Cross-Talk between Peroxisome Proliferator-Activated Receptor (PPAR)
and Liver X Receptor (LXR) in Nutritional Regulation of Fatty Acid Metabolism. II. LXRs Suppress Lipid Degradation Gene Promoters through Inhibition of PPAR Signaling
Tomohiro Ide,
Hitoshi Shimano,
Tomohiro Yoshikawa,
Naoya Yahagi,
Michiyo Amemiya-Kudo,
Takashi Matsuzaka,
Masanori Nakakuki,
Shigeru Yatoh,
Yoko Iizuka,
Sachiko Tomita,
Ken Ohashi,
Akimitsu Takahashi,
Hirohito Sone,
Takanari Gotoda,
Jun-ichi Osuga,
Shun Ishibashi and
Nobuhiro Yamada
Department of Internal Medicine (T.I., H.S., T.M., M.N., S.Y., A.T., H.S., N.S.), Institute of Clinical Medicine, University of Tsukuba, Ibaraki 305-8575, Japan; and Department of Metabolic Diseases (T.Y., N.Y., M.A.-K., Y.I., S.T., K.O., T.G., J.O., S.I.), Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan
Address all correspondence and requests for reprints to: Hitoshi Shimano, M.D., Ph.D., Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. E-mail: shimano-tky{at}umin.ac.jp.
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ABSTRACT
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Fatty acid metabolism is transcriptionally regulated by two reciprocal systems: peroxisome proliferator-activated receptor (PPAR)
controls fatty acid degradation, whereas sterol regulatory element-binding protein-1c activated by liver X receptor (LXR) regulates fatty acid synthesis. To explore potential interactions between LXR and PPAR, the effect of LXR activation on PPAR
signaling was investigated. In luciferase reporter gene assays, overexpression of LXR
or ß suppressed PPAR
-induced peroxisome proliferator response element-luciferase activity in a dose-dependent manner. LXR agonists, T0901317 and 22(R)-hydroxycholesterol, dose dependently enhanced the suppressive effects of LXRs. Gel shift assays demonstrated that LXR reduced binding of PPAR
/retinoid X receptor (RXR)
to peroxisome proliferator response element. Addition of increasing amounts of RXR
restored these inhibitory effects in both luciferase and gel shift assays, suggesting the presence of RXR
competition. In vitro protein binding assays demonstrated that activation of LXR by an LXR agonist promoted formation of LXR/RXR
and, more importantly, LXR/PPAR
heterodimers, leading to a reduction of PPAR
/RXR
formation. Supportively, in vivo administration of the LXR ligand to mice and rat primary hepatocytes substantially decreased hepatic mRNA levels of PPAR
-targeted genes in both basal and PPAR
agonist-induced conditions. The amount of nuclear PPAR
/RXR heterodimers in the mouse livers was induced by treatment with PPAR
ligand, and was suppressed by superimposed LXR ligand. Taken together with data from the accompanying paper (Yoshikawa, T., T. Ide, H. Shimano, N. Yahagi, M. Amemiya-Kudo, T. Matsuzaka, S. Yatoh, T. Kitamine, H. Okazaki, Y. Tamura, M. Sekiya, A. Takahashi, A. H. Hasty, R. Sato, H. Sone, J. Osuga, S. Ishibashi, and N. Yamada, Endocrinology 144:12401254) describing PPAR
suppression of the LXR-sterol regulatory element-binding protein-1c pathway, we propose the presence of an intricate network of nutritional transcription factors with mutual interactions, resulting in efficient reciprocal regulation of lipid degradation and lipogenesis.
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INTRODUCTION
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RECENT PROGRESS IN studying transcriptional regulation of lipid catabolism has unveiled the functions of orphan nuclear receptors such as peroxisome proliferator-activated receptors (PPARs) and liver X receptors (LXRs), whereas roles by sterol regulatory element-binding protein (SREBP) family have been established as lipid synthetic regulators. PPARs are members of a nuclear receptor superfamily and are structurally related to other members such as thyroid hormone receptor (TR) and vitamin D3 receptor (VDR) (1). PPARs are known to regulate expression of numerous genes involved in fatty acid metabolism and adipocyte differentiation (2, 3). PPAR
is abundantly expressed in tissues, which have high lipid catabolic activity, such as liver, kidney, heart, skeletal muscle, and brown adipose tissue (4, 5). PPAR
is activated by fatty acids, eicosanoids, and fibrates, a known class of hypolipidemic drugs. Like other nuclear receptors such as: TR, VDR, and retinoic acid receptor (RAR) (6, 7, 8, 9), PPAR
forms a heterodimer with RXR
, which enhances its binding to DNA sequence elements termed peroxisome proliferator response elements (PPRE) (10, 11, 12). PPREs have been recently identified in the 5'-flanking sequences of genes involved in lipid degradation such as the ACO [acyl-coenzyme A (CoA) oxidase] (13), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMG-CoA Syn) (14), L-carnitine palmitoyltransferase (CPTI) (15), and L-fatty acid binding protein genes (16). Studies using PPAR
-deficient mice have established that PPAR
plays a crucial role in fatty acid degradation (17, 18, 19).
