, 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.TABLE 1Sequence primers used for qPCRGene (accession number)5′–3′ sequencesSize of the ampliconSource/Referencebpabca1 (NM_013454)GCT CTG GGA GAG GAT GCT GA (forward)106This studyCGT TTC CGG GAA GTG TCC TA (reverse)abcg1 (NM_009593)GCT GTG CGT TTT GTG CTG TT (forward)83Ref. 51.Barish G.D. Downes M. Alaynick W.A. Yu R.T. Ocampo C.B. Bookout A.L. Mangelsdorf D.J. Evans R.M. Mol. Endocrinol. 2005; 19: 2466-2477Crossref PubMed Scopus (194) Google ScholarTGC AGC TCC AAT CAG TAG TCC TAA (reverse)ERα (NM_007956)TAT GCC TCT GGC TAC CAT TA (forward)183This studyATG GTG CAT TGG TTT GTA GC (reverse)ERRα (NM_007953)CAA ACG CCT CTG CCT GGT CT (forward)113Ref. 52.Horard B. Rayet B. Triqueneaux G. Laudet V. Delaunay F. Vanacker J.M. J. Mol. Endocrinol. 2004; 33: 87-97Crossref PubMed Scopus (47) Google ScholarACT CGA TGC TCC CCT GGA TG (reverse)fas (NM_007988)CCC CAA CCC TGA GAT CCC A (forward)82This studyTTG ATG CCC ACG TTG CC (reverse)ldlr (AF_425607)AAG ACT CAT GCA GCA GGA AC (forward)160This studyGCC TCC ACA GCT GAA TTG AT (reverse)lpl (NM_008509)AGG ACC CCT GAA GAC ACA GCT (forward)148This studyGCC ACC CAA CTC TCA TAC ATT CC (reverse)srebp1c (NM_011480)GGA GCC ATG GAT TGC ACT TT (forward)189This studyGCT TCC AGA GAG GAG GCC AG (reverse)lxrα (AJ_132601)GGG AGG AGT GTG TGC TGT CAG (forward)192This studyGAG CGC CTG TTA CAC TGT TGC (reverse)lxrβ (NM_009473)AAG CAG GTG CCA GGG TTC T (forward)140Ref. 27.Cummins C.L. Volle D.H. Zhang Y. McDonald J.G. Sion B. Lefrancois Martinez A.M. Caira F. Veyssiere G. Mangelsdorf D.J. Lobaccaro J.M.A. J. Clin. Invest. 2006; 116: 1902-1912Crossref PubMed Scopus (139) Google ScholarTGC ATT CTG TCT CGT GGT TGT (reverse)oxtr (D86599.1)TTC TTC GTG CAG ATG TGG AG (forward)114This studyTGT AGA TCC ATG GGT TGC AG (reverse)PR (NM_008829)GTC AGG CTG GCA TGG TCC TT (forward)161This studyAGG GCC TGG CTC TCG TTA GG (reverse)SM22α (U36588)CGA CCA AGC CTT CTC TGC C (forward)51This studyTGC CGT AGG ATG GAC CCT T (reverse)Telokin (AF314149)CCC GGA GAT GAA ATC CCG (forward)106This studyGGC ATA GCT GCT TTT GTG GG (reverse)Cyclophilin (NM_011149)GGA GAT GGC ACA GGA GGA A (forward)75Ref. 27.Cummins C.L. Volle D.H. Zhang Y. McDonald J.G. Sion B. Lefrancois Martinez A.M. Caira F. Veyssiere G. Mangelsdorf D.J. Lobaccaro J.M.A. J. Clin. Invest. 2006; 116: 1902-1912Crossref PubMed Scopus (139) Google ScholarGCC CGT AGT GCT TCA GCT T (reverse)scd1 (NM_009127.3)CCG GAG ACC CCT TAG ATC GA (forward)88This studyTAG CCT GTA AAA GAT TTC TGC AAA CC (reverse)scd2 (NM_009128.1)TTC CTC ATC ATT GCC AAC ACC (forward)54This studyGGG CCC ATT CAT ACA CGT CA (reverse) Open table in a new tab 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), ERα (estrogen receptor α) (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA), ERRα (estrogen-related receptor α) (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 (PGF2α receptor) (1:500; Cayman Chemical), or β-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.Dalle M. Dauprat-Dalle P. Barlet J.P. Arch. Int. Physiol. Biochim. Biophys. 1992; 100: 251-254PubMed Google Scholar) 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 PGF2α (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. Loss of LXRβ 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 lxrα-/- mice (0.37% ± 0.04, n = 4), the lxrβ-/- mice (0.40% ± 0.02, n = 7), and the lxrα;β-/- 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 (×400) did not reveal any perturbation of the endometrium structure (data not shown), whereas vacuoles were visible in layers of myometrium from lxrβ-/- (Fig. 1A, g) and lxrα;β-/- 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 lxrβ-/- (Fig. 1A, k) and lxrα; β-/- (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 LXRβ-deficient mice as young as 1 month old (data not shown). Since lxrα-/- mice appeared to have no lipid-rich vacuole (Fig. 1A, j), we concluded that the phenotype was due to the absence of LXRβ.TABLE 2Surface 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.GenotypeEndometriumCLLLWild type (15.Smith R.D. Babiychuk E.B. Noble K. Draeger A. Wray S. Am. J. Physiol. 