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Liver X Receptor- Gene Expression Is Positively Regulated by Thyroid Hormone

Department of Medicine and Molecular Science, Graduate School of Medicine, Gunma University, Maebashi, Gunma 371-8511, Japan

Address all correspondence and requests for reprints to: Koshi Hashimoto, M.D., Ph.D., Department of Medicine and Molecular Science, Graduate School of Medicine, Gunma University, 3-39-22 Showa-machi Maebashi, Gunma, Japan 371-8511. E-mail: khashi{at}med.gunma-u.ac.jp.

	Abstract

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The nuclear oxysterol receptors, liver X receptors (LXRs), and thyroid hormone receptors (TRs) cross talk mutually in many aspects of transcription, sharing the same DNA binding site (direct repeat-4) with identical geometry and polarity. In the current study, we demonstrated that thyroid hormone (T₃) up-regulated mouse LXR-, but not LXR-ß, mRNA expression in the liver and that cholesterol administration did not affect the LXR- mRNA levels. Recently, several groups have reported that human LXR- autoregulates its own gene promoter through binding to the LXR response element. Therefore, we examined whether TRs regulate the mouse LXR- gene promoter activity. Luciferase assays showed that TR-ß1 positively regulated the mouse LXR- gene transcription. Analysis of serial deletion mutants of the promoter demonstrated that the positive regulation by TR-ß1 was not observed in the –1240/+30-bp construct. EMSA(s) demonstrated that TR-ß1 or retinoid X receptor- did not bind to the region from –1300 to –1240 bp (site A), whereas chromatin-immunoprecipitation assays revealed that TR-ß1 and retinoid X receptor- were recruited to the site A, indicating the presence of intermediating protein between the nuclear receptors and DNA site. We also showed that human LXR- gene expression and promoter activities were up-regulated by thyroid hormone. These data suggest that LXR- mRNA expression is positively regulated by TR-ß1 and thyroid hormone at the transcriptional level in mammals. This novel insight that thyroid hormone regulates LXR- mRNA levels and promoter activity should shed light on a cross talk between LXR- and TR-ß1 as a new therapeutic target against dyslipidemia and atherosclerosis.

	Introduction

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LIVER X RECEPTORS (LXRs) are nuclear receptors that play pivotal roles in the transcriptional control of lipid and carbohydrate metabolism (1, 2). In addition to their function in lipid homeostasis, LXRs have also modulated immune and inflammatory responses in macrophages (3). LXR exists in two isoforms, LXR- and -ß (also referred to as Nr1h3 and Nr1h2, respectively) (4, 5). LXR- is highly expressed in the liver, and expressed at lower levels in the adrenal glands, intestine, adipose tissue, macrophages, lung, and kidney, whereas LXR-ß is ubiquitously expressed (6). The LXRs are ligand-dependent transcription factors that form heterodimers with the retinoid X receptor (RXR). The RXR/LXR heterodimers bind to LXR responsive elements (LXREs) consisting of direct repeats (DRs) of the core sequence AGGTCA separated by four nucleotides (DR-4) (4, 5, 7, 8). Thyroid hormone receptors (TRs) also possess two isoforms, TR- and -ß (Nr1a1 and Nr1a2), and each isoform exists as two or three subtypes, respectively (1, 2, ß1, ß2, and ß3) (9, 10). TR- plays a key role in postnatal development and cardiac metabolism, whereas TR-ß regulates multiple steps in hepatic metabolism as well as thyroid hormone levels (9). Although the LXRs and TRs belong to two distinct receptor subgroups with respect to ligand-binding affinity (11), the two receptor systems show similarity with respect to molecular mechanisms, target genes, and physiological roles (11, 12). Both TR and LXRs form heterodimers with RXR, and bind to DR-4 with identical geometry and polarity (11, 12, 13, 14). We recently showed that TR-ß and LXR- interact on the mouse cholesterol 7 -hydroxylase (CYP7A1) gene promoter, suggesting the possibility of cross talk between the two receptors (15). Another group (16) reported that TR and LXR compete for binding at the DR-4 site of the ATP-binding cassette transporter A1 gene promoter. These data indicate that LXR and TR could cross talk at the transcription level. Recently, several groups (17, 18, 19) have reported that LXR- autoregulates the human LXR- gene promoter through binding to the LXRE. Considering the structural similarity between LXRs and TRs, we examined whether TRs regulate the expression of the mouse LXR- gene in the liver. In the current study, we obtained evidence suggesting that mouse LXR- mRNA expression is positively regulated by TR at the transcriptional level, and that a cross talk pathway between LXR- and TR-ß exists in the autoregulation of the LXR- gene. We also demonstrated that human LXR- mRNA expression and promoter activity are positively regulated by thyroid hormone. Our data suggest that TR-ß regulates LXR- mRNA expression in mammals, and a cross talk between TR-ß and LXR- could be a new therapeutic target against dyslipidemia and atherosclerosis.