LXRs (LXR
/NR1H3 and LXRß/NR1H2) were identified as orphan nuclear receptors and are now thought to regulate the metabolism of several important lipids, including cholesterol and bile acid (20, 21). LXRs regulate intracellular cholesterol levels by induction of the gene expression of cholesterol 7
-hydroxylase (22, 23), which is the rate-limiting enzyme of the classic bile acid synthesis pathway, and ATP-binding cassette transporter A1 (ABCA1) (24), which modulates apolipoprotein mediated-efflux of cholesterol. Further evidence supporting an important role of LXR
in lipid homeostasis is provided by the loss of capacity to regulate catabolism of dietary cholesterol in LXR
deficient mice, an effect for which the isoform, LXRß, could not compensate (25). Differences in the physiological functions between LXR
and LXRß including target and tissue specificity have been suggested (26). LXRß is ubiquitously expressed, whereas LXR
is restricted to metabolically active tissues, such as liver, kidney, intestines, and adrenal glands (27, 28). Recently, we (29) and others (30) reported that both LXR
and ß are dominant activators for SREBP-1c/adipocyte determination and differentiation 1 (ADD1). Previous in vivo studies established that SREBP-1c plays a crucial role in the dietary regulation of most hepatic genes of fatty acid synthetic enzymes (31, 32, 33, 34). Therefore, LXRs could be also important in fatty acid synthesis (35).
As PPAR
and LXR-SREBP-1c are reciprocal regulators for fatty acid metabolism, nuclear receptors could interact with each other as was previously observed in a cross-talk between PPAR
and TR (36, 37). The current study examined the effects of LXR activation on PPAR
signaling. The results demonstrate that LXR
ligand activation represses PPAR
signaling through reduction of stimulated-PPAR
/RXR heterodimerization in the liver. Taken together with the accompanying paper (38) describing PPAR suppression of the LXR-SREBP-1c pathway, we propose a novel aspect of nutritional regulation with these mutual interactions forming a network of transcription factors regulating fatty acid metabolism.
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RESULTS
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LXR Activation Represses PPAR
-Mediated Transactivation
To estimate the effect of LXR/RXR
activation on PPAR
signaling, transfection studies with human embryonic kidney (HEK) 293 cells were performed using luciferase (Luc) reporter gene assays containing a PPRE from the ACO gene promoter [pPPRE-Luc (PPRE-Luc plasmid)], a representative PPAR
target. As shown in Fig. 1
, the Luc activity of PPRE-Luc was markedly (60-fold) induced by cotransfection of PPAR
due to lack of endogenous PPAR expression in HEK293 cells (data not shown). The PPAR
-inducible expression of Luc activity was slightly, but dose dependently suppressed by coexpression of LXR
(Fig. 1A
) (50% inhibition at a DNA dose ratio of 1:10). Expression level of transfected PPAR
or LXR gene in HEK293 cells were roughly comparable to that in mouse liver as estimated by Northern blotting, and thus was within a physiological range (data not shown). This observation is consistent with a previous report describing LXR interaction with PPAR
signaling (39). More interestingly, addition of LXR ligands such as 22RHC and T0901317 markedly enhanced the inhibitory effects of LXR
. LXRß alone, without an LXR ligand, substantially inhibit PPAR
activation of PPRE-Luc in a dose-dependent manner (Fig. 1B
). A 50% inhibition was observed at a DNA ratio of 1:5. Additional LXR ligands further augmented the inhibitory effect of LXRß. These effects by LXRs and LXR ligands are not due to direct inhibition of Luc activity because Luc activity from constitutive expression of Luc gene by TK promoter-Luc construct was not affected by LXR coexpression, by addition of LXR ligand, or a combination of both (Fig. 1C
). These data demonstrate that LXRs and their ligands inhibit PPAR signaling with overexpressing nuclear receptors system.
