Destabilization of TNF-alpha mRNA by retinoic acid in hepatic macrophages: implications for alcoholic liver disease

Kenta Motomura1, Mitsuru Ohata1, Michael Satre3, and Hidekazu Tsukamoto1,2,4

Departments of 1 Medicine and 2 Pathology, University of Southern California School of Medicine, Los Angeles 90033; 3 Department of Nutrition, University of California, Davis 95616; and 4 Veterans Affairs Greater Los Angeles Healthcare System, Sepulveda, California 91343


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Retinoic acid (RA) inhibits hepatic macrophage (HM) cytokine expression, and retinoids are depleted in alcoholic liver disease (ALD). However, neither the causal link between the two nor the mechanism underlying RA-mediated HM inhibition is known. The aim of the present study was to determine the mechanism of RA-induced inhibition of HM tumor necrosis factor (TNF)-alpha expression and the relevance of this regulation to ALD. Treatment with all-trans RA (500 nM) caused a 50% inhibition in lipopolysaccharide (LPS)-stimulated TNF-alpha expression by cultured normal rat HM. The mRNA levels for inducible nitric oxide synthase, interleukin (IL)-6, IL-1alpha , and IL-1beta were also reduced, whereas those for transforming growth factor-beta 1, MMP-9, and membrane cofactor protein-1 were unaffected. The inhibitory effect on TNF-alpha expression was reproduced by LG268, a retinoid X receptor (RXR)-specific ligand, but not by TTNPB, an RA receptor (RAR)-specific ligand. RA did not alter LPS-stimulated NF-kB and activation protein-1 binding but significantly decreased TNF-alpha mRNA stability in HM. HM isolated from the ALD model showed significant decreases in all-trans RA (-48%) and 9-cis RA (-61%) contents, RA response element (RARE) binding, and mRNA levels for RARbeta , RXRalpha , and cytosolic retinol binding protein-1, whereas TNF-alpha mRNA expression was induced. TNF-alpha mRNA stability was increased in these cells, and an ex vivo treatment with all-trans RA normalized both RARbeta and TNF-alpha mRNA levels. These results demonstrate the RA-induced destabilization of TNF-alpha mRNA by cultured HM and the association of RA depletion with increased TNF-alpha mRNA stability in HM from experimental ALD. These findings suggest that RA depletion primes HM for proinflammatory cytokine expression in ALD, at least in part, via posttranscriptional regulation.

Kupffer cells; tumor necrosis factor-alpha mRNA stability; retinoic acid receptors; retinoid X receptors; retinoic acid response element


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TUMOR NECROSIS FACTOR (TNF)-alpha is a pleiotropic cytokine with diverse biological effects on all mammalian cells. Expression of this cytokine is induced during endotoxemia, infection, and tissue injury. This response is an integral component of host defense mechanisms as exemplified by the acute-phase response (25) and the role of comitogen fulfilled by this cytokine in liver regeneration (9). On the other hand, excessive or sustained TNF-alpha expression is implicated in cell death, matrix remodeling, microcirculation disturbances, and acute and chronic inflammation. TNF-alpha is considered the central mediator in liver damage induced by a variety of hepatotoxic conditions, because administration of antibodies against TNF-alpha (7, 16) or soluble receptor for this cytokine (8) offers hepatoprotective effects under these conditions. Hepatic macrophages (HM) serve as one of the major sources of TNF-alpha , and its expression by HM is upregulated in liver injury (21, 29). In the homeostatic response to injury or infection, expression of acute-phase cytokines such as TNF-alpha , and interleukin (IL)-1 is induced but also tightly regulated by negative feedback mechanisms to allow downregulation of the cytokines after achieving physiological or immunological effects. However, as in other chronic inflammatory diseases, such as rheumatoid arthritis and Crohn's disease, TNF-alpha induction is often sustained in chronic liver disease. This may be due to the persistent presence of causal factors for hepatocellular injury (e.g., viral infection, cholestasis, alcohol) resulting in sustained inflammatory responses. Alternatively, HM cytokine expression may be continuously upregulated due to dysregulated control mechanisms. For the latter possibility, several hypotheses have been proposed, including TNF-alpha promoter polymorphism (1, 13) and defective release of IL-10 (26). However, these postulates still remain to be validated, and the molecular mechanisms responsible for sustained TNF-alpha induction by HM in chronic liver disease are still poorly defined.

Vitamin A and its metabolites are essential for regulation of cell proliferation and differentiation (see Ref. 44 for review). This lipid-soluble vitamin is stored mainly in liver, and its level is decreased in chronic liver disease (27). Besides the potent antiproliferative effects on various cell types (30), retinoic acids (RA) are known to exert anti-inflammatory effects (3, 42). These effects appear to be mediated through downregulation of Th1 cytokines such as interferon-gamma (5), TNF-alpha (32, 34), and IL-12 (35). RA also inhibits transcription of metalloproteinase genes (37, 43), thereby contributing to its anti-inflammatory effects. Most, if not all, of these RA-induced effects are considered to be mediated by antagonistic cross-coupling of transcription factors such as activation protein (AP)-1 (37, 43) or nuclear factor (NF)-kappa B (35) with RA receptors (RAR) and/or retinoid X receptors (RXR).

