 |
INTRODUCTION |
Liver alcohol dehydrogenase
(ADH,1 alcohol:NAD
oxidoreductase, EC 1.1.1.1.) is principally responsible for ethanol
oxidation. Immobilization stress in rats, which stimulates the
hypothalamo-hypophyseal-adrenocortical axis, increases liver ADH
activity and ethanol elimination (1). The enzyme activity is regulated
by hormones and increases in the activity of the enzyme, resulting in
increased formation of metabolites of ethanol such as acetaldehyde,
which are important in the pathogenesis of alcoholic liver disease
(2).
CCAAT/enhancer binding protein
, upstream regulatory factor (USF),
and signal transduction and activator of transcription 5b (STAT5b) are
transcription factors that bind to and activate the ADH promoter in
transfection experiments. C/EBP
binds principally between
22 and
11 (3), USF between
60 and
52 (4), and STAT5b between
211 and
203 (5) relative to the start site of transcription. C/EBP
also
binds to a second site adjacent to the STAT5b binding site (5). The
action of growth hormone (GH) in enhancing the ADH promoter activity is
mediated by both C/EBP
(6) and STAT5b (5).
Endotoxin originating from intestinal bacteria is an important mediator
of hepatocellular inflammation in the intragastric-feeding rat model of
alcoholic liver disease (7, 8). Lipopolysaccharide (LPS), the endotoxin
component of Gram-negative bacteria, leads to the production of a
variety of inflammatory cytokines such as tumor necrosis factor
and
interleukin-1, the formation of oxygen radicals, and the translocation
of nuclear factor-
B (NF-
B) to the cell nucleus (9, 10).
LPS activates the hypothalamo-hypophyseal-adrenocortical axis, and this
is manifested principally by increases in ACTH and cortisol secretion
(11). The effect of LPS on GH is species-dependent with
increases in the human but decreases in the rat (11).
The purpose of the study was to determine whether LPS with its
associate acute phase metabolic response influences ADH and whether
such an effect on ADH is mediated by NF-
B.
 |
EXPERIMENTAL PROCEDURES |
Animals and Materials--
Male Sprague-Dawley rats were
obtained from Charles River Laboratories (Wilmington, MA). All animals
received humane care in compliance with the guidelines from the Animal
Care and Use Committee of The Johns Hopkins University. LPS from
Escherichia coli was obtained from Sigma. Dulbecco's
modified Eagle's medium (DMEM), fetal bovine serum, and agarose were
purchased from Invitrogen. Plastic 752 tissue
culture flasks were purchased from BD Biosciences.
[
-32P]dATP and [
-32P]dCTP were
purchased from PerkinElmer Life Sciences. An oligonucleotide containing
the consensus binding sequence of NF-
B was obtained from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA).
Animal Treatment--
Rats received intraperitoneal injections
of LPS (100 µg/100g body weight) daily for 3 days, while control rats
received isovolumetric amounts of saline. The animals were sacrificed
2 h after the last injection. Approximately 400-500 mg of the
liver was homogenized in 4 volumes of 0.25 M sucrose in 0.1 M Tris-HCl buffer, pH 7.4, centrifuged at 10,000 × g for 10 min. The resulting supernatant was used for the
determination of ADH activity and ADH protein. One section of 1.0-1.2
gm of the liver was processed for RNA isolation, and the remainder of
the liver was used for preparation of nuclear protein extracts as
described previously (4). The nuclear protein extracts were aliquoted
and stored under nitrogen at
100 °C. Protein content of the
cytosol and nuclear extract was determined by the method of Lowry
et al. (12).
Alcohol Dehydrogenase Activity--
ADH activity was determined
in the liver cytosol at 37 °C by the method of Crow et
al. (13). The reaction mixture was 1.0 ml and consisted of 0.5 M Tris-HCL, pH 7.2, 18 mM ethanol, 2.8 mM NAD+, and 0.03 ml of the liver cytosol. One
unit of enzyme activity is defined as the formation of 1 µmol
NADH per min. Lactate dehydrogenase activity was determined by the
method of Plagemann et al. (14).
Immunoreactive Protein of Alcohol
Dehydrogenase--
Immunoreactive protein of ADH was determined by
quantitative enzyme-linked immunosorbent assay as described previously
(15).
