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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
-Dependent Regulation of Lipid Homeostasis and Inflammatory Cytokine Expression Contributes to Alcohol-Induced Liver InjuryDepartments of Pharmacology, Toxicology, and Therapeutics (M.A.G., L.H., Y.-J.Y.W.) and Pathology and Laboratory Medicine (I.D.), University of Kansas Medical Center, Kansas City, Kansas; and Department of Pathology, Harbor-University of California Los Angeles Medical Center, Torrance, California (S.W.F.)
Received for publication
September 27, 2007
Accepted
October 31, 2007.
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Abstract |
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(RXR)-deficient mice are more sensitive to ethanol toxicity than wild-type mice. Because RXR-mediated pathways are implicated in lipid homeostasis and the inflammatory response, we hypothesized that a compromise in lipid metabolism and associated production of proinflammatory mediators are responsible for the hepatotoxicity observed in ethanol-treated hepatocyte RXR-deficient mice. Wild-type and hepatocyte RXR-deficient mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks. After ethanol treatment, serum ALT levels increased significantly (4-fold) in hepatocyte RXR-deficient mice, but not in the wild-type mice. Hepatic liver fatty acid binding protein (L-FABP) mRNA and protein levels were reduced due to RXR deficiency. Ethanol induced L-FABP mRNA and protein in wild-type mice and provided protection against nonesterified fatty acid toxicity; however, this effect was absent in the mutant mice. Accordingly, hepatic nonesterified fatty acid level was increased in ethanol-fed mutant mice. Ethanol increased nuclear factor (NF)-
B binding activity in hepatocyte RXR-deficient mice, but not in wild-type mice. In agreement, hepatic mRNA levels of proinflammatory cytokines and chemokines were increased to a greater extent in the mutant than in wild-type mice. Furthermore, signal transducer and activator of transcription factor (STAT) 3 and associated Bcl-xL induction was observed in ethanol-fed wild-type mice but not in ethanol-fed hepatocyte RXR-deficient mice. Taken together, after ethanol treatment, hepatocyte RXR deficiency results in lack of L-FABP induction, increased hepatic free fatty acids, NF-B activation, and proinflammatory cytokines production and a lack of STAT3 activation, which in part may contribute to alcohol-induced liver damage.
(RXR), is the most abundant among the three RXR isoforms (, β, and 
) (Mangelsdorf et al., 1992
). RXR dimerizes with class II nuclear hormone receptors including retinoic acid receptors, thyroid hormone receptors, vitamin D receptor, farnesoid X receptors, liver X receptors (LXRs), and peroxisome proliferator-activated receptors (PPARs) (Blumberg and Evans, 1998).
Previously, we have shown that many nuclear receptor-mediated pathways, including the lipid-sensing PPAR-mediated pathway, are compromised when hepatocyte RXR is deficient (Wan et al., 2000a,b, 2003; Cai et al., 2002). Recent reports indicate that these nuclear receptors play important role in the inflammatory process; therefore, it is important to study how RXR regulates inflammatory signaling and alcoholic liver disease (ALD).
Recent data from our laboratory indicated that a deficiency in the expression of RXR in hepatocytes caused a reduction of S-adenosylmethionine (SAM) and glutathione levels, resulting in more serious alcohol-induced liver injury (Dai et al., 2003). In addition, we established that hepatocyte RXR controls the metabolism of ethanol (Gyamfi et al., 2006). Hepatocyte RXR deficiency leads to induction of alcohol dehydrogenase activity and reduction of aldehyde dehydrogenase and glutathione S-transferase activities (Gyamfi et al., 2006). These changes resulted in acetaldehyde accumulation in the hepatocyte RXR-deficient mice after ethanol ingestion (Gyamfi et al., 2006). Thus, RXR is not only important for regulating lipid homeostasis; it also plays a crucial role in ethanol and acetaldehyde detoxification. Furthermore, hepatic retinoid content is depleted in alcoholic liver disease (Leo and Lieber, 1983). Taken together, it is highly likely that retinoid-mediated signaling pathways contribute to the protection of liver from alcohol-induced liver injury. Accordingly, RXR could serve as a target for the prevention and treatment of alcohol-induced liver injury.
