Chronic Ethanol Increases Lipopolysaccharide-stimulated Egr-1 Expression in RAW 264.7 Macrophages

Increased production of tumor necrosis factor α (TNFα) is associated with the development of alcoholic liver disease. Culture of RAW264.7 macrophages with 25 mm ethanol for 48 h increased lipopolysaccharide (LPS)-stimulated accumulation of tumor necrosis factor α (TNFα) peptide and mRNA by 2-fold. We investigated whether chronic ethanol-induced increases in the DNA binding and/or promoter activity of the key transcription factors regulating LPS-stimulated TNFα promoter activity contribute to increased TNFα expression. Binding of Egr-1 to theTNFα promoter was increased by 2.5-fold after ethanol exposure, whereas NFκB binding was decreased to 30% of control. AP-1 binding was not affected. Changes in binding activity were paralleled by an increased contribution of the Egr-1 binding site and a decreased contribution of the NFκB site to LPS-stimulated TNFα promoter activity. Overexpression of dominant negative Egr-1 prevented the ethanol-induced increase in LPS-stimulated TNFα mRNA accumulation. Chronic ethanol exposure enhanced LPS-stimulatedEgr-1 promoter-driven CAT expression and transcription ofEgr-1. Induction of Egr-1 is dependent on ERK1/2 activation in other systems. Therefore, we investigated whether the ERK1/2 pathway mediated the chronic ethanol-induced increases in Egr-1 and TNFα. Increased Egr-1 promoter activity and TNFα mRNA accumulation after chronic ethanol were both prevented by overexpression of dominant negative ERK1/2. LPS-stimulated ERK1/2 phosphorylation was increased 2-fold in cells cultured with ethanol compared with controls. These results demonstrate that enhanced LPS-dependent activation of Egr-1 contributes to increased TNFα production after chronic ethanol exposure.

Increased production of tumor necrosis factor ␣ (TNF␣) is associated with the development of alcoholic liver disease. Culture of RAW264.7 macrophages with 25 mM ethanol for 48 h increased lipopolysaccharide (LPS)stimulated accumulation of tumor necrosis factor ␣ (TNF␣) peptide and mRNA by 2-fold. We investigated whether chronic ethanol-induced increases in the DNA binding and/or promoter activity of the key transcription factors regulating LPS-stimulated TNF␣ promoter activity contribute to increased TNF␣ expression. Binding of Egr-1 to the TNF␣ promoter was increased by 2.5-fold after ethanol exposure, whereas NFB binding was decreased to 30% of control. AP-1 binding was not affected. Changes in binding activity were paralleled by an increased contribution of the Egr-1 binding site and a decreased contribution of the NFB site to LPS-stimulated TNF␣ promoter activity. Overexpression of dominant negative Egr-1 prevented the ethanol-induced increase in LPS-stimulated TNF␣ mRNA accumulation. Chronic ethanol exposure enhanced LPS-stimulated Egr-1 promoter-driven CAT expression and transcription of Egr-1. Induction of Egr-1 is dependent on ERK1/2 activation in other systems. Therefore, we investigated whether the ERK1/2 pathway mediated the chronic ethanol-induced increases in Egr-1 and TNF␣. Increased Egr-1 promoter activity and TNF␣ mRNA accumulation after chronic ethanol were both prevented by overexpression of dominant negative ERK1/2. LPS-stimulated ERK1/2 phosphorylation was increased 2-fold in cells cultured with ethanol compared with controls. These results demonstrate that enhanced LPS-dependent activation of Egr-1 contributes to increased TNF␣ production after chronic ethanol exposure.
Activation of macrophages by endotoxin/lipopolysaccharide (LPS), 1 a component of the cell wall of Gram-negative bacteria, leads to the production of a variety of inflammatory cytokines, including tumor necrosis factor ␣ (TNF␣) and interleukin 1␤, as well as reactive oxygen species. LPS-stimulated TNF␣ expression is a highly regulated process that involves both transcriptional and post-transcriptional mechanisms (1). LPS binds to a cell surface receptor, CD14, which, via interactions with additional plasma membrane proteins, such as the toll-like receptor 4 (2), stimulates a complex array of signal transduction cascades (2,3). Stimulation of macrophages with LPS activates tyrosine kinases, protein kinase C, nuclear factor B (NFB), as well as members of the mitogen-activated protein kinase family, including ERK1/2, p38, and c-Jun N-terminal kinase (JNK) (3). Increased TNF␣ expression in response to LPS requires the activation of a distinct set of transcription factors binding to at least two regions of the TNF␣ promoter (4,5). Although the exact array of transcription factors interacting with the TNF␣ promoter is to some extent cell-and speciesspecific (6), recruitment of NFB and early growth response 1 (Egr-1), as well as increased c-Jun binding, appears to be required for full activation of TNF␣ expression in most types of macrophages (4,5). LPS-mediated activation of specific signaling cascades translates into the activation of these transcription factors. NFB activation results in its translocation to the nucleus and binding to the TNF␣ promoter (3). Similarly, activation of JNK mediates c-Jun phosphorylation and activity (7) and ERK1/2 is required for enhanced Egr-1 binding to the TNF␣ promoter (8,9).
