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Molecular Endocrinology, doi:10.1210/me.2007-0187
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*Breast Cancer
Molecular Endocrinology 21 (8): 1781-1790
Copyright © 2007 by The Endocrine Society

Estrogen Deprivation and Inhibition of Breast Cancer Growth in Vivo through Activation of the Orphan Nuclear Receptor Liver X Receptor

Haibiao Gong, Ping Guo, Yonggong Zhai, Jie Zhou, Hirdesh Uppal, Michael J. Jarzynka, Wen-Chao Song, Shi-Yuan Cheng and Wen Xie

Center for Pharmacogenetics and Department of Pharmaceutical Sciences (H.G., Y.Z., J.Z., H.U., W.X.), University of Pittsburgh, Pittsburgh, Pennsylvania 15261; University of Pittsburgh Cancer Institute and Department of Pathology (P.G., M.J.J., S.-Y.C.), Pittsburgh, Pennsylvania 15213; Biomedicine Research Institute (Y.Z.), Beijing Normal University, Beijing 100875, China; and Institute for Translational Medicine and Therapeutics and Department of Pharmacology (W.-C.S.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Dr. Wen Xie, Center for Pharmacogenetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. E-mail: wex6{at}pitt.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen plays an important role in normal physiology. It is also a risk factor for breast cancer, and antiestrogen therapies have been shown to be effective in the treatment and prevention of breast cancers. The liver is important for estrogen metabolism, and a compromised liver function has been linked to hyperestrogenism in patients. In this report, we showed that the liver X receptor (LXR) controls estrogen homeostasis by regulating the basal and inducible hepatic expression of estrogen sulfotransferase (Est, or Sult1e1), an enzyme critical for metabolic estrogen deactivation. Genetic or pharmacological activation of LXR resulted in Est induction, which in turn inhibited estrogen-dependent uterine epithelial cell proliferation and gene expression, as well as breast cancer growth in a nude mouse model of tumorigenicity. We further established that Est is a transcriptional target of LXR, and deletion of the Est gene in mice abolished the LXR effect on estrogen deprivation. Interestingly, Est regulation by LXR appeared to be liver specific, further underscoring the role of liver in estrogen metabolism. Activation of LXR failed to induce other major estrogen-metabolizing enzymes, suggesting that the LXR effect on estrogen metabolism is Est specific. In summary, our results have revealed a novel mechanism controlling estrogen homeostasis in vivo and may have implications for drug development in the treatment of breast cancer and other estrogen-related cancerous endocrine disorders.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN PLAYS A CRUCIAL role in a wide array of physiological events in mammals, ranging from sexual development, to reproductive cycle, fertility, and maintenance of pregnancy (1, 2, 3). A dramatic lowing of estrogen levels during menopause has been associated with osteoporosis and cardiovascular diseases. This observation has led to the development of the estrogen-containing hormone replacement therapy, which has been widely used to treat menopause-related syndromes (4). Estrogen also plays an important role in the initiation and progressive growth of human breast cancers (1, 2, 5, 6). Accordingly, strategies to down-regulate estrogen activities, such as those that use antiestrogens and aromatase inhibitors, have been proven effective to treat and prevent breast cancers. The traditional antiestrogenic agents, such as tamoxifen, although effective, are known to have side effects, such as risk for endometrial cancer and deep vein thrombosis. As such, an outstanding challenge is to develop novel and effective estrogen deprivation strategies.

A critical metabolic pathway to deactivate estrogens is through the estrogen sulfotransferase (EST, or SULT1E1)-mediated sulfation. The sulfonated estrogens cannot bind to and activate the estrogen receptor, and thus lose their estrogenic activities (7). EST belongs to the Phase II sulfotransferase family of drug metabolizing enzymes. As the primary sulfotransferase isoform responsible for estrogen sulfation at physiological concentrations (8), EST plays an important role in the metabolic deactivation of estrogens in both humans and rodents (7). Mice deficient of Est exhibited phenotypes in their reproductive system and/or reproductive functions (9, 10). In addition to the reproductive tissues, Est is also expressed in the mouse liver, but at a substantially lower level (7). EST has also been shown to express in the human liver and this expression exhibited marked interindividual variation (11). However, the effect of hepatic Est expression on estrogen homeostasis has not been reported.

The liver X receptors (LXRs), including the {alpha}- and ß-isoforms, were defined as sterol sensors because they can be activated by cholesterol-derived oxysterols (12). In addition to being activated by endogenous oxysterols, LXRs can also be activated by synthetic agonists, such as T0901317 (TO1317) (13) and GW3965 (14). LXRs were initially shown to have an overall antiatherosclerotic effect by increasing hepatic cholesterol catabolism (12, 15). In rodents, LXR{alpha} increases cholesterol catabolism by inducing cholesterol 7{alpha}-hydroxylase (Cyp7a1), a rate-limiting enzyme that catalyzes the conversion of cholesterol to bile acids (16). In the intestine, LXRs increase the expression of ATP-binding cassette transporters, resulting in cholesterol efflux and blockade of intestinal cholesterol absorption (17). In macrophages, LXRs promote reverse cholesterol transport by up-regulating ATP-binding cassette transporters and apolipoproteins E, C-I, C-IV, and C-II (15). In addition, LXRs inhibit macrophage inflammatory responses and impact antimicrobial responses (18, 19). Despite their promise as antiatherosclerotic targets, LXRs have also been linked to prolipogenic side effects by activating the lipogenic transcriptional factor Srebp-1c and a battery of lipogenic enzymes (13, 20).