Inflammation plays an important role in the development of ALD. Nuclear receptors are known to have regulatory effects on the inflammatory process. Both PPARs and LXRs are mediators of lipid metabolism and inflammatory gene expression in cells of the artery wall including macrophages (Tontonoz and Mangelsdorf, 2003; Evans et al., 2004). Lipid accumulation in the macrophages leads to the transcriptional activation of PPARs and LXRs by providing the cell with oxidized fatty acid and oxysterol ligands, respectively (Nagy et al., 1998; Tontonoz and Mangelsdorf, 2003). PPARs and LXRs can antagonize the nuclear factor (NF)-B signaling pathway and inhibit induction of gene expression in response to lipopolysaccharide (LPS) (Ricote et al., 1998; Joseph et al., 2003). Lack of PPAR and LXR in macrophages accelerates atherosclerosis in rodents (Tangirala et al., 2002). Therefore, PPARs and LXRs are negative regulators of inflammatory gene expression in macrophages. Like hepatocyte RXR-deficient mice, PPAR-null mice are also more susceptible to alcohol-induced liver injury (Nakajima et al., 2004).
Evidence also indicates that RXR plays a role in the inflammatory process in the liver. For example, tumor necrosis factor (TNF) and interleukin (IL)-1 decrease the expression of RXR, PPAR, PPAR, LXR, and their cofactors in human hepatoma Hep3B cells (Kim et al., 2007). The acute phase response induced by LPS is associated with decreased hepatic proteins involved in lipid metabolism concomitant with the reduction of hepatic RXR level in both the mouse and the hamster (Beigneux et al., 2000; Ghose et al., 2004). The down-regulation of RXR in inflamed liver is due to nuclear export and degradation (Zimmerman et al., 2006). The RXR-mediated pathways could also be inhibited due to direct interaction between NF-B p65 and the RXR DNA binding domain and thus may prevent the binding of RXR to consensus DNA sequences (Gu et al., 2006). These findings clearly demonstrate a down-regulation of RXR signaling during the inflammatory process. However, the role of RXR in regulating inflammatory cytokines and its relationship with fatty acid metabolism remains to be elucidated.
Using hepatocyte RXR-deficient mice, we investigated the role of hepatocyte RXR in regulating lipid homeostasis and inflammatory cytokines and their impact upon ALD. We document that alcohol-induced liver injury in hepatocyte RXR-deficient mice is associated with dysregulation of lipid homeostasis, NF-B activation, and a robust induction of hepatic mRNA levels of IL-6, IL-1β, TNF, macrophage inflammatory protein (MIP)-2, plasminogen activator inhibitor type-1 (PAI-1), and collagen 1. In addition, ethanol-induced signal transducer and activator of transcription factor (STAT) 3 and Bcl-xL activation were not observed in hepatocyte RXR-deficient mice. These findings provide the first evidence for a direct link between hepatocyte RXR and inflammatory processes in the development of ALD.
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Materials and Methods |
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, 2003) and hepatocyte RXR-deficient mice (10–12 weeks old) were used in all the experiments. Hepatocyte RXR-deficient mice were generated by specifically mutating the RXR gene in hepatocytes using cre/lox-mediated recombination as described previously (Wan et al., 2000a). The mice were housed individually in steel microisolator cages at 22°C with a 12/12-h light/dark cycle. After 2 days of feeding mice control liquid diet, mice were randomized into ethanol- or pair-fed groups (n = 6). The ethanol-fed group was allowed free access to ethanol-containing diets with increasing concentrations of ethanol over a 7-day period as described previously (McMullen et al., 2005). The ethanol concentration was kept thereafter at 5% for an additional 5 weeks. Ethanol comprised 27.5% of the total caloric intake of mice in this group. Liquid diets were based upon the Lieber-DeCarli ethanol formulation and provide 1 kcal/ml (purchased from DYETS Inc., Bethlehem, PA). Protein content accounted for 18.9% of total calories, fat comprised 16.5% of total calories, and 64.5% of calories were derived from carbohydrates. Control mice were pair-fed diets that isocalorically substituted cornstarch for ethanol during the entire study period. All procedures were conducted in accordance with the Institute of Laboratory Animal Resources (1996) and were approved by the Kansas University Medical Center Institutional Animal Care and Use Committee. After 6 weeks of feeding, mice were fasted overnight and sacrificed. Blood samples were centrifuged at 3000 rpm for 15 min to collect serum. Livers were rapidly excised and weighed, and then aliquots were snap-frozen in liquid nitrogen and kept at –70°C for RNA, nuclear protein, and lipid extraction or Oil Red O staining. The remaining fresh liver tissue was immediately homogenized for isolation of microsomes or fixed in 10% formalin for hematoxylin and eosin (H&E) staining. Serum ALT Activity. Serum was stored at –70°C and used for the alanine aminotransferase (ALT) activity assay. Serum ALT activity was determined using the Liquid ALT Reagent kit (Pointe Scientific Inc., Brussels, Belgium).