RAR and RXR are members of the steroid/thyroid hormone receptor superfamily of ligand-dependent transcription factors. RAR-dependent signaling is transduced via a heterodimeric complex of RAR/RXR activated by binding of all-trans RA or 9-cis RA to RAR. RXR-dependent signaling is transduced by a ligand-activated homodimer of RXR or RXR heterodimerized with one of other nuclear receptors, including vitamin D, estrogen, thyroid hormone, peroxisome proliferator-activated (PPAR) receptors, and chicken ovalbumin upstream promotor (COUP), (see Ref. 31 for review). To better understand the mechanism of RA-mediated regulation in HM, we have recently demonstrated the expression of RAR and RXR subtypes and RA response element (RARE) binding in HM isolated from normal rat (40). This study revealed an ~5- to 10-fold higher expression of RXRalpha and -beta mRNA in HM compared with hepatic stellate cells and other nonparenchymal liver cell types, suggesting the potential biological importance of this class of receptors in HM. In support of this notion, RA-mediated suppression of TNF-alpha secretion by HM was recently shown to be RXR dependent (34).

The present study was undertaken to investigate the molecular mechanism by which RA suppresses TNF-alpha expression by cultured rat HM. Furthermore, the relevance of this regulation to the in vivo biology was examined by studying HM isolated from a rat model of alcoholic liver disease (ALD), in which induced TNF-alpha expression and vitamin A depletion were expected. Our study reveals a novel finding of RA-mediated destabilization of TNF-alpha mRNA in cultured HM. Furthermore, the analysis of HM from the in vivo model demonstrates concomitant RA depletion and increased TNF-alpha mRNA stability, suggesting a mechanistic link between them. These results thus support the possibility that HM from experimental ALD are primed for TNF-alpha upregulation, due at least in part to diminished RA-mediated destabilization of TNF-alpha mRNA.


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Animals. Normal male Wistar rats weighing 500-650 g (5-6 mo old) were used for isolation of HM as described below. This age of the animals was selected to approximate the age of Wistar rats at the end of 9-wk alcohol feeding, as described in HM isolation and culture. A detailed description of the rat model of ALD has been reported elsewhere (53). In brief, male Wistar rats weighing 350-375 g were surgically implanted with long-term gastrostomy catheters to enable continuous intragastric infusion of a high-fat diet (35% cal fat as corn oil at an ethanol dose of 8 g · kg-1 · day-1) plus an increasing concentration of ethanol or isocaloric dextrose solution. Because an increase in ethanol intake was achieved by adding ethanol to the basal diet, the total caloric intake increased and the caloric percentages of macronutrients changed during the experiment. At the 9th wk, the ethanol dose was increased to 13.5 g · kg-1 · day-1, the total caloric intake increased to 208 cal · kg-1 · day-1, and the macronutrient distributions of the calories were 20.2% as protein, 6.6% as carbohydrate, 28.7% as fat, and 44.5% as ethanol. This regimen was previously shown to result in induction of macro- and microvesicular steatosis, balloon cell degeneration, focal necrosis, and mononuclear cell infiltration with increased proinflammatory cytokine gene expression by HM (54). The animal protocol described in this study was approved by the Institutional Animal Care and Use Committee of the University of Southern California and was performed in accordance with the guidelines of the National Institutes of Health.

HM isolation and culture. HM were isolated from normal chow-fed, ethanol-fed, and pair-fed control rats by a modification of the method previously described (21). The liver was sequentially digested with pronase and type IV collagenase by in situ perfusion. Parenchymal cells were removed by centrifugation of the digest at 50 g for 2 min, and nonparenchymal cells were laid on top of the four density gradients of arabinogalactan (Larcoll, Sigma Chemical, St. Louis, MO). The gradients were centrifuged at 21,400 rpm for 45 min at 25°C using a Beckman SW41 Ti rotor (Beckman Instruments, Fullerton, CA). A relatively pure fraction of HM was recovered from the interface between 8 and 12% arabinogalactan. The purity of HM was assessed by phase contrast microscopy and latex bead (1 µm) phagocytosis and exceeded 87%. Viability was assessed by the trypan blue exclusion test and always exceeded 95%. For in vitro experiments to test effects of RA on HM, the cells isolated from normal rats were seeded at 30 × 106 cells/100-mm dish and were further purified by removing nonadherent cells after a 3-h incubation in RPMI 1640 with 10% fetal calf serum (FCS). The medium was changed to RPMI 1640 with 5% FCS thereafter, and the cells were cultured for 24-36 h until the experiments. This adherence method achieved a purity that was always >95%. Before the experiments, the cells were washed twice with PBS and incubated in a serum-free medium. To investigate the positive signaling of RA, the cells were treated with all-trans RA (500 nM) or with DMSO as a vehicle control, and total RNA and nuclear proteins were extracted at the indicated time points over 24 h. To examine the effects of RA on cytokine expression by cultured HM, the cells were pretreated with all-trans RA at 500 nM or DMSO for 16 h and stimulated with lipopolysaccharide (LPS; 10 ng/ml). At 1, 4, and 6 h after LPS stimulation, total RNA was extracted for RT-PCR or Northern blot analysis. To assess the role of RAR and RXR in RA-mediated effects, an RAR-specific ligand, TTNPB (Ro 13-7410), and an RXR-specific ligand, LG268, were also used at equimolar concentration (500 nM). TTNPB and LG268 were kindly provided to us by Dr. M. Klaus, Hoffmann-La Roche, Basel, Switerland, and Dr. R. Heyman, Ligand Pharmaceuticals, San Diego, CA, respectively.