TNF␣ has important protective functions in mediating host defenses against infection and tumor formation. However, increased production of TNF␣ has been implicated in the pathogenesis of a number of inflammatory diseases, including alcoholic liver disease (10). Treatment of rats with antibodies to TNF␣ prevents liver damage resulting from chronic gastricinfusion of ethanol (10). Similarly, transgenic mice lacking the TNF␣ receptor I gene are resistant to chronic-ethanol induced liver damage (11). Although the importance of TNF␣ to the progression of alcoholic liver disease is clear, the mechanism(s) by which ethanol increases TNF␣ production are not well understood. One contributing factor to enhanced TNF␣ production is an increased exposure to LPS after ethanol consumption. LPS levels are increased in the blood of alcoholics (12,13) and rats exposed to ethanol via gastric infusion (14). Recent data indicate that long term ethanol exposure also increases the sensitivity of macrophages to LPS activation. For example, long term ethanol consumption results in an increased susceptibility to endotoxin-induced liver injury (15). Moreover, LPSstimulated TNF␣ accumulation in hepatic macrophages is increased after chronic ethanol feeding (16,17).
Both short and long term ethanol exposure can disrupt a number of hormone-and neurotransmitter-dependent signal transduction pathways, including tyrosine kinases, NFB, protein kinase C, and ERK1/2 activation (18). Because LPS utilizes many of these same ethanol-sensitive signal transduction pathways, we hypothesized that chronic ethanol exposure enhances TNF␣ production by macrophages by disrupting LPSdependent signal transduction. Using RAW 264.7 cells, a macrophage-like cell line, here we show that chronic ethanol exposure in culture enhances accumulation of bioactive TNF␣ in response to LPS. Of the three transcription factors required for maximal activation of TNF␣ expression in response to LPS, we found that chronic ethanol enhanced only Egr-1 binding to the TNF␣ promoter, whereas NFB binding was decreased and AP-1 binding was unchanged. These changes in the binding of trans-acting factors were paralleled by an increased contribution of Egr-1 and a decreased contribution of NFB to TNF␣ promoter activity. Because induction of Egr-1 requires activation of ERK1/2 in other cell types (9, 19 -21), we investigated whether up-regulation of the ERK1/2 pathway by chronic ethanol contributed to increased Egr-1 binding activity and LPSstimulated TNF␣ production. Using dominant negative ERK1/2 and Egr-1 constructs, as well as specific inhibitors of ERK1/2 activation, we demonstrate that enhanced LPS-stimulated Egr-1 expression and DNA-binding activity contribute to increased TNF␣ production after chronic ethanol exposure.
Cell Culture-The RAW 264.7 macrophage-like cell line was obtained from the American Type Culture Collection and routinely cultured in Dulbecco's modified Eagle's media (DMEM) with 10% fetal bovine serum (FBS) and penicillin-streptomycin at 37°C and 5% CO 2 . For experiments, RAW 264.7 cells were seeded at 3.4 ϫ 10 4 /cm 2 in 6-well (for ERK1/2 activation, RNA extraction), 96-well (for TNF␣ bioassay) or 100-mm dishes (for nuclear extracts). After overnight culture, medium was changed to DMEM plus 10% FBS with or without ethanol. Control and ethanol-treated plates were wrapped with Parafilm to prevent evaporation of ethanol; parafilm wrapping had no effect on the pH of the cell culture media over the 48 h in culture (data not shown). The concentration of ethanol in media was measured by enzymatic assay (Sigma Chemical Co., St. Louis MO) and was within 80% of the starting concentration after 24 h(data not shown).
For transfections, RAW264.7 macrophages were grown in 100-mm dishes to 60% confluency and were transiently transfected with control and expression vectors using TransFast transfection reagent (Promega), according to the manufacturer's instructions. Transfected cells were subcultured and then treated or not with 25 mM ethanol for 48 h as described above.
TNF␣ Bioassay-RAW 264.7 cells were cultured with or without ethanol in 96-well plates for 48 h and then stimulated with or without 100 ng/ml LPS for 0 -4 h. TNF␣ peptide accumulation reaches a peak between 4 and 6 h of LPS stimulation, with levels maintained over 24 h (data not shown). TNF␣ peptide accumulated in the media was measured by bioassay as previously described (16).