	Materials and Methods

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Animals
Four-week-old male C57/BL6 mice were used for the study. All aspects of animal care were approved by the Institutional Animal Care and Use Committee of Gunma University Graduate School of Medicine (Maebashi, Gunma, Japan). Animals were maintained on a 12-h light/12-h dark schedule (lights on at 0600 h), and fed laboratory chow as indicated and given water ad libitum. A 2% cholesterol diet (15) was purchased from Oriental Bioservice (Tokyo, Japan). The LXR ligand, TO901317 (20) (Cayman Chemical, Ann Arbor, MI), was administered to the mice at a concentration of 50 mg/kg·d in dimethyl sulfoxide (DMSO) as vehicle by ip injection for 2 wk (21). The mice were rendered hypothyroid by the inclusion of 0.1% methimazole (MMI) in the drinking water and 1% (wt/wt) propylthiouracil (PTU) in the chow for 21 d (15, 22, 23). To introduce a thyrotoxic status, the mice were injected daily with 10 µg/body of T₃ for an additional 5-d period (15, 24). The number of mice receiving each treatment is indicated in the figure legends 1 and 5. Serum free-T₄ levels were determined using a GammaCoat RIA kit (DiaSorin Inc., Stillwater, MN), and free-T₃ levels were determined using an AMERLEX-MAB kit (Amersham Intl., Buckinghamshire, UK); we confirmed the mouse serum thyroid hormone levels as described in Ref. 24 . The serum thyroid hormone data were reported in our previous report (24).

Plasmids
The mouse LXR- promoter (–3000/+30 bp) plasmid, which contained the region from –3000 to +30 bp of the mouse LXR- gene, was generated by genomic PCR using 5'-GTGTGGTACCTAGCCCTTCTCTGCGAACCTTCCT-3' as a sense primer and 5'-GTGTGGTACCTACCTAATCCCAGCAGCCT-3' as an antisense primer (15). An ASP718 restriction enzyme site was introduced into the primer sequences so that the PCR product could be subcloned into the pGL4-Luc vector (Promega Corp., Madison, WI). The human LXR- promoter (–2625/+384 bp) plasmid, which contained the region from –2625 to +384 bp of the human LXR- gene, was generated by genomic PCR using 5'-GTGTGGTACCAGCAGGAAACTGGGAATG-3' as a sense primer and 5'-GTGTGGTACCTGTCCAGAAGTCTCGGT-3' as an antisense primer (15). An ASP718 restriction enzyme site was also introduced into the primer sequences for subcloning into the pGL4-Luc vector (Promega). We excised the –3000/+30-bp plasmid with Pst1 and EcoR1 restriction enzyme to make the site A deletion construct (–1391 to –1252-bp region was deleted.). The –1240 to +30-bp construct of the mouse LXR- gene was generated using PCR site-directed mutagenesis (24). The pGL3 promoter vector (also referred as SV40-Luc) (Promega) was used to make site A-SV40-Luc construct. The site A double-stranded oligonucleotides (–1300 to –1240-bp region) were ligated into the SV40-Luc at ASP718 and Bgl2 sites. All human TR-ß1, RXR-, and murine LXR- cDNAs were placed into an SV40 expression construct, pSG5 (15). All PCR-generated constructs were verified by sequencing the DNA.

