The Acute Phase Response Is Associated with Retinoid X Receptor Repression in Rodent Liver*

The acute phase response (APR) is associated with decreased hepatic expression of many proteins involved in lipid metabolism. The nuclear hormone receptors peroxisome proliferator-activated receptor α (PPARα) and liver X receptor (LXR) play key roles in regulation of hepatic lipid metabolism. Because heterodimerization with RXR is crucial for their action, we hypothesized that a decrease in RXR may be one mechanism to coordinately down-regulate gene expression during APR. We demonstrate that lipopolysaccharide (LPS) induces a rapid, dose-dependent decrease in RXRα, RXRβ, and RXRγ proteins in hamster liver. Maximum inhibition was observed at 4 h for RXRα (62%) and RXRβ (50%) and at 2 h for RXRγ (61%). These decreases were associated with a marked reduction in RXRα, RXRβ, and RXRγ mRNA levels. Increased RNA degradation is likely responsible for the repression of RXR, because LPS did not decreaseRXRβ and RXRγ transcription and only marginally inhibited (38%) RXRα transcription. RXR repression was associated with decreased LXRα and PPARα mRNA levels and reduced RXR·RXR, RXR·PPAR and RXR·LXR binding activities in nuclear extracts. Furthermore, LPS markedly decreased both basal and Wy-14,643-induced expression of acyl-CoA synthetase, a well characterized PPARα target. The reduction in hepatic RXR levels alone or in association with other nuclear hormone receptors could be a mechanism for coordinately inhibiting the expression of multiple genes during the APR.

genes could be coordinately decreased during the APR is by the reduction of the levels of specific transcription factors. Because heterodimerization with RXR is crucial for the action of several nuclear hormone receptors including PPAR and LXR, we hypothesized that a decrease in RXR levels in the liver may occur during APR. Previous studies by Sugawara et al. (27) showed that TNF decreases the expression of a RXR␤ promoter construct in rat GH3 cells. Here we report that RXR␣, RXR␤, and RXR␥ proteins and mRNA levels decline during the APR in hamster. RXR repression is associated with LXR␣ and PPAR␣ repression, resulting in an overall decreased ability of RXR⅐RXR homodimers, RXR⅐PPAR, and RXR⅐LXR heterodimers to bind to their respective response elements.

EXPERIMENTAL PROCEDURES
Materials-LPS (Escherichia coli 55:B5) was obtained from Difco Laboratories and freshly diluted to desired concentration in pyrogenfree 0.9% saline. Human TNF-␣ (specific activity, 5 ϫ 10 7 units/mg) was provided by Genentech, Inc. Recombinant human IL-1␤ (specific activity, 4 ϫ 10 8 units/mg) was a gift from Dr. Charles A. Dinarello (University of Colorado, Denver, CO). The cytokines were freshly diluted to desired concentrations in pyrogen-free 0.9% saline containing 0.1% human serum albumin. Oligo(dT)-cellulose type 77F was from Amersham Pharmacia Biotech. Wy-14,643 was purchased from Sigma and freshly resuspended in corn oil at the appropriate concentration.
Animals-Male Syrian hamsters (140 -160 g) were purchased from Simonsen Laboratories (Gilroy, CA). The animals were maintained in a normal-light-cycle room and were provided with rodent chow and water ad libitum. Anesthesia was induced with halothane. To assess the effect of the acute phase response on RXR, hamsters were injected intraperitoneally with 0.1-100 g/100 g of body weight (BW) LPS, 25 g/100 g of BW TNF-␣, or 1 g/100 g of BW IL-1␤ in 0.5 ml of saline or with saline alone. To assess the effect of LPS treatment on PPAR␣ activation, hamsters were injected IP daily with Wy-14,643 at a dosage of 5 mg/100 g of BW or with corn oil alone for 5 days. On the fifth day, 100 g/100 g of BW LPS or saline alone was administered IP. Food was withdrawn at the time of injection because LPS and cytokines induce marked anorexia in rodents (28). Livers were removed after treatment at the times indicated below. The doses of LPS used in this study have significant effects on triglyceride and cholesterol metabolism without causing death (23,29). Similarly, the nonlethal doses of TNF-␣ and IL-1␤ used in this study reproduce many of the effects of LPS on lipid metabolism, causing marked changes in serum lipid and lipoprotein levels (9,30).