2005; 288: C982-C988Crossref PubMed Scopus (90) Google Scholar)43.5 ± 1.714.6 ± 0.541.9 ± 2.0lxrα–/– (12.Brizzi P. Tonolo G. Esposito F. Puddu L. Dessole S. Maioli M. Milia S. Am. J. Obstet. Gynecol. 1999; 181: 430-434Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar)39.4 ± 1.214.3 ± 0.846.3 ± 1.7lxrβ–/– (16.Buxton I.L. Vittori J.C. Proc. West Pharmacol. Soc. 2005; 48: 126-128PubMed Google Scholar)43.0 ± 3.517.1 ± 1.239.9 ± 2.7lxrα;β–/– (15.Smith R.D. Babiychuk E.B. Noble K. Draeger A. Wray S. Am. J. Physiol. 2005; 288: C982-C988Crossref PubMed Scopus (90) Google Scholar)43.4 ± 1.114.0 ± 0.642.6 ± 1.4 Open table in a new tab 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 PPARα and -γ (peroxisomal proliferator-activated receptors α and γ) (see Fig. 4A). 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 lxrα;β-/- 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 LXRβ regulates lipid homeostasis in uterine smooth muscle. LXRβ 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.Davies J.D. Carpenter K.L. Challis I.R. Figg N.L. McNair R. Proudfoot D. Weissberg P.L. Shanahan C.M. J. Biol. Chem. 2005; 280: 3911-3919Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), 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 lxrβ-/- and lxrα;β-/- mice, compared with wild-type mice; p < 0.0005) and 12 months of age (27.6- and 66.5-fold in lxrβ-/- and lxrα;β-/- mice, compared with wild-type mice; p < 0.0005) (Fig. 2). lxrα-/- 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 lxrβ-/- and lxrα; β-/- uterus was due to the accumulation of cholesteryl esters. Whatever the age considered, cholesteryl ester concentration was significantly higher in lxrα;β-/- uterus than in lxrβ-/-. This could suggest a mechanism of a slight redundancy between the two isoforms, where LXRα could partially reverse the drastic phenotype induced by absence of LXRβ. No significant differences in free cholesterol (Fig. 2, white bars), triacylglycerol, and phospholipid contents (data not shown) were observed among the genotypes. Both LXRα and LXRβ 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 LXRβ. We wondered whether this specificity in the LXR isoform was due to the exclusive expression of LXRβ in the tissue. The presence of LXRα and LXRβ mRNA was checked by qPCR on whole uterus as well as mucosa and muscular parts. As shown in Fig. 3, lxrα and lxrβ 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. 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 LXRβ-deficient mice, gene expression was examined by qPCR from whole uteri of wild-type and lxrα; β-/- 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). Not surprisingly, transcripts of the low density lipoprotein receptor ldlr (low density lipoprotein receptor) was significantly lower in lxrα;β-/- mice, since this gene is known to be regulated by the intracellular concentration of oxysterols through a negative regulation loop (34.Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4519) Google Scholar). Interestingly, although the basal level of abca1 was unchanged, basal accumulation of abcg1 was significantly lower in lxrα;β-/- 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). Interesti" @default.
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• W2077671696 title "Oxysterol Nuclear Receptor LXRβ Regulates Cholesterol Homeostasis and Contractile Function in Mouse Uterus" @default.
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