Hepatic Nonesterified Fatty Acid, Triglyceride, and Cholesterol Levels. Total liver lipids were extracted from 50 to 100 mg of liver homogenate using methanol and chloroform as described previously (Folch et al., 1957; Zhou et al., 2006). Hepatic nonesterified fatty acid (NEFA) level was determined using the NEFA C test kit (Wako Pure Chemical Industries, Richmond, VA).
Hepatic triglyceride was quantified using a Triglyceride test kit (Wako Pure Chemical Industries). The hepatic cholesterol content was quantified using a Cholesterol E-test kit (Wako Pure Chemical Industries).
Oil Red O and H&E Staining of Liver Sections. Frozen liver sections (10 µm) were stained with Oil Red O and counterstained with H&E for lipid content determination. Following fixation of the livers with 10% formalin/phosphate-buffered saline, livers were sliced and stained with H&E for histological examination. Apoptotic cells in 10 random x 400 fields/liver section were counted by a pathologist blinded to the study.
Hepatic CYP2E1 Activity. Microsomal fractions were separated from fresh liver tissue as described previously (Gyamfi et al., 2006). CYP2E1 activity in liver microsomes was estimated colorimetrically by measuring the hydroxylation of p-nitrophenol to 4-nitrocathecol (Reinke and Moyer, 1985).
SAM and S-Adenosylhomocysteine. The liver homogenate was prepared as described previously (Dai et al., 2003) and mixed with an equal volume of 10% trichloroacetic acid solution, then centrifuged at 10,000 rpm for 20 min at 4°C. The supernatant was collected and stored at –80°C. The levels of SAM and S-adenosylhomocysteine (SAH) were measured as described previously (She et al., 1994).
Preparation of Cytosolic and Nuclear Extracts, Western Blot Analysis, and Enzyme-Linked Immunosorbent Assay-Based NF-B Binding Assay. Frozen livers were homogenized in lysis buffer [10 mM HEPES, pH 7.9, 100 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.5% Nonidet P-40, and a protease inhibitor cocktail (Pierce, Rockford, IL)] at 4°C, followed by centrifugation at 10,000g for 30 min. The supernatant was collected as cytosolic fraction. The nuclear pellet obtained was resuspended in nuclear extraction buffer at 4°C. The suspension was kept on ice and vortexed intermittently for 30 min, followed by centrifugation at 10,000g for 10 min. The supernatant was collected as nuclear extracts. The cytosolic fraction (30 µg/lane) and liver homogenate (25–50 µg/lane) were mixed in Laemmli loading buffer containing β-mercaptoethanol, boiled for 5 min, and then subjected to Western blot analysis as described previously (Gyamfi et al., 2006). Liver homogenate and cytosolic fraction were separated by 10 or 15% SDS-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membrane. The cytosolic fractions were immunoblotted with phosphospecific anti-STAT3 (Tyr705) (Cell Signaling Technology, Boston, MA) and liver fatty acid binding protein (L-FABP) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The liver homogenate were probed with anti-Bcl-xL (Cell Signaling Technology) and Bcl-2 (Santa Cruz Biotechnology). Blots were then incubated with the appropriate peroxidase-conjugated anti-rabbit or donkey anti-rabbit IgG (Santa Cruz Biotechnology, Inc.) secondary antibodies diluted in Tris-buffered saline/Tween 20 plus 1% milk for 60 min at room temperature. After probing, blots were stripped and reprobed with anti-STAT3 (Cell Signaling Technology) and anti-β-actin antibodies (Santa Cruz Biotechnology, Inc.). Proteins were viewed using enhanced chemiluminescence. The intensities of the bands were quantified using the Quantity One 1-D Analyzer Software (Bio-Rad Laboratories, Hercules, CA). To quantify activated NF-B binding to its consensus binding site, the transcription factor enzyme-linked immunosorbent assay NF-B p65 kit was used (Panomics, Inc., Fremont, CA). Equal amounts of nuclear protein extracts (10 µg) were used and performed according to the manufacturer's protocol. Protein contents in the liver homogenate, cytosol, nuclear extracts, and microsomes were determined by the Bradford method (Bradford, 1976).