In this study, we revealed a novel function of LXRs in estrogen homeostasis. We showed that activation of LXR induced Est expression, which in turn promoted the metabolic deactivation of estrogens. The LXR-mediated Est regulation constitutes a novel strategy of estrogen deprivation and may have implications in the prevention and treatment of breast cancer and other estrogen-related endocrine disorders.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of LXR Induced the Hepatic Expression of Est and Decreased Circulating Level of Estrogens
To examine the in vivo roles of LXR, we have recently created transgenic mice that express the activated LXR{alpha} (VP-LXR{alpha}) in the liver and intestine under the control of the rat liver fatty acid-binding protein (FABP) promoter (21). Created by fusing the VP16 activation domain of the herpes simplex virus to the amino-terminal of mouse LXR{alpha}, VP-LXR{alpha} shares the same DNA binding specificity as its wild-type counterpart, and cotransfection with VP-LXR{alpha} activated the LXR-responsive reporter gene in the absence of an agonist (21). The transgene expression in a panel of female tissues and male testis was evaluated by Northern blot analysis using the transgene-specific simian virus 40 poly (A) cDNA probe. As shown in Fig. 1AGo, top panel, in addition to its expression in the liver and intestine, VP-LXR{alpha} mRNA was also detected at lower levels in the ovary and testis, but not in the kidney, lung, white fat, brain, uterus, and mammary gland. The testicle transgenic expression, confirmed by real-time PCR analysis (data not shown), is consistent with an earlier report of low expression of endogenous liver FABP in this tissue (22). The transgene had little effect on the expression of endogenous LXR{alpha} in the liver (data not shown). Our preliminary Affymetrix gene expression analysis showed that the hepatic expression of Est was induced in the transgenic mice (data not shown), and this result was confirmed by Northern blot analysis (Fig. 1AGo, top panel). The basal expression of Est in the livers of wild-type mice was very low. The Est induction in transgenic mice was so dramatic that its expression reached to a level similar to that of the male testis, the tissue known to have the highest constitutive Est expression (7). Interestingly, LXR-induced Est expression appeared to be liver specific, as the transgene failed to induce testicle Est expression (Fig. 1AGo, top panel). All other tissues examined in Fig. 1AGo, top panel, did not have detectable expression of Est, regardless of the transgene expression.


Figure 1
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  		Fig. 1. Activation of LXR Induced the Hepatic Expression of Est and Decreased Circulating Level of Estrogens

A (top panel), Expression of the transgene as revealed by Northern blot analysis. All tissues, except testes, are from female mice. Membrane was first probed with the transgene-specific simian virus 40 poly (A) cDNA probe. The membrane was subsequently stripped and reprobed with Est and Gapdh as a loading control. Lanes represent pooled samples from three mice per genotype. Relative expressions of VP-LXR{alpha} and Est are labeled with expression levels in the transgenic liver arbitrarily set as 1. A (bottom panel), Up-regulation of Est in the livers of VP-LXR{alpha} transgenic and TO1317 or 22(R)-hydroxycholesterol-treated wild-type mice as revealed by Northern blot analysis. Lanes represent individual mice. B, The TO1317 effect on Est expression was abolished in the LXR DKO mice but not in the PXR null mice as revealed by real-time PCR analysis. C, The basal expression of Est was reduced in female LXR DKO mice as revealed by real-time PCR analysis. D, TO1317 treatment in wild-type mice had no effect on Est expression in the testis (left panel) or mammary gland (right panel). E, Activation of LXR increased estrogen sulfation. Cytosolic liver extracts from transgenic or TO1317-treated wild-type mice were subjected to sulfation assay using estrone as the substrate. [35S]PAPS was used as the sulfate donor. Radioactivity was measured by scintillation counter after separation and removal of free [35S]PAPS. F, Four-week-old virgin transgenic females or wild-type females treated with TO1317 were measured for endogenous E2 levels. When drug treatments were used, mice received a single ip dose of TO1317 (TO) or 22(R)-hydroxycholesterol [22(R)] (50 mg/kg each) 24 h before being killed, except mice in panel F who received three daily ip dose of TO1317 (50 mg/kg). Lanes in B–F represent four to six mice per group, and results are presented as averages and SD. WT, Wild type; TG, transgenic; DMSO, dimethylsulfoxide. *, P < 0.05; **, P < 0.01; #, P > 0.05, compared with the vehicle and/or wild-type controls. Student’s t test was used to calculate P values.

 
The induction of Est by LXR was further analyzed. Northern blot analysis showed that the liver expression of Est was markedly up-regulated in transgenic mice of both sexes (Fig. 1AGo, bottom panel). The Est induction was also seen in wild-type mice treated with LXR agonists TO1317 or 22(R)-hydroxycholesterol (Fig. 1AGo, bottom panel). The TO1317 effect was abolished in the LXR {alpha} and ß double knockout (LXR DKO) mice (16) (Fig. 1BGo). The LXR DKO females also showed decreased basal expression of Est (Fig. 1CGo), suggesting that LXR is required for both the basal and inducible expression of Est. The Est regulation appeared to be LXR specific. The loss of pregnane X receptor (PXR) had no effect on the TO1317-induced Est expression (Fig. 1BGo), and the expression of Est was not induced in transgenic mice expressing the activated PXR (23) (data not shown). TO1317 is also known to activate PXR (24), the intact regulation of Est in the PXR null mice suggested that the TO1317 effect on Est expression was mediated by LXRs. Consistent with results in transgenic mice, the LXR ligand effect also appeared to be liver specific. TO1317 had little effect on Est expression in the testis (Fig. 1DGo, left panel) and mammary gland (Fig. 1DGo, right panel), although Est and LXRs are expressed in both tissues (25, 26).

The effects of genetic and pharmacological activation of LXR on Est expression were also confirmed by a sulfotransferase enzymatic assay using estrone as the substrate (Fig. 1EGo). Consistent with the regulation of Est expression and activity, 4-wk-old virgin female transgenic mice or wild-type mice treated with TO1317 for 3 d exhibited significantly reduced endogenous 17ß-estradiol (E2) level (Fig. 1FGo). Of note, the LXR agonists induced Est expression and activity as efficiently as the VP-LXR{alpha} transgene (Fig. 1AGo, bottom panel, and panels E and F).