Preparation of Nuclear Extracts-Fresh liver (1.5-2 g) was homogenized in 10 mM HEPES (pH 7.9), 25 mM KCl, 0.15 mM spermine, 1 mM EDTA, 2 M sucrose, 10% glycerol, 50 mM NaF, 2 mM sodium metavanadate, 0.5 mM dithiothreitol, and 1% protease inhibitor mixture (Sigma) at the times indicated below after LPS or saline treatment. Immediately following homogenization, nuclear proteins were extracted as described by Neish et al. (31), except that 1 mM NaF, 0.1 mM metavanadate, and 1% protease inhibitor mixture (Sigma) were added to all buffers. Nuclear protein content was determined by the Bradford assay (Bio-Rad), and yields were similar in control and LPS-treated groups.
Western Blot Analysis-Denatured nuclear protein (25 g) was loaded onto 10% polyacrylamide precast gels (Bio-Rad) and subjected to electrophoresis. After electrotransfer onto polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), blots were blocked with phosphate-buffered saline containing 0.10% Tween and 5% dry milk for 1 h at room temperature and incubated for 1 h at room temperature , and the animals were sacrificed at the time indicated after LPS administration. Hepatic nuclear extracts were prepared, and Western blot analysis was carried out as described under "Experimental Procedures." Data (means Ϯ S.E., n ϭ 5) are expressed as a percentage of controls. *, p Ͻ 0.05; ***, p Ͻ 0.005 versus control.
with the following polyclonal rabbit antibodies (Santa Cruz Biotechnology) at a dilution of 1:5000: anti-RXR␣, anti-RXR␤, and anti-RXR␥. Immune complexes were detected using horseradish peroxidase-linked donkey anti-rabbit IgG (dilution 1:20,000) according to the ECL Plus Western blotting kit (Amersham Pharmacia Biotech). Immunoreactive bands obtained by autoradiography were quantified by densitometry.
RNA Isolation and Northern Blot Analysis-Total RNA was isolated from 300 -400 mg of snap-frozen whole liver tissue by a modified acid guanidinium thiocyanate-phenol-chloroform method (32) as described earlier (21). Poly(A)ϩ RNA was purified using oligo(dT) cellulose and quantified by measuring absorption at 260 nm. Ten micrograms of poly(A)ϩ or 20 g of total RNA were denatured and electrophoresed on a 1% agarose/formaldehyde gel. The uniformity of sample applications was checked by UV visualization of the acridine orange-stained gel before electrotransfer to Nytran membrane (Schleicher & Schuell), or when indicated, p18S was used as a control probe. We and others have found that LPS increases actin mRNA levels in liver by 2-5-fold in rodents (29,33). TNF and IL-1 produced a 2-fold increase in actin mRNA levels. LPS also produces a 2-fold increase in glyceraldehyde-3phosphate dehydrogenase and a 2.6-fold increase in cyclophilin mRNA (20). Thus, the mRNA levels of actin, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin, which are widely used to normalize data, cannot be used to study LPS or cytokine-induced regulation of proteins in liver. However, the differing direction of the changes in mRNA levels for specific proteins after LPS or cytokine administration, the magnitude of the alterations, and the relatively small standard error of the mean make it unlikely that the changes observed were due to unequal loading of mRNA (20,23,24,29,34). Prehybridization, hybridization, and washing procedures were performed as described previously (21).