Quantification of mRNA Levels Using Real-Time Polymerase Chain Reaction. Total RNA was isolated from frozen liver tissues using the TRIzol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). RNA concentration and quality were determined spectrophotometrically at 260 nm and by the A260/ A280 ratio, respectively. Total RNA (1 µg) was reversed transcribed into cDNA in a total reaction volume of 50 µl with the use of 2.5 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen) and 6.25 ng of oligo(dT)15 (Promega, Madison, WI) as a primer. The samples were placed in a Thermo Cycler and heated to 42°C for 15 min and then to 95°C for 15 min. cDNA was then diluted 10-fold with water and subjected to real-time polymerase chain reaction (PCR) to quantify the mRNA level of MIP-2, IL-1β, IL-6, TNF, PAI-1, collagen 1, L-FABP, β-actin, and GAPDH. Primers and probes (Table 1) were designed using Primer Express 2.0 (Applied Biosystems, Foster City, CA). The primers and probes were designed to cross introns to ensure that only cDNA and not genomic DNA was amplified. The fluorogenic MGB probe was labeled with the reporter dye 5-carboxyfluorescein. TaqMan Universal PCR Master Mix (Applied Biosystems) was used to prepare the PCR mix. Primers and probes were added to a final concentration of 909 and 125 nmol, respectively, in a total volume of 20 µl. The amplification reactions were carried out in the ABI Prism 7900 sequence detection system (Applied Biosystems) with initial hold steps (50°C for 2 min, followed by 95°C for 10 min) and 40 cycles of a two-step PCR (92°C for 15 s, 60°C for 1 min). The fluorescence intensity of each sample was measured at each temperature change to monitor amplification of the target gene. The comparative CT method was used to determine -fold differences between samples. The amount of mRNA was calculated using the comparative CT method, which determines the amount of target normalized to an endogenous reference. Each gene was normalized to either GAPDH or β-actin after a validation experiment to verify that efficiency of the target gene amplification is equal to that of the endogenous reference.
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Statistical Analysis. Data are presented as means ± S.E.M. (n = 4–6). Statistical analysis was performed using one-way analysis of variance followed by post hoc tests. A P value of <0.05 was considered statistically significant.
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Results |
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-deficient mice (Fig. 1a). The increase in serum ALT levels in the mutant mice fed ethanol was 3.1-fold compared with ethanol-fed wild-type mice (Fig. 1a). In contrast, serum ALT levels were not induced by ethanol in the wild-type mice (Fig. 1a). This finding indicates that using the Lieber-DeCarli ethanol model, hepatocyte RXR-deficient mice are more susceptible to alcohol-induced liver injury, which is consistent with our previous finding using the intragastric ethanol infusion model (Dai et al., 2003). Furthermore, apoptotic cells were found in ethanol-fed mutant mice (Fig. 1, b and d) but not noted in ethanol-fed wild-type mice. Ethanol-fed hepatocyte RXR-deficient mice also revealed the presence of mitotic cells, suggesting that apoptosis and cell proliferation are operative to prevent severe hepatocyte injury (Fig. 1, b–d). The increase in apoptosis in ethanol-fed mutant mice might in part contribute to the observed liver injury seen in hepatocyte RXR-deficient mice (Fig. 1, a and e).
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Lipid content was evaluated by analyzing the nonesterified fatty acid (Fig. 2a), triglyceride (Fig. 2b), and cholesterol (Fig. 2c) levels in the liver. Hepatic free fatty acids levels were not different between wild-type and hepatocyte RXR-deficient mice fed a control diet (Fig. 2a). However, ethanol treatment significantly increased hepatic free fatty acid levels in the mutant mice, but not in wild-type mice (Fig. 2a). Ethanol feeding significantly increased hepatic triglyceride and cholesterol levels in both mutant and wild-type mice (Fig. 2, b and c). The increase in hepatic triglycerides levels by ethanol was not different between the two genotypes (Fig. 2b). However, the increase in hepatic free fatty acids and cholesterol levels by ethanol was higher in hepatocyte RXR-deficient mice compared with wild-type mice fed ethanol (Fig. 2, a and c). In addition, the basal hepatic cholesterol levels were higher in the hepatocyte RXR-deficient mice than the wild-type mice.
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target-gene, binds fatty acids and stimulates their esterification, thereby attenuating fatty acid accumulation and toxicity (Shevchuk et al., 1991; Lieber, 2004). Chronic ethanol ingestion increases the concentration of L-FABP in the liver (Shevchuk et al., 1991; Lieber, 2004). The levels of L-FABP mRNA were quantified in ethanol treated wild-type and hepatocyte RXR-deficient mice (Fig. 2d). The basal level of L-FABP mRNA level was reduced when hepatocyte RXR was deficient due to a defective RXR/PPAR-mediated pathway (Fig. 2d) (Wan et al., 2000a). Consistent with these results, it was observed that the protein levels of L-FABP were decreased by 50% in the untreated mutant mice (Fig. 2e). Ethanol-feeding increased L-FABP mRNA and protein levels by 6- and 2.5-fold, respectively, in wild-type mice (Fig. 2, d and e). In contrast, ethanol ingestion increased neither L-FABP mRNA nor protein levels in the mutant mice (Fig. 2, d and e). Compared with ethanol-fed wild-type mice, both L-FABP mRNA and protein levels were decreased in hepatocyte RXR-deficient mice fed ethanol (Fig. 2, d and e). This lack of L-FABP mRNA induction in the livers of the mutant mice may be responsible for the observed ethanol-induced accumulation of nonesterified fatty acids in the mutant mice after ethanol ingestion (Fig. 2a). However, a 2-fold reduction of both L-FABP mRNA and protein levels due to hepatocyte RXR deficiency did not alter the basal nonesterified fatty acid level compared with wild-type mice (Fig. 2a).
Histological analysis of the livers by Oil Red O staining revealed lipid accumulation in the ethanol-fed mouse livers (Fig. 2, h and i), whereas lipid droplets were rare in the livers of the control group (Fig. 2, f and g). H&E staining of liver section from wild-type mice fed ethanol revealed minimal neutrophil infiltration (data not shown), whereas neutrophil infiltration was prominent in hepatocyte RXR-deficient mice fed ethanol (Fig. 2j).
Ethanol induced CYP2E1 activity in the wild-type mice (21.1 ± 1.7 nmol/mg), compared with wild-type mice fed control diet (13.9 ± 0.7 nmol/mg). However, CYP2E1 activity was not induced by ethanol in the mutant mice (13.5 ± 1.9 nmol/mg), compared with hepatocyte RXR-deficient mice fed the control diet (13.1 ± 1.0 nmol/mg). This finding suggests that CYP2E1 did not contribute to alcohol-induced liver injury in hepatocyte RXR-deficient mice.
In addition to dysregulation of lipid metabolism, abnormal methionine metabolism leading to decreased SAM levels is important in the pathogenesis of alcohol-induced liver injury (Dai et al., 2003; McClain et al., 2005). SAM down-regulates the production of toxic proinflammatory cytokines while increasing the levels of beneficial anti-inflammatory cytokines (McClain et al., 2005). Hepatocyte RXR-deficiency results in reduction of hepatic SAM level (Fig. 3a). Ethanol ingestion significantly decreased SAM levels in both wild-type and hepatocyte RXR-deficient mice (Fig. 3a). Compared with ethanol-fed wild-type mice, SAM levels decreased in the mutant mice after ethanol ingestion (0.76-fold) (Fig. 3a). SAH levels were significantly increased by ethanol feeding of wild-type mice (Fig. 3b). The SAM/SAH ratio was significantly lower in ethanol-fed wild-type mice compared with wild-type mice fed the control diet (Fig. 3c). Although ethanol-fed hepatocyte RXR-deficient mice also showed an increase in SAH levels, this induction did not reach statistical significance (Fig. 3b). As a result, ethanol did not decrease the SAM/SAH ratio in hepatocyte RXR-deficient mice (Fig. 3c). SAH levels and the SAM/SAH ratio after ethanol treatment were not different between wild-type and hepatocyte RXR-deficient mice (Fig. 3, b and c). This finding suggests that ethanol-induced ALT is not due to reduction of the SAM/SAH.
