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J. Biol. Chem., Vol. 275, Issue 36, 28285-28290, September 8, 2000

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Peroxisome Proliferator-activated Receptor -mediated Pathways Are Altered in Hepatocyte-specific Retinoid X Receptor -deficient Mice^*

Yu-Jui Yvonne Wan^§, Yan Cai, William Lungo, Paul Fu, Joseph Locker^¶, Samuel French, and Henry M. Sucov

From the  Department of Pathology, Harbor-UCLA Medical Center, Torrance, California 90509, the ^¶ Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, and the  Department of Biochemistry & Molecular Biology and Cell & Neurobiology, Institute for Genetic Medicine, Keck School of Medicine, University Southern California, Los Angeles, California 90033

Received for publication, February 4, 2000, and in revised form, June 19, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Retinoid x receptor  (RXR) serves as an active partner of peroxisome proliferator-activated receptor (PPAR). In order to dissect the functional role of RXR and PPAR in PPAR-mediated pathways, the hepatocyte RXR-deficient mice have been challenged with physiological and pharmacological stresses, fasting and Wy14,643, respectively. The data demonstrate that RXR and PPAR deficiency are different in several aspects. At the basal untreated level, RXR deficiency resulted in marked induction of apolipoprotein A-I and C-III (apoA-I and apoC-III) mRNA levels and serum cholesterol and triglyceride levels, which was not found in PPAR-null mice. Fasting-induced PPAR activation was drastically prevented in the absence of hepatocyte RXR. Wy14,643-mediated pleiotropic effects were also altered due to the absence of hepatocyte RXR. Hepatocyte RXR deficiency did not change the basal acyl-CoA oxidase, medium chain acyl-CoA dehydrogenase, and malic enzyme mRNA levels. However, the inducibility of those genes by Wy14,643 was markedly reduced in the mutant mouse livers. In contrast, the basal cytochrome P450 4A1, liver fatty acid-binding protein, and apoA-I and apoC-III mRNA levels were significantly altered in the mutant mouse livers, but the regulatory effect of Wy14,643 on expression of those genes remained the same. Wy14,643-induced hepatomegaly was partially inhibited in hepatocyte RXR-deficient mice. Wy14,643-induced hepatocyte peroxisome proliferation was preserved in the absence of hepatocyte RXR. These data suggested that in comparison to PPAR, hepatocyte RXR has its unique role in lipid homeostasis and that the effect of RXR, -, and - is redundant in certain aspects.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferators including herbicides, plasticizers, hypolipidemic drugs (fibrates), and leukotriene D4 inhibitors play a crucial role in hepatocyte proliferation. The most potent peroxisome proliferator is Wy14,643. These agents cause profound peroxisome proliferation in hepatocytes resulting in hepatomegaly and hepatoma and a rapid transcription of genes encoding the enzymes involved in fatty acid metabolism (for reviews, see Refs. 1-4). Peroxisome proliferators exert their pleitropic responses via PPAR,¹ a member of the nuclear hormone receptor superfamily (5-10). Besides peroxisome proliferator, PPAR can also be activated by certain conditions such as starvation, high fat diet, and diabetes mellitus under which increased fatty acids are delivered to the liver (11-13).

RXRs are the required active heterodimeric partners of PPARs (14). Thus, RXR, PPAR, and their ligands are all actively involved in regulating liver gene expression, fatty acid metabolism, lipid transport, and hepatocyte proliferation. Among the three types of RXR, RXR is the predominant one expressed in the liver.

Absence of PPAR expression in knockout mice prevents the induction of hepatocyte peroxisome proliferation and of fatty acid synthesizing enzymes and  oxidizing enzymes by Wy14,643 (15-17). In addition, PPAR deficiency leads to elevated serum cholesterol levels in young adult mice and increased serum triglyceride levels and steatosis in aging mice (18). There is no in vivo model available with which to compare the role of RXR with PPAR, because of embryonic lethality caused by a fetal cardiac phenotype in RXR-null mice (19-21). RXR and RXR-null mice have no apparent consequence on the liver (21). Furthermore, RXR is strongly implicated in postnatal liver physiology and regulation of liver gene (22-28). To understand the biological role of RXR in the liver, we have generated hepatocyte-specific RXR knockout mice using a cre/loxP recombination system (29). In this study, we further characterized the impact of RXR in PPAR-mediated pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mouse-- A line of mice in which the RXR gene is conditionally mutated by introduction of loxP sites into introns flanking exon 4 of the RXR gene was provided by Dr. K. Chien (University of California, San Diego) (30). This modified "floxed" allele is fully functional, in those animals which are homozygous for this allele are normal and viable. Moreover, this mutated allele was used to specifically ablate RXR function in the cardiomyocyte lineage (30). To abolish RXR function in the hepatocytes, the albumin promoter/enhancer was employed to express cre recombinase. Dr. M. A. Magnuson (Vanderbilt Medical Center) provided this albumin-cre transgenic line, which provides liver-specific expression. Heptocyte-specific RXR knockout was established by crossing albumin-cre transgene with the RXR flox/flox background (29). PPAR-null mice were generously provided by Dr. Frank Gonzalez (15).