Plasmids--
The full-length cDNA plasmid encoding the rat
class I ADH and the rat class I 240ADH-CAT construct, which were
obtained from Dr. David W. Crabb of Indiana University School of
Medicine (Indianapolis, IN), have been described previously (3). The
NF-
B expression vectors RSV-NF-
B (p50) and RSV-Rel (p65) and the
empty vector K1 were obtained from Professor Guidalberto Manfioletti,
from the University of Trieste, Italy. The STAT5b expression vector pCMX-STAT5b and the empty vector pCMV5 were gifts from Dr. Gregorio Gil
of the Medical College of Virginia (Richmond, VA). Luciferase constructs of the ADH promoter were prepared as described previously (5).
Isolation and Quantitation of Messenger RNA--
Total cellular
RNA was isolated using the procedure of Chomcynski and Sacchi (16). RNA
quality and concentration were verified by agarose-formaldehyde gel
electrophoresis with ethidium bromide staining. Northern blots for rat
liver ADH and mouse
-actin were performed as described previously
(17). The amount of ADH mRNA hybridized was visualized by
autoradiography and quantitated by densitometry.
Electrophoretic Mobility Shift Assays (EMSA)--
The sequences
of the ADH oligonucleotides used for EMSA are shown in Table
I. Complimentary strands of each
oligonucleotide were annealed, and the double-stranded oligonucleotides
were labeled with [
-32P]dATP and
[
-32P]dCTP using Klenow enzyme according to the method
of Feinberg and Vogelstein (18). DNA-protein binding reactions were
performed with nuclear extracts (8 µg of protein) following the
previously described EMSA procedure (3). For "supershift" EMSA
experiments, rabbit polyclonal antibodies to NF-
B p50, NF-
B p65,
USF-1, USF-2, STAT5b, and C/EBP
, obtained from Santa Cruz
Biotechnology, Inc., were used. These antibodies were added separately
to the reaction at the completion of DNA-protein binding, incubated for
an additional 30 min at room temperature, and resolved on an 8%
nondenaturing polyacrylamide gel.
View this table:
[in this window]
[in a new window]
|
Table I
ADH oligonucleotides used for EMSA
The C/EBP, USF, and STAT regulatory motifs, respectively, in the ADH-E,
ADH-U, and ADHwt oligonucleotides are underlined.
|
|
Transient Transfection and Luciferase Assay--
Transient
transfection experiments were carried out in cultured HepG2 cells using
the calcium phosphate precipitation method (19). HepG2 cells were
seeded on 75-cm2 polystyrene flasks and allowed to grow to
60-70% confluency in DMEM containing 10% fetal bovine serum. The
medium was renewed 1 h prior to transfection. To each flask 10 µg of pGL3-ADH, 5 µg of
-galactosidase vectors, and 5 µg of
salmon sperm DNA were added in the form of calcium phosphate
precipitates. For each experiment, pGL3-basic and pGL3-control
luciferase vectors were used as a negative and a positive control.
After overnight incubation, the cells were shocked with 10%
Me2SO in DMEM for 3 min and then washed and refed
with fresh DMEM containing 10% fetal bovine serum. At 40 h after
transfection, the cells were harvested and subjected to one freeze-thaw
cycle in 200 µl of the reporter lysis buffer (Promega). Luciferase
activity and
-galactosidase activity were determined by respective
chemiluminescent assays (20, 21).
Ultraviolet Cross-linking of Nuclear Proteins to Oligonucleotides
and Immunoblot Analysis--
The binding of nuclear proteins to the
oligonucleotide probe was performed as for EMSA. Reactions using 8 µg
of nuclear protein and 50 fmol of radioactively labeled
oligonucleotides were used. Following the binding reaction, UV
cross-linking was performed as described previously (4, 5). The
membranes were then incubated with rabbit polyclonal antibody to
NF-
B p50, NF-
B p65, USF-1, USF-2, STAT 5b (1:200 dilution), or to
C/EBP
(C-19, 1:500 dilution) (Santa Cruz Biotechnology Inc.) for one
h. After repeated washing for 1 h, the membranes were incubated
with horseradish peroxidase-conjugated goat anti-rabbit IgG (1: 50,000 dilution) (Amersham Biosciences) for one h. The membranes were again
washed for 1 h and then immersed in lumigen PS-3 acridan substrate
solution (ECL Plus, Amersham Biosciences) for 5 min. The antigens
were visualized by exposing the membrane to x-ray film.