Northern Blot Analysis and Ribonuclease Protection Assay-After 48 h culture with or without ethanol, cells were treated with or without 100 ng/ml LPS in DMEM/10% FBS, and total RNA were isolated with TRIzol reagent following the manufacturer's procedure (Invitrogen, Grand Island, NY). For Northern blot analysis, 10 g of total RNA was electrophoresed through 1.2% agarose-formaldehyde gels, transferred to GeneScreen Plus membranes (PerkinElmer Life Sciences, Boston, MA) and UV-cross-linked. Based on previously published sequences, four antisense oligonucleotides corresponding to different regions of mature mRNA of murine TNF␣ gene were designed as probes. They were: T1, 5Ј-TTGACCACAGCGCTGAGTTGGTCCCCCTTCTCCAGCT-GGAAGACT-3Ј; T2, 5Ј-AAAGTAGACCTGCCCGGACTCCGCAAAGTC-TAAGTACTT-3Ј; T3, 5Ј-GTGAGGAGCACGTAGTCGGGGCAGCCTTG-TCCC-3Ј; T4, 5Ј-AGACATAGGCACCGCCTGGAGTTCTGGAAGCCCC-CC-3Ј. The sequences of the probes for 18 S rRNA were 5Ј-ATGGCTT-AATCTTTGAGACAAGCATATGCTACTGGCAGC-3Ј and 5Ј-TGCACG-CATCCCCCCCCGGGAAGGGGGGTCAGCGCC-3Ј. Probes were endlabeled with [␥-32 P]ATP (PerkinElmer Life Sciences). The membranes were prehybridized with Church-Gilbert buffer at 55°C for 2 h and then hybridized with the same buffer containing the probes overnight. For ribonuclease protection assays, mouse cytokine multiprobe DNA templates (BD PharMingen) and CAT-linearized DNA template (Promega) were used to synthesize in vitro transcribed antisense riboprobes. Ribonuclease protection assays were carried out according to the manufacturer's instructions (Ambion). Samples were then run on 5% sequencing gels, dried, and autoradiographed.
ERK1/2 Activation-After culture with or without 25 mM ethanol for 48 h, cells were washed once with 2 ml of DMEM/10% FBS. Cells were then treated with or without 100 ng/ml LPS in DMEM/10% FBS. At the end of each treatment, cells were moved to ice, washed with 2 ml of ice-cold phosphate-buffered saline buffer containing 1 mM sodium orthovanadate and 2 mM EDTA and then lysed in lysis buffer (20 mM Tris-HCl, pH 8, 1% Triton X-100, 100 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, and protease inhibitors (Complete, Roche Molecular Biochemicals, Indianapolis, IN)) for 30 min at 4°C. Lysates were centrifuged at 14,000 ϫ g for 15 min at 4°C. 50 -80 g of protein were separated by SDS-PAGE, transferred to polyvinylidene difluoride and probed using anti-phospho ERK1/2 antibodies. Membranes were then stripped and reprobed with anti-ERK1/2 antibodies.
Nuclear Isolation and Extraction-After culture with and without 25 mM ethanol for 48 h, nuclei were isolated and either extracted with 0.4 M NaCl (26) (high salt extract) or lysed in lysis buffer (total nuclear extract). After extraction, nuclei were centrifuged at 14,000 ϫ g for 15 min, and the supernatants were used for Western blot analysis of Egr-1 and PU.1 expression (total nuclear extract) or electrophoretic mobility shift assay (EMSA) (high salt extract).
Electrophoretic Mobility Shift Assays-An oligonucleotide corresponding to the Egr-1 binding site in the promoter region of murine TNF␣ gene (5Ј-AACCCTCTGCCCCCGCGATGGAG-3Ј) was used to measure the DNA binding activity of Egr-1. Oligonucleotides for the consensus NFB and AP-1 binding sites were used to assess NFB and AP-1 DNA binding activity, respectively. After annealing, the doublestranded oligonucleotides were end-labeled with [␥-32 P]ATP. 20,000 -50,000 cpm of 32 P-labeled oligonucleotides were used in each binding reaction (20 l), which contained 0.2 pmol of DNA probes, 3-5 g of nuclear extracts and binding buffer (5% glycerol, 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 100 ng/ml poly(dI-dC)⅐poly(dI-dC)). After incubation on ice for 20 min, the mixtures were loaded onto 6% nondenaturing polyacrylamide gels pre-run with TBE (20 mM Tris base, pH 8.3, 20 mM boric acid, and 0.5 mM EDTA) buffer with 1% glycerol and 4 mM MgCl 2 at 120 V for 30 min. The gels were run at 120 V, dried, and autoradiographed. Equal loading of labeled oligonucleotide was confirmed by examining the density of unlabeled oligonucleotide at the bottom of each lane. Controls were run with increasing concentrations of unlabeled oligonucleotide to confirm the specificity of the gel shifts (data not shown).
Luciferase Assays-RAW 264.7 macrophages were transiently transfected with TNF␣ promoter-luciferase constructs or empty vector, along with pRLTK, which expresses Renilla luciferase (Promega). In some experiments, cells were also co-transfected with pRC/CMV IB S32A/ S36A (IB superrepressor) (25) or empty vector. Cells were then subcultured into 96-well plates and cultured with or without 25 mM ethanol for 48 h. Cells were then stimulated or not with 1000 ng/ml LPS for 4 h. Samples from replicate 96-well plates were pooled for preparing cell lysates. All assays were done in triplicate. Reporter firefly luciferase and Renilla luciferase were measured using the Dual Luciferase Reporter Assay System (Promega). Data were normalized for transfection efficiency by dividing firefly luciferase activity with that of Renilla luciferase.