4-Chloro-6-(2,3-sylidine)-pyrimidinylthio)acetic acid (Wy14,643) was purchased commercially (ChemSyn Science Laboratories, Lenexa, KS). Pelleted mouse chow, which was composed of 21.4% protein, 55% carbohydrates, 4% fat, 6.7% ash, 4% fiber, and less than 10% moisture, was commercially prepared containing either 0.0% (control) or 0.1% (w/w) Wy14,643 (Bioserv, Frenchtown, NJ). For all the experiments 10-16-week-old male mice were used. Mice were fed either control or Wy14,643 diet ad libitum for 10 days. For the starvation experiment, mouse chow was removed from mice for 48 h. Animals were housed in groups of two or three in plastic microisolator cages at 25 °C with a 12-h light/12-h dark cycle.

At the end of the treatment, animals were weighed and anesthetized with pentobarbital (60 mg/kg, intraperitoneally). Blood samples were obtained by intracardiac puncture. Blood triglycerides and cholesterol levels were determined by automated analysis. The liver was removed immediately, weighed, frozen in liquid nitrogen, and processed for RNA extraction. Part of liver was fixed by formalin and 1.5% glutaraldehyde for light and electron microscopy analysis, respectively.

Northern Blot Hybridization-- Molecular aspects of hepatocyte-specific RXR mutation were evaluated by Northern blotting analysis of RNA levels in the liver for the expression of PPAR target genes. The gene probes used were apoA-I and apoC-III (provided by Dr. J. Auwerx), liver fatty acid-binding protein (provided by Dr. J. Gordon), malic enzyme (provided by Dr. G. Brent), acyl-CoA oxidase (31) (provided by Dr. T. Osumi), medium chain acyl-CoA dehydrogenase (12) (provided by Dr. D. Kelly), CYP4A1 (32) (provided by Dr. F. Gonzalez), and catalase (33) (purchased from American Type Culture Collection).

For Northern analysis, hepatocyte and liver total RNA was extracted by the guanidinium isothiocyanate method (34). Twenty µg of total RNA per lane was resolved by electrophoresis on a 1.2% agarose gels containing 2.2 M formaldehyde and then transferred to nylon membranes by capillary blotting. cDNA fragments were labeled by random priming and hybridized to membranes in 7% (w/v) SDS, 0.5 M sodium phosphate, pH 6.5, 1 mM EDTA, and 1 mg/ml bovine serum albumin at 68 °C overnight. The membranes were washed twice in 1% SDS, 50 mM NaCl, and 1 mM EDTA at 68 °C for 15 min each and autoradiographed using intensifying screens. Four animals from each group were studied for each gene. The amount of mRNA expressed was quantitated by densitometry and then normalized with the level of 18 S rRNA to obtain mean and standard deviation. Statistical relevance of discrepancies between groups was evaluated by Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibition of Fasting-induced PPAR Activation in Hepatocyte RXR-deficient Mouse-- Prolonged starvation induces dramatic changes in metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by fatty acid oxidation in the liver. It has been demonstrated that PPAR mediates the adaptive response to fasting (11-13). To analyze the role of RXR in fasting-activated PPAR pathways, PPAR-null mic, hepatocyte RXR-deficient mice, and wild-type controls were deprived from food for 48 h and then the expression of PPAR target genes in the livers was examined by Northern hybridization.