Data Analysis--
All data points are expressed as means ± S.E. The differences between means of paired groups and between
means of more than two paired groups were examined by Student's
t test and by two-way analysis of variance plus appropriate
multiple comparisons, respectively.
 |
RESULTS |
LPS Enhances ADH--
The administration of LPS resulted in a mean
2.9-fold increase in liver ADH activity (Table
II) but in no significant change in
lactate dehydrogenase activity. ADH immunoprotein was also increased
after LPS administration from 7.49 ± 4.12 ng/mg cytosol protein
in the controls to 10.91 ± 0.70 ng/mg cytosol protein after LPS
(p < 0.05). LPS administration did not change liver weight or protein concentration in the liver cytosol. The effect of LPS
in increasing ADH activity and protein was accompanied by an increase
in ADH mRNA. The relative densitometric readings of ADH
mRNA/
actin mRNA ratios for the autoradiographs of the Northern blots were increased to 164 ± 12 (n = 5)
after LPS treatment as compared with 100 ± 24 (n = 5) for controls (p < 0.05).
View this table:
[in this window]
[in a new window]
|
Table II
Effect of LPS administration on hepatic alcohol dehydrogenase (ADH) and
lactate dehydrogenase (LDH) activities
LPS was administered intraperitoneally in a daily dose of 100 µg/100
gm body weight for 3 days. Enzyme activities are expressed per liver
cytosol protein. All values are expressed as means ± S.E. of six
animals.
|
|
NF-
B Binds to the ADH Promoter and This Binding Activity Is
Increased by LPS--
LPS increased the binding activity of NF-
B to
an oligonucleotide specifying the NK-
B binding consensus sequence
(Fig. 1). Furthermore, NF-
B in nuclear
extracts from rat liver was found to bind to the oligonucleotide ADHwt.
specifying a region from
226 to
194 of the ADH promoter (Table I).
EMSA with the nuclear extracts from control rats shows four protein-DNA
complexes with the ADHwt oligonucleotide (Fig.
2A, arrows). These
complexes were previously identified (5) as binding by STAT5b
(upper complex) and C/EBP
(two middle
complexes). Nuclear extract from LPS-treated rats resulted in the
formation of two additional upper protein-DNA complexes with the ADHwt
oligonucleotide (Fig. 2A, arrowheads). Antibody
to NF-
B p50 decreased the uppermost protein-DNA complex and resulted
in the formation of a supershifted complex, whereas antibody to NF-
B
p65 resulted in a smaller supershifted complex (Fig. 2B).
LPS did not significantly change the binding activity of nuclear STAT5b
and C/EBP
to the ADHwt oligonucleotide from that obtained with
nuclear extracts of control rats. LPS, however, resulted in a decrease
in the formation of the protein-DNA complex with the ADH-E
oligonucleotide (Fig. 3), which
represents C/EBP
binding to the more proximal site (Table I) of the
ADH promoter (3).

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 1.
EMSA showing the binding of
NF- B in rat liver nuclear extracts
(NE) from five control and five LPS-treated rats to
the NF- B consensus sequence. LPS was
administered intraperitonealy in a dose of 100 µg/100 g body weight
for 3 days. The EMSA was performed with 8 µg of NE and the labeled
oligonucleotide. The arrow indicates the protein-DNA
complexes. F indicates the position of the free probe.
|
|

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 2.
A, EMSA showing the binding of rat liver
nuclear extract (NE) from control and LPS-treated rats to
the ADHwt oligonucleotide. LPS was administered intraperitonealy in a
dose of 100 µg/100 g body weight for 3 days. The EMSA was performed
with 8 µg of NE and the labeled oligonucleotide. The
arrows indicate the protein-DNA complexes.
Arrowheads indicate additional protein-DNA complexes present
in NE of LPS-treated rats. B, EMSA with supershift. The same
reaction mixture was incubated with preimmune serum (PI) or
antibodies to NF- B p65 and NF- B p50 (1:200 dilution). The
arrowhead indicates the supershifted band. F
indicates the position of the free probe.
|
|

View larger version (77K):
[in this window]
[in a new window]
|
Fig. 3.
EMSA showing the binding of rat liver nuclear
extracts (NE) from control and LPS-treated rats to the
ADHU oligonucleotide. LPS was administered intraperitonealy in a
dose of 100 µg/100 g body weight for 3 days. The EMSA was performed
with 8 µg of NE and the labeled oligonucleotide. The arrow
indicates the protein-DNA complex. F indicates the position
of the free probe.
|
|
The NF-
B (p65) and the NF-
B (p50) Expression Vector Inhibit
the Activity of the ADH Promoter--
To determine whether the action
of LPS in increasing ADH activity was mediated by NF-
B, we
determined the effects of NF-
B expression vectors on the
cotransfected ADH promoter. The NF-
B (p65) and NF-
B (p50)
expression vectors resulted in inhibition of the activity of the ADH
promoter (pGL-ADH) (Table III). The combination of the NF-
B expression vectors did not result in additional inhibition over that obtained with NF-
B (p65) alone. The
NF-
B (p65) expression vector also markedly inhibited the activation
of pGL-ADH by the STAT5b expression vector (Table
IV).
View this table:
[in this window]
[in a new window]
|
Table III
Effect of NF- B on activity of the alcohol dehydrogenase (ADH)
promoter
Data are expressed as means ± S.E. at four determinations. 15 µg of pGL3-ADH and of each of the expression vectors was transfected.
The control was transfected with the empty vector k1.
|
|
View this table:
[in this window]
[in a new window]
|
Table IV
Effect of NF- B (p65) on the activity of the alcohol dehydrogenase
(ADH) promoter in the absence and presence of the STAT5b expression
vector
Data are expressed as means ± S.E. of four determinations. The
control was transfected with the empty vector k1. ND, not done.
|
|
LPS Increases the Binding of USF Proteins to the ADH
Promoter--
USF was previously shown by us to bind to and activate
the ADH promoter (4). UV cross-linking with immunoblots was done to
further define the effects of LPS on NF-
B and C/EBP
binding and
to determine the effect of LPS on USF binding to ADH oligonucleotides. LPS increased the binding of NF-
B p65 (Fig.
4) but had no effect on the minimal
binding of NF-
B p50. LPS decreased the binding of the 35-kDa
C/EBP
to ADH-E (Fig. 5). By contrast,
LPS resulted in moderate increases in the binding of the 43-kDa USF-1
and 44-kDa USF-2 proteins and a marked increase in the binding of the
17-kDa USF protein to the ADH-U oligonucleotide (Fig.
6).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Immunoblot showing the presence of
NF- B proteins in nuclear extracts
(NE) from control and LPS-treated rat liver UV
cross-linked to the labeled ADHwt oligonucleotide. The blots were
incubated with antibodies to NF- B p65 and NF- B p50 (1:200
dilution) or with preimmune rabbit serum. Preimmune rabbit serum
detected no protein bands. Arrows indicate the
location of the bound NF- B proteins with their molecular mass
(kDa). Molecular mass was determined from standards with
adjustment for the molecular mass of the oligonucleotide.
|
|

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 5.
Immunoblot showing the presence of
C/EBP in nuclear extracts
(NE) from control and LPS-treated rat liver UV
cross-linked to the labeled ADHU oligonucleotide. The blots were
incubated with antibody to C/EBP (1:500 dilution) or with preimmune
rabbit serum. Preimmune rabbit serum detected no protein bands.
Arrows indicate the location of the bound C/EBP proteins
with their molecular masses (kDa). Molecular mass was
determined from standards with adjustment for the molecular mass of the
oligonucleotide.
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 6.
Immunoblot showing the presence of USF in
nuclear extracts (NE) from control and LPS-treated rat
liver UV cross-linked to the labeled ADHU oligonucleotide. The
blots were incubated with antibodies to USF1 and -2 (1:200 dilution) or
with preimmune rabbit serum. Preimmune rabbit serum detected no protein
bands. Arrows indicate the location and molecular mass of
the bound USF proteins with their molecular mass (kDa).