Transfections and luciferase assay
For the luciferase assay, we used CV-1 cells, which were derived from the kidney of an African green monkey. Two micrograms of the reporter plasmid and human RXR- and human TR-ß1 or mouse LXR- in pSG5 (otherwise indicated) were transfected per well of a six-well plate into CV-1 cells using the calcium-phosphate method. Sixteen hours after transfection, cultures were treated with serum-free DMEM for 8 h in the absence or presence of 10^–8 M T₃, 10 µM 22(R)hydroxy(OH)cholesterol (25), or 1 µM TO901317 (20). 22(R)(OH)cholesterol (Sigma-Aldrich, St. Louis, MO) was dissolved in ethanol, and TO901317 (Cayman) was dissolved in DMSO and added to the cells after transfection in medium containing 10% resin charcoal double-stripped fetal bovine serum. All transfections were equalized for the same total amount of expression vector using an empty vector as needed. We performed ß-gal assays to confirm the transfection efficiency of the luciferase assay for each experiment at least once and found no significant difference in transfection efficiency among the plates. Data are presented as fold basal activation expressed as fold induction over vector (pSG5) in the absence of ligand stimulation ± SEM otherwise as indicated. Luciferase activity was expressed as arbitrary light units per microgram of cellular protein. All transfection experiments were repeated at least twice with triplicate determinations.

Western blotting
For analysis of the protein expression of LXR-, 30 µg whole cell extracts from mouse liver were subjected to SDS-PAGE. Western blotting was performed using a rabbit anti-LXR- polyclonal antibody (sc-13068; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-Cyclophilin A (07–313; Upstate Biotechnology, Lake Placid, NY) as a control. The bands were quantitatively measured using Adobe Photoshop 4.0 (Adobe Systems Corp., San Jose, CA) and National Institutes of Health (NIH) Image (Scion Corp., Frederick, MD), and standardized against cyclophilin controls. All Western blotting experiments were repeated at least three times with similar results and a representative result is shown.