Nuclear Run-on-Nuclear run-on experiments to measure nascent RNA transcripts were essentially performed as described elsewhere (27). Briefly, RAW264.7 cells were pretreated or not with 25 mM ethanol for 48 h and stimulated with 0 or 100 ng/ml LPS for 30 -60 min. Following stimulation, nuclei were isolated from 1-2 ϫ 10 7 cells/treatment group and were incubated with 2ϫ reaction buffer (10 mM Tris-Cl, 5 mM MgCl 2 , 0.3 mM KCl, 10 mM each of ATP, GTP, CTP, 1 mM dithiothreitol) in the presence of [␣-32 P]UTP at 30°C for 30 min and were further incubated with RNase-free DNase I (1 mg/ml) for 5 min at 30°C and with Proteinase K (20 mg/ml) for an additional 30 min at 42°C. Labeled RNA was harvested and hybridized with mouse Egr-1 and glyceraldehyde-3-phosphate dehydrogenase cDNAs immobilized on nitrocellulose filters for 36 h at 65°C. Membranes were thoroughly washed and processed for autoradiography.
Statistical Analysis-Values are expressed as means Ϯ S.E.; in some graphs the magnitude of the S.E. is too small to see on the graph. Student's t test was used to compare between groups.

RESULTS
Long term ethanol feeding enhances LPS-stimulated TNF␣ accumulation by Kupffer cells isolated from rats (16). Here we first investigated whether this increased sensitivity to LPS was also observed after treatment of macrophages with ethanol in culture. RAW264.7 macrophages were cultured with and without 25 mM ethanol for 48 h and then stimulated or not with 100 ng/ml LPS for 2-4 h. Although LPS increased TNF␣ production in control cells, secretion of TNF␣ in response to LPS was increased by 1.7-to 2.2-fold in cells cultured with ethanol compared with controls (Fig. 1A). This increase was maintained up to 10 h after LPS stimulation (data not shown). LPS stimulation of TNF␣ secretion was associated with increased TNF␣ mRNA accumulation. Increased TNF␣ mRNA in response to LPS followed a similar time course in both control and ethanol-treated cells, with maximal accumulation observed between 45 and 60 min (Fig. 1B). However, the quantity of TNF␣ mRNA was 2.0-to 2.2-fold greater in ethanol-treated cells compared with controls at each of the time points examined (Fig. 1B).
Although LPS-induced TNF␣ production is controlled at transcriptional, post-transcriptional, and post-translational levels (28), increased transcription is the initial response to LPS. Recruitment of NFB and Egr-1, as well as increased c-Jun binding to a CRE/AP-1 site, on the TNF␣ promoter are required for full activation of TNF␣ expression in macrophages (4,5). Therefore, we asked whether chronic ethanol exposure impacted on the activation of NFB, AP-1, and Egr-1 binding to DNA in response to LPS. Gel-shift assays revealed that culture of RAW264.7 macrophages with 25 mM ethanol for 48 h increased binding of nuclear protein to the Egr-1 site in the TNF␣ promoter ( Fig. 2A). LPS increased Egr-1 binding to the TNF␣ promoter by 2.2 Ϯ 0.5-fold over baseline in control cells compared with 5.7 Ϯ 0.6-fold over baseline after chronic ethanol treatment (p Ͻ 0.05, n ϭ 3). In contrast, chronic exposure to ethanol decreased binding of nuclear proteins to an NFB consensus DNA binding site ( Fig. 2A). LPS increased NFB binding by 11.8 Ϯ 3.2-fold over baseline in controls compared with 3.9 Ϯ 0.8-fold after ethanol (p Ͻ 0.05, n ϭ 3). Chronic ethanol exposure had no effect on the binding of nuclear proteins to an oligonucleotide for the AP-1 binding site. LPS increased AP-1 binding by 1.7 Ϯ 0.2-fold over baseline in controls, compared with 1.9 Ϯ 0.5-fold after ethanol exposure (n ϭ 3) ( Fig. 2A).