LXR Cannot Bind to PPRE, but Inhibits PPAR
/RXR
Binding to PPRE
The inhibitory effect of LXR was estimated in the light of the dose dependency of LXR ligands in Fig. 2
. As a positive control, a dose-response curve of LXRE (LXR response element)-Luc activation by T0901317 and 22RHC with coexpression of LXR
is shown in the upper panel, which is consistent with previous data (29). With a similar efficiency, these LXR agonists inhibited PPAR
activation of PPRE-Luc, making both curves a mirror image of each other. LXRß coexpression gave similar results with a slightly higher efficiency.
To further investigate the mechanism for inhibition of PPAR
by LXR, gel mobility shift assays were performed. The binding specificity of LXR/RXR
to LXRE, and PPAR
/RXR
to PPRE was confirmed in the experiments shown in Fig. 3
, A and B. RXR
is required for specific binding of both LXR and PPAR. Neither LXR
/RXR
nor LXRß/RXR
bound to PPRE within the sensitivity of this assay. Coincubation of PPAR
and LXR
or ß essentially caused no shift using PPRE probe. These data exclude the possibility that LXR/RXR
competes with PPAR
/RXR
binding to a response element. Next, PPAR
/RXR
binding to PPRE was estimated in the presence of LXR and/or its ligand (Fig. 4
). PPAR
/RXR
binding to PPRE was partially inhibited by addition of LXR protein. LXR ligand enhanced this inhibition. These data are consistent with results from reporter assays, suggesting that suppression of PPAR
activity by LXR is mediated at least partly through inhibition of PPAR
/RXR
binding to PPRE.
RXR Restores LXR Repression of PPAR
/RXR
-Mediated Transactivation
Because PPAR
and LXRs share RXR
as a heterodimer partner for their specific functions, it is conceivable that LXR inhibition of PPAR
activation on PPRE-Luc is mediated through competition for RXR
when RXR
is in limiting amounts in cultured cells. Thus, the effect of supplementation with RXR
on the LXR inhibition of PPRE-Luc activity was evaluated (Fig. 5
). By cotransfection of an increasing amount of RXR
, PPAR
-activated PPRE-Luc activity was enhanced, indicating that the amount of RXR
is not saturated for PPAR
/RXR
activation (Fig. 5A
). Figure 5B
shows effect of each amount of RXR
on the percent inhibition of PPAR
-induced PPRE-Luc values by LXR
activation (LXR
coexpression with or without an LXR ligand, T0901317 or 22RHC). The inhibitory efficiency of LXR activation was attenuated by coexpressed RXR
in a dose-dependent manner (Fig. 5B
). Similar results were obtained in the repression by LXRß (Fig. 5C
).
Next, we compared the inhibitory action of LXR on PPAR signaling with other RXR heterodimer partners, TR and farnesoid X receptor (FXR) (Fig. 6A
). TRß strongly suppressed PPRE-Luc activity, which is consistent with a previous report (37). Inhibition by FXR was modest. The ability of inhibitory action on PPAR
-mediated transactivation appears to be different among LXR, TRß, and FXR, suggesting their constitutive activity based on levels of intracellular natural ligands may influence cross-talk in heterodimeric receptors with RXR
.

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Figure 6. Effects of RXR Heterodimers (A) and CBP/p300 (B) on PPRE-Luc Activity
A, pPPRE-Luc and pSV-renilla Luc were cotransfected into HEK293 cells with indicated expression plasmids: pCMV-mPPAR (0.25 µg), pCMV-LXRß (0.25 µg), pCMV-TRß (0.25 µg), and pCMV-FXR (0.25 µg). After incubation for 24 h, Luc activity was measured and normalized to renilla Luc activity. B, pPPRE-Luc and pSV-renilla Luc were cotransfected into HEK293 cells with indicated expression plasmids: pCMV-PPAR (0.25 µg), pCMV-LXR (0.25 µg), pCMV-CBP (0.5 µg), pCMV-p300 (0.5 µg), and pCMV-RXR (0.5 µg). Cells were treated with or without 3 µM Wy14,643 and/or 3 µM T0901317. After incubation for 24 h, Luc activity was measured and normalized to renilla Luc activity. All results are means ± SE from three independent experiments.
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cAMP response element binding protein-binding protein (CBP)/p300 is known to be involved in ligand-dependent activation of PPARs (40, 41, 42). However, unlike RXR, overexpression of CBP/p300 did not restore on LXR suppression of PPAR
-induced PPRE-Luc activity (Fig. 6B
).