RNA preparation, Northern blot analysis
Total RNA was extracted from mouse liver and HepG2 cells using ISOGEN (Nippon Gene, Tokyo, Japan), and 20 µg total RNA of HepG2 cells was subjected to Northern blot analysis, as indicated in the Fig. 7 legend. The probe for cyclophilin was purchased from Ambion Inc. (pTRI-cyclophilin-mouse antisense control template; Ambion Inc., Austin, TX). The probe for LXR- was prepared by transcribing mouse LXR- cDNA subcloned into pGEM-T easy (Promega) using T7 RNA polymerase. Northern blot analysis was performed using [-³²P] UTP-labeled antisense riboprobes. The hybridization bands were quantitatively measured using Adobe Photoshop 4.0 (Adobe Systems) and NIH Image (Scion), and standardized against cyclophilin controls. All Northern blotting experiments were repeated at least three times with similar results and a representative result is shown.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Thyroid hormone induced LXR- mRNA in HepG2 cells and human LXR- gene promoter activity. A, HepG2 cells were cultured in 10-cm diameter dishes, and T3 was added to the medium as indicated. Total RNA was isolated, and 20 µg total RNA was subjected to Northern blot analysis using mouse LXR- riboprobes. Numbers in the box represent relative OD levels controlled for cyclophilin mRNA levels. B, RT quantitative PCR (real-time PCR) was performed using HepG2 cellular cDNA. Results (mean ± SE; n = 3) are normalized by GAPDH mRNA levels and showed as fold-expression correlated to the level in the absence of T3, which was set to one. An asterisk indicates that the difference between the denoted pairs is significant at a confidence level of *, P < 0.01, by t testing. C, The human LXR- promoter (–2625/+384 bp) coupled to the luciferase reporter construct (pGL4) was cotransfected into CV-1 cells in the presence of an expression vector for TR-ß1 and RXR- (0.5 µg/well in a six-well format). Relative luciferase activity (mean ± SE; n = 3) represents the luciferase activity of the reporter construct in the absence of T3 (10–8 M). An asterisk indicates that the difference between the denoted pairs is significant at a confidence level of *, P < 0.01, by t testing.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Thyroid hormone induced LXR-, but not LXR-ß, mRNA and protein expression in the mouse liver. LXR agonist did not induce LXR- mRNA in the mouse liver. A–C, C57/B6 mice (4-wk-old male) were first rendered hypothyroid (H) with MMI and PTU diet, and then were treated with T3 (T). They were also fed a 2% cholesterol diet (C) as indicated. They were also treated with TO901317 at a concentration of 50 mg/kg·d in DMSO as vehicle by ip injection for 2 wk (B). Each treatment involved six mice. Liver total RNA was isolated, and 1 µg total RNA was subjected to RT. RT quantitative PCR (real-time PCR) analysis was performed using mouse liver cDNA. Relative values (mean ± SE; n = 6) normalized by GAPDH mRNA levels compared with "Basal (B)" are shown as relative expression (fold) (A and C). Relative values (mean ± SE; n = 6) normalized by GAPDH mRNA levels compared with DMSO are shown as relative expression (fold) (B). An asterisk indicates that the difference compared with "Basal (B)" is significant at a confidence level of *, P < 0.01; **, P < 0.001, by t testing (A). D, Western blot analysis using mouse liver whole cell extract. Representative Western blots for LXR- are shown. Relative OD (mean ± SE; n = 6) was controlled for cyclophilin levels using NIH image software. OD levels in mice fed normal chow (Basal) were assigned a value of 100% for each group. An asterisk indicates that the difference compared with "Basal" is significant at a confidence level of *, P < 0.01, by t testing. B, Basal status (control).

View larger version (18K): [in this window] [in a new window]   FIG. 4. Neither RXR- nor TR-ß bound to site A (the –1300 to –1240-bp region). A, Mouse LXR- promoter sequences around the –1300 to –240-bp region. We divided the sequences into two parts (LXR1 and LXR2). The LXR1 and LXR2 sequences are boxed. Italics represent mutations in LXR1 and LXR2. B, Four microliters of in vitro translated human TR-ß1, and/or human RXR- protein or unprogrammed rabbit reticulocyte lysates were incubated with 32P-radiolabeled DNA probes (DR-4, LXR1). cold oligo, Nonradiolabeled double-stranded oligonucleotides; mouse IgG, mouse normal IgG; mt, mutant; n.s., nonspecific bands; Probe, free radiolabeled probes; RXR-, anti-RXR- antibody; TR-ß1, anti-TR-ß1 antibody; Unprogrammed Reticulocyte lysates, reticulocyte lysates were in vitro translated without template plasmids as mock controls; wt, wild type.

Statistical analyses
Statistical analysis was performed using ANOVA. Values are expressed as the mean ± SEM. The significance of differences between the mean values was evaluated using the unpaired Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
To examine whether thyroid hormone regulates mouse LXR- gene expression, we performed real-time RT-PCR using mouse liver steady-state total RNA. For this purpose we first rendered the mice in a hypothyroid state with an MMI/PTU diet, then injected them with T₃ ip to make them thyrotoxic. We also fed them a 2% cholesterol diet (Fig. 1A). As shown in Fig. 1A, thyroid hormone induced and hypothyroid treatment reduced the mouse LXR- gene expression by about 3.3 and 0.26-fold, respectively, compared with the basal level. On the other hand, cholesterol administration did not increase the mouse LXR- mRNA levels in the liver. Next, we administered TO901317, which is a synthetic ligand for LXRs, to C57/BL6 mice ip. We performed real-time RT-PCR using the mouse liver RNA to examine LXR- gene expression. As shown in Fig. 1B, the ligand did not increase the mouse LXR- mRNA level. LXR-ß mRNA levels showed no difference among the treatments, indicating that thyroid hormone did not affect LXR-ß gene expression (Fig. 1C). Western blot analysis using mouse liver whole cell extract demonstrated that thyrotoxic treatment increased and hypothyroid treatment reduced the LXR- protein level by about 3-fold and 60% reduction, respectively (Fig. 1D).