Using a series of TNF␣ promoter reporter constructs, we next tested whether the ethanol-induced changes in binding of nuclear proteins to the TNF␣ promoter were associated with changes in TNF␣ promoter activity. There was no difference in LPS stimulation of luciferase activity when the full-length TNF␣ promoter was linked to the luciferase reporter between control and ethanol-treated cells (1.8 Ϯ 0.1 relative luciferase activity in control, compared with 1.5 Ϯ 0.4 after chronic ethanol, n ϭ 6). This is consistent with our previous finding that chronic ethanol has no net effect on total LPS-stimulated TNF␣ transcription using nuclear run-on assays (29). However, chronic ethanol changed the relative contributions of the Egr-1 and NFB site to LPS-stimulated TNF␣ promoter activity. Removal of a region from Ϫ615 to Ϫ161, which contains the Egr-1 site, decreased LPS stimulation for reported activity by 49% in control cells, consistent with previously published reports using this truncation (5). However, after chronic ethanol, truncation of the Egr-1 site resulted in a 84% decrease in promoter activity (Fig. 2B). Similarly, selective mutation of the Egr-1 binding site in the TNF␣ promoter decreased LPS-stimulated luciferase activity by 56% in control cells, compared with 78% after chronic ethanol. In contrast, selective mutation of the NFB-3 site in the TNF␣ promoter decreased LPS-stimulated luciferase activity by 55% in control cells but had no effect on LPS-stimulated TNF␣ promoter activity after chronic ethanol exposure (Fig. 2B). To further investigate this loss of NFB activity after chronic ethanol, we compared the effects of overexpression of an IB superrepressor (IB-AA) on transcription from the full-length TNF␣ promoter. In control cells, inhibition of NFB activity by overexpression of the IB superrepressor decreased LPS-stimulated luciferase activity by 53% (Fig. 2C). In contrast, after chronic ethanol, the IB superrepressor had no effect on LPS-stimulated transcription from the TNF␣ promoter (Fig. 2C). These results demonstrate that the contribution of the Egr-1 site and NFB site to LPS-stimulated transcription from the TNF␣ promoter are different between control and ethanol-treated cells. After chronic ethanol, NFB function is lost, and this is compensated for by an increase in Egr-1 binding.
Because chronic ethanol exposure increased both LPS-stimulated Egr-1 binding to the TNF␣ promoter, as well as the functional contribution of the Egr-1 site to promoter activity, we hypothesized that Egr-1 was essential in mediating increased TNF␣ mRNA accumulation in response to chronic ethanol exposure. To test this hypothesis, RAW264.7 macrophages were transiently transfected with a plasmid, pCMV ETTL, which contains a truncated form of the Egr-1 protein lacking the N-terminal activation domain (dominant negative) (21), or empty vector, cultured for 48 h with or without 25 mM ethanol and then stimulated or not with LPS for 60 min. In cells transfected with empty vector, TNF␣ mRNA accumulation, measured by ribonuclease protection assay, was increased in both control and ethanol-treated cells in response to LPS; TNF␣ mRNA was 2.0-fold higher in ethanol-treated cells (Fig.  3). In contrast, LPS-stimulated TNF␣ mRNA accumulation was blunted in cells overexpressing the dominant negative Egr-1, and there was no stimulatory effect of chronic ethanol exposure (Fig. 3).
We next investigated the mechanism by which chronic ethanol increased Egr-1 activity. LPS treatment increased the quantity of Egr-1 protein in the nucleus (Fig. 4). After culture with 25 mM ethanol for 48 h, LPS stimulation of Egr-1 protein accumulation in the nucleus was increased 1.9-fold over controls (Fig. 4). Increased Egr-1 protein accumulation suggested that ethanol exposure increased the expression of Egr-1 in response to LPS stimulation. To test this hypothesis, RAW264.7 macrophages were transfected with an Egr-1 promoter-CAT reporter construct or pCAT control vector and then cultured with or without 25 mM ethanol for 48 h. Cells were then stimulated or not with 100 ng/ml LPS for 60 min. LPS did not increase CAT mRNA expression in either control or ethanol-treated cells transfected with pCAT control vector (Fig. 5). In contrast, LPS increased CAT mRNA 3-fold over basal in control cells expressing the Egr-1 promoter-CAT construct (Fig.  5). After chronic ethanol exposure, LPS stimulation of CAT mRNA expression was increased by 8.3-fold over basal (Fig. 5). Increased Egr-1 promoter activity was associated with increased rates of Egr-1 transcription after chronic ethanol (Fig.  6). Higher rates of Egr-1 transcription were observed after 30- to 60-min stimulation with LPS.
LPS activates a complex array of signal transduction cascades leading to increased TNF␣ production by macrophages (3). Activation of ERK1/2 is required for full activation of LPSstimulated TNF␣ production (4,8,9). In endothelial and epithelial cells, activation of ERK1/2 regulates Egr-1 expression (30,31). Recently, we have found that LPS-dependent activation of ERK1/2 mediates Egr-1 expression in RAW264.7 macrophages (9). To investigate the potential role of ERK1/2 in mediating the chronic effects of ethanol on LPS-stimulated Egr-1 activity and TNF␣ production, we first asked whether chronic ethanol-induced increases in Egr-1 promoter activity were dependent on ERK1/2 activity. RAW264.7 macrophages transfected with Egr-1 promoter CAT construct were co-transfected with vectors expressing dominant negative ERK1/2 or empty vector. In cells overexpressing dominant negative ERK1/2, LPS treatment did not increase phosphorylation of ERK1/2 (data not shown) or Egr-1 promoter-dependent CAT expression (Fig.  5), indicating that ERK1/2 activation was required for mediating LPS-stimulated Egr-1 expression.