Activation of LXR Inhibited Uterine Estrogen Responses in an Est-Dependent Manner
Estrogens are known to promote uterine epithelial proliferation. To determine the effect of LXR-mediated Est activation on estrogen deprivation, ovariectomized wild-type and VP-LXR{alpha} transgenic females were subjected to uterine estrogen response measurements that include epithelial proliferation by bromodeoxyuridine (BrdU) labeling and estrogen-responsive gene expression by real-time PCR. As shown in Fig. 2AGo, E2 treatment in wild-type mice increased BrdU labeling as expected (Fig. 2A-bGo). However, the E2 effect was compromised in both the TO1317 pretreated wild-type (Fig. 2A-cGo) and mock-treated VP-LXR{alpha} transgenic (Fig. 2A-eGo) mice. The transgene (Fig. 2A-dGo) or TO1317 treatment (data not shown) alone, in the absence of E2, had little effect on the basal proliferation. When the TO1317 treatment was performed in the Est null mice (26) (Fig. 2A-fGo) or when the VP-LXR{alpha} transgene was bred into the Est null background (data not shown), the TO1317 and transgenic effects on the E2 responses were abolished, demonstrating that Est expression is required for the LXR effect. Activation of LXRs also led to an inhibition of estrogen-responsive uterine gene expression. The mRNA expression of progesterone receptor (Pgr), lactoferrin (Ltf), and insulin-like growth factor 1 (Igf-1) was induced, and thioredoxin-interacting protein (Txnip) was suppressed by E2 in ovariectomized wild-type female mice as expected (Fig. 2BGo) (27, 28, 29). Consistent with the cell proliferation results, the E2 effects on the expression of these E2-responsive genes were abolished in the TO1317-treated wild-type and mock-treated VP-LXR{alpha} transgenic mice, and the TO1317 effect was abolished in the Est null mice (Fig. 2BGo). In another estrogen-dependent uterotropic bioassay (30), estrogen treatment caused typical uterine enlargement (water imbibition) in the wild-type female mice, but this uterotropic effect was abolished in the VP-LXR{alpha} transgenic mice (Fig. 2Go, C and D).


Figure 2
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  		Fig. 2. Activation of LXR Inhibited Uterine Estrogen Responses

A, Immunostaining of BrdU incorporation on uterine paraffin sections from mice of different genotypes and drug treatments. All mice were ovariectomized 7 d before the estrogen treatment. Mice received a single sc injection of vehicle or E2 (20 mg/kg body weight) 20 h before being killed. All mice were labeled for BrdU (60 mg/kg) for 2 h before being killed. Percentages of BrdU-positive nuclei are quantitated and labeled. Mice in A-c and A-f were pretreated with TO1317 (TO) for 3 d before the E2 treatment. Three to five mice were used for each group, and representative fields are shown. The original magnification is x200 for all panels. B, Regulation of uterine gene expression as measured by real-time PCR. Pgr, Progesterone receptor; Ltf, lactoferrin; Igf-1, insulin-like growth factor 1; Txnip, thioredoxin interacting protein. C and D, Appearance (C) and quantitation (D) of the uterotropic bioassay results in the wild-type and transgenic mice with four to six mice per group. WT, Wild type; TG, transgenic; VEH, vehicle. **, P < 0.01, compared with the vehicle control.

 
Est Is a Transcriptional Target of LXR{alpha}
To understand the molecular mechanism by which LXR regulates Est, we cloned up to 4.2 kb of the 5'-regulatory sequences of the mouse Est gene and examined their regulation by LXR{alpha}. The 4.2-kb promoter was activated by the wild-type LXR{alpha} in the presence of TO1317 (Fig. 3AGo). Deletion analyses located the LXR-responsive region to nucleotide –200 bp to –400 bp (Fig. 3AGo). Inspection of this 200-bp sequence revealed a DR-4 (direct repeats spaced by four nucleotides) type nuclear receptor response element (Fig. 3BGo). EMSAs revealed that the LXR{alpha}/retinoic X receptor (RXR) heterodimers can bind to Est/DR-4, and this binding can be efficiently competed by excess unlabeled Srebp-1c/DR-4 (20) or Est/DR-4, but not by the mutant Est/DR-4 (Fig. 3BGo). Chromatin immunoprecipitation (ChIP) assay was used to demonstrate the recruitment of LXR{alpha} onto the Est promoter. In this experiment, the hemagglutinin (HA)-tagged mouse LXR{alpha} or the HA vector control plasmid was transfected into the livers of the wild-type mice in the presence or absence of TO1317 treatment. ChIP assay was performed with the use of an anti-HA antibody (31). As shown in Fig. 3CGo, treatment with TO1317 resulted in the recruitment of HA-LXR{alpha} onto the Est promoter. ChIP on the Srebp-1c gene promoter was included as the positive control. Consistent with EMSA results, thymidine kinase (tk)-Est/DR-4, an Est/DR-4 containing synthetic tk reporter, but not its mutant variant, was activated by LXR{alpha} in the presence of TO1317 (Fig. 3DGo, left panel). The DR-4 element was also necessary in the context of the 0.4-kb natural promoter, because mutation of this element abolished LXR{alpha} transactivation (Fig. 3AGo). The 2.1-kb natural promoter was also activated by TO1317 when transfected into the mouse liver by a hydrodynamic gene delivery system (31) (Fig. 3DGo, right panel).


Figure 3
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  		Fig. 3. Est Is a Transcriptional Target of LXR{alpha}

A, Luciferase reporter genes that contain the mEst gene promoter (EstP) sequences were transfected into HepG2 cells together with the LXR{alpha} expression vector. The transfection efficiency was normalized against the ß-gal activities from a cotransfected CMX-ß-gal vector. Fold inductions of TO1317 over solvent control are labeled. B, The LXR{alpha}/RXR{alpha} heterodimers bind to Est/DR-4 as revealed by EMSA. Shown on the left is the partial DNA sequence of the EstP. The DR-4 element is capitalized, and the mutant variant is shown with the mutated nucleotides underlined. The LXRE from the Srebp-1c promoter is also shown. The binding of LXR{alpha}/RXR heterodimers to Est/DR-4 was efficiently competed by Est/DR-4 itself and Srebp/LXRE, but not by the mutant Est/DR-4. C, Recruitment of mLXR{alpha} onto the mEst promoter as revealed by ChIP analysis. HA-tagged mLXR{alpha} or the HA vector control was used to transfect wild-type mouse livers by a hydrodynamic gene delivery method. Mice were treated with vehicle or TO1317 for 9.5 h before being killed. ChIP was performed with the use of an anti-HA antibody. ChIP on the Srebp-1c gene promoter was included as the positive control. Lanes represent individual mice. D, LXR{alpha} activates the tk-Est/DR4 promoter in HepG2 cells (left panel) and the 2.1-kb promoter in the mouse livers (right panel). Cell transfection results shown are fold induction over solvent and represent the averages and SD from triplicate assays. For the promoter liver transfection experiment, n = 6 per group. DMSO, Dimethylsulfoxide; LXRE, LXR response element.