We also subcloned the mouse LXR- gene promoter (–3000 to +30 bp) and ligated it into pGL4 Luciferase reporter plasmid. We used CV-1 cells for luciferase assays. As shown in Fig. 2, neither TO901317 nor 22(R) hydroxycholesterol, which is also a ligand for LXRs, induced the promoter activity. However, T₃ significantly induced the mouse LXR- promoter activity (3-fold), indicating that thyroid hormone positively regulates the mouse LXR- gene expression at the transcriptional level.

View larger version (13K): [in this window] [in a new window]   FIG. 2. T3 but not LXR agonists induced the mouse LXR- promoter activity. The mouse LXR- promoter (–3000/+30 bp) coupled to the luciferase reporter construct (pGL4) were cotransfected into CV-1 cells in the presence of an expression vector (pSG5) for RXR- and TR- ß1 or LXR- (0.5 µg/well in a six-well format). Relative luciferase activity (mean ± SE; n = 3) represents the luciferase activity of the reporter construct (–3000/+30 pGL4) in the absence of ligands. Vehicle for T3(10–8 M),22(R)OH cholesterol and TO901317 was distilled water buffered with 100 mM HEPES (pH 7.5), 100% ethanol, and DMSO, respectively. An asterisk indicates that the difference between the denoted pairs is significant at a confidence level of **, P < 0.01 by t testing.

View larger version (9K): [in this window] [in a new window]   FIG. 3. The –3000/+30 reporter or the deletion mutant reporters were cotransfected into CV-1 cells in the presence of an expression vector for TR-ß1 together with RXR- in pSG5 (0.5 µg/well in a six-well format). A schematic representation of the deletion mutants of the LXR- promoter is shown in the left panel. Relative luciferase (LUC) activity (mean ± SE; n = 3) represents the luciferase activity of each reporter construct in the absence of T3 (10–8 M). An asterisk indicates that the difference between the denoted pairs is significant at a confidence level of *, P < 0.05 or **, P < 0.01, by t testing.

View larger version (21K): [in this window] [in a new window]   FIG. 5. TR-ß1 and RXR- are recruited to site A in a T3-dependent manner in vivo. In vivo ChIP assays were performed using mouse liver tissue for site A. To verify legitimacy of the experiment, we also performed the assays for DR-4 sites in mouse SREBP-1c promoter. The location of the PCR primers is indicated in cartoons above the data. C57/B6 mice (4-wk-old males) were rendered thyrotoxic with T3 (T) or hypothyroid with MMI and PTU diet (H). Some of them were fed normal chow only (B). Each treatment involved six mice. Liver tissue homogenate from each treatment group was immunoprecipitated with a TR-ß1 antibody (TR-ß) or an RXR- antibody (RXR-), or mouse normal IgG as a NC. The input was a nonimmunoprecipitated sample used as a positive control. Relative OD (mean ± SE; n = 3) was controlled for input levels by NIH Image software. No detectable bands are observed in assays with mouse normal IgG. OD of input in the three treatments was almost identical with no significant differences. The asterisk indicates that the difference between the denoted pairs is significant at a confidence level of *, P < 0.01, by t testing. N.D., Not detectable.