The RXR restoration of LXR-dependent suppression was also evaluated in gel mobility shift assays. Without LXR, the signal of PPRE shifted by PPAR
was enhanced with increasing amounts of RXR
, suggesting that the amount of RXR
is a limiting factor in the range of PPAR
/RXR
ratio used here (Fig. 7A
). With the lowest amount of RXR
, addition of LXRs and T0901317 caused a significant decrease in PPAR
/RXR
binding to PPRE (Fig. 7
, A and B). The inhibitory effect of LXR
activation was abolished with increasing concentrations of RXR
. In the case of LXRß, RXR restoration of the binding was partial even at the highest amount, reflecting stronger LXRß inhibitory effect. These data are consistent with the results from cell reporter assays (Fig. 5
), providing additional evidence for the hypothesis that LXR inhibits PPAR
activation by competing with it for heterodimerization with RXR
.
LXR Can Bind to PPAR
and Inhibits Formation of PPAR
/RXR
Heterodimer
To explore the heterodimerization of the three nuclear receptors, mutual protein-protein interactions were investigated (Fig. 8
). First, LXR
protein was labeled with 35S-methionine, incubated with unlabeled RXR
protein, and precipitated with anti-RXR
antibody. Resolution of immunocomplex on SDS-PAGE detected labeled LXR
, indicating formation of an LXR
/RXR
heterodimer. The complex formation was markedly enhanced by addition of T0901317, suggesting that LXR ligand increases affinity for LXR
/RXR
heterodimerization (Fig. 8A
). We also tested the possibility of formation of PPAR
/LXR
heterodimers, as was previously described (39). Labeled LXR
was incubated with PPAR
and immunoprecipitated with anti-PPAR
antibody. PPAR
/LXR
complex was detected with an intensity comparable to LXR
/RXR
, suggesting that LXR
can heterodimerize with PPAR
with similar efficiency to RXR
(Fig. 8C
). T0901317 enhanced this interaction. LXRß gave very similar results (Fig. 8
, B and D). The gel shift assay (Fig. 3B
) showed no retardation of the PPRE probe in the presence of both PPAR
and LXR
, indicating that PPAR
/LXR
heterodimers do not bind to PPRE. Therefore, LXR
could show inhibition of PPAR
signaling by forming not only LXR
/RXR
, but also PPAR
/LXR
in a competition with functional PPAR
/RXR formation. To show this, effects of LXRs on PPAR
/RXR
interaction were estimated in this protein interaction assay. PPAR
protein was labeled, incubated with RXR
in the presence or absence of LXR
, precipitated with anti-RXR
antibody, and run on gels. PPAR
/RXR
heterodimer formation was inhibited by addition of LXR
or LXRß with T0901317 (Fig. 8
, E and F), suggesting that PPAR
/LXR as well as LXR/RXR
could prevent formation of PPAR
/RXR
and participate in repressing PPAR
signaling.
LXR Activation Inhibits PPAR
/RXR
Binding to PPRE in Hepatic Nuclear Extracts
To examine suppressive action of LXR on PPAR/RXR binding to PPRE in a more physiological condition, gel mobility shift assays were performed using hepatic nuclear extracts from fasted mice. In nuclear extracts from fasted mice, LXR
and RXR
were abundant on immunoblot analysis, although PPAR
was decreased after refeeding (Fig. 9A
) as well as at mRNA level. Gel shift mobility assays demonstrated that the PPRE probe was shifted by coincubation with hepatic nuclear extracts. The signal of the complexes was stronger from fasted mice than in a refed state, was enhanced by addition of Wy14,643 and declined after addition of anti-PPAR
or anti-RXR
antibody, confirming the presence of PPAR
/RXR
in the hepatic nuclear extracts from fasted mice and its specific binding to PPRE (Fig. 9B
). The PPRE complex also contained a small amount of unidentified RXR heterodimer(s). T0901713 suppressed the signal of the PPAR
/RXR
-PPRE complex induced by Wy14,643 (Fig. 9C
). Thus, these data indicate that LXR ligand suppressed PPAR
ligand-induced PPAR
/RXR
-PPRE complexes in a condition with physiological levels and spectrum of various nuclear receptors.