Membranes were probed with [␣-32 P]dCTP labeled cDNAs using the random priming technique. mRNA levels were detected by exposure of the membrane to x-ray film and quantified by densitometry. hRXR␣ cDNA was a gift from Dr. Daniel D. Bikle (University of California, San Francisco, CA). mouse RXR␤, mouse RXR␥, human LXR␣, and human LXR␤ cDNAs were kindly provided by Dr. David J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). RACS cDNA was kindly provided by Dr. Pamela J. Smith (Ross Products Division, Abbott Laboratories, Columbus, OH). rPPAR␣, mPPAR␦, and mPPAR␥ cDNAs were a gift from Dr. Anthony Bass (University of California San Francisco, CA).
Nuclear Run-on Transcription Assay-Isolation of liver nuclei from fresh tissue, the procedure for in vitro nuclear transcription and hybridization was essentially as described by Clarke et al. (35). Briefly, nuclei were incubated with 200 Ci of [␣-32 P]UTP, and after labeling nascent transcripts for 30 min at 30°C, total RNA was recovered according to Chomczynski and Sacchi (32). After prehybridization, all of the in vitro labeled RNA isolated (2-9 ϫ 10 6 cpm total) from nuclei of control and LPS-treated hamsters were hybridized to prepared nylon membrane (Schleicher & Schuell). After being washed and autoradiographed, the filters were air-dried, and the amount of in vitro labeled RNA that hybridized to each dot containing 10 g of cDNA for RXR␣, RXR␤, RXR␤, actin, and vector pUC19 was measured by liquid scintillation counting.
Statistical Analysis-Data are expressed as mean Ϯ S.E. of experiments from 3-5 animals per group for each time point. The difference between two experimental groups was analyzed using the unpaired t test. Differences among multiple groups were analyzed using one-way analysis of variance with the Dunnett's post-test correction. A p value Ͻ 0.05 was considered significant.

LPS and Cytokines
Decrease RXR Levels-We initially determined the effect of LPS administration on RXR␣ protein levels in the nuclei from liver of Syrian hamsters. RXR␣ is the most abundant RXR isoform in liver. As shown in Fig. 1A, LPS (100 g/100 g of BW) produced a maximum decrease (62%) in RXR␣ protein levels at 4 h. A similar decrease was also present at 8 h following LPS treatment, but by 16 h, RXR␣ protein was returning toward normal levels (23% decrease at 16 h). As shown in Fig. 2A, the LPS-induced decrease in RXR␣ protein levels was dose-dependent, with the half-maximal effect occurring at approximately 2 g/100 g of BW. Thus, LPS at relatively low doses (LD 50 for LPS in rodents is approximately 5 mg/100 g of BW) rapidly decreases RXR␣ protein levels in the liver of Syrian hamsters.
We next determined whether this decrease in RXR␣ protein levels was associated with alterations in RXR␣ mRNA levels in the liver. As shown in Fig. 3A, LPS administration resulted in a marked reduction (97%) in RXR␣ mRNA levels in the liver of Syrian hamsters at 4 h. To determine whether this decrease in mRNA levels was due to an inhibition of transcription, nuclear run-on assays were performed on nuclei prepared from hamster liver 4 h after LPS or saline injection. As shown in Fig. 4A, LPS treatment resulted in a 38% decrease in RXR␣ transcription compared with control. Therefore, the decrease in RXR␣ protein levels following LPS administration is associated with a decrease in mRNA levels that is partially accounted for by LPS inhibition of RXR␣ gene transcription. However, the modest reduction in transcription compared with the marked decrease in mRNA levels suggests that post-transcriptional factors in addition to inhibition of transcription contribute to the LPS-induced decrease in RXR␣ mRNA levels.
Because cytokines, such as TNF and IL-1, mediate many of the changes induced by LPS administration, we next examined the effect of TNF and/or IL-1 on RXR␣ mRNA levels. As shown in Fig. 3B, 2 h after the administration of TNF, IL-1, or TNF plus IL-1, there was a 63, 60, and 80% reduction in RXR␣ mRNA levels, respectively. Thus, the combination of TNF and IL-1 can reproduce the effects of LPS.
Both RXR␤ and RXR␥ are also present in the liver but are expressed at lower levels than RXR␣. As shown in Fig. 1, B and C, LPS treatment (100 g/100 g of BW) resulted in a decrease in RXR␤ and RXR␥ protein levels in the liver of Syrian hamsters. RXR␥ was decreased by 61% as early as 2 h after LPS administration but returned to normal by 8 h (Fig. 1C). RXR␤ protein levels also rapidly decreased following LPS treatment, but in contrast to RXR␥, this decrease was sustained for at least 16 h (Fig. 1B). The decrease in protein levels of RXR␥ and RXR␤ induced by LPS was a sensitive response, with the halfmaximal effect seen at less than 1 g of LPS/100 g of BW for RXR␥ (Fig. 2C) and approximately 1 g of LPS/100 g of BW for RXR␤ (Fig. 2B). Thus, LPS treatment not only decreases RXR␣ protein levels but also decreases the levels of RXR␤ and RXR␥, which are less abundant isoforms of RXR.
To determine whether the decreases in RXR␤ and RXR␥ could be due to changes in mRNA levels, we next measured hepatic mRNA levels in the liver following LPS treatment. At 4 h after LPS treatment, there was a 76 and 90% reduction in the hepatic mRNA levels of RXR␤ and RXR␥, respectively (Fig.  3A). In contrast to RXR␣, the decreases in RXR␤ and RXR␥ mRNA levels were not associated with a decrease in transcription (Fig. 4, B and C).
We next examined the effect of TNF and/or IL-1 on RXR␤ and RXR␥ mRNA levels in the liver. As shown in Fig. 3B, cytokine treatment reduced RXR␤ and RXR␥ mRNA levels by approximately 50%. In contrast to the effect of cytokines on RXR␣ mRNA levels, cytokine administration did not reduce RXR␤ or RXR␥ mRNA levels to the degree seen following LPS treatment.

FIG. 4. Effect of LPS treatment on RXR␣ (A), RXR␤ (B), and RXR␥ (C) gene transcription rates in hamster liver.
Syrian hamsters were injected IP with either saline or LPS (100 g of LPS/100 g of BW). Four hours later, livers were removed, and nuclei for in vitro transcription were prepared as described under "Experimental Procedures." Data (means Ϯ S.E., n ϭ 5) are expressed as a percentage of controls. ***, p Ͻ 0.005 versus control.

LPS Treatment Also Reduces LXR␣ and PPAR␣ Expression
in Liver-To determine whether the decrease in RXR expression is associated with an alteration in LXR and PPAR expression, LXR and PPAR mRNA levels were measured in hamster liver following LPS treatment. LXR␣ is abundantly expressed in liver and in tissues playing an important role in lipid metabolism (39), whereas LXR␤, which is also present in the liver, displays a more widespread pattern of expression (40). PPAR␣ has been shown to be the major isoform in human (39), rat (41), and mouse (42) liver. PPAR␦ and PPAR␥ are present in the liver but at a lower level of expression than PPAR␣ (39,41,42). Four hours after LPS administration, there was an 89% reduction in mRNA levels of LXR␣ and PPAR␣ (Fig. 5, A and B,  respectively). The level of expression of the minor isoforms was reduced by 84% in the case of PPAR␥ (Fig. 5B) and was not significantly altered in the case of PPAR␦ (Fig. 5B) and LXR␤ (Fig. 5A). Thus, in contrast to the overall inhibition of RXR species, LPS treatment lead to the specific inhibition of LXR␣, PPAR␣, and PPAR␥ but not LXR␤ or PPAR␦.