In mice fed the control diet, PPAR deficiency caused a reduction in the level of acyl-CoA oxidase (AOX) and cytochrome P450 4A1 (CYP4A1) mRNA encoding the key enzymes involved in fatty acid - and -oxidation pathways (Fig. 1). PPAR deficiency also resulted in a decreased expression of liver fatty acid-binding protein (LFABP) mRNA and a weak induction of apoA-I mRNA. In comparison, RXR deficiency resulted in inhibition of expression of CYP4A1 and LFABP mRNA. The level of AOX and medium chain acyl-CoA dehydrogenase (MCAD) mRNA remained unchanged. The most striking difference between the PPAR- and RXR-deficient mice was that the expression of apoA-I and apoC-III mRNA was markedly increased in the absence of RXR, whereas the induction was very weak, if there was any, in the PPAR-null mice (Fig. 1).

View larger version (88K): [in this window] [in a new window]   Fig. 1.   Inhibition of starvation-induced PPAR target gene expression in PPAR-null mice and hepatocyte RXR-deficient mice. Representative Northern blots demonstrate the expression of AOX, MCAD, CYP4A1, LFABP, apoA-I, and apoC-III mRNA in the livers of wild-type, PPAR-deficient, and hepatocyte RXR-deficient mice fed regular diet (control) or starved for 48 h (fasting). Total RNA (20 µg) from mouse livers were electrophoresed and hybridized with the indicated cDNA probes. The relative fold changes of the message levels after normalization to 18 S rRNA level are indicated below each panel.

In wild-type mice, starvation caused significant induction of the PPAR target gene except for the apoC-III gene (Fig. 1). PPAR deficiency completely abolished PPAR target gene activation induced by starvation. The reduced expression of AOX, MCAD, CYP4A1, and LFABP genes in fasted PPAR-null mice suggested that the transcription of these genes was dependent on PPAR in the fasting state. RXR deficiency had a similar effect; starvation induced PPAR activation was prevented in the absence of RXR. Starvation only caused a weak induction of MCAD and CYP4A1 mRNA in RXR-deficient mice (1.8- and 4-fold induction, respectively) compared with wild-type mice (10- and 20-fold induction, respectively); this weak effect caused by starvation probably was due to the presence of RXR and -. These data unambiguously proved that in vivo in the hepatocyte, the effect of PPAR and RXR is coupled. In addition, hepatocyte RXR has a unique effect in regulating the expression of apolipoprotein genes in vivo.

Alteration of Peroxisome Proliferator-induced PPAR Activation in Hepatocyte RXR-deficient Mouse-- To further analyze the role of RXR in PPAR/RXR-mediated pathways; the expression of the PPAR/RXR target genes was examined in Wy14,643-treated mice. Wild type and RXR-deficient mice were treated with Wy14,643 (0.1%, w/w) for 10 days. Total liver RNA was extracted for analyzing the expression of PPAR target genes. The results of two representative mouse liver samples from two mice are shown in Fig. 2. Consistent with the data demonstrated in Fig. 1, the basal AOX, MCAD, and malic enzyme mRNA level remained unchanged in mutant mouse livers. After Wy14,643 treatment, the expression of AOX, MCAD, and malic enzyme mRNA (× 50, × 20, and × 50, respectively) in the wild-type mouse livers was significantly induced (Fig. 2). In contrast, the inductions were markedly reduced due to hepatocyte RXR deficiency (only 2-5-fold induction). The expression of the catalase gene was not affected by Wy14,643 in wild-type and mutant mouse livers (Fig. 2). These data indicate that at the physiological level, PPAR/RXR or RXR/RXR do not regulate the basal transcription of the AOX, MCAD, and malic enzyme gene in vivo and that only exogenous ligand (Wy14,643)-activated PPAR/RXR can regulate the expression of these genes. The weak residual inducibility of these genes by Wy14,643 in the mutant mouse livers may be due to the presence of RXR and -.

View larger version (76K): [in this window] [in a new window]   Fig. 2.   Inhibition expression of AOX, MCAD, and malic enzyme mRNA induced by Wy14,643 in hepatocyte RXR-deficient mouse livers. Representative Northern blots demonstrate the expression of AOX, MCAD, malic enzyme, and catalase mRNA in the livers of wild-type and hepatocyte RXR-deficient mice fed control or Wy14,643 (0.1%) rodent diet for 10 days. Total RNA (20 µg) from mouse livers were electrophoresed and hybridized with the indicated cDNA probes. The relative fold changes of the message levels after normalization to 18 S rRNA level are indicated below each panel.