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Figure 9. PPAR Activator Inhibits LXR Ligand-Induced LXR/RXR Binding to PPRE in Hepatic Nuclear Extracts
A, Protein and mRNA levels of PPAR , LXR , and RXR in hepatic nuclear extracts from fasted or refed mice. For fasting and refeeding treatment, mice were fasted for 24 h and fed a high sucrose/fat-free diet for 12 h. Equal protein amounts (20 µg) in nuclear extracts were subjected to SDS-PAGE, and immunoblotted with an anti-PPAR , anti-LXR , or anti-RXR antibody. Total RNA was isolated from the livers of mice from each group, pooled, and subjected to Northern blot analysis with the indicated cDNA probes. B, Confirmation of presence of PPAR in Wy14,643 induced-complexes of nuclear proteins/ACO-PPRE in nuclear extracts from fasted mice. The 32P-labeled ACO-PPRE probe and hepatic nuclear extracts (1 mg) from fasted mice were incubated with Wy14,643 in the presence or absence of anti-PPAR (H-98, sc-9000, Santa Cruz Biotechnology Inc.) or anti-RXRa (D-20, sc-553, Santa Cruz Biotechnology Inc.) antibody. The DNA-protein complexes were resolved in a 4.5% PAGE. C, Wy14,643 induced-PPAR /RXR binding to PPRE was inhibited by addition of T0901317. 32P-labeled ACO-PPRE probe and hepatic nuclear extracts from fasted mice were incubated with Wy14,643 and/or T0901317. The DNA-protein complexes were resolved in a 4.5% PAGE.
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LXR Activation Represses PPAR
Target Gene in Primary Hepatocytes and Mouse Livers
To extend these findings to a system with more physiological relevance, LXR interference with PPAR
signaling was estimated using their ligands in rat primary hepatocytes. Hepatocytes were incubated with increasing amounts of T0901317 and expression of related target genes was estimated by Northern blot analysis (Fig. 10
). LXR activation was confirmed by a dose-dependent increases in SREBP-1c and ABCA1 mRNA levels (Fig. 10A
). LXR ligand dose dependently suppressed a representative PPAR
gene, mHMG-CoA syn mRNA level. In the presence of Wy14,643 at a concentration sufficient to induce mHMG-CoA syn, a clear dose-dependent suppression by T0901317 was still observed (Fig. 10B
). These data demonstrate that LXR activation by its ligand suppresses PPAR
-target genes, in a competitive manner with PPAR
activation, supporting LXR interference with PPAR
signaling in primary hepatocytes. To further evaluate the in vivo physiological significance of LXR interference to PPAR
-mediated transactivation, similar conditions in mouse livers were recreated. Mice were fasted to induce hepatic endogenous PPAR
and its downstream genes (17, 31) and were treated with T0901317 to activate LXR. Various PPAR
-target genes in the livers such as mHMG CoA Syn, ACO, CPTI, and cytochrome P450A2 were estimated by Northern blot analysis. Wy14,643 was also administered to fasted mice as a positive control for activation of PPAR
target genes. As shown in Fig. 11A
, hepatic mRNA levels of these PPAR
-regulated genes from T0901317 treated animals were considerably decreased, whereas Wy14643 treatment increased expression of these genes. The activation of LXR by T0901317 was confirmed by observational increases in SREBP-1 and ABCA1 mRNA levels, both of which are well-known LXR target genes (Fig. 11B
). These data demonstrate that LXR activation by LXR ligand can affect PPAR
signaling in mouse livers. In contrast, Wy14,643 alone slightly increased ABCA1 mRNA (Fig. 11B
), which is consistent with recently reports of PPAR-LXR-ABCA1 pathway in macrophages (43, 44, 45). Moreover, we studied LXR interference in mice treated with both PPAR
and LXR ligands. As estimated by Northern blot analysis of mHMG-CoA syn and ACO mRNA levels, these PPAR
-target genes were suppressed by T0901317 in both Wy14,643-induced and untreated conditions (Fig. 11D
). Immunoprecipitation assays of liver nuclear extracts from these mice demonstrated that PPAR
/RXR
heterodimers were increased by Wy14,643 treatment and this induction was suppressed by T0901317. These results suggested that LXR
ligand could repress hepatic expression of the PPAR
target genes via reduction of PPAR
/RXR
formation in liver nucleus (Fig. 11E
), extending the hypothesis from transfection studies to in vivo conditions.

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Figure 10. LXR Ligand Suppresses PPAR -Target Gene Expression in Rat Primary Hepatocytes
A, T0901317 inhibited gene expression of mHMG-CoA Syn in rat primary hepatoctyes. The cells were incubated with the indicated concentration of T0901317 for 24 h. B, T0901317 suppressed induction of mHMG-CoA Syn gene expression by Wy14,643 treatment. The hepatocytes were incubated with the indicated concentration of T0901317 in the absence or presence of Wy14643 for 24 h. Total RNA (10 µg) was extracted and Northern blot analysis was performed with the indicated cDNA probes. Fold changes of expression relative to corresponding with controls are shown.
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Figure 11. Effect of LXR Ligand on PPAR Target Gene Expression in the Livers of Fasted Mouse
AC, Mice (n = 5) were fasted and treated with T0901317 (50 mg/kg) or Wy14,643 (50 mg/kg) for 18 h. D, Mice (n = 3) were fasted and treated with T0901317 (50 mg/kg), Wy14,643 (50 mg/kg), or both these ligands for 18 h. Total RNA was isolated from the livers of mice from each group, pooled, and subjected to northern blot analysis with the indicated cDNA probes. Fold increases of expression relative to corresponding with vehicle-treated controls are shown. E, T0901317 inhibited Wy14,643-induced LXR/RXR heterodimer formation in the liver from C57BL6 mice. Hepatic nuclear extracts were prepared from each group. Equal protein amounts of nuclear extracts were subjected to immunoprecipitation by using anti-PPAR antibody, which had been coupled to protein G-sepharose beads. Immunoprecipitates were subjected to SDS-PAGE, and immunoblotted with an anti-RXR antibody. Arrowheads represent RXR or IgG signal.
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DISCUSSION
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The current studies demonstrate that LXR activation by either overexpression of LXR or its ligand activation causes suppression of PPAR
signaling. The Luc, gel shift, and protein-protein binding assays show that LXR activation inhibits formation of PPAR
/RXR
, and thus, their binding to PPRE. RXR
supplementation restored this LXR inhibition of PPAR action, indicating that the mechanism could be at least partially explained by RXR
competition between PPAR and LXR. Protein-protein interaction experiments established the formation of PPAR
/LXR
and PPAR
/LXRß as well as LXR/RXR heterodimers. Direct binding between PPAR
and LXR
has been found in a two-hybrid system, and involvement of LXR
in PPAR signaling has been implicated (39). We further found that PPAR
/LXR formation is enhanced by LXR ligand. PPAR
/LXR complex cannot bind to either PPRE or LXRE. Therefore, this heterodimer can interfere with both PPAR and LXR action and can contribute to PPAR inhibition of the LXR-SREBP-1c system as proposed in the accompanying paper (38) as well as LXR inhibition of PPAR signaling as described in the current study. Consistently, the combined treatment with both PPAR
and LXR agonists resulted in simultaneous reduction of both PPAR
/RXR
and LXR/RXR
[Fig. 11E
in this paper and Fig. 9B
in the accompanying paper (38)], implicating an important contribution of PPAR
/LXR formation in reciprocal inhibition of LXR and PPAR
signaling. This can also explain the restoration by supplementation with RXR, not only by providing sufficient RXR for functional RXR heterodimers, but also by preventing nonfunctional PPAR
/LXR formation.
LXR inhibition of PPAR
signaling observed from transfection studies with cultured cells is likely to be extendable to the in vivo regulation of the hepatic energy metabolism. It is noteworthy that administration of LXR agonist reduced hepatic nuclear PPAR
/RXR and impaired expression of hepatic fatty acid degradation enzyme genes both in rat primary hepatocytes and livers of fasted mice (Figs. 10
and 11
). In a fasted state, hepatic PPAR
expression is highly induced and fatty acids are recruited as ligands for PPAR
activation (17, 18). Nutritional condition does not markedly change the expression of hepatic LXR
(29). Therefore, LXR ligand can activate hepatic LXR
and cause cross-talk with PPAR
/RXR leading to suppression of expression of PPAR
target genes involved in lipid degradation. These data demonstrate that LXR interference with PPAR signaling can occur in vivo. In the accompanying paper (38), we observed a mirror image observation, that PPAR
activation inhibits ligand-induced LXR signaling in hepatocytes.
The triangle relationship among LXR, PPAR, and RXR could be crucial for mutual regulation of both LXR and PPAR activities, and thus nutritional regulation of their downstream genes. PPAR
is involved in fatty acid degradation as an adaptic control of energy depletion. Meanwhile, SREBP-1c, whose expression is dominated by LXR is involved in fatty acid synthesis for storage of excess energy. These opposite nutritional regulators are reciprocally up- and down-regulated depending upon energy states. As summarized in Fig. 12
, our findings suggest that the mutual suppression efficiently facilitates these reciprocal actions of PPAR
and LXR-SREBP-1c systems. These studies should open up a new paradigm of a novel cross-talk of nutritional transcription factors in energy metabolism where the nuclear concentrations of each receptor and ligand are crucial for nutritional regulation for fatty acid metabolism. It is also important to investigate whether the cross-talk of these receptors involves a potential competition for their coactivators such as CBP/p300 (42, 46, 47), steroid receptor coactivator-1 (48, 49, 50), and PPAR
coactivator-1 (51, 52). Further studies should focus on the precise mechanisms for this network.

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Figure 12. Mutual Interactions between PPAR and LXR-SREBP-1c in Reciprocal Regulation of Fatty Acid Metabolism
Current cross-talk of PPAR , LXR, and SREBP-1c in nutritional regulation is schematized, based upon the current study and the accompanying paper (38 ).
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MATERIALS AND METHODS
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Materials
Anti-PPAR
sc-9000, RXR
D20 sc-553, and LXR
C-19 sc-1201 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), Redivue [
-32P]deoxy-CTP (6,000 Ci/mmol) and Protein G Sepharose from Amersham Biosciences Inc. (Uppsala, Sweden), in vitro transcription and translation kit (TNT Coupled Reticulocyte Lysate System) from Promega (Madison, WI), and other chemicals from Sigma (St. Louis, MO). T0901317 (N-methyl-N-[4-(2, 2, 2- trifluoro-1-hydroxy-1-trifluoromethylethyl)-phenyl]-benzenesulfonamide) was provided by Kyorin Pharmaceutical Co. Ltd. (Tochigi, Japan).
Plasmids
Cytomegalovirus (CMV) promoter expression plasmid of human RXR
and the Luc reporter gene construct, PPRE from ACO gene promoter fused upstream from thymidine kinase (TK) promoter (pPPRE-Luc), and pCMV-RXR were kind gifts from Dr. D. J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). pCMV-FXR, pCMX-TRß, and pCMV-CBP were from Dr. H. Fujii (Graduate School of Agricultural and Life Science, The University of Tokyo, Tokyo, Japan), Dr. R. M. Evans (The Salk Institute for Biological Studies, La Jolla, CA), and Dr. T. Nakajima (St. Marianna University School of Medicine, Kawasaki, Japan), respectively. The LXRE of SREBP-1c promoter-Luc construct (pLXRE-Luc), pCMV-mLXR
and pCMV-mLXRß were prepared as previously described (29). CMV or T7 promoter expression plasmid of mouse (m) PPAR
(1468, amino acid) (16) was prepared by PCR, and the cDNA was introduced into pCMV7 or pBluescript II SK vector, respectively. T7 promoter expression plasmids of mLXR
and mLXRß were prepared using pBluescript II SK vector.
Transfections and Luc Assays
HEK293 were grown at 37 C in an atmosphere of 5% CO2 in DMEM containing 25 mM glucose, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate supplemented with 10% fetal bovine serum (FBS). Transfection studies were carried out with cells plated on 12-well plates as previously described (29). The indicated amount of each expression plasmid was transfected simultaneously with a Luc reporter plasmid (0.25 µg) and simian virus 40-ß-galactosidase (ß-gal) plasmid (0.2 µg) or (0.05 µg). The total amount of DNA in each transfection was adjusted to 1.5 µg/well with the vector DNA, pCMV7. 22RHC and T0901713 were dissolved in ethanol. Each agent was treated to the cells immediately after transfection in DMEM with 10% FBS, and incubated for 24 h. After incubation, the amount of Luc activity in transfectants was measured and normalized to the amount of ß-gal activity.
Gel Mobility Shift Assays
Gel mobility shift assays were performed as previously described (53). Briefly, mLXR
, mLXRß, mPPAR
, and human (h) RXR
proteins were generated from the expression vectors using a coupled in vitro transcription/translation system (Promega). Double-stranded oligonucleotides used in gel mobility shift assays were the LXREb of the SREBP-1c promoter (29) and PPRE of the ACO promoter (16). These were then labeled with [
-32P]deoxy-CTP by klenow enzyme, followed by purification on sephadex G50 columns. The labeled probes (30,000100,000 cpm) were incubated with lysates (1 to 4 µl), or hepatic nuclear extract (1 µg) in a mixture (20 µl) containing 10 mM HEPES (pH 7.6), 50 mM KCl, 0.05 mM EDTA, 2.5 mM MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.4 µg/ml poly(deoxyinosine-deoxycytidine), 0.1% Triton X-100, and 1 mg/ml nonfat milk for 60 min on ice. The DNA-protein complexes were resolved on a 4.5% PAGE at 100 V for 2 h at 4 C. Gels were dried and exposed to the filter of BAS2000 with BAStation software (Fuji Photo Film, Kanagawa, Japan).
Coimmunoprecipitation of Receptors
In vitro translated [35S]methionine-labeled receptors with unlabeled receptors or hepatic nuclear extracts from ligand-treated mice were brought to a final volume of 20 or 200 µl with buffer containing 10 mM HEPES (pH 7.6), 50 mM KCl, 0.05 mM EDTA, 2.5 mM MgCl2, 8.5% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100, and 1 mg/ml nonfat milk for 2 h at 4 C and incubated with 10 µl of rabbit or goat polyclonal antibodies binding to protein G-sepharose for overnight at 4 C. The precipitations were washed with PBS containing 0.2% Tween-20 and 3% BSA. After microcentrifugation, the pellet was washed four times with 1 ml of ice-cold PBS containing 0.2% Tween-20. Twenty microliters of SDS-PAGE sample buffer were added to the final pellet and boiled for 5 min at 95 C. The supernatant was subjected to electrophoresis on 10% SDS-PAGE.
Hepatocyte Isolation and Culture
Primary hepatocytes were isolated from male Sprague- Dawley rats (160180 g, Japan Clea, Tokyo, Japan) using the collagenase perfusion method as described previously (54). The viability of isolated cells was over 90% as determined by the trypan blue. Cells were resuspended in DMEM containing 100 U/ml penicillin and 100 µg/ml streptomycin sulfate supplemented with 5% FBS, seeded on collagen-coated dishes 100 mm at a final density of 4 x 104 cells/cm2. After an attachment for 4 h, cells were cultured with medium containing the indicated agonists for 24 h.
Animals
Male mice (C57BL/6J) were obtained from Charles River Japan (Yokohama, Japan). All mice were given a standard diet and tap water ad libitum. All institutional guidelines for animal care and use were applied in this study. Vehicle (0.5% carboxymethyl-cellulose) T0901317 (50 mg/kg), Wy14,643 (50 mg/kg), or both their agonists was orally administered to the mice before 18 h fasting. For fasting and refeeding treatment, mice were fasted for 24 h and fed a high sucrose/fat free diet for 12 h as described (55). Hepatic nuclear protein were prepared from the livers as previously described (56), were subjected to immunoblotting with the anti-PPAR
, anti-LXR
, or anti RXR
antibodies.
Northern Blot Analysis
Total RNA was extracted from livers and rat primary hepatocytes using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA). Equal aliquots of total RNA from mice in each group were pooled (total 10 µg), subjected to formalin-denatured agarose electrophoresis, and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech, Uppsala, Sweden). Blot hybridization was performed with the cDNA probes labeled with [
-32P]CTP (6000 Ci/mmol) using the Megaprime DNA Labeling System (Amersham Biosciences Inc.). The cDNA probes for SREBP-1, fatty acid synthase, ACO, cytochrome P450A2, PPAR
, LXR
and ß, ABCA1 and acidic ribosomal phosphoprotein PO (36B4) were prepared as previously described (29, 31). The cDNA probes for L-FABP, CPTI, and mHMG-CoA Syn were provided by Kyorin Pharmaceutical Co. LTD. Each signal was analyzed with BAS2000 and BAStation software (Fuji Photo Film).
 |
ACKNOWLEDGMENTS
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We are grateful to A. H. Hasty for critical reading of the manuscript. We also thank T. Kitamine, H. Okazaki, Y. Tamura, and M. Sekiya for helpful discussion.
 |
FOOTNOTES
|
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This study was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research.
T.I. and H.S. equally contributed to this work.
Abbreviations: ABCA1, ATP-binding cassette transporter A1; ACO, acyl-CoA oxidase; 36B4, acidic ribosomal phosphoprotein PO; CBP, cAMP-response element binding protein-binding protein; CMV, cytomegalovirus; CoA, coenzyme A; CPTI, L-carnitine palmitoyltransferase; FBS, fetal bovine serum; FXR, farnesoid X receptor; ß-gal, ß-galactosidase; HEK, human embryonic kidney; Luc, luciferase; LXR, liver X receptor; LXRE, liver X receptor response element; m, mouse; mHMG-CoA syn, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase; PGC-1, peroxisome proliferator-activated receptor
coactivator-1; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator responsive element; pSV, simian virus 40 promoter plasmid; 22RHC, 22(R)-hydroxycholesterol; RXR, retinoid X receptor; SREBP-1c, sterol regulatory element-binding protein 1c; TK, thymidine kinase; TR, thyroid hormone receptor; VDR, vitamin D3 receptor.
Received for publication May 23, 2002.
Accepted for publication April 14, 2003.
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