Endotoxin Enhances Liver Alcohol Dehydrogenase by Action through Upstream Stimulatory Factor but Not by Nuclear Factor-kappa B*

James J. Potter, Lynda Rennie-Tankersley, and Esteban MezeyDagger

From the Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, October 2, 2002, and in revised form, November 15, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Liver alcohol dehydrogenase (ADH) is increased by physiological stress and by chronic administration of growth hormone (GH). Endotoxin plays a role in the pathogenesis of alcoholic liver disease. The effect of lipopolysaccharide (LPS), the endotoxin component of Gram-negative bacteria, was determined on liver ADH. LPS given daily to rats for 3 days increased ADH mRNA, ADH protein, and ADH activity. Nuclear factor-kappa B (NF-kappa B) in the liver nuclear extracts bound to an oligonucleotide specifying region -226 to -194 of the ADH promoter, whereas upstream stimulatory factor (USF) was shown previously to bind to a more proximal site. LPS increased NF-kappa B and USF binding to the ADH promoter. The NF-kappa B (p65) and NF-kappa B (p50) expression vectors inhibited the transfected ADH promoter activity, which contrasts with the previously demonstrated stimulation by an USF expression vector. The binding activities of STAT5b and of C/EBPbeta , which mediate the effect of GH on ADH, were not changed or decreased, respectively, by LPS, indicating that GH plays no intermediary role in the effect of LPS. This study shows that LPS increases ADH and that this effect is mediated by increased binding of USF to the ADH promoter and not by NF-kappa B, which has an inhibitory action.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta , 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/EBPbeta 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/EBPbeta 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/EBPbeta (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 alpha  and interleukin-1, the formation of oxygen radicals, and the translocation of nuclear factor-kappa B (NF-kappa 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-kappa B.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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. [alpha -32P]dATP and [alpha -32P]dCTP were purchased from PerkinElmer Life Sciences. An oligonucleotide containing the consensus binding sequence of NF-kappa 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-kappa B expression vectors RSV-NF-kappa 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 gamma -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 [alpha -32P]dATP and [alpha -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-kappa B p50, NF-kappa B p65, USF-1, USF-2, STAT5b, and C/EBPbeta , 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.

                              
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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 beta -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 beta -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-kappa B p50, NF-kappa B p65, USF-1, USF-2, STAT 5b (1:200 dilution), or to C/EBPbeta (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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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/gamma 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).

                              
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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-kappa B Binds to the ADH Promoter and This Binding Activity Is Increased by LPS-- LPS increased the binding activity of NF-kappa B to an oligonucleotide specifying the NK-kappa B binding consensus sequence (Fig. 1). Furthermore, NF-kappa 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/EBPbeta (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-kappa B p50 decreased the uppermost protein-DNA complex and resulted in the formation of a supershifted complex, whereas antibody to NF-kappa B p65 resulted in a smaller supershifted complex (Fig. 2B). LPS did not significantly change the binding activity of nuclear STAT5b and C/EBPbeta 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/EBPbeta binding to the more proximal site (Table I) of the ADH promoter (3).


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Fig. 1.   EMSA showing the binding of NF-kappa B in rat liver nuclear extracts (NE) from five control and five LPS-treated rats to the NF-kappa 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.


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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-kappa B p65 and NF-kappa B p50 (1:200 dilution). The arrowhead indicates the supershifted band. F indicates the position of the free probe.


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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-kappa B (p65) and the NF-kappa 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-kappa B, we determined the effects of NF-kappa B expression vectors on the cotransfected ADH promoter. The NF-kappa B (p65) and NF-kappa B (p50) expression vectors resulted in inhibition of the activity of the ADH promoter (pGL-ADH) (Table III). The combination of the NF-kappa B expression vectors did not result in additional inhibition over that obtained with NF-kappa B (p65) alone. The NF-kappa B (p65) expression vector also markedly inhibited the activation of pGL-ADH by the STAT5b expression vector (Table IV).

                              
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Table III
Effect of NF-kappa 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.

                              
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Table IV
Effect of NF-kappa 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-kappa B and C/EBPbeta binding and to determine the effect of LPS on USF binding to ADH oligonucleotides. LPS increased the binding of NF-kappa B p65 (Fig. 4) but had no effect on the minimal binding of NF-kappa B p50. LPS decreased the binding of the 35-kDa C/EBPbeta 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).


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Fig. 4.   Immunoblot showing the presence of NF-kappa 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-kappa B p65 and NF-kappa 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-kappa B proteins with their molecular mass (kDa). Molecular mass was determined from standards with adjustment for the molecular mass of the oligonucleotide.


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Fig. 5.   Immunoblot showing the presence of C/EBPbeta 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/EBPbeta (1:500 dilution) or with preimmune rabbit serum. Preimmune rabbit serum detected no protein bands. Arrows indicate the location of the bound C/EBPbeta proteins with their molecular masses (kDa). Molecular mass was determined from standards with adjustment for the molecular mass of the oligonucleotide.


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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study shows that LPS increases the message, protein, and activity of liver ADH. LPS activates NF-kappa B, and NF-kappa B in turn is a known regulator of the gene expression of tumor necrosis factor alpha  and other inflammatory cytokines. NF-kappa 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-kappa B activity and in increased NF-kappa B binding to the ADH promoter, the effect of LPS in enhancing the ADH was not mediated by NF-kappa B. Indeed, the NF-kappa B expression vectors resulted in inhibition of the activity of the ADH promoter. Furthermore, the NF-kappa 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 Ikappa Balpha and to inhibit the nuclear translocation of NF-kappa B p65, resulting in decreased NF-kappa B binding to the NF-kappa B consensus oligonucleotide (22). Hence, it is possible that any inhibitory effect of NF-kappa 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/EBPbeta (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/EBPbeta 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/EBPalpha and the 35-kDa C/EBPbeta proteins in nuclear extracts from mice and their binding to the C/EBP binding site of the alpha 1-acid glycoprotein promoter (25). The 20-kDa C/EBPbeta 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-kappa 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/EBPbeta and USF negate a possible inhibitory effect of NF-kappa B. Also of note is that in vivo the accumulation of NF-kappa B p50 homodimers is greater than that of NF-kappa B p50-p65 heterodimers in rat hepatocytes after LPS administration (29) and, as shown in this study, the inhibitory action of NF-kappa B p50 was less than that of NF-kappa 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-kappa 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.

    FOOTNOTES

* This work was supported by Grant AA00626 from the United States Public Health Service.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.

Dagger To whom correspondence should be addressed: 921 Ross Research Bldg., The Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-955-7856; Fax: 410-955-9677; E-mail: emezey@jhmi.edu.

Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M210097200

    ABBREVIATIONS

The abbreviations used are: ADH, alcohol dehydrogenase; LPS, lipopolysaccharide; HPA, hypothalamo-hypophyseal-adrenocortical; NF-kappa B, nuclear factor-kappa B; C/EBP, CCAAT/enhancer binding protein; STAT, signal transducers and activators of transcription; USF, upstream stimulatory factor; GH, growth hormone; NE, nuclear extract; DMEM, Dulbecco's modified Eagle medium; EMSA, electrophoretic mobility shift assay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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