LPS Administration Decreases the Binding of RXR Homodimers and RXR⅐PPAR and RXR⅐LXR Heterodimers to Specific Response Elements-Electrophoretic gel mobility assays were carried out to determine whether the decrease in RXR expression resulted in a decline in RXR binding to DNA. Nu-clear hormone receptors recognize derivatives of a direct hexanucleotide repeat. The orientation of the half-sites and the number of nucleotides spacing the two half-sites determine the specificity of the response element (3,5). We used a 32 P-labeled DNA oligonucleotide containing the RXR response element, a direct repeat spaced by one nucleotide (DR1), from the retinolbinding protein type II (CRBPII) promoter, which preferentially binds to RXR homodimers (37). As shown in Fig. 6A, three major RXR complexes were observed in the control samples. Competition with a 100-fold molar excess of specific oligonucleotide, but not of mutated oligonucleotide, demonstrated the specificity of the three complexes. In addition, a portion of these complexes was supershifted after incubation of control nuclear extract with anti-RXR␣ and anti-RXR␤ antibodies (Fig.  6C). Specifically, incubation with RXR␤ antisera markedly decreased the higher molecular weight complex, suggesting that this complex is mainly composed of RXR␤ homodimers. In our hands, RXR␥ antiserum was unable to supershift any of the three complexes. The formation of RXR-DNA complexes was not affected by nonspecific IgG. At 4 h, LPS treatment induced an overall 74% (p Ͻ 0.005) decrease in RXR homodimer binding, with the two higher molecular weight complexes being the most affected (Fig. 6, A and B). Thus, the decrease in RXR nuclear protein levels is associated with a decline in RXR binding activity during endotoxemia.
Because RXR is the obligatory partner for high affinity binding of PPAR and LXR to their response elements, we next carried out electrophoretic gel mobility assays to determine whether the down-regulation in RXR species along with PPAR␣ and LXR␣ was associated with a decrease in PPAR and/or LXR binding activity. The following oligonucleotides were used: PPAR response element (DR1), derived from the PPAR response element present in the acyl-CoA oxidase promoter; and CYP7-LXR response element (DR4), derived from the LXR response element of the CYP7A gene. As shown in Fig.  6, A and B, 4 h after LPS administration, RXR⅐PPAR and RXR⅐LXR binding were reduced by 90% (p Ͻ 0.005) and 58% (p Ͻ 0.05) compared with control, respectively. Therefore, LPS treatment leads to a global decrease in RXR, PPAR, and LXR dimer binding activity in the liver.
To determine whether the decrease in RXR⅐PPAR binding that we found in hepatic nuclear extracts from LPS treated animals could be associated with a decreased expression of a PPAR␣ regulated gene, we next examined the effect of LPS on ACS mRNA level in hamsters pretreated with a specific PPAR␣ agonist, Wy-14,643 (43). As reported previously (21), LPS treatment alone resulted in a marked decrease (72%) in ACS mRNA levels in liver at 4 h (Fig. 7). Five days of treatment with Wy-14,643 markedly up-regulated (2-fold) ACS mRNA levels, as expected (11,44). Most importantly, LPS administration led to a marked reduction in ACS mRNA levels in hamsters pretreated with Wy-14,643, indicating that LPS, possibly by decreasing RXR⅐PPAR heterodimer binding, can block the stimulatory effect of PPAR␣ activators in liver. DISCUSSION Infection, inflammation, and trauma induce a wide array of metabolic changes in the liver that constitute the APR (45). The FIG. 6. Effect of LPS treatment on RXR, PPAR, and LXR binding to their specific response element. Syrian hamsters were injected IP with either saline or LPS (100 g of LPS/100 g of BW). Four hours later, hepatic nuclear extracts were prepared as described under "Experimental Procedures." Ten micrograms of nuclear extracts were incubated with radiolabeled oligonucleotides representing binding sites for RXR homodimers, and RXR⅐PPAR and RXR⅐LXR heterodimers. A, representative electrophoretic gel mobility shift assays. Unlabeled specific (100Xwt) and nonspecific (100Xmut) competing oligonucleotides were included at 100-fold excess 1 h prior to the addition of the labeled probes. Arrows represent specific bound complexes. B, densitometric analysis of hepatic DNA-binding proteins. Data (means Ϯ S.E., n ϭ 5) are expressed as a percentage of controls. *, p Ͻ 0.05; ***, p Ͻ 0.005 versus control. C, electrophoretic mobility shift assay using a nuclear extract from a control hamster performed in the presence of antibodies raised against RXR␣ (lane 2), RXR␤ (lane 3), RXR␥ (lane 4), and rabbit IgG (lane 5). SS1 and SS2 represent the complexes supershifted by the RXR␣ and RXR␤ antibodies, respectively. APR is mediated by cytokines, particularly TNF, IL-1, and IL-6 (46,47). The hepatic synthesis of certain proteins, such as C-reactive protein and serum amyloid A, is increased (positive acute phase proteins), whereas the synthesis of other proteins, such as albumin and transferrin, is inhibited (negative acute phase proteins) (45).
The mechanism by which gene transcription is stimulated during the APR has been extensively studied. Class I acute phase proteins (APPs) are stimulated by IL-1 type cytokines, whereas class II APPs are stimulated by the IL-6 family of cytokines (46,47). IL-1-induced activation of CEBP and NFB is thought to mediate the increase in transcription of class I APP genes, whereas activation of CEBP and members of the STAT family of transcription factors is thought to mediate the IL-6-induced stimulation of class II APP gene expression (46,47).
Much less is known about the molecular mechanisms responsible for the repression of negative APPs. Down-regulation of specific hepatic nuclear factors, such as HNF-1 and HNF-4, during APR (48) has been implicated in the regulation of certain negative APPs. For example, a decrease in HNF-1 is thought to be responsible for the reduced transcription of albumin (49), the microsomal triglyceride transfer protein (50) and the sodium-dependent bile acid transporter (51), whereas a decrease in HNF-4 could account for the decline in apoCIII levels (52,53).
In the present paper, we demonstrate that the induction of the APR by either LPS or cytokine administration decreases the levels of all three RXR isoforms in the liver. The decrease in RXR␣, RXR␤, and RXR␥ protein levels occurs rapidly, within 2-4 h, and is sustained for as long as 16 h for the most abundant isoform of RXR␣ and also for RXR␤. Moreover, this decrease in RXR protein levels is induced by low doses of LPS (half-maximal effect occurring at approximately 1-2 g of LPS/ 100 g of BW, compared with a LD 50 of approximately 5 mg/100 g of BW), indicating that this reduction in RXR is a very sensitive response to LPS. The decrease in RXR protein levels is accompanied by a marked reduction in RXR mRNA levels, suggesting that a decrease in protein synthesis account for the reduction in protein levels.
Interestingly, the decrease in RXR mRNA levels does not appear to be entirely due to a decrease in RXR gene transcription. Nuclear run-on assays did not demonstrate a change in RXR␤ and RXR␥ transcription and showed only a very modest 38% decrease in RXR␣ transcription, which is not sufficient to account for the marked reduction in RXR␣ mRNA levels following LPS administration. It therefore appears that the decrease in RXR mRNA levels is primarily due to an LPS-induced specific degradation of RXR mRNA. Recent studies have suggested that LPS also reduces connexin 32 mRNA levels in the liver by increasing their degradation rate (54). Unfortunately, using in vivo models such as these, it is difficult to carry out studies to directly demonstrate that LPS accelerates RXR mRNA degradation. In addition to the usual difficulties of measuring RNA degradation in vivo, degradation studies typically use actinomycin D, and it should be recognized that this compound dramatically increases the sensitivity to LPS (55), which will make interpretation of the results difficult. Definitive studies of the mechanism by which RXR mRNA levels are decreased during the APR await the development of an in vitro model.