View larger version (18K): [in this window] [in a new window]   FIG. 8. A, Deductive mechanism by which thyroid hormone and TR-ß1 regulate mouse LXR- gene promoter. Based on data in EMSAs and in vivo ChIP assays, TR-ß1 and RXR- are recruited to site A upon T3 administration. We speculate about an intermediating protein (indicated as "X" in cartoon), which would bind to site A, and interact with both TR-ß1 and RXR-. B, Contribution of T3 to the hepatic lipid metabolism. Schematic diagram illustrating the relationship between T3 and key factors in hepatic lipid metabolism. T3 induces LXR- gene expression, and LXR- induces CYP7A1 mRNA levels, which promote cholesterol excretion to the bile acids. In human, LXR- autoregulate LXR- gene expression (17 18 19 ), however, similar regulation is not observed in mice. T3 regulates CYP7A1 gene expression, and there are "species differences" in the way of the gene regulation (34 ). LXR- positively regulates SREBP-1c mRNA levels (35 36 37 ), whereas thyroid hormone negatively regulates SREBP-1c gene expression (24 ). Therefore, T3 fine-tunes SREBP-1c gene expression directly or via modulating of LXR- mRNA levels.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Site A is functionally responsible for T3-induction of mouse LXR- promoter. A, A mouse LXR- gene promoter (–3000/+30 bp) mutant in which site A was deleted coupled to the luciferase (LUC) reporter construct (pGL4-Luc) or the –1240/+30 promoter construct in pGL4-Luc (A) was cotransfected into CV-1 cells with an expression vector for wild-type human TR-ß1, as indicated in a six-well (w) format together with RXR- in pSG5 (0.5 µg/well in six-well format). B, Double-stranded oligonucleotides for site A were ligated into pGL3 promoter plasmid (also referred as SV40-Luc). Site A-SV40-Luc or SV40-Luc plasmid was cotransfected into CV-1 cells as mentioned previously. Relative luciferase activity (mean ± SE; n = 3) represents the luciferase activity of the wild-type (–3000 to +30 bp) reporter construct (A) or SV40-Luc plasmid in the absence of T3 (10–8 M). Asterisks indicate that the difference between the denoted pairs is significant at a confidence level of P < 0.05 or P < 0.01 by t testing.

We treated HepG2 cells, which were derived from human hepatocytes, with thyroid hormone (T₃) and then subjected them to Northern blot analysis using mouse antisense LXR- riboprobe. As shown in Fig. 7A, LXR- mRNA expression was increased in a T₃ dose-dependent manner. This indicated that T₃ also induces human LXR- gene expression. We also performed real-time RT-PCR for human LXR- mRNA expression using total RNA of HepG2 cells and confirmed these data (Fig. 7B). We subcloned the human LXR- gene promoter (–2625/+384 bp) by genomic PCR and prepared a –2625/+384-bp pGL4 construct (Fig. 7C). As shown in Fig. 7C, as expected based on the RNA data (Fig. 7, A and B), the human LXR- promoter was also activated by T₃ administration in CV-1 cells. These data suggest that T₃ could positively regulate LXR- mRNA expression at the transcriptional level in mammals.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the current study, we demonstrated that mouse LXR- mRNA and protein expression in the liver were up-regulated by thyroid hormone, and also showed that the gene promoter was activated by T₃. Furthermore, we demonstrated that LXR agonists such as 22(R) (OH)cholesterol and TO901317 did not increase mouse LXR- mRNA levels in the liver or mouse LXR- promoter activities in CV-1 cells, in agreement with data previously reported by another group (13). This was in accord with the fact that no cognitive LXREs or DR-4 sites have been identified in the mouse LXR- gene promoter (14). Nonetheless, our data showed that LXR- mRNA expression in the mouse liver was clearly up-regulated by T₃ (Fig. 1). This prompted us to explore if the mouse LXR- promoter would be regulated by TRs and thyroid hormone. We found neither half-sites of TREs nor those of LXREs in the mouse LXR- promoter sequence. A serial deletion study of the promoter indicated that a putative TRE might exist between –1300 and –1240 bp. Although EMSAs revealed that either TR-ß or RXR- bound to the site specifically, in vivo ChIP assays clearly demonstrated that TR-ß and RXR- were recruited to the site. These data proposed an existence of unknown proteins that bind to the site directly and assist the nuclear receptor recruitment (Fig. 8A), and should be explored in further study.