In contrast to the AOX, MCAD, and malic enzyme genes, the basal transcription of the CYP4A1 and LFABP genes can be controlled by PPAR/RXR at the physiological level. CYP4A1 and LFABP mRNA level was reduced about 3-fold in RXR-deficient mouse livers compared with the wild-type livers (Fig. 3). However, the inducibility of these two genes by Wy14,643 remained the same in mutant mouse livers (Fig. 3). After Wy14,643 administration, there was a 50- and 10-fold induction of CYP4A1 and LFABP mRNA level, respectively, in both wild-type and mutant mouse livers. These data suggest that the basal transcription of the CYP4A1 and LFABP genes is constitutively regulated by PPAR/RXR or RXR/RXR through endogenous ligands such as polyunsaturated fatty acids or 9-cis-retinoic acid in vivo. Therefore, in the absence of RXR, these genes are expressed at a reduced level. However, when pharmacological levels of exogenous ligands are present, the availability of RXR and - is sufficient to mediate the inductive effect of Wy14,643.

View larger version (75K): [in this window] [in a new window]   Fig. 3.   Reduction of basal CYP4A1 and LFABP mRNA level in hepatocyte RXR-deficient mouse livers. Total RNA (20 µg) was extracted from representative livers of wild-type and hepatocyte RXR-deficient mice fed control or Wy14,643 (0.1%) rodent diet for 10 days. Northern blot hybridization was performed using the indicated cDNA probes. The relative fold changes of the message levels after normalization to 18 S rRNA level are indicated below each panel.

To further understand the role of RXR in regulating cholesterol and lipid homeostasis, the expression of apoA-I and apoC-III mRNA was examined in Wy14,643-treated mice. In normal cells, PPAR agonists suppress the expression of these genes. RXR is involved in the basal transcription of the apolipoprotein genes because the basal mRNA levels in normally fed mice were increased in the absence of RXR (Fig. 4). However, the inhibitory effect of Wy14,643 on apolipoprotein gene expression remained in the absence of hepatocyte RXR.

View larger version (66K): [in this window] [in a new window]   Fig. 4.   The expression of the apoA-I and apoC-III genes in wild-type and hepatocyte RXR-deficient mouse livers. Representative Northern blots demonstrate the expression of apoA-I and apoC-III mRNA in the livers of wild-type and hepatocyte RXR-deficient mice fed control or Wy14,643 (0.1%) rodent diet for 10 days. Total RNA (20 µg) was electrophoresed and hybridized with the indicated cDNA probes. The relative fold changes of the message levels after normalization to 18 S rRNA level are indicated below each panel.

Taken together, the expression pattern of these PPAR target genes can be divided into two groups. In the first group exemplified by the AOX, MCAD, and malic enzyme genes, these genes' basal mRNA level remains unchanged in mutant mouse liver, but the inducibility of the gene by Wy14,643 is decreased remarkably. In the second group, which includes the CYP4A1, LFABP, apoA-I, and apoC-III genes, the basal mRNA level is altered in the absence of RXR, but the regulatory effect of Wy14,643 on gene expression remains unchanged in mutant mouse liver.

Reduction of Serum Cholesterol and Triglyceride Level by Wy14,643 in Hepatocyte-specific RXR-deficient Mouse-- As a hypolipidemic drug (1-4), Wy14,643 reduces serum cholesterol and triglyceride level. These effects were tested in the hepatocyte RXR-deficient mice. As shown in Fig. 5, basal serum triglyceride and cholesterol levels were elevated in the RXR-deficient mice, which is consistent with the Northern data (Figs. 1 and 4) demonstrating the induction of apoA-I and apoC-III mRNA in the mutant mouse livers. Administration of Wy14,643 reduced serum triglyceride and cholesterol level not only in wild-type but also in mutant mice. Therefore, Wy14,643 still can exert its hypolipidemic effect even when RXR is not expressed in the hepatocyte.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Triglycerides and cholesterol levels after Wy14, 643 treatment in RXR (+/+) and (/) mice. Each value represents the mean ± S.D. of four mice. Statistically significant differences between treated and untreated animals of the same genotype (*), as well as between wild-type and deficient mice (**) are indicated by asterisks (p < 0.05).