One potential mechanism by which ethanol could enhance LPS-stimulated Egr-1 expression would be via an enhancement of LPS-stimulated ERK1/2 activation. To address this question, LPS-stimulated ERK1/2 phosphorylation measured in control and ethanol-treated cells. RAW264.7 macrophages were cultured with 50 mM ethanol for 48 h and then stimulated with 100 ng/ml LPS. LPS-induced activation of ERK1/2 phos-phorylation reached a maximum between 40 and 50 min (Fig.  7) and returned to baseline after 2 h (data not shown). Enhanced LPS-induced ERK1/2 phosphorylation after chronic exposure to ethanol could be detected as early as 20 min after activation. Increased phosphorylation was maintained over 40 min after stimulation with LPS (Fig. 7). Ethanol-induced increases in LPS-stimulated ERK1/2 phosphorylation were dosedependent; exposure to 10 mM ethanol had little effect on LPSdependent responses (data not shown), whereas exposure to 25 or 50 mM ethanol increased ERK1/2 phosphorylation by 2.1 Ϯ 0.3-fold (25 mM ethanol) and 2.5 Ϯ 0.4-fold (50 mM ethanol, n ϭ 6) compared with cells not cultured with ethanol. Chronic exposure to ethanol had no effect on the total immunoreactive ERK1/2 quantity (87 Ϯ 17 and 141 Ϯ 20 arbitrary units of density for ERK1 and ERK2, respectively, in controls compared with 104 Ϯ 21 and 116 Ϯ 26 after culture with 25 mM ethanol for 48 h, n ϭ 6).
We next investigated whether LPS-stimulated ERK1/2 activation mediated chronic ethanol-induced increases in Egr-1 binding to the TNF␣ promoter. Overexpression of kinase-dead ERK1/2 prevented the chronic ethanol-induced increase in Egr-1 binding to the TNF␣ promoter (Fig. 8A). Overexpression of dominant negative ERK1/2 decreased TNF␣ mRNA accumulation in both control and ethanol-treated cells; however, overexpression of dominant negative ERK1/2 eliminated the difference in TNF␣ mRNA accumulation between control and ethanol-treated cells (Fig. 8B). A similar normalization of TNF␣ peptide secretion was observed after pre-treatment of RAW264.7 macrophages with PD98059, an inhibitor of MAPK kinase (MEK), which prevents phosphorylation of ERK1/2 (Fig.  8C). PD98059 inhibits LPS-stimulated ERK1/2 phosphorylation with an IC 50 of ϳ20 M in RAW 264.7 macrophages (9). Pre-treatment with 20 M PD98059 decreased TNF␣ secretion in both control and ethanol-treated cells; however, LPS-stimulated TNF␣ secretion was no longer increased in cells chronically exposed to ethanol compared with control. DISCUSSION Increased production of TNF␣ is required for the progression of alcoholic liver disease (10). Although long term ethanol con-sumption is associated with increased TNF␣ in the circulation of both humans and animal models (32,33), the mechanisms by which chronic ethanol exposure increases TNF␣ production are FIG. 5. Chronic ethanol increases LPS-stimulated Egr-1 promoter activity via an ERK1/2-dependent mechanism. RAW264.7 macrophages were transfected with pCAT control vector or co-transfected with Egr-1 promoter-CAT reporter construct with dominant negative ERK1/2 expression vector or its empty vector control and then subcultured in the presence or absence of 25 mM ethanol for 48 h. Cell culture media was then removed and replaced with fresh DMEM with 10% FBS (without ethanol) and stimulated or not with 100 ng/ml LPS for 60 min. RNA was harvested and analyzed for CAT and ␤-actin mRNA expression by ribonuclease protection assay. Values represent means Ϯ S.E. and are expressed as percentage of LPS-treated cells not cultured with ethanol and transfected with empty vector, n ϭ 3. A representative autoradiograph is shown.
FIG. 6. LPS-stimulated Egr-1 transcription is increased after chronic ethanol. RAW264.7 macrophages were cultured in the presence or absence of 25 mM ethanol for 48 h. Cells were then stimulated with 100 ng/ml LPS for 30 min. Nuclei were isolated and analyzed for de novo transcribed Egr-1 and glyceraldehyde-3-phosphate dehydrogenase mRNA by nuclear run-on experiments. Values represent means Ϯ S.E., n ϭ 3. A representative autoradiograph is shown. FIG. 7. Chronic ethanol exposure increases LPS-stimulated ERK1/2 phosphorylation. RAW 264.7 macrophages were cultured with 0 or 50 mM ethanol for 48 h. Cell culture media was then removed and replaced with fresh DMEM with 10% FBS (without ethanol) and then stimulated or not with 100 ng/ml LPS for 0 -40 min. Cells were then lysed and activated ERK1/2 assayed by Western blot analysis using antibodies specific for phosphorylated ERK1/2. The same membranes were stripped and then used to measure total ERK1/2 protein with antibodies recognizing total ERK1/2 protein. Values represent means Ϯ S.E., n ϭ 5; *, p Ͻ 0.01 compared with cells not cultured with ethanol. Insets show representative Western blots. not well understood. We have previously reported that long term ethanol consumption by rats increases the sensitivity of hepatic macrophages to LPS-stimulated TNF␣ secretion (16,17). Here we have found that chronic ethanol exposure during culture also increased the sensitivity of RAW264.7 macrophages to LPS-stimulated TNF␣ secretion (Fig. 1). This in vitro response to chronic ethanol exposure in culture suggests that changes in TNF␣ production by macrophages observed after long term ethanol exposure in vivo are not due solely to systemic responses, such as increased exposure to endotoxin/LPS (12)(13)(14) or changes in retinoic acid status (34), but are due, at least in part, to a direct effect of ethanol exposure on macrophage function.