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NF-B is a central proinflammatory transcription factor known to induce proinflammatory cytokine synthesis. Ethanol increased NF-B p65 binding activity in hepatocyte RXR-deficient mice (1.7-fold), but not in wild-type mice (Fig. 4). The activation of NF-B suggested increased expression of NF-B-regulated cytokines and chemokines in the mutant mice. This finding suggests that NF-B activity can be controlled by multiple regulatory processes including RXR. To determine the effect of RXR deficiency on the expression of inflammatory mediators during ethanol exposure, mRNA levels of several proinflammatory cytokines and chemokines were measured. TNF, IL-6, and IL-1β mRNA levels were increased by 8.6-, 52.0-, and 6.9-fold, respectively, in hepatocyte RXR-deficient mice after ethanol feeding, whereas only the IL-6 mRNA level was increased in wild-type mice after ethanol treatment (2.5-fold) (Fig. 5, a–c). A significant decrease in the basal TNF mRNA level was observed in hepatocyte RXR-deficient mice compared with wild-type mice (Fig. 5a), suggesting that RXR may be important for TNF expression. Ethanol ingestion induced MIP-2 mRNA level by 3.5-fold in hepatocyte RXR-deficient mice; such induction did not reach statistical significance in wild-type mice (Fig. 5d). Ethanol ingestion also caused a significantly greater induction of the profibrogenic factor PAI-1 mRNA level in hepatocyte RXR-deficient (5.5-fold) compared with wild-type (2.3-fold) mice (Fig. 5e). A significant increase in the collagen-1 mRNA (1.6-fold) level was observed by ethanol feeding of hepatocyte RXR-deficient mice, but not in wild-type mice (Fig. 5f). Because IL-6 mRNA levels were highly induced by ethanol in the mutant mice, we also examined protein levels. Consistent with the mRNA levels, ethanol-treated mutant mice had the highest level of IL-6 protein (Fig. 6, a and b). However, the -fold induction in protein levels was not as high in comparison with the changes at the mRNA level (Fig. 6, a and b).
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The STAT3 is a transcription factor implicated in the regulation of inflammatory signaling in the liver that is potently activated by IL-6 (Hong et al., 2002a). IL-6-induced STAT3 phosphorylation is a known antiapoptotic pathway that protects the liver from ethanol-induced liver injury (Hong et al., 2002a). Because IL-6 mRNA was induced by ethanol in the livers of wild-type and hepatocyte RXR-deficient mice, phosphorylated STAT3 was quantified by Western blot. Ethanol increased the level of phosphorylated STAT3 in wild-type mice, but not in the hepatocyte RXR-deficient mice (Fig. 7, a and b). IL-6 is highly induced by ethanol in the hepatocyte RXR-deficient mice, but the associated STAT-3 activation was not found. Thus, hepatocyte RXR deficiency alters many signaling pathways, including IL-6-mediated STAT3 activation. The underlying mechanism remains to be investigated.
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deficiency. Ethanol induced the level of Bcl-xL protein (2.7-fold) in the wild-type mice, but not in the mutant mice (Fig. 8, a and b). Furthermore, ethanol decreased Bcl-2 protein level in hepatocyte RXR-deficient mouse livers to 0.1-fold compared with the level in the mutant mice fed control diet (Fig. 8, a and c). Bcl-2 protein level in ethanol-fed wild-type was 0.6-fold compared with wild-type mice fed control diet. Although there was no difference between Bcl-xL protein expression in the two genotypes of ethanol-fed mice, Bcl-2 protein was significantly decreased in ethanol-fed mutant compared with wild-type mice treated with ethanol (Fig. 8, a and c). The induction of Bcl-xL protein by ethanol in the wild-type mice suggests that antiapoptosis and liver repair might have occurred in the wild-type mice after ethanol treatment. In contrast, the lack of induction of Bcl-xL and the marked decrease in Bcl-2 by ethanol in the mutant mice may account for the apoptotic effect of ethanol in the mutant mice (Fig. 1, b, d, and e). This is consistent with the morphological findings (Fig. 1, b–e).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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deficiency increases the susceptibility to alcohol-induced liver damage (Dai et al., 2003; Gyamfi et al., 2006). In earlier reports, we found that decreased hepatic SAM and reduced glutathione levels as well as increased alcohol clearance associated with decreased acetaldehyde elimination in hepatocyte RXR-deficient mice contributed to a greater susceptibility to ethanol-induced toxicity in the mutant compared with the wild-type mice (Dai et al., 2003; Gyamfi et al., 2006). In the present study, we find that the pathologic events during chronic ethanol feeding of the hepatocyte RXR-deficient mice might involve lack of L-FABP gene induction and elevated hepatic NEFA levels accompanied by NF-B activation and markedly up-regulation of proinflammatory cytokine and chemokine gene expression. In addition, ethanol-induced STAT3 and Bcl-xL activation were suppressed due to hepatocyte RXR deficiency. Although ethanol-fed wild-type mice had steatotic livers and higher CYP2E1 activity than ethanol-treated mutant mice, chronic alcohol feeding did not induce liver damage in the wild-type mice, most probably due to induction of the L-FABP gene, Bcl-xL protein, and minimal up-regulation of inflammatory cytokines and chemokines in wild-type mice.