The decrease in RXR protein levels in the liver during the APR may affect the transcription of a variety of genes. In the present study, we demonstrate that RXR binding to RXR⅐RXR response element is decreased following LPS treatment. In addition to forming homodimers, RXR is an obligate partner in heterodimers formed with several nuclear hormone receptors, such as PPAR and LXR. We further demonstrate that the FIG. 7. LPS effect on the expression of ACS, a PPAR␣ target gene, after activation by a specific PPAR␣ agonist. Hepatic total RNAs were prepared 4 h after saline or LPS administration (100 g/100 g of BW) from hamsters pretreated with saline or Wy-14,643 (5 mg/100 g of BW) for 5 days. Northern blots were prepared as described under "Experimental Procedures." Data (means Ϯ S.E., n ϭ 4 -5) are expressed as a percentage of controls after normalization to individual 18 S data. **, p Ͻ 0.01 versus control. expression of LXR␣, PPAR␣, and PPAR␥, along with the binding of nuclear extracts from acute phase liver to RXR⅐PPAR (DR-1) and RXR⅐LXR (DR-4) regulatory elements, is reduced. Because LPS treatment did not significantly modify the level of expression of LXR␤ and PPAR␦, it would be of interest to determine whether the expression and/or the binding of other nuclear hormone receptors, such as RAR, thyroid hormone receptor, vitamin D receptor, and farnesoid X receptor, that also form heterodimers with RXR, is also reduced.
Given the variety of nuclear hormone receptors that form obligate heterodimers with RXR and the large number of genes that they regulate, a decrease in RXR and some of its partners in the liver during the APR could provide a mechanism to coordinately decrease the expression of a large number of different proteins. Looking at one model gene, ACS, which is regulated by PPAR␣ (11), we demonstrate that LPS administration decreases ACS mRNA levels not only in normal animals, but also in animals in which ACS expression was induced by prior treatment with the PPAR␣ ligand, Wy-14,643. These data suggest that induction of the APR can inhibit the stimulation of transcription induced by PPAR␣ activators. However, to understand the relative importance of RXR repression and LXR␣ or PPAR␣ repression for the decreases in gene transcription that occur during the APR, one will have to develop transgenic models in which RXR levels are maintained during the APR.
The APR results in marked alterations in lipid metabolism in the liver (9). Whereas hepatic fatty acid uptake is increased and fatty acids are preferentially esterified to form triglycerides, there is a concomitant decrease in fatty acid oxidation and in bile acid synthesis. Many of the enzymes and transporters involved in these metabolic changes, such as carnitine palmitoyltransferase I, 3-hydroxy-3-methylglutaryl-CoA synthase, acyl-CoA oxidase, ACS, FA transport protein, FA-binding protein, and CYP7A are known to be regulated by PPAR or LXR (10 -19). It is possible that during the APR, the reduced availability of RXR protein and possibly of other nuclear hormone receptors represents a mechanism to coordinately regulate these metabolic changes in liver. Additionally, it has recently been recognized that orphan receptors PXR and CAR form heterodimers with RXR and modulate drug metabolism by regulating the expression of CYP2 and CYP3 P450 enzymes (56). A decrease in RXR could by itself explain the well characterized decrease in P450 enzymes and inhibition of drug metabolism that occurs during the APR (57). Lastly, one would anticipate that genes regulated by other nuclear hormone receptors that form heterodimers with RXR might also be downregulated during the APR. In fact, prior studies have shown that the expression of the malic enzyme, which is regulated by PPAR (58) and thyroid hormone receptor (59), is decreased after liver injury (60), supporting this hypothesis.
In summary, the present manuscript demonstrates that the APR is associated with a decrease in mRNAs coding for RXR proteins, resulting in a marked reduction in RXR protein levels in the liver. This reduction in RXR species appears to be primarily due to an increase in RNA degradation rate. RXR repression is associated with reduced LXR␣ and PPAR␣ expression levels, resulting in an overall decreased binding activity to regulatory elements that recognize RXR⅐RXR, RXR⅐PPAR, and RXR⅐LXR dimers in nuclear extracts from acute phase liver. It can be hypothesized that the reduction in RXR levels, along with levels of other nuclear hormone receptors in the liver, could be a mechanism to coordinately down-regulate the expression of a large number of genes during the APR.