Reduced Hepatomegaly in the Hepatocyte RXR-deficient Mice Fed Wy14,643 Diet-- It is well characterized that Wy14,643 causes liver enlargement due to hypertrophy and hyperplasia (hepatomegaly) of hepatocytes (1-4). Furthermore, clofibrate- and Wy14,643-induced hepatomegaly is not found in PPAR-null mice (15). In our system, the data was reproducible where Wy14,643 also produced a marked increase in liver weight in the wild-type mouse. The liver/body weight ratio of the wild-type mice increased 2.4-fold after 10 days of Wy14,643 feeding compared with mice fed a standard control diet (Table I). In contrast, the liver/body ratio of hepatocyte RXR-deficient mouse only increased by 1.6-fold after Wy14,643 treatment. Therefore, the hepatomegaly caused by treatment with the peroxisome proliferator was partially prevented when RXR was absent.

View this table: [in this window] [in a new window]   Table I Liver/body weight ratio of wild-type (RXR +/+) and hepatocyte RXR-deficient (RXR /) mice Mice were fed either a control diet or 0.1% Wy14,643 for 10 days. Results are the mean ± S.D. of four determinations.

Morphological Analysis of Hepatocyte-specific RXR-deficient Mice Fed Control and Wy14,643 Diet-- Using light and electron microscopy, the liver morphology of the wild-type and RXR-deficient mice was evaluated (Fig. 6). Compared with wild-type mouse livers, RXR-deficient mouse livers had normal morphology under light and electron microscope (Fig. 6, a-d). Treatment of wild-type mice with Wy14,643 resulted in pale pink staining of enlarged cells which had increased homogeneous cytoplasm. The cytoplasmic rough endoplasmic reticulum was strikingly reduced (Fig. 6e). Furthermore, the number and size of peroxisome were significantly increased after the administration of Wy14,643 as demonstrated by electron microscopy (Fig. 6f). In contrast, under light microscopy, the mutant mouse liver contain both normal and enlarged cells after administration of Wy14,643 (Fig. 6g). Electron microscopy revealed that Wy14,643 still induced hepatocyte peroxisome proliferation in RXR-deficient mice (Fig. 6h).

View larger version (76K): [in this window] [in a new window]   Fig. 6.   Light (× 364) and electron micrographs (× 2,652) of livers from wild-type (a, b, e, and f) and hepatocyte RXR-deficient (c, d, g, and h) mice fed control (a, b, c, and d) and Wy14,643 (0.1%, w/w, e, f, g, and h) rodent diet for 10 days.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Using biochemical and morphological analyses, we have analyzed the hepatic role of RXR and demonstrated both essential and redundant effects of RXR in RXR/PPAR-mediated pathways. Hepatocyte RXR is crucial for basal lipid and cholesterol homeostasis since serum cholesterol and triglyceride levels are elevated in normally fed mice lacking RXR. RXR deficiency can partially prevent the hepatomegaly effect of peroxisome proliferator. Hepatocyte RXR is essential for maintaining the physiological level of CYP4A1, LFABP, apoA-I, and apoC-III. Hepatocyte RXR deficiency also significantly prevents starvation and Wy14,643-induced PPAR activation. Mice with hepatocyte RXR deficiency are unable to increase the capacity for cellular fatty acid utilization in the context of short-term starvation. However, hepatocyte RXR deficiency neither prevents hepatocyte peroxisome proliferation nor the hypolipidemic effect of the peroxisome proliferators. Since the RXR, -, and - genes are expressed in different types of liver cells including parenchyma, endothelial, Kupffer, and stellate cells (35, 36), the presence of RXR in the liver cells other than hepatocytes as well as the redundant role of RXRs could explain why hepatocyte RXR-deficient mice are still responsive to Wy14,643.

The hepatocyte-specific RXR-deficient mice allow us to compare the functional roles of RXR with PPAR. Phenotype comparison between the hepatocyte RXR-deficient and PPAR-null mice (15-18) is summarized in Table II. In PPAR knockout mice, basal serum cholesterol level is elevated to the same extent (1.6-fold induction) as in the hepatocyte-specific RXR knockout mice. However, young adult male PPAR-null mice have normal serum triglyceride and apoC-III level (16, 18). Serum triglyceride level only elevates in aged animals (6-12-month-old), and the level is higher in females (2-fold induction) than males (1.5-fold induction) (18). In contrast, in hepatocyte RXR-deficient mice, a 1.7-fold induction of serum triglyceride level and a remarkable induction of apoC-III gene expression were observed in 2-month-old male mice. The early induction in serum triglyceride level defines the unique and important role of hepatocyte RXR in controlling lipid homeostasis. It is possible that the effect of RXR in regulating apoC-III gene expression and serum triglyceride level is mediated through dimerization with PPAR rather than PPAR.