Fatty acids are a major energy source and comprise an integral structural component of cell membranes as esterified phospholipids. Intracellular accumulation of free fatty acids may damage the cell membrane, thereby contributing to necrosis, inflammation, and progression to fibrosis and cirrhosis (Lieber, 2004). FABPs are proposed to function in the transport, metabolism, and storage of fatty acids (Desvergne et al., 1998) and to protect other proteins and membranes from the toxic effects of high fatty acid concentrations. Reports indicate that chronic ethanol ingestion increases concentrations of L-FABP (Shevchuk et al., 1991; Lieber, 2004). Deleterious accumulation of fatty acids in ethanol-fed female rats and in alcoholic women is the result of inadequate induction of cytosolic L-FABP, which might contribute to the sex differences in ethanol-induced toxicity (Shevchuk et al., 1991). Consistent with the published findings, we also observed induction of L-FABP mRNA and protein in wild-type mice by ethanol (Shevchuk et al., 1991; Lieber, 2004). The basal L-FABP mRNA level was significantly reduced and not inducible by ethanol in the hepatocyte RXR-deficient mice. L-FABP is a direct PPAR target gene (Landrier et al., 2004). The down-regulation of L-FABP gene expression is apparently due to a defect in the RXR/PPAR-mediated pathway (Wan et al., 2000a). Reduced L-FABP gene expression in the mutant mice was associated with elevated hepatic free fatty acids levels in the mutant mice after ethanol ingestion, which might contribute to the ethanol-induced liver injury we observed in the mutant mice. It was also observed that ethanol ingestion increased hepatic triglyceride and cholesterol levels in both mutant and wild-type mice; however, the ethanol-induced steatosis in the wild-type mice was not associated with liver injury. Our data are consistent with recent reports in the literature of a distinction between ethanol-induced steatosis and hepatocyte injury/inflammation observed in PAI-1 knockouts and in the different responses of complement knockouts fed ethanol (Bergheim et al., 2006; Pritchard et al., 2007).
Our data found that the SAM level was decreased after ethanol ingestion in wild-type and hepatocyte RXR-deficient mice. SAM is a precursor of reduced glutathione, a major cellular antioxidant, and the decrease in SAM levels may exacerbate oxidative stress induced by ethanol ingestion. Because the basal SAM level is much lower in hepatocyte RXR-deficient mice than wild-type mice, the reduced hepatic SAM level might be another priming factor besides reduced basal L-FABP levels for increased susceptibility of hepatocyte RXR-deficient mice to ethanol-induced liver injury.
The activation and translocation of NF-B plays a central role both in inflammatory responses and in liver injury (Tilg and Diehl, 2000). Induction of TNF and IL-6 genes is partially regulated through the NF-B sequence localized in their promoter region (Drouet et al., 1991; Galien et al., 1996). Consistent with the observation that ethanol-fed hepatocyte RXR-deficient mice had increased TNF and IL-6 mRNA expression compared with wild-type mice fed ethanol, we observed increased NF-B p65 activation in ethanol-fed hepatocyte RXR-deficient mice compared with ethanol-fed wild-type mice. In agreement with the observed up-regulation in NF-B activation, the gene expression of IL-1β, MIP-2, and PAI-1 controlled by NF-B was coordinately induced by ethanol in hepatocyte RXR-deficient mice, which may contribute to liver injury.
Another intriguing finding is the decreased basal TNF gene expression and its high inducibility by ethanol in the absence of hepatocyte RXR. TNF is produced in Kupffer cells as well as other sinusoidal cells in the liver following alcohol administration (Thurman, 1998). TNF promotes inflammation, leukocyte infiltration, tissue fibrosis, and cytokine production. One of the cell targets for TNF is parenchymal cells as verified by the ability of hepatocytes to express TNF receptors and chemokines in response to TNF (Liu et al., 2000). If TNF is produced by nonparenchymal cells exclusively, then the presence of cross talk between hepatocyte RXR and Kupffer cells needs to be examined. However, because TNF mRNA is detectable in unstimulated mouse liver, it is very likely that the TNF gene is expressed in hepatocytes. If this is the case, our findings suggest that RXR in hepatocyte might directly or indirectly control TNF gene expression in hepatocytes and Kupffer cells. The likelihood of an indirect effect is high, particularly given the increase in inflammatory cells in the livers of the mutant mice after ethanol ingestion.