It is of interest to note that mouse LXR-ß gene expression is not induced by thyroid hormone. Similarly, human LXR-ß is not induced by LXR ligands (19). Although there are several similarities between LXR- and -ß, there are also several differences. Both isoforms share fairly well-conserved ligand binding domains (78% amino acid homology), and both respond to endogenous oxysterol ligands (4, 5, 12). However, their expression patterns are different. Furthermore, LXR- plays a pivotal role in hepatic cholesterol metabolism, whereas LXR-ß does not have a comparable role (28). Here, we highlight another difference between the two isoforms: LXR- is inducible by thyroid hormone, whereas LXR-ß is not.

We examined if the T₃ induction of the mouse LXR- promoter activity was species dependent. For this purpose we used the human LXR- promoter and demonstrated that the human LXR- promoter was also inducible by thyroid hormone in CV-1 cells. In very recent studies, it was shown that LXR- autoregulates the human LXR- promoter (17, 18, 19). The human LXR- promoter contains three cognitive LXREs in its upstream region; therefore, the autoregulation by LXR- is probably exerted through the LXREs. Because TR-ß1 and LXR- share the same binding element, DR-4, we speculate that TR-ß also regulates the human LXR- promoter via the LXREs. This will have to be examined in detail in future studies.

We have recently analyzed TR-ß knock-in mice having a natural mutation in TR-ß that prevents ligand binding, causing thyroid hormone resistance (29), and reported that LXR- mRNA levels were increased in the knock-in mice to the same or somehow higher degree as in the wild-type mice in response to T₃ (15)_. We speculate that TR- could compensate for TR-ß function to increase LXR- mRNA levels in the knock-in mice.

In conclusion, we have discovered a novel regulation of LXR- by thyroid hormone, which occurs in the mouse liver. Because the human LXR- promoter is also induced by T₃ in CV-1 cells, there is a possibility that LXR- mRNA expression could be up-regulated by thyroid hormone, especially in the liver in mammals. As shown in Fig. 8B, thyroid hormone induces LXR- mRNA expression, leading to the induction of 7-hydroxylase (CYP7A1), which is the rate-limiting enzyme in the elimination of cholesterol through the synthesis of bile acids. This pathway could be related to the reduction of serum total cholesterol levels by thyroid hormone. However, there could be species differences in the way in which T₃ regulates CYP7A1 gene expression (30, 31, 32, 33, 34). LXR- is known to induce SREBP-1c gene expression (35, 36, 37), whereas we (24) reported that thyroid hormone negatively regulates SREBP-1c gene expression. Therefore, we speculate that T₃ via TR could fine-tune SREBP-1c mRNA levels, as shown in Fig. 8B. Although it has been said that TR does not appear to affect the lipid metabolic cascade (38), several recent reports indicated that TR and LXR cross talk mutually in lipid homeostasis (15, 16, 21, 24). Thus, our finding that T₃ could regulate LXR- mRNA expression suggests that a cross talk between TR-ß and LXR- could be a new therapeutic target against dyslipidemia and atherosclerosis.

	Footnotes

 
This work was supported by a grant from the Japan Intractable Disease Research Foundation and Yamaguchi Endocrine Disease Research Foundation (to K.H.).

Disclosure Statement: The authors have nothing to declare.

First Published Online July 12, 2007

Abbreviations: ChIP, Chromatin immunoprecipitation; CYP7A1, cholesterol 7-hydroxylase; DMSO, dimethyl sulfoxide; DR, direct repeat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LXR, liver X receptor; LXRE, liver X receptor responsive element; MMI, methimazole; NC, negative control; PTU, propylthiouracil; RXR, retinoid X receptor; SREBP-1c, sterol response element-binding protein 1c; TR, thyroid hormone receptor; TRE, thyroid hormone response element.

Received February 1, 2007.

Accepted for publication July 2, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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