View this table: [in this window] [in a new window]   Table II Phenotype comparison between hepatocyte-specific RXR-deficient and PPAR-null mice

In PPAR-null mice, peroxisome proliferators such as clofibrate and Wy14,643 are completely unable to induce hepatomegaly and hepatocyte peroxisome proliferation, and have no effect in regulating the expression of PPAR target genes including AOX, bifunctional enzymes, CYP4A1, CYP4A3, LFABP, apoA-I, and apoC-III (15-17). These data suggest that the effect of PPAR is unique in peroxisome proliferator-mediated pathways, and that PPAR and - cannot replace PPAR. In contrast, in vivo, the roles of RXR, -, and - appear to be at least partially redundant.

Based on our results, the PPAR/RXR target genes can be categorized into several groups. The first group of genes includes AOX and malic enzyme. The basal transcriptional rate of these genes is controlled by PPAR, but not by RXR. The second group of genes is CYP4A1, LFABP, and apoA-I. Within this group, the basal transcriptional rate of the genes is constitutively maintained by PPAR as well as by RXR through endogenous ligands. The third group of genes include apoC-III. The basal transcriptional rate of the apoC-III gene is controlled by RXR, but not by PPAR. Since RXR controls the basal transcription of the CYP4A1, LFABP, and apoA-I genes, but has no effect on the AOX, MCAD, and malic enzyme genes, these data suggest that in vivo at the physiological level RXR is crucial for microsomal -hydroxylation of fatty acids, fatty acid transport, and cholesterol and fatty acid homeostasis, whereas RXR may only become important for AOX- and MCAD-mediated fatty acid -oxidation and malic enzyme-mediated lipogenesis when pharmacological dose of PPAR ligand is employed.

Even though RXR and - are able to substitute RXR, the total amount of RXRs is critical in mediating the action of RXRs because in the absence of RXR, fatty acid is not utilized efficiently in response to starvation and Wy14,643 cannot fully exert its effects. RXR dimerizes with more than 10 different kinds of receptor. Activation one of these RXR-mediated pathways might alter other pathways in opposite directions. When the pool of RXRs is decreased, many RXR-mediated regulatory pathways may be impaired. Based on our data, it seems that the level of RXR, rather than the type of RXR, has a major impact in mediating the effect of peroxisome proliferator. It is crucial to understand the regulation of the RXR genes.

RXR can be freely activated in permissive heterodimers with PPAR (37) although it also can be silent in nonpermissive heterodimers with the thyroid hormone receptor or the vitamin D receptor (38). It would be interesting to test if 9-cis-retinoic acid has the same effect as Wy14,643 on RXR-deficient mice. 9-cis-Retinoic acid can activate RXR/RAR and RXR/RXR, and that would further deprive the availability of RXR to PPAR. Therefore, challenge the mutant mice with 9-cis-retinoic acid may produce more phenotypes.

Taken together, nuclear factors might have unique, redundant, synergistic, or antagonistic effects. These effects depend on the relative level of the receptors, presence of hormones, or the pathological condition. Comprehension of the regulation of liver gene transcription provides insight into the understanding of the molecular mechanisms leading to liver physiology, function, development, and differentiation, as well as proliferation.

	ACKNOWLEDGEMENTS

We thank Drs. Frank Gonzalez, Kenneth Chien, and Mark Magnuson for providing PPAR knockout mice, mice which carrying floxed RXR alleles, and albumin-cre transgenic mice, respectively. We also thank all the investigators listed under "Experimental Procedures" for providing cDNA clones.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant CA53596.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Pathology, Harbor-UCLA Medical Center, 1000 West Carson St., Torrance, CA 90509. Tel.: 310-222-3876; Fax: 310-782-6649; E-mail: agarose@ucla.edu.

Published, JBC Papers in Press, June 23, 2000, DOI 10.1074/jbc.M000934200

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor ; RXR, retinoid X receptor; AOX, acyl-CoA oxidase; LFABP, liver fatty acid-binding protein; MCAD, medium chain acyl-CoA dehydrogenase; CYP4A1, cytochrome P450 4A1; apoA-I, apolipoprotein A-I; apoC-III, apolipoprotein G-III.

	REFERENCES

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