STAT3 mediates the expression of a variety of genes in response to cytokines and thus plays a key role in hepatocyte proliferation and apoptosis prevention after liver injury (Cressman et al., 1996; Hong et al., 2002a). In response to cytokines and growth factors, including interferons, epidermal growth factor, IL-5, IL-6, and hepatocyte growth factor, STAT3 can be activated through phosphorylation by Janus kinase and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators (Desrivieres et al., 2006). IL-6 is readily detected in patients with ALD, and its concentration correlates with the extent of the disease (Gonzalez-Quintela et al., 1999). Furthermore, IL-6-deficient mice are more prone to ethanol-induced apoptosis in the liver (Hong et al., 2002b). Although IL-6 gene and protein expression were induced in ethanol-fed hepatocyte RXR-deficient mice, elevated IL-6 did not result in activation of STAT3 in the mutant mice as found in ethanol-fed wild-type mice. Consistent with this observation, although ethanol induced the antiapoptotic Bcl-xL protein in wild-type mice, such an increase was not seen in the mutant mice. Additionally, the Bcl-2 protein was not only significantly decreased in untreated hepatocyte RXR-deficient mice but further decreased by ethanol. Indeed, apoptosis does occur in the mutant mice, but not in wild-type mice. A greater production of TNF in female liver was responsible for a greater percentage of apoptotic cells in female rats than male rats fed ethanol (Colantoni et al., 2003). Taken together, it is suggested that the varied effect of ethanol on Bcl-xL and Bcl-2 as well as increased TNF gene expression in the mutant mice indicate that liver repair that includes regeneration, and antiapoptosis might be impaired in the hepatocyte RXR-null mice leading to liver injury. Further investigation on how RXR regulates STAT3 activation and inhibition of ethanol-induced apoptosis is warranted.
Retinoic acid (RA) inhibits hepatic macrophage TNF expression and is known to have anti-inflammatory effects (Motomura et al., 2001). LG268, which is an RXR-specific ligand, inhibits TNF mRNA expression (Motomura et al., 2001). Although anti-inflammatory action of RA was found to involve destabilization of the TNF mRNA, our current results suggest a prominent role by the RA receptor, RXR (Motomura et al., 2001). Our results demonstrating the important role of RXR in inflammation is also supported by findings that PPAR-deficient mice had prolonged inflammatory response after arachidonic acid or leukotriene B4 administration (Devchand et al., 1996). PPAR-deficient mice also display an exacerbated inflammatory response to LPS as well as to chronic high-fat diet ingestion (Delerive et al., 1999; Stienstra et al., 2007). It remains to be investigated which RXR-mediated pathway is important for alcohol-induced liver injury. Furthermore, it also remains to be elucidated whether RXR or the heterodimeric partner of RXR is a better target to treat and prevent the ethanol-induced inflammatory response.
In summary, the present study is the first to address the role of hepatocyte RXR in the expression of proinflammatory cytokines and chemokines in response to alcohol ingestion. Taken together, our results revealed dysregulation of lipid metabolism as well as an increased inflammatory response due to hepatocyte RXR deficiency. Reduced L-FABP mRNA and protein levels, elevated hepatic free fatty acid level and reduced hepatic SAM content might prime the hepatocyte RXR-deficient mice to be susceptible to ethanol toxicity. Enhanced activation of NF-B, a pleiotropic transcription factor that triggers the production of other cytokines and chemokines and suppression of STAT3 phosphorylation coupled with increased apoptosis in the mutant mice, is a critical mechanism leading to ethanol-induced liver injury in hepatocyte RXR-deficient mice.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: RXR, retinoid X receptor ; LXR, liver X receptor; PPAR, peroxisome proliferator-activated receptor; ALD, alcoholic liver disease; SAM, S-adenosylmethionine; NF, nuclear factor; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL, interleukin; MIP, macrophage inflammatory protein; PAI-1; plasminogen activator inhibitor type-1; STAT, signal transducer and activator of transcription factor; H&E, hematoxylin and eosin; ALT, alanine aminotransferase; NEFA, nonesterified fatty acid; SAH, S-adenosylhomocysteine; L-FABP, liver fatty acid binding protein; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RA, retinoic acid.
Address correspondence to: Dr. Yu-Jui Yvonne Wan, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7417. E-mail: ywan{at}kumc.edu
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) or ethanol (
) for 6 weeks. ALT level (a) and assessment of apoptosis by H&E-stained liver sections (b–e) were determined. Liver sections showing apoptotic cells (200x) and insert (400x) (indicated by arrows, b); mitotic cells, 400x (indicated by arrowheads, c); and apoptotic and mitotic cells, 400x (indicated by arrows and arrowheads, d) in hepatocyte RXR
, P < 0.05 versus wild-type mice fed ethanol diet. ##, P < 0.01; #, P < 0.05 versus hepatocyte RXR