Messenger RNA stability assay. Cultured HM were stimulated with LPS (10 ng/ml) after overnight pretreatment with RA or vehicle alone, as described above. After 120 min, actinomycin D (5 µg/ml) was added to the cells, and total RNA was collected at the indicated time points. The RNA samples were analyzed for TNF-alpha and beta -actin mRNA levels by Northern blot analysis, as described in Northern blot analysis. To assess TNF-alpha mRNA stability in HM from the ethanol-fed and pair-fed control animals, the isolated HM (25 × 106 cells) were immediately incubated in serum-free RPMI 1640 with all-trans RA (100 nM) or DMSO for 4 h in a 50-ml sterile Falcon tube in a tissue culture incubator. This ex vivo method was used instead of culturing the cells on a plastic dish to assess the in vivo changes while minimizing artifactual effects caused by cellular activation seen after the latter method. After the incubation, total RNA was extracted and used for Northern blot analysis as described in detail in Northern blot analysis. Densitometric data of TNF-alpha mRNA from autoradiograms were standardized by those of 18S rRNA, and the mRNA decay was assessed by expressing the data as the percentage of the mRNA level at time 0 for each group. These data were then entered into the SAAM II program (SAAM Institute, Univ. of Washington, Seattle, WA) to produce the single-exponential fitting and to calculate the half life.

Retinoid extraction and analysis. Freshly isolated HM (50 × 106 cells) were immediately subjected to retinoid extraction by use of the method previously described (39, 45). Briefly, all manipulations of samples were carried out in a darkened room under yellow light to prevent photooxidation. Individual samples of HM were placed in amber tubes, rinsed once with phosphate-buffered saline at 4°C, sonicated in 1.5 vol of deionized water containing 0.01% sodium dodecyl sulfate (SDS), and extracted sequentially with 2 volume of butanol and acetonitrile (1:1) containing 1 mg/ml butylated hydroxytoluene and with 0.25 vol of saturated K2HPO4. After centrifugation, the organic phase was carefully removed, filtered into a chilled amber Eppendorf tube, and taken to dryness under argon gas. The sample residue was then reconstituted in 150 µl of ethanol-acetonitrile (3:1) and stored at -80°C until assayed. Before extraction, each sample was spiked with a known concentration of synthetic retinoid (RO13-7410; Hoffman-LaRoche, Nutley, NJ) as an internal standard to assess extraction efficiency. Analysis of retinoids was carried out by high-performance liquid chromatography (HPLC) using a system consisting of a Bio-Rad ValueChrom computer workstation and software (Bio-Rad Laboratories, Hercules, CA) and a model SPD-10A UV-visible detector (Shimazu Scientific Instruments, Colombia, MD), with a reverse-phase Nova-Pac C18 analytic column (Waters Associates, Milford, MA). The samples were eluted under isocratic conditions with the use of an elution mobile phase consisting of methanol-tetrahydrofuran-acetonitrile-0.01 M ammonium acetate (60:10.5:3.5:26) with detection at 350 nm/0.002 absorbance units full scale, with quantification by computer integration of peak areas with reference to standard curves from authentic retinoid standards. Retinoids used as standards were obtained from Fluka Chemical (Ronkonkoma, NY) and Gohman-La Roche. The final results were standardized by the cell number.

RNA and nuclear protein extraction. The purified fraction of isolated HM was immediately subjected to total RNA extraction by the guanidium-phenol-chloroform method (6) and nuclear protein extraction by the method of Shreiber et al. (46). RNA and nuclear protein concentrations were determined by absorbance at 260 nm and a Bradford assay, respectively.

RT-PCR. Total RNA (3 µg) was reverse transcribed into cDNA with the use of 600 U of Molony murine leukemia virus reverse transcriptase and oligo(dT)15 as a primer at 37°C for 60 min. The synthesized cDNA for retinoid receptors (RARbeta and RXRalpha ), cytosolic retinol binding protein (CRBP)-I, cytokines [TNF-alpha , transforming growth factor (TGF)beta , and ILs), chemokine [macrophage inflammatory protein (MIP)-1], matrix metalloproteinase (MMP) 9, inducible nitric oxide synthase (iNOS), and beta -actin were amplified using specific sets of primers (17, 36, 39, 47, 54, 56). Each PCR mixture contained 0.4 µM of a specific set of primers, 0.2 mM of each dNTP, 2.5 U Taq polymerase, and 1.5 mM MgCl2 in a PCR buffer. The PCR procedure consisted of 18-35 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 30 s, and extension at 72°C for 90 s, with initial denaturation of sample cDNA at 94°C for 3 min before PCR and an additional extension period of 10 min after the last cycle. To assure that the PCR amplification did not reach the plateau phase, PCR with each set of primers was performed using at least 2-3 different cycles. The number of cycles shown to be optimal for each gene was as follows: RARbeta , 25 or 30; RXRalpha , 35; CRBP-1, 29; TNF-alpha , 18 for cultured HM and 25 for HM from the alcohol model; IL-1alpha , IL-1beta , and membrane cofactor protein (MCP)-1, 18; IL-6, iNOS, and MMP-9, 20; and TGFbeta 1, 22.

Northern blot analysis. RNA samples amounting to 10-20 µg were electrophoresed on formaldehyde-containing agarose gels and transferred to nylon membranes (Nytran; Suhleicher & Schuell, Keene, NH). Ethidium bromide staining was used to assess the equal loading and the intact nature of RNA samples. Northern blot hybridization was performed with complementary DNA for rat TNF-alpha (21, 29) and human RARbeta (2), which were kindly provided by Dr. Karl Decker, University of Freiburg, Germany, and Dr. Ronald Evans, The Salk Institute, San Diego, CA, respectively. The random priming method was used to label cDNA by [32P]dCTP. The filters were prehybridized and hybridized at 52°C for TNF-alpha and 18S rRNA and at 42°C for RARbeta in 10× Denhart's solution, 0.5% SDS, 50 µM Tris, 5 µM EDTA, 5× standard sodium citrate (SSC), 150 mg/ml sonicated salmon sperm DNA, and 10% dextran sulfate. After 18 h of hybridization, the filters were washed twice at room temperature in 2× SSC and 0.1% SDS; twice at 50°C for TNF-alpha and 18S rRNA or at 40°C for RARbeta in 2× SSC and 1% SDS; and twice at 50°C for TNF-alpha and 18S rRNA or at 40°C for RARbeta in 0.1× SSC and 0.1% SDS, with each washing period lasting 30 min. Autoradiography was performed with Kodak XAR film (Eastman Kodak, Rochester, NY) at -80°C and phosphoimager screen (Molecular Dynamics, Sunnyvale, CA) at room temperature. The densitometric analysis of mRNA levels was performed by the computer software ImageQuant for Power Macintosh v1.2 (Molecular Dynamics) and was standardized by the phosphoimager data of 18S rRNA hybridization.

Electrophoretic mobility shift assay. Nuclear protein extract (5-10 µg) was incubated with a reaction mixture [20 mM HEPES, pH 7.6, 100 mM KCl, 0.2 mM EDTA, 2 mM dithiothreitol, 20% glycerol, 200 µg/ml poly(dI-dC)] on ice for 10 min. One to two nanograms of a 32P-labeled double-stranded RARE, NF-kappa B, or AP-1 oligonucleotide were added to the mixture and incubated for an additional 20 min. The reaction mixture was then resolved on a 6% nondenaturing polyacrylamide gel in 0.5 × 45 mM tris(hydroxymethyl)aminomethane, 45 mM boric acid, 1 mM EDTA. The gel was dried and subjected to autoradiography at -80°C. The sequences of the RARE, NF-kappa B, and AP-1 were previously described (29, 39). For supershift assay [electrophoretic mobility shift assay (EMSA)], we used polyclonal antibodies against human (h)RARalpha and hRXRalpha (kindly provided by Dr. Ronald M. Evans) and those against P65, P50, c-Fos, and c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA).

TNF-alpha protein determination. The culture medium was collected 6 h after addition of LPS (10 ng/ml) from DMSO- or all-trans RA-treated HM in culture, and the concentration of TNF-alpha protein was determined using an ELISA kit for mouse TNF-alpha (Quantikine M, R&D Systems, Minneapolis, MN).

Statistical analysis. All data are expressed as means ± SD. The significance of the difference between the groups was assessed using standard or paired t-test.


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Before investigating negative regulation by RA of HM cytokine gene expression, it was important to establish that HM possess RA-induced positive transcriptional regulation. Cultured HM from normal rats were treated with all-trans RA (500 nM), and RARE binding and RARbeta and CRBP-1 mRNA levels were examined as the positive controls. As shown in Fig. 1A, addition of all-trans RA caused increased RARE binding from 15 min and lasting for 2 h. The specificity of the RARE binding was verified by complete competition with a 100× excess amount of the unlabeled RARE probe (data not shown) and a supershift assay using antibodies against RARalpha or RXRalpha (Fig. 1B). This change was accompanied by induced mRNA expression of RARbeta and RXRalpha (Fig. 2), which are known to contain functional RARE (50) or to be induced by RA (55), respectively. In particular, RARbeta was selected because this gene has the strongest RARE promoter activity (50). Thus these results served as evidence for the existence of RA-mediated positive transcriptional regulation in HM.


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Fig. 1.   Retinoic acid (RA)-mediated positive transcriptional regulation in cultured rat hepatic macrophages (HM). A: RA response element (RARE) binding was examined using nuclear extracts prepared from cultured rat HM treated with all-trans RA (500 nM) for the indicated time. Note that increased RARE binding started at 15 min after RA addition and lasted for 2 h (arrow). B: supershift assays confirm that the binding complex contains RA receptor (RAR)alpha and retinoid X receptor (RXR)alpha .



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Fig. 2.   Induced RARbeta and RXRalpha mRNA by RA in cultured HM. Treatment of rat cultured HM with all-trans RA (500 nM) for 1 h increased RARbeta and RXRalpha mRNA levels as shown by RT-PCR analysis. Semiquantitative densitometric data were standardized with beta -actin and compared (bottom). Values indicated in bars are means of 3 separate experiments.

RA was also previously shown to inhibit LPS-induced TNF-alpha secretion by murine peritoneal macrophages (32) and HM (34). Indeed, we confirmed this finding by demonstrating a 50% inhibition in LPS-stimulated release of this cytokine by cultured HM exposed to 500 nM all-trans RA (Fig. 3). In addition, we also showed that this inhibition was accompanied by a concomitant reduction in TNF-alpha mRNA level (Fig. 4A), demonstrating that the RA-mediated effect was at the pretranslational level. To assess whether the observed effect of RA is mediated via RAR, RXR, or both, the cultured HM were treated with an equimolar concentration of TTNPB (Ro 13-7410), an RAR-specific agonist, or LG268, an RXR-specific agonist, and LPS-stimulated TNF-alpha mRNA expression was examined. As shown in Fig. 4B, TTNPB caused no changes, whereas LG268 reproduced the inhibitory effect observed with all-trans RA. Standardization of densitometric data for TNF-alpha mRNA levels with those for 18S rRNA revealed 33 and 57% reductions in TNF-alpha mRNA expression by 100 and 500 nM LG268, respectively. Thus this result suggests that RA-induced inhibition of TNF-alpha expression is mediated via RXR but not via RAR.


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Fig. 3.   RA reduces lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF)-alpha production by HM. Pretreatment of cultured rat HM with all-trans RA (500 nM) inhibited TNF-alpha secretion stimulated with LPS (10 ng/ml) for 6 h, as determined by a commercially available ELISA kit. Values shown are means ± SD from 4 sets of experiments.



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Fig. 4.   RA reduces LPS-induced TNF-alpha mRNA levels in HM. A: total RNAs were extracted from the RA (500 nM) or vehicle (DMSO)-treated HM after stimulation with LPS (10 ng/ml) for 1 h and analyzed for TNF-alpha mRNA levels by Northern blot analysis. Note suppressed TNF-alpha mRNA levels in the RA-treated cells. 18S rRNA was used as an internal control. B: RA-mediated inhibition of TNF-alpha expression is RXR dependent. HM cultures were treated with DMSO (Control), TTNPB (500 nM), an RAR-specific ligand, or LGD (LG268), an RXR-specific ligand (100 or 500 nM) for 16 h, followed by LPS stimulation for 1 h. TNF-alpha mRNA levels were similarly analyzed by Northern blot analysis. Note that LG268, but not TTNPB, reproduced the all-trans RA-mediated inhibitory effect on TNF-alpha mRNA.

Next, we examined whether the RA-induced inhibition is specific for TNF-alpha or is also observable for other HM-specific genes. To address this question, we performed semiquantitative RT-PCR to screen the effects of RA on various genes. RA selectively decreased LPS-induced mRNA levels for TNF-alpha , iNOS, IL-1alpha , and IL-6, but other genes, such as anti-inflammatory cytokines (TGFbeta and IL-10), MMP-9, and MCP-1 were unaffected (Fig. 5). Similar differential effects were observed for the cells stimulated with LPS for 1 h (Fig. 5) and 6 h (data not shown). Thus the demonstrated differential effects were not likely due to RA-induced changes in the kinetics of mRNA expression for these diverse genes.


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Fig. 5.   Differential effects of RA on LPS-stimulated HM gene expression. To screen for the effects of RA (500 nM) on other HM-specific genes, semiquantitative RT-PCR analyses were performed using total RNA prepared after LPS stimulation for 1 h. Densitometric analysis and standardization with beta -actin showed significantly reduced mRNA levels for TNF-alpha , inducible nitric oxide synthase, interleukin (IL)-6, and IL-1alpha , whereas other genes were not affected. MMP-9, matrix metalloproteinase 9; MCP-1, membrane cofactor-1. Values shown are means ± SD from 4 separate sets of experiments. *P < 0.05, P < 0.01 vs. DMSO control.

Because the inflammatory mediators inhibited by RA are known to be regulated predominantly by transcription factors NF-kappa B and AP-1, we examined DNA binding of these factors in nuclear extracts collected from the RA-treated HM that were stimulated with LPS for 1, 4, or 6 h. RA treatment caused no differences in these parameters at any time points. Thus these results demonstrate that LPS-induced binding of these two transcription factors is not affected by RA.

Posttranscriptional regulation has increasingly been recognized as one of the pivotal mechanisms by which regulation of TNF-alpha synthesis is attained. To test this possibility for the observed RA effect, we examined TNF-alpha mRNA stability in the RA-treated HM. The TNF-alpha mRNA level was already reduced in RA-treated HM at time 0 compared with the DMSO-treated cells. Densitometric data for TNF-alpha mRNA were standardized with 18S rRNA data and were expressed as the percentage of the initial (time 0) values. The single-exponential fitting of the decay data based on standardized TNF-alpha mRNA levels for each group revealed a significantly enhanced degradation of TNF-alpha mRNA in the RA-treated cells after the addition of actinomycin D compared with the DMSO (vehicle)-treated control cells (Fig. 6). Thus this result demonstrates that RA treatment reduces TNF-alpha mRNA stability and suggests that this posttranscriptional effect is, at least in part, responsible for RA-mediated inhibition of TNF-alpha expression by LPS-stimulated cultured HM.


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Fig. 6.   RA destabilizes TNF-alpha mRNA in HM. Cultured HM were treated with DMSO or all-trans RA (500 nM, RA) for 16 h and subsequently stimulated with LPS (10 ng/ml). After 2 h, actinomycin D was added to the cultures to stop RNA synthesis, and the decay of TNF-alpha over a 2-h period was assessed by Northern blot analysis (A). Densitometric data standardized with 18S rRNA were expressed as percent initial (time 0) TNF-alpha mRNA level in each group (B). Results shown are means ± SD from 4 separate sets of experiments. The SAAM II program was used to produce the single-exponential fits to the data for each group and to derive half-life (t1/2) values. Note an accelerated decay of TNF-alpha mRNA in RA-treated HM () compared with DMSO controls (open circle ). *P < 0.05. The calculated t1/2 for TNF-alpha mRNA in the RA-treated cells (1.27 ± 0.22 h) was significantly reduced vs. that in the control cells (2.01 ± 0.26 h).

We then asked how relevant this finding is to the in vivo biology of HM. It is well known that retinoid levels are severely reduced in various types of liver disease such as fulminant hepatitis and ALD (27), where induction of TNF-alpha is a common feature (21). We hypothesized that this association may reflect the reverse situation of the effect of RA demonstrated in cultured HM. To test this hypothesis, we isolated HM from the rat model of alcoholic liver injury. The HPLC analysis of retinoid extracts demonstrated significant 48 and 61% decreases in the content of all-trans and 9-cis RA, respectively, compared with the cells from pair-fed controls (Table 1). The retinol content was also significantly reduced. These results established that HM from the model have the reduced RA contents.

                              
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Table 1.   Retinoid content in HM

Next, we examined whether the demonstrated RA depletion in the HM from the alcohol-fed animals resulted in functional consequences. For this question, we first analyzed the RARE binding of nuclear extracts prepared from the cells. As depicted in Fig. 7, the EMSA results clearly demonstrated diminished RARE binding in the HM nuclear extracts from the alcohol-fed animals. Furthermore, semiquantitative RT-PCR analysis of total RNA prepared from these cells showed coordinated reductions in the mRNA levels of RARbeta , RXRalpha , and CRBP-1, which contain functional RARE (Fig. 8, left), whereas the TNF-alpha mRNA level as determined by Northern blot analysis was expectedly upregulated (Fig. 8, right). Therefore, these results support the notion that, due to RA deficiency, RARE binding decreased and RA-mediated positive regulation of genes such as RARbeta , RXRalpha , and CRBP-1 was suppressed in HM from the alcohol model and that RA-mediated negative regulation of TNF-alpha expression was also inhibited, resulting in TNF-alpha upregulation.


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Fig. 7.   RARE binding is diminished in HM from alcohol-fed animals. Nuclear extracts prepared from alcohol- and pair-fed control animals were analyzed for RARE binding by electrophoretic mobility shift assay. Note apparent diminution of RARE binding in the HM from alcohol-fed animals.



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Fig. 8.   Messenger RNA expression of RARE-containing genes is reduced in HM from alcohol-fed animals. Total RNA extracted from alcohol-fed and control animals were analyzed for RARbeta , RXRalpha , and cytosolic retinol binding protein (CRBP)-I by RT-PCR and for TNF-alpha by Northern blot analysis. This bar graph summarizes standardized densitometric data. Note significantly decreased mRNA levels of all 3 RARE-containing genes and expected TNF-alpha induction in alcohol-fed animals. Values are means ± SD from 4-5 pairs of animals.

To further test the role of RA depletion in TNF-alpha induction in the HM from the alcohol-fed animals, the cells were treated overnight with all-trans RA. This ex vivo treatment normalized both suppressed RARbeta and induced TNF-alpha mRNA expression, as shown in Fig. 9. In our in vitro experiment with the RA-treated cultured HM, the decreased mRNA stability was shown to be responsible, at least in part, for the suppressed TNF-alpha expression. Therefore, we examined next whether the induced TNF-alpha expression seen in the RA-depleted HM from the alcohol-fed rats was associated with increased TNF-alpha mRNA stability. After the first experiment, we learned that the TNF-alpha mRNA decay was much faster in the freshly isolated HM compared with cultured HM and that more early time points were required for accurate analysis. We repeated the experiment with the time points of 0.25, 0.5, 1, and 1.5 h (Fig. 10). The fitting of the data revealed that the mRNA decay in the alcohol group was significantly delayed (t1/2: 1.24 ± 0.04 vs. 0.88 ± 0.04, P < 0.05).


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Fig. 9.   Ex vivo RA treatment normalizes RARbeta and TNF-alpha mRNA levels in HM from alcohol-fed animals. HM from alcohol-fed animals were treated ex vivo with DMSO (-RA) or all-trans RA (100 nM, +RA) for 4 h, and steady-state mRNA levels for RARbeta , TNFalpha , and 18S rRNA were examined by Northern blot analysis. Note that RA treatment increases RARbeta mRNA and reduces TNF-alpha mRNA levels.



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Fig. 10.   Increased TNF-alpha mRNA stability in HM from alcohol-fed rats. A: HM isolated from alcohol-fed and pair-fed control animals were incubated with actinomycin D for the indicated duration, and the decay of TNF-alpha mRNA was analyzed by Northern blot analysis. Fitting analysis of the decay data for the 2 groups revealed the significantly increased half-life in HM from the alcohol-fed animals (1.24 ± 0.04 h) vs. cells from the controls (0.88 ± 0.04). B: Data shown are means ± SD from 4 pairs of animals.


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DISCUSSION
REFERENCES

Key findings of the present study can be summarized as follows. 1) RA inhibits TNF-alpha expression by HM in an RXR-dependent manner; 2) this effect is mediated, at least in part, via destabilization of TNF-alpha mRNA; 3) the relevance of this regulatory mechanism to the in vivo biology is supported by the demonstration of the reverse situation in HM from experimental alcoholic liver injury, where the cellular depletion of RA is closely associated with the increased TNF-alpha mRNA stability and steady-state level; and 4) the ex vivo treatment of these cells with RA normalizes TNF-alpha expression. These findings are novel and have important therapeutic implications for immune modulation of alcoholic liver injury.

The synthesis of TNF-alpha is regulated at the transcriptional and posttranscriptional and translational and posttranslational levels (15, 24). In particular, posttranscriptional control has recently received much interest as it was shown to play a critical role in various immunological regulation including IL-10-mediated downregulation of TNF-alpha expression (23), as well as in induction of this cytokine in response to trauma (12). We may now add alcoholic liver injury and RA treatment to this list. The 3' untranslated region (UTR) of TNF-alpha mRNA contains multiple repeats of an AUUU motif (adenine uracil-rich element, ARE) similar to that identified in other cytokine and proto-oncogene mRNAs (14, 19). Binding of proteins to the AREs destabilizes the mRNAs and/or exerts translational repression, as seen in resting macrophages. Upon stimulation of macrophages with LPS, this repression is overcome, and TNF-alpha mRNA is efficiently translated (14). The biological significance of the posttranscriptional control is underscored by the spontaneous induction of severe systemic inflammatory disorders in transgenic mice expressing TNF-alpha lacking its 3'-UTR (22) or mice deficient in tristetraprolin, a zinc-binding protein shown to destabilize TNF-alpha mRNA via its binding to the ARE (51). It is currently unknown whether the ARE-dependent mechanism mediated the destabilization of TNF-alpha mRNA in RA-treated HM or whether an inhibition of this mechanism was responsible for the increased TNF-alpha mRNA stability seen in HM from the rat model of alcoholic liver injury. These are intriguing possibilities that need to be tested in future studies.

Retinoids are known to modulate functions of macrophages and lymphocytes (5, 32, 34, 35). The hepatic content of retinoids is depleted in ALD (27), where immunological mechanisms are considered to play pathogenetic roles. The results from the present study offer a mechanistic link to these associations by putting forward the hypothesis that RA deficiency in HM primes the cells for induction of TNF-alpha synthesis in alcoholic liver injury via diminished RA-mediated destabilization of TNF-alpha mRNA. Effects of retinoids on gene regulation take place at different levels. Transcriptional regulation by retinoids has been studied extensively and appears to occur in intricate and diverse manners depending on the gene and host cell type. Inhibition of AP-1 by natural or synthetic retinoids may underlie their antiproliferative effects (11) or suppressed transcription of MMP-1 (43) and MMP-3 (37). This negative effect on AP-1 may be mediated via downregulation of c-fos expression (4) or antagonistic direct interaction with AP-1 (37, 43). RA also inhibits IL-12 expression in murine macrophages through interaction of RXR with NF-kappa B (35), but RA-mediated inhibition of tissue factor transcription in LPS-stimulated human monocytes does not appear to involve AP-1 or NF-kappa B (38). Our results also did not demonstrate RA-induced effects on NF-kappa B and AP-1 binding in LPS-stimulated HM. However, these results simply reflect the status of binding to the cis-regulatory elements and do not provide assessment on the TNF-alpha promoter activity governed by these elements. Moreover, our results demonstrating the posttranscriptional regulation of TNF-alpha expression by RA do not exclude the possibility of RA-mediated transcriptional effects on HM either in vitro or in alcoholic liver injury. In fact, the most recent results from our ongoing study demonstrate RA-induced inhibition of TNF-alpha promoter activity in cultured HM (unpublished observation). Thus it is probable that RA has both transcriptional and posttranscriptional effects on the TNF-alpha gene. The present study had its main focus on demonstration of the latter mechanism. Our ongoing study investigates whether RA-mediated inhibition of TNF-alpha promoter activity is accompanied by inhibition of TNF-alpha transcription and how RA facilitates these transcriptional effects.

In contrast, RA has been shown to induce IL-1 gene expression by human peripheral blood mononuclear cells, the effect that was attributed to transcriptional induction but shown to be regulated at the posttranscriptional level (20). In that same study, TNF-alpha mRNA expression was also induced, but in a fluctuating manner. Reasons for the discrepancy in RA-induced effects between that and our studies cannot be ascertained at the present time; however, they may include the difference in the cell type used (blood mononuclear cells vs. fully differentiated HM). In fact, RA, specifically an RXR ligand, promotes monocyte/macrophage differentiation as a ligand to an active partner (RXR) for PPARgamma heterodimerization (52). This effect on differentiation is characterized by induction of CD14, CD11b, and CD18. On the other hand, we did not observe any effects of RA on CD14 expression by cultured HM (unpublished observation). Thus RA may exert differential effects on the cells depending on the state of monocyte differentiation. Indeed, our preliminary results comparing the transcriptional effects of RA on HM and a murine macrophage cell line, RAW 264.7, demonstrate differential effects of RA on TNF-alpha promoter activity, and these effects appear related to the difference in cellular differentiation state (unpublished observation).

In vivo effects of RA on liver injury are complex. Pretreatment of mice with RA protects them from lethality associated with fulminant hepatitis induced by P-acnes and LPS (33), the response that exemplifies RA-mediated antinflammatory effect on HM (32, 34). Vitamin A administration suppresses experimental liver fibrosis induced by CCl4 without affecting hepatocellular injury (49), whereas vitamin A deficiency promotes liver fibrogenesis induced by this hepatotoxic drug (48). Paradoxically, vitamin A can promote acute CCl4 hepatotoxicity (10) and alcoholic liver fibrogenesis (28). These differential and contradictory responses likely reflect diverse effects of retinoids on different functional aspects of the different liver cell types, as well as the higher doses used in the latter studies (10, 28). For instance, the potentiation of hepatotoxicity by vitamin A is a likely consequence of enhanced oxidative injury rendered by metabolism of retinoids via induced cytochrome P-450 in hepatocytes (10, 28). On the other hand, the antifibrogenic responses of retinoids are considered to be mediated via their primary effects on hepatic stellate cells rather than on hepatocytes (49). To add to these complexities, 9-cis RA (41) and 9,13-di-cis RA (18) were shown to activate latent TGFbeta via induction of plasminogen activator in cultured stellate cells and to exacerbate experimental liver fibrosis (41). The present study focused on the mechanistic relationship between the cellular content and signaling of RA and TNF-alpha expression in HM. Our results demonstrate a novel mechanism of RA-induced TNF-alpha mRNA destabilization that we believe underlies suppressed TNF-alpha expression by RA-exposed HM in culture. The in vivo results also support the inverse relationship between endogenous RA levels and TNF-alpha mRNA stability and a notion that diminished RA signaling may be a causal factor for sustained upregulation of TNF-alpha expression by HM in alcoholic liver injury. The concentration of RA used in our study (500 nM) is higher than the known concentrations in different tissues (from 10 to ~100 nM). However, we have also observed that lower concentrations of RA (10~100 nM) inhibit TNF-alpha expression in cultured HM (unpublished results), suggesting that our findings on RA-mediated regulation can be rendered by physiological to pharmacological RA concentrations. In light of the complexities and difficulties in predicting the in vivo effects of retinoids on the pathological livers, an RA therapy that is directly and specifically targeted to HM may prove efficacious for the treatment of liver diseases in which HM-derived TNF-alpha and other proinflammatory cytokines are known to play pivotal roles.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Drs. Hong-yan Li, Min Lin, and Jin May Yang for technical assistance.


    FOOTNOTES

This study was supported by National Institutes of Health Grants R37-AA-06603 (H. Tsukamoto); USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases (P50-AA-11999); Molecular Biology, Tissue Culture, and Mathematical Analysis-modeling Core Facilities of USC Research Center for Liver Diseases (P30-DK-48522); and Medical Research Service of Department of Veterans Affairs (H. Tsukamoto).

Address for reprint requests and other correspondence: H. Tsukamoto, Dept. of Pathology, USC Keck School of Medicine, MMR 402, 1333 San Pablo St., Los Angeles, CA 90033 (E-mail: htsukamo{at}hsc.usc.edu).

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.

Received 27 February 2001; accepted in final form 17 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 281(3):E420-E429