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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ABSTRACT |
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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)- 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-
expression by
cultured normal rat HM. The mRNA levels for inducible nitric oxide
synthase, interleukin (IL)-6, IL-1
, and IL-1
were also reduced,
whereas those for transforming growth factor-
1, MMP-9, and membrane
cofactor protein-1 were unaffected. The inhibitory effect on TNF-
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-
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
RAR
, RXR
, and cytosolic retinol binding protein-1, whereas TNF-
mRNA expression was induced. TNF-
mRNA stability was
increased in these cells, and an ex vivo treatment with
all-trans RA normalized both RAR
and TNF-
mRNA levels.
These results demonstrate the RA-induced destabilization of TNF-
mRNA by cultured HM and the association of RA depletion with increased
TNF-
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- mRNA stability; retinoic
acid receptors; retinoid X receptors; retinoic acid response element
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INTRODUCTION |
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TUMOR NECROSIS
FACTOR (TNF)- 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-
expression is implicated in cell death, matrix remodeling, microcirculation disturbances, and acute and chronic inflammation. TNF-
is considered the central mediator in liver damage induced by a
variety of hepatotoxic conditions, because administration of antibodies
against TNF-
(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-
, 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-
, 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-
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-
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-
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- (5), TNF-
(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)-
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 RXR and -
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-
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- 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-
expression and vitamin A
depletion were expected. Our study reveals a novel finding of
RA-mediated destabilization of TNF-
mRNA in cultured HM.
Furthermore, the analysis of HM from the in vivo model demonstrates concomitant RA depletion and increased TNF-
mRNA stability,
suggesting a mechanistic link between them. These results thus support
the possibility that HM from experimental ALD are primed for TNF-
upregulation, due at least in part to diminished RA-mediated
destabilization of TNF-
mRNA.
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METHODS |
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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 · kg1 · 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- and
-actin mRNA levels by Northern blot
analysis, as described in Northern blot analysis. To assess
TNF-
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-
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 (RAR and RXR
), cytosolic retinol binding
protein (CRBP)-I, cytokines [TNF-
, transforming growth factor (TGF)
, and ILs), chemokine [macrophage inflammatory protein (MIP)-1], matrix metalloproteinase (MMP) 9, inducible
nitric oxide synthase (iNOS), and
-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: RAR
, 25 or 30; RXR
, 35; CRBP-1, 29; TNF-
,
18 for cultured HM and 25 for HM from the alcohol model; IL-1
,
IL-1
, and membrane cofactor protein (MCP)-1, 18; IL-6, iNOS, and
MMP-9, 20; and TGF
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- (21, 29) and human
RAR
(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-
and 18S rRNA and at
42°C for RAR
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-
and 18S rRNA or at
40°C for RAR
in 2× SSC and 1% SDS; and twice at 50°C for
TNF-
and 18S rRNA or at 40°C for RAR
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-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-
B, and AP-1 were previously described
(29, 39). For supershift assay [electrophoretic mobility
shift assay (EMSA)], we used polyclonal antibodies against human
(h)RAR
and hRXR
(kindly provided by Dr. Ronald M. Evans) and
those against P65, P50, c-Fos, and c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA).
TNF- 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-
protein was determined using an ELISA kit
for mouse TNF-
(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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RESULTS |
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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 RAR 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 RAR
or RXR
(Fig. 1B). This change
was accompanied by induced mRNA expression of RAR
and RXR
(Fig.
2), which are known to contain functional RARE (50) or to be induced by RA (55),
respectively. In particular, RAR
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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RA was also previously shown to inhibit LPS-induced TNF- 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-
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-
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-
mRNA levels with those for 18S rRNA revealed 33 and 57%
reductions in TNF-
mRNA expression by 100 and 500 nM LG268,
respectively. Thus this result suggests that RA-induced inhibition of
TNF-
expression is mediated via RXR but not via RAR.
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Next, we examined whether the RA-induced inhibition is specific for
TNF- 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-
, iNOS, IL-1
, and IL-6, but other genes, such
as anti-inflammatory cytokines (TGF
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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Because the inflammatory mediators inhibited by RA are known to be
regulated predominantly by transcription factors NF-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- synthesis is
attained. To test this possibility for the observed RA effect, we
examined TNF-
mRNA stability in the RA-treated HM. The TNF-
mRNA
level was already reduced in RA-treated HM at time 0 compared with the DMSO-treated cells. Densitometric data for TNF-
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-
mRNA levels for each group revealed a significantly enhanced degradation of TNF-
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-
mRNA stability and suggests that this
posttranscriptional effect is, at least in part, responsible for
RA-mediated inhibition of TNF-
expression by LPS-stimulated cultured
HM.
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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- 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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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 RAR, RXR
, and CRBP-1, which contain functional RARE
(Fig. 8, left), whereas the
TNF-
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 RAR
,
RXR
, and CRBP-1 was suppressed in HM from the alcohol model and that RA-mediated negative regulation of TNF-
expression was also
inhibited, resulting in TNF-
upregulation.
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To further test the role of RA depletion in TNF- 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 RAR
and induced TNF-
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-
expression.
Therefore, we examined next whether the induced TNF-
expression seen
in the RA-depleted HM from the alcohol-fed rats was associated with increased TNF-
mRNA stability. After the first experiment, we learned that the TNF-
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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DISCUSSION |
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Key findings of the present study can be summarized as follows.
1) RA inhibits TNF- expression by HM in an RXR-dependent manner; 2) this effect is mediated, at least in part, via
destabilization of TNF-
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-
mRNA stability and steady-state
level; and 4) the ex vivo treatment of these cells with RA
normalizes TNF-
expression. These findings are novel and have
important therapeutic implications for immune modulation of alcoholic
liver injury.
The synthesis of TNF- 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-
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-
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-
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-
lacking
its 3'-UTR (22) or mice deficient in tristetraprolin, a
zinc-binding protein shown to destabilize TNF-
mRNA via its binding
to the ARE (51). It is currently unknown whether the
ARE-dependent mechanism mediated the destabilization of TNF-
mRNA in
RA-treated HM or whether an inhibition of this mechanism was
responsible for the increased TNF-
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- synthesis in alcoholic liver injury via
diminished RA-mediated destabilization of TNF-
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-
B (35), but RA-mediated inhibition of tissue factor transcription in
LPS-stimulated human monocytes does not appear to involve AP-1 or
NF-
B (38). Our results also did not demonstrate
RA-induced effects on NF-
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-
promoter activity governed by these elements. Moreover, our
results demonstrating the posttranscriptional regulation of TNF-
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-
promoter activity in
cultured HM (unpublished observation). Thus it is probable that RA has
both transcriptional and posttranscriptional effects on the TNF-
gene. The present study had its main focus on demonstration of the
latter mechanism. Our ongoing study investigates whether RA-mediated
inhibition of TNF-
promoter activity is accompanied by inhibition of
TNF-
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- 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 PPAR
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-
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 TGF 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-
expression in HM. Our results demonstrate a novel mechanism of
RA-induced TNF-
mRNA destabilization that we believe underlies
suppressed TNF-
expression by RA-exposed HM in culture. The in vivo
results also support the inverse relationship between endogenous RA
levels and TNF-
mRNA stability and a notion that diminished RA
signaling may be a causal factor for sustained upregulation of TNF-
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-
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-
and other proinflammatory cytokines are known to play pivotal roles.
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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.
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