Using this cultured macrophage model system, we investigated the molecular mechanisms by which ethanol enhances LPS-stimulated TNF␣ expression. Here we have shown that increased TNF␣ production after chronic ethanol is dependent on Egr-1 activity. Egr-1, a member of the immediate early gene family, is a zinc finger transcription factor thought to play a role in mediating cellular responses to environmental stress such as ischemia, mechanical injury, and ionizing radiation (35). Egr-1 is rapidly induced upon LPS treatment in murine peritoneal macrophages (36), as well as RAW264.7 macrophages (9) (Fig. 4). Here we report that chronic ethanol exposure further increases LPS-stimulated Egr-1 expression in RAW264.7 macrophages. Importantly, we show that overexpression of a dominant negative form of Egr-1 ameliorates the effects of ethanol on TNF␣ mRNA accumulation. Up-regulation of Egr-1 expression after chronic ethanol was mediated by enhanced LPS-stimulated ERK1/2 phosphorylation, leading to activation of the Egr-1 promoter and increased Egr-1 transcrip-tion. These studies demonstrate that long term ethanol exposure exacerbates LPS-mediated activation of Egr-1, contributing to chronic ethanol-induced increases in LPS-stimulated TNF␣ production.
LPS-stimulated TNF␣ production is a complex and highly regulated process. Regulation occurs at transcriptional and post-transcriptional levels (3), and it is likely that ethanol acts at multiple steps in the regulation of TNF␣ production. For example, we have recently reported that chronic ethanol exposure stabilizes TNF␣ mRNA and that this stabilization contributes to increased TNF␣ production after chronic ethanol exposure (29). Although TNF␣ production is regulated at both transcriptional and post-transcriptional mechanisms, activation of TNF␣ transcription is a required first step in response to LPS (3). Activation of TNF␣ transcription is under complex control mechanisms, involving the activation of a number of transcription factors that bind to the TNF␣ promoter (4,5). In most macrophages, recruitment of NFB, AP-1 and Egr-1 appears to be required for full activation of TNF␣ expression (4,5). We hypothesized that a modulation of the binding of these key transcription factors to the TNF␣ promoter after chronic ethanol could contribute to increased TNF␣ production. Using gel shift assays and TNF␣ promoter-reporter constructs, we found that chronic ethanol exposure had profound effects on binding and functional activity of both the NFB and Egr-1 binding sites in the TNF␣ promoter. Binding of nuclear proteins to an NFB binding site after stimulation with LPS was reduced to only 30% of control values after chronic ethanol exposure. Deletion of the NFB-3 binding site had no effect on promoter activity after chronic ethanol, compared with the 55% lower promoter activity in control cells when this site was deleted. Similarly, inhibition of NFB activity with the IB superrepressor had no effect on transcription from the TNF␣ promoter after chronic ethanol exposure, compared with a 53% lower promoter activity in control cells. This decrease in NFB binding after chronic ethanol exposure in culture is similar to a decrease in LPS-stimulated NFB binding after chronic ethanol feeding reported in both alveolar macrophages (37) and Kupffer cells (17).
In contrast to the loss in NFB binding, Egr-1 binding to the TNF␣ promoter was increased 2.5-fold after chronic ethanol (Fig. 2). Moreover, loss of the Egr-1 binding site in the TNF␣ promoter, either due to a truncation or point mutation in the promoter, decreased LPS-stimulated promoter activity to a much greater degree in ethanol-treated cells compared with control. Interestingly, LPS-stimulated reporter activity driven by the full-length TNF␣ promoter did not differ between control and ethanol-treated cells. This is consistent with our previous observation that chronic ethanol has no net effect on total LPS-stimulated TNF␣ transcription, measured by nuclear run-on assays (29). However, from the results reported here, it is clear that the contributions of Egr-1 and NFB to LPSstimulated transcription from the TNF␣ promoter are different in control versus ethanol-treated cells. After chronic ethanol, NFB function is lost, and this is compensated by an increase in Egr-1 binding. Overexpression of an Egr-1 protein lacking the activation domain decreased TNF␣ mRNA accumulation in both control and ethanol-treated cells and prevented the ethanol-induced increase in TNF␣ mRNA. Overexpression of dominant negative Egr-1 decreased LPS-stimulated TNF␣ transcription by 50% in control RAW 264.7 macrophages (data not shown). Furthermore, we have previously reported that inhibition of ERK1/2, either by pre-treatment with PD98059 or overexpression of dominant negative ERK1/2, has no effect on LPS-stimulated TNF␣ mRNA stability after chronic ethanol exposure (29). Taken together, these data indicate that increased Egr-1 activity was critical to the maintenance of TNF␣ transcription after chronic ethanol exposure, acting to compensate for the decrease in NFB binding and promoter activity (Fig. 2).