Molecular mass was determined from standards with adjustment for the
molecular mass of the oligonucleotide.
|
|
 |
DISCUSSION |
This study shows that LPS increases the message, protein, and
activity of liver ADH. LPS activates NF-
B, and NF-
B in turn is a
known regulator of the gene expression of tumor necrosis factor
and
other inflammatory cytokines. NF-
B in nuclear protein extracts from
rat liver was shown in this study to bind to the ADH promoter. Although
LPS resulted in the expected increase of NF-
B activity and in
increased NF-
B binding to the ADH promoter, the effect of LPS in
enhancing the ADH was not mediated by NF-
B. Indeed, the NF-
B
expression vectors resulted in inhibition of the activity of the ADH
promoter. Furthermore, the NF-
B (p65) expression vector inhibited
the activation of the ADH promoter by the STAT5b expression vector.
Acetaldehyde in vitro was shown to diminish LPS-stimulated
degradation of I
B
and to inhibit the nuclear translocation of
NF-
B p65, resulting in decreased NF-
B binding to the NF-
B
consensus oligonucleotide (22). Hence, it is possible that any
inhibitory effect of NF-
B on ADH may be markedly decreased or
abrogated by the acetaldehyde produced during ethanol metabolism.
GH enhances ADH, an effect mediated by both C/EBP
(6) and STAT5b
(5). The effect of LPS on increasing ADH, however, is not mediated by
GH. LPS increases GH secretion in the human but decreases GH secretion
in the rat (11, 23). Furthermore, LPS did not affect the binding of
STAT5b and decreased the binding of C/EBP
to oligonucleotides
specifying their binding sites on the ADH promoter. These findings are
in agreement with prior observations showing that LPS down-regulated
the STAT5-mediated GH-responsive gene serine protease inhibitor 2 (24).
Also, LPS decreased the 42-kDa C/EBP
and the 35-kDa C/EBP
proteins in nuclear extracts from mice and their binding to the C/EBP
binding site of the
1-acid glycoprotein promoter (25).
The 20-kDa C/EBP
and its binding were increased acutely after LPS
but decreased to baseline values after 48 h (25).
This study indicates that the effect of LPS in increasing ADH is
mediated by increased binding of USF to the ADH promoter. In a previous
study, we demonstrated that USF activated the promoter of the rat
ADH gene (4). The binding to the ADH promoter of the
43-kDa USF-1 and the 44-kDa USF-2 isoforms, which are principally responsible for transcriptional activity in mammalian cells (26), was
increased by LPS. In addition, the binding of a smaller 17-kDa USF
polypeptide was also increased by LPS. The 17-kDa polypeptide is
recognized by USF-1 antibody and binds to DNA as both a homodimer and a
heterodimer with full-length USF-2 (26, 27). Heat shock and LPS were
previously found to increase binding of nuclear proteins to the E-box
of the murine-inducible nitric oxidase gene, and these proteins were
supershifted with antibodies to USF-1 and -2. However, mutation of the
E-box did not affect the activation of the promoter by LPS (28).
Despite the inhibitory effect of NF-
B on ADH in transfection
experiments, LPS administration in vivo resulted in an
increase in ADH message, protein, and activity, indicating that
activators such as C/EBP
and USF negate a possible inhibitory effect
of NF-
B. Also of note is that in vivo the accumulation of
NF-
B p50 homodimers is greater than that of NF-
B p50-p65
heterodimers in rat hepatocytes after LPS administration (29) and, as
shown in this study, the inhibitory action of NF-
B p50 was less than that of NF-
B p65 in transfection experiments. The greater increase in ADH activity than in ADH protein and mRNA after LPS
administration suggests that the effect of LPS is not only on
transcription but possibly also on activation of ADH.
In summary, this study shows that LPS, the endotoxin component of
Gram-negative bacteria, increases ADH activity and that this effect is
mediated by increased binding of USF to the ADH promoter and not by
NF-
B, which has an inhibitory action. An increased rate of formation
of acetaldehyde caused by an enhanced ADH activity may contribute to
worsening of alcoholic liver injury caused by endotoxin.