 
Activation of LXR Failed to Induce Other Major Hepatic Estrogen Metabolizing Enzymes
In addition to Est, several other hepatic enzymes have also been shown to affect estrogen metabolism. These include the Phase I cytochrome P450 Cyp3a and 1a2 enzymes and the Phase II UDP-glucuronosyltransferase 1a1 (Ugt1a1) (for a review, see Ref. 32). Affymetrix microarray analysis showed that the hepatic expression of Cyp3a11 was not induced but rather suppressed, and the expression of Cyp1a2 and Ugt1a1 was not affected in the VP-LXR{alpha} transgenic mice (data not shown). The suppression of Cyp3a11 (Fig. 4AGo) and lack of Ugt1a1 regulation (Fig. 4BGo) in the transgenic mice was confirmed by real-time PCR and Northern blot analysis, respectively. The lack of Ugt1a1 activation in the livers of transgenic mice was also confirmed by the unchanged E2 glucuronidation as revealed by an in vitro UGT enzymatic assay using E2 as the substrate (data not shown). Similar suppression of Cyp3a11 and lack of Ugt1a1 and Cyp1a2 regulation were also seen in wild-type mice treated with LXR agonists 22(R)-hydroxycholesterol or GW3965 (Fig. 4CGo). The suppression of Cyp3a11 by LXR activation was consistent with a recent report that the basal expression of Cyp3a11 was increased in LXR DKO mice (33). The mechanism for LXR-mediated Cyp3a11 suppression remains to be determined.


Figure 4
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  		Fig. 4. Activation of LXR Failed to Induce Other Major Hepatic Estrogen Metabolizing Enzymes

A, Suppression of hepatic Cyp3a11 mRNA expression in female VP-LXR{alpha} transgenic mice as revealed by real-time PCR; n = 5 for each group. B, Lack of Ugt1a1 regulation in female TG mice as revealed by Northern blot analysis. The expression of unchanged pregnane X receptor (Pxr) (22 ) was included as control. Lanes represent individual mice. C, Suppression of Cyp3a11 and lack of Ugt1a1 and Cyp1a2 regulation in wild-type male mice treated with three daily dose of LXR agonists 22(R)-hydroxycholesterol (40 mg/kg) or GW3965 (20 mg/kg). LXR target gene Scd-1 was included as a positive control. WT, Wild type; TG, transgenic; VEH, vehicle. *, P < 0.05; **, P < 0.01; #, P > 0.05, compared with the wild type (A) or vehicle (C) controls.

 
LXR Agonist Inhibited Estrogen-Dependent Breast Cancer Cell Tumorigenicity in Nude Mice
The inhibition of estrogen activity prompted us to examine whether the activation of LXRs can inhibit estrogen-promoted breast cancer growth in xenograft models using ovariectomized nude mice. We chose the estrogen receptor-positive and estrogen-responsive breast cancer MCF-7 cells. But instead of the parent MCF-7 cells, we used MCF-7/VEGF cells that overexpress the vascular endothelial growth factor (VEGF). We have previously reported that, under our experimental conditions, the parent MCF-7 cells had low tumorigenicity, developing tumors only in 40–50% of E2-treated mice, with tumor volumes as small as 200–300 mm3 45 d after inoculation. In contrast, the MCF-7/VEGF cells exhibited a full penetrance of both estrogen-independent and -dependent growth with substantially increased tumor volumes in the presence of E2 (34). The estrogen effects on the tumorigenicity of the MCF-7/VEGF cells were evaluated and compared in the absence or presence of TO1317. As shown in Fig. 5Go, the MCF-7/VEGF cells were highly tumorigenic in E2-treated mice, consistent with our previous report (34). However, TO1317 treatment significantly attenuated the E2-enhanced tumor growth, leading to tumor growth kinetics similar to that in the absence of E2 treatment (Fig. 5AGo). The inset in Fig. 5AGo shows a pair of representative E2-induced tumors in the absence or presence of TO1317. The tumor-inhibitory effect was associated with a decreased tumor cell BrdU labeling index (Fig. 5BGo) and circulating E2 levels (Fig. 5CGo) in the TO1317-treated tumor-bearing mice. The hepatic mEst expression in the TO1317-treated nude mice was significantly induced compared with their mock-treated counterparts (Fig. 5DGo, left panel), whereas TO1317 had little effect on hEST expression in the MCF-7 tumor tissues (Fig. 5DGo, right panel), suggesting that the inhibitory effect is due to the EST activation in the liver but not in the tumor tissues. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay showed little differences in the percentage of apoptotic cells between vehicle- and TO1317-treated tumors (data not shown), suggesting that increased apoptosis may not explain the inhibitory effect. The inhibition was specific for the E2-dependent breast tumor growth, because TO1317 had little effect on the growth of MDA-MB-231, an ER-negative and E2-independent breast cancer cell line (35), in nude mice (Fig. 5EGo). TO1317 had no effect on in vitro proliferation of MCF-7 cell in the presence or absence of E2 (data not shown).


Figure 5
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  		Fig. 5. LXR Agonist TO1317 Inhibited Estrogen-Dependent But Not Estrogen-Independent Human Breast Cancer Cell Tumorigenicity in Ovariectomized Nude Mice

A, Growth kinetics of the MCF-7/VEGF tumors in the presence of indicated hormone and drug treatments. The TO1317 group received daily treatment of TO1317 (15 mg/kg) by gavage. Tumor volumes were measured at the indicated times. Results are presented as averages and SD. Each group contains at least nine mice. **, P < 0.01, compared with the TO1317 treatment group. The inset shows pairs of representative E2-induced tumors in vehicle- or TO1317-treated mice. B, Quantitation of BrdU-positive nuclei in the E2-induced MCF-7/VEGF tumors 25 d post inoculation. Mice were labeled with BrdU (60 mg/kg) for 2 h before being killed. Insets are representative fields of BrdU immunostaining. **, P < 0.01. C, Serum levels of E2 from mice in panel A at the time of euthanasia (35 d post inoculation). *, P < 0.05. D, Increased expression of Est in the mouse livers (left panel) but not in the MCF-7 tumors (right panel) of the tumor-bearing nude mice. **, P < 0.01. E, TO1317 had little effect on the estrogen-independent growth of MDA-MB-231 cells. The TO1317 group received daily treatment of TO1317 (15 mg/kg) by gavage. The inset shows representative MDA-MB-231 tumors; n = 6 per group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The current study showed that LXRs regulate Est and control estrogen homeostasis. The endocrine role of LXR is distinct to the known functions of this receptor in cholesterol and lipid homeostasis and inflammation (12). Based on our observations that: 1) Est is a transcriptional target of LXR; 2) Est is necessary for the estrogen deprivation effect of LXR; and 3) Activation of LXR fails to induce other major estrogen-metabolizing enzymes, we conclude that activation of Est plays an important role in LXR-mediated estrogen deprivation. However, we cannot exclude the possibility that additional LXR target genes may have also contributed to the phenotype. These additional target genes could have also contributed to the potential estrogen-independent growth-inhibitory effect of LXR.

Est is believed to be the primary sulfotransferase isoform responsible for estrogen sulfation at physiological concentrations due to its low Km value for E2 (8). Consistent with the notion that Est plays a critical role in estrogen deactivation, disruption of Est in mice resulted in structural and functional lesions in the male reproductive system (9). Female Est knockout mice exhibited spontaneous fetal loss caused by placental thrombosis due to insufficient estrogen deactivation (10). EST is highly expressed in both rodent and human normal mammary epithelial cells, but its expression is diminished in human breast cancer cells (36). An ectopic expression of EST in MCF-7 cells suppresses the estrogen response, suggesting an important role for this enzyme in regulating estrogen-dependent breast cancer cell growth (37, 38). The identification of Est as a transcriptional target of LXR may establish this nuclear receptor as a therapeutic target to down-regulate estrogen activity. Indeed, the inhibition of estrogen-dependent MCF-7 tumor growth by LXR agonist supports the practicality of this therapeutic strategy. However, because TO1317 is also known to activate PXR (24) and thus potentially induce other estrogen-metabolizing enzymes such as Cyp3a11 and Ugt1a1, we cannot exclude the possibility that TO11317 target(s) other than LXR may have also contributed to the phenotype in the xenograft model. In the transgenic model, it is clear that activation of hepatic Cyp3a11 and Ugt1a1 did not contribute to estrogen deprivation because these two enzymes were not induced by the transgene (Fig. 4Go, A and B). The lack of Cyp3a11 and Ugt1a1 activation by LXR was also confirmed in wild-type mice treated with two PXR-neutral LXR agonists GW3965 and 22(R)-hydroxycholesterol (Fig. 4CGo).

There is substantial evidence to support Est as a local estrogen modulator, such as its enriched expression in the testis and placenta (26). The basal expression of Est in many other tissues, including liver and intestine, is either very low or beyond detection. The current study suggests that the expression of this enzyme is also highly inducible in the liver, and this liver-inducible expression of Est is likely to play an important role in systemic estrogen deprivation. In the transgenic model, although the FABP promoter was also targeted to the intestine, this tissue has no detectable expression of Est (data not shown). In the pharmacological model, the LXR agonist effect on Est expression also appeared to be liver specific. The mechanism for tissue-specific Est regulation remains to be determined. In the nude mouse tumorigenic model, treatment with TO1317 induced hepatic Est expression but had little effect on hEST expression in the MCF-7 cell tumors. Therefore, it is likely that the liver is primarily responsible for the estrogen deprivation effect of LXRs. However, we cannot exclude the possibility that other LXR-expressing tissues may have also contributed to the phenotypes. The significance of hepatic Est regulation in estrogen homeostasis is also consistent with clinical observations. Compromised liver functions in male patients, such as those seen during liver cirrhosis, have been linked to hyperestrogenism (39, 40). It would be interesting to know whether or not a compromised LXR-EST regulatory pathway has contributed to the hyperestrogenism in certain liver diseases.

Although Est expression was clearly required for the LXR effect, we noticed that changes in serum E2 levels in transgenic and TO1317-treated wild-type mice were not as dramatic as changes in Est mRNA expression. One potential explanation is that animals may have compensatory mechanisms to maintain the circulating E2 at a higher than expected level. Such a compensatory effect in steroid hormone homeostasis is not unusual. We recently reported that PXR-mediated increase in glucocorticoid metabolism was actually associated with an increased serum concentration of glucocorticoid (41). The E2 level in LXR-activated mice was 50–60% of that of the control animals. We found that in the xenograft model, the tumor volume reduced to less than half when E2 pellets of a half-dose were implanted (data not shown). Therefore, we believe the E2 reduction in LXR-activated mice is functionally relevant.

In addition to being implicated in breast cancer, estrogen is also known to have diverse physiological functions, among which pregnancy and mammary gland development are two most significant estrogen-dependent processes (1, 5). Although activation of LXR was sufficient to reduce the endogenous circulating E2 levels in young female mice, we did not notice obvious reproductive phenotypes or loss of fetus in VP-LXR{alpha} transgenic or LXR agonist-treated wild-type mice, suggesting that the increased estrogen metabolism in the transgenic mice was within the normal capacity of the endogenous estrogen production. However, future studies are necessary to determine whether or not the activation of LXR will influence pregnancy and the development of mammary glands. It has also been suggested that E2 may decrease LXR mRNA expression in macrophages (42) or mouse adipose tissue (43). It is unknown whether the LXR suppression also exists in the liver, and if so, it is tempting to speculate that mammals may have an estrogen-dependent and LXR-mediated feedback mechanism to control the estrogen homeostasis.

Although the regulation of mouse Est by LXR was robust, it remains to be determined whether this regulation is conserved in humans and to what extent. Results in Fig. 5CGo showed that EST was not activated by TO1317 in the MCF-7/VEGF tumor, despite the expression of LXRs in this cell type. The lack of mammary Est induction was also seen in TO1317-treated wild-type females (Fig. 1DGo). In primary human hepatocytes and HepG2 cells, treatment with TO1317 failed to induce EST (data not shown). However, the lack of TO1317 effect in human hepatocytes alone cannot conclude the lack of EST regulation, because primary mouse hepatocytes maintained in regular hepatocyte medium also failed to respond to TO1317 (data not shown). It is unclear whether the culture conditions have prevented the hepatocyte response. It is not unusual that primary hepatocyte cultures lose certain differentiated liver functions, which could have explained the lack of TO1317 effect on mouse hepatocytes despite the robust effect on intact mouse livers. However, we cannot exclude the possibility that like Cyp7a1 (44), Est regulation may be rodent specific.

Estrogens are known to play an important role in many physiological and disease states. These include mammary development, breast and lung cancer, and osteoporosis. Moreover, most, if not all, hormone replacement therapy regimens contain estrogens either alone or in combination with progestins (4). The potential drug-hormone interactions caused by LXR agonists suggest that it would be desirable to avoid sustained LXR activation in patients undergoing hormone replacement therapies. In summary, the LXR-mediated estrogen deprivation may have broad implications in estrogen homeostasis, endocrine therapies, and tumorigenesis, including a potential use in breast cancer prevention and treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mouse Models
To create FABP-VP-LXR{alpha} transgene, the VP-LXR{alpha} cDNA was placed downstream of the rat FABP promoter (45). The production of this transgenic line has been described previously (21). The LXR DKO (16), Est null (26), and PXR null (23) mice have been described previously. All transgenic mice have a mixed background of C57BL/6J and 129/SvImJ. The ovariectomized nude mice were purchased from Taconic Farms, Inc. (Germantown, NY). The use of mice in this study has complied with all relevant federal guidelines and institutional policies.

Measurement of Uterine Estrogen Response
Five-week old virgin females were subjected to ovariectomies. Mice were given a single sc injection of E2 (20 µg/kg body weight) 7 d after the surgery. Mice were given a single ip injection of BrdU (60 mg/kg) 18 h after the E2 injection and killed 2 h after. One uterine horn was harvested for histology and measurement of cell proliferation by BrdU immunostaining (46), and the other was harvest for RNA extraction and gene expression analysis by real-time PCR. In the uterotropic bioassay, 3-wk-old virgin female mice received daily sc injections of vehicle or E2 (5 µg/kg/d) for 3 d (47). Mice were then killed 24 h after the last E2 dose, and the uteri were dissected, weighed, and photographed. When necessary, mice were subjected to daily treatment of TO1317 (50 mg/kg, ip injection) starting 3 d before the E2 treatment and continued until the completion of the experiments.

Sulfotransferase Assay
Sulfotransferase assay was carried out using [35S] phosphoadenosine phosphosulfate (PAPS) (PerkinElmer, Wellesley, MA) as previously described (48). In brief, 20 µg/ml of total liver cytosolic extract was used with 1 µM of estrone substrate. After the reactions, free [35S]PAPS was removed by extracting with ethyl acetate. The aqueous phase was then counted in scintillation counter for radioactivity.

Cloning of mEst Promoter, Transient Transfections, and DNA-Binding Analysis
The 4.2-kb (–4164 bp to +46 bp) 5'-regulatory sequences of the mEst gene were cloned by PCR using a template of mEst-containing bacterial artificial chromosome clone (identification no. RP24–571N6) from the Children’s Hospital Oakland Research Institute BACPAC Resource Center (Oakland, CA). Deletion mutants were generated by PCR-mediated mutagenesis. HepG2 cells were transfected with the reporter constructs and LXR{alpha} expression vector in 48-well plates as previously described (45). When necessary, cells were treated with TO1317 (10 µM) for 24 h before luciferase assay. The transfection efficiency was normalized against the ß-galactosidase (ß-gal) activities from a cotransfected CMX-ß-gal vector. The hydrodynamic liver transfection was performed as we previously described (31). EMSA were performed using TNT in vitro transcribed and translated proteins as described previously (49).

ChIP Assay
Wild type female mice (4 wk old) received an ip injection of dimethylsulfoxide or TO1317 (50 mg/kg) 30 min before being hydrodynamically transfected with the pCMX-HA-LXR{alpha} or pCMX-HA control plasmid. The liver transfection and ChIP assays were performed the same as we described previously (31). The primers for mEst/DR-4 are: 5'-CCAAAGGGGAGAAACAGCTG-3' and 5'-GAGAAGGAGGCAGAGACTAAC-3'; primers for mSrebp-1c/DR-4 are: 5'-CTCTTTTCGGGGATGGTTG-3'and 5'-GTTTCTCCCGGTGCTCT-3'. The PCR products of Est and Srebp-1c are 142 bp and 141bp, respectively.

Northern Blot Analysis and Real-Time PCR
Total RNAs were extracted using the TRIZOL Reagent. Northern hybridization was carried out as described elsewhere (45). When necessary, quantification was performed with the NIH Image software. Real-time PCR using predesigned Assay-On-Demand TaqMan reagents or SYBR Green-based assays was performed with the ABI 7300 Real-Time PCR System (31). Sequences of the real-time probes are available upon request.

Nude Mice Tumorigenicity
MCF-7 and MDA-MB-231 breast tumors were established in the mammary fat pads of ovariectomized female nude mice as described previously (34, 50). Briefly, 1 x 107 of MCF-7/VEGF cells (34) were inoculated into the mammary fat pads of 8-wk-old ovariectomized female nude mice that were implanted with E2 pellets (0.72 mg/60-d release) or placebo pellets (Innovative Research of America, Sarasota, FL). The E2-treated mice were randomly divided into two groups, with one group receiving daily treatment of TO1317 (15 mg/kg by gavage) and the other receiving vehicle. MDA-MB-231 cells were inoculated at 5 x 106 per animal, and the mice received TO1317 or vehicle but not E2. The volumes of the tumors were measured using a caliper every 5 d. Mice were labeled with BrdU 30 min before being killed. The serum concentrations of active E2 were measured using the ACTIVE ESTRADIOL EIA kit from Diagnostic Systems Laboratories (Webster, TX) according to the manufacturer’s instruction.


   ACKNOWLEDGMENTS
 
We thank Dr. David Mangelsdorf for LXR null mouse RNA samples, Ms. Bethany Everson for her help with the EST null mice, and Drs. Ramalinga Kuruba and Song Li for synthesizing GW3965.


   FOOTNOTES
 
This work was supported in part by National Institutes of Health Grants ES014626, ES012479, and CA107011 (to W.X.); HD042767 (to W.-C.S.); and Department of Defense Breast Cancer Program Grants DAMD17-01-1-0375 and DAMD-17-02-1-0584 (to S.Y.C.). H.G. is supported by a Postdoctoral Fellowship (PDF0503458) from the Susan G. Komen Breast Cancer Foundation. Y.Z. is supported by the Chinese Government Research Grant 2004AA221060.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 29, 2007

Abbreviations: BrdU, Bromodeoxyuridine; ChIP, chromatin immunoprecipitation; DKO, double knockout; DR-4, direct repeats spaced by four nucleotides; E2, 17ß-estradiol; EST, estrogen sulfotransferase; FABP, fatty acid-binding protein; ß-gal, ß-galactosidase; HA, hemagglutinin; LXR, liver X receptor; PAPS, phosphoadenosine phosphosulfate; PXR, pregnane X receptor; RXR, retinoic X receptor; tk, thymidine kinase; VEGF, vascular endothelial growth factor

Received for publication April 13, 2007. Accepted for publication May 22, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. O’Malley BW 2005 A life-long search for the molecular pathways of steroid hormone action. Mol Endocrinol 19:1402–1411[Abstract/Free Full Text]
  2. Yager JD, Davidson NE 2006 Estrogen carcinogenesis in breast cancer. N Engl J Med 354:270–282[Free Full Text]
  3. Nilsson S, Gustafsson JA 2002 Biological role of estrogen and estrogen receptors. Crit Rev Biochem Mol Biol 37:1–28[CrossRef][Medline]
  4. Warren MP 2004 A comparative review of the risks and benefits of hormone replacement therapy regimens. Am J Obstet Gynecol 190:1141–1167[CrossRef][Medline]
  5. Jensen EV 2004 From chemical warfare to breast cancer management. Nat Med 10:1018–1021[CrossRef][Medline]
  6. Deroo BJ, Korach KS 2006 Estrogen receptors and human disease. J Clin Invest 116:561–570[CrossRef][Medline]
  7. Song WC 2001 Biochemistry and reproductive endocrinology of estrogen sulfotransferase. Ann NY Acad Sci 948:43–50[CrossRef][Medline]
  8. Kakuta Y, Pedersen LC, Chae K, Song WC, Leblanc D, London R, Carter CW, Negishi M 1998 Mouse steroid sulfotransferases: substrate specificity and preliminary X-ray crystallographic analysis. Biochem Pharmacol 55:313–317[CrossRef][Medline]
  9. Qian YM, Sun XJ, Tong MH, Li XP, Richa J, Song WC 2001 Targeted disruption of the mouse estrogen sulfotransferase gene reveals a role of estrogen metabolism in intracrine and paracrine estrogen regulation. Endocrinology 142:5342–5350[Abstract/Free Full Text]
  10. Tong MH, Jiang H, Liu P, Lawson JA, Brass LF, Song WC 2005 Spontaneous fetal loss caused by placental thrombosis in estrogen sulfotransferase-deficient mice. Nat Med 11:153–159[CrossRef][Medline]
  11. Song WC, Qian Y, Li AP 1998 Estrogen sulfotransferase expression in the human liver: marked interindividual variation and lack of gender specificity. J Pharmacol Exp Ther 284:1197–1202[Abstract/Free Full Text]
  12. Tontonoz P, Mangelsdorf DJ 2003 Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol 17:985–993[Abstract/Free Full Text]
  13. Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, Schwendner S, Wang S, Thoolen M, Mangelsdorf DJ, Lustig KD, Shan B 2000 Role of LXRs in control of lipogenesis. Genes Dev 14:2831–2838[Abstract/Free Full Text]
  14. Collins JL, Fivush AM, Watson MA, Galardi CM, Lewis MC, Moore LB, Parks DJ, Wilson JG, Tippin TK, Binz JG, Plunket KD, Morgan DG, Beaudet EJ, Whitney KD, Kliewer SA, Willson TM 2002 Identification of a nonsteroidal liver X receptor agonist through parallel array synthesis of tertiary amines. J Med Chem 45:1963–1966[CrossRef][Medline]
  15. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ 2001 Nuclear receptors and lipid physiology: opening the X-files. Science 294:1866–1870[Abstract/Free Full Text]
  16. Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, Hammer RE, Mangelsdorf DJ 1998 Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR {alpha}. Cell 93:693–704[CrossRef][Medline]
  17. Repa JJ, Turley SD, Lobaccaro JA, Medina J, Li L, Lustig K, Shan B, Heyman RA, Dietschy JM, Mangelsdorf DJ 2000 Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science 289:1524–1529[Abstract/Free Full Text]
  18. Joseph SB, Bradley MN, Castrillo A, Bruhn KW, Mak PA, Pei L, Hogenesch J, O’Connell R M, Cheng G, Saez E, Miller JF, Tontonoz P 2004 LXR-dependent gene expression is important for macrophage survival and the innate immune response. Cell 119:299–309[CrossRef][Medline]
  19. Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P 2003 Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med 9:213–219[CrossRef][Medline]
  20. Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I, Shan B, Brown MS, Goldstein JL, Mangelsdorf DJ 2000 Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXR{alpha} and LXRß. Genes Dev 14:2819–2830[Abstract/Free Full Text]
  21. Uppal H, Saini SP, Moschetta A, Mu Y, Zhou J, Gong H, Zhai Y, Ren S, Michalopoulos GK, Mangelsdorf DJ, Xie W 2007 Activation of LXRs prevents bile acid toxicity and cholestasis in female mice. Hepatology 45:422–432[CrossRef][Medline]
  22. Gordon JI, Elshourbagy N, Lowe JB, Liao WS, Alpers DH, Taylor JM 1985 Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins. J Biol Chem 260:1995–1998[Abstract/Free Full Text]
  23. Xie W, Barwick JL, Downes M, Blumberg B, Simon CM, Nelson MC, Neuschwander-Tetri BA, Brunt EM, Guzelian PS, Evans RM 2000 Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406:435–439[CrossRef][Medline]
  24. Shenoy SD, Spencer TA, Mercer-Haines NA, Alipour M, Gargano MD, Runge-Morris M, Kocarek TA 2004 CYP3A induction by liver x receptor ligands in primary cultured rat and mouse hepatocytes is mediated by the pregnane X receptor. Drug Metab Dispos 32:66–71[Abstract/Free Full Text]
  25. Robertson KM, Schuster GU, Steffensen KR, Hovatta O, Meaney S, Hultenby K, Johansson LC, Svechnikov K, Soder O, Gustafsson JA 2005 The liver X receptor-ß is essential for maintaining cholesterol homeostasis in the testis. Endocrinology 146:2519–2530[Abstract/Free Full Text]
  26. Tong MH, Christenson LK, Song WC 2004 Aberrant cholesterol transport and impaired steroidogenesis in Leydig cells lacking estrogen sulfotransferase. Endocrinology 145:2487–2497[Abstract/Free Full Text]
  27. Das SK, Tan J, Johnson DC, Dey SK 1998 Differential spatiotemporal regulation of lactoferrin and progesterone receptor genes in the mouse uterus by primary estrogen, catechol estrogen, and xenoestrogen. Endocrinology 139:2905–2915[Abstract/Free Full Text]
  28. Deroo BJ, Hewitt SC, Peddada SD, Korach KS 2004 Estradiol regulates the thioredoxin antioxidant system in the mouse uterus. Endocrinology 145:5485–5492[Abstract/Free Full Text]
  29. Klotz DM, Hewitt SC, Ciana P, Raviscioni M, Lindzey JK, Foley J, Maggi A, DiAugustine RP, Korach KS 2002 Requirement of estrogen receptor-{alpha} in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk. J Biol Chem 277:8531–8537[Abstract/Free Full Text]
  30. Korach KS 1994 Insights from the study of animals lacking functional estrogen receptor. Science 266:1524–1527[Abstract/Free Full Text]
  31. Zhou J, Zhai Y, Mu Y, Gong H, Uppal H, Toma D, Ren S, Evans RM, Xie W 2006 A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway. J Biol Chem 281:15013–15020[Abstract/Free Full Text]
  32. Tsuchiya Y, Nakajima M, Yokoi T 2005 Cytochrome P450-mediated metabolism of estrogens and its regulation in human. Cancer Lett 227:115–124[CrossRef][Medline]
  33. Gnerre C, Schuster GU, Roth A, Handschin C, Johansson L, Looser R, Parini P, Podvinec M, Robertsson K, Gustafsson JA, Meyer UA 2005 LXR deficiency and cholesterol feeding affect the expression and phenobarbital-mediated induction of cytochromes P450 in mouse liver. J Lipid Res 46:1633–1642[Abstract/Free Full Text]
  34. Guo P, Fang Q, Tao HQ, Schafer CA, Fenton BM, Ding I, Hu B, Cheng SY 2003 Overexpression of vascular endothelial growth factor by MCF-7 breast cancer cells promotes estrogen-independent tumor growth in vivo. Cancer Res 63:4684–4691[Abstract/Free Full Text]
  35. Bartucci M, Morelli C, Mauro L, Ando S, Surmacz E 2001 Differential insulin-like growth factor I receptor signaling and function in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. Cancer Res 61:6747–6754[Abstract/Free Full Text]
  36. Falany JL, Macrina N, Falany CN 2002 Regulation of MCF-7 breast cancer cell growth by ß-estradiol sulfation. Breast Cancer Res Treat 74:167–176[CrossRef][Medline]
  37. Qian Y, Deng C, Song WC 1998 Expression of estrogen sulfotransferase in MCF-7 cells by cDNA transfection suppresses the estrogen response: potential role of the enzyme in regulating estrogen-dependent growth of breast epithelial cells. J Pharmacol Exp Ther 286:555–560[Abstract/Free Full Text]
  38. Falany JL, Falany CN 1997 Regulation of estrogen activity by sulfation in human MCF-7 breast cancer cells. Oncol Res 9:589–596[Medline]
  39. Adlercrueuta H 1974 Hepatic metabolism of estrogens in health and disease. N Engl J Med 290:1081–1083[Medline]
  40. Korenman SG, Perrin LE, McCallum TP 1969 Estrodiol in human plasma: demonstration of elevated levels in gynecomastia and in cirrhosis. J Clin Invest 48:45a
  41. Zhai Y, Pai HV, Zhou J, Amico JA, Vollmer RR, Xie W 2007 Activation of pregnane X receptor disrupts glucocorticoid and mineralocorticoid homeostasis. Mol Endocrinol 21:138–147[Abstract/Free Full Text]
  42. Kramer PR, Wray S 2002 17ß-Estradiol regulates expression of genes that function in macrophage activation and cholesterol homeostasis. J Steroid Biochem Mol Biol 81:203–216[CrossRef][Medline]
  43. Lundholm L, Moverare S, Steffensen KR, Nilsson M, Otsuki M, Ohlsson C, Gustafsson JA, Dahlman-Wright K 2004 Gene expression profiling identifies liver X receptor {alpha} as an estrogen-regulated gene in mouse adipose tissue. J Mol Endocrinol 32:879–892[Abstract]
  44. Chiang JY, Kimmel R, Stroup D 2001 Regulation of cholesterol 7{alpha}-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXR{alpha}). Gene 262:257–265[CrossRef][Medline]
  45. Gong H, Singh SV, Singh SP, Mu Y, Lee JH, Saini SP, Toma D, Ren S, Kagan VE, Day BW, Zimniak P, Xie W 2006 Orphan nuclear receptor pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells. Mol Endocrinol 20:279–290[Abstract/Free Full Text]
  46. Xie W, Paterson AJ, Chin E, Nabell LM, Kudlow JE 1997 Targeted expression of a dominant negative epidermal growth factor receptor in the mammary gland of transgenic mice inhibits pubertal mammary duct development. Mol Endocrinol 11:1766–1781[Abstract/Free Full Text]
  47. Padilla-Banks E, Jefferson WN, Newbold RR 2001 The immature mouse is a suitable model for detection of estrogenicity in the uterotropic bioassay. Environ Health Perspect 109:821–826[Medline]
  48. Saini SP, Sonoda J, Xu L, Toma D, Uppal H, Mu Y, Ren S, Moore DD, Evans RM, Xie W 2004 A novel constitutive androstane receptor-mediated and CYP3A-independent pathway of bile acid detoxification. Mol Pharmacol 65:292–300[Abstract/Free Full Text]
  49. Saini SP, Mu Y, Gong H, Toma D, Uppal H, Ren S, Li S, Poloyac SM, Xie W 2005 Dual role of orphan nuclear receptor pregnane X receptor in bilirubin detoxification in mice. Hepatology 41:497–505[CrossRef][Medline]
  50. Tester AM, Waltham M, Oh SJ, Bae SN, Bills MM, Walker EC, Kern FG, Stetler-Stevenson WG, Lippman ME, Thompson EW 2004 Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and experimental metastasis of MDA-MB-231 human breast cancer cells in nude mice. Cancer Res 64:652–658[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   LXRα  |  RXRα
Ligands:   22α-Hydroxycholesterol  |  T0901317  |  GW 3965  |  17β-Estradiol



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