Although it is clear that Egr-1 binding is required for full activation of TNF␣ gene expression both in vitro (5) and in vivo (38), the signal transduction pathways leading from the interaction of LPS with cell surface receptors on macrophages to increased Egr-1 expression are not well understood. Hypoxia induces Egr-1 expression in vascular tissue (35). Ethanol exposure can result in hypoxia in cells, which rapidly metabolize ethanol, such as hepatocytes (39). However, it is unlikely that ethanol-induced hypoxia is involved in the increase in Egr-1 expression reported here, because 1) there is no effect of ethanol on Egr-1 activity in cells not treated with LPS and 2) ethanol is not present during LPS stimulation/Egr-1 induction. Egr-1 induction in response to a number of stimuli, including shear stress in endothelial cells (20) and leptin stimulation of the hypothalamus (40), involves ERK1/2 activation. We have recently found that ERK1/2 activation is also required for LPSstimulated Egr-1 activation in RAW264.7 macrophages (9). Thus, although it is clear from all the data presented that ERK1/2 is involved in LPS-stimulated TNF␣ production in both control and ethanol-treated cells, we have found that chronic ethanol exposure enhances ERK1/2 activation by LPS (Fig. 7). Inhibition of ERK1/2 activity decreased LPS-stimulated TNF␣ production in both control and ethanol-treated cells. However, upon inhibition of ERK1/2 activity, TNF␣ mRNA accumulation and peptide secretion were no longer different between control and ethanol-treated cells, demonstrating that ERK1/2 pathways contribute to the overproduction of LPS-stimulated TNF␣ production after chronic ethanol.
Acute and chronic ethanol exposure impairs the function of a number of signal transduction cascades, including members of MAPK signaling pathways (18). However, very little is known regarding the effects of chronic ethanol on LPS-stimulated signal transduction. Here we have found that long term culture of RAW264.7 macrophages with 25-50 mM ethanol increased LPS-stimulated ERK1/2 phosphorylation. Long term culture of PC12 cells with 100 mM ethanol also enhances nerve growth factor (NGF)-induced activation of ERK1/2 (41). Similarly, culture of hepatocytes with 100 -200 mM ethanol for 16 -24 h increases ERK1/2 phosphorylation in response to a number of agonists (42,43). In contrast, chronic ethanol feeding to rats decreases ERK1/2 activation by epidermal growth factor, hepatocyte growth factor, and insulin in isolated hepatocytes (44). Ethanol-induced increases in ERK1/2 activation are associated with changes in cell function. In PC12 cells, ethanol-mediated increases in ERK1/2 were associated with increased NGF-stimulated neurite outgrowth (41), whereas in the present study, increased ERK1/2 activation was associated with increased Egr-1 expression and greater production of TNF␣. Interestingly, NGF-induced neurite outgrowth in PC12 cells has been reported to be dependent on ERK1/2-mediated induction of Egr-1 (45,46); however, it is not known whether the effects of ethanol on NGF-stimulated ERK1/2 are also associated with changes in Egr-1 expression.
The mechanism by which ethanol potentiates LPS-stimulated activation of ERK1/2 is not known. We have recently reported that chronic ethanol exposure, both after ethanol feeding to rats or exposure to macrophages in culture, also increases LPS-stimulated p38 phosphorylation (33). We are currently investigating whether ethanol acts at a common upstream signaling intermediate linking LPS-receptor activation to both these members of the MAP kinase family. Alternatively, ethanol could also enhance both ERK1/2 and p38 activity by decreasing MAP kinase phosphatase-1 activity, which can inactivate both phosphorylated ERK1/2 and p38.
The essential role for Egr-1 in mediating the chronic effects of ethanol on LPS-stimulated TNF␣ mRNA was clearly demonstrated, because overexpression of a truncated form of Egr-1 lacking the N-terminal activation domain prevented chronic ethanol-induced increases in TNF␣ mRNA (Fig. 3). Using deletion mutations of the TNF␣ promoter, several studies have demonstrated that Egr-1 binding is required for full activation of TNF␣ gene expression (4,5). Furthermore, Egr-1 knock out mice show reduced TNF␣ expression in lung, as well as lower airway inflammation, in response to inflammatory stimuli (38). However, Egr-1 knock out mice maintain LPS-stimulated cytokine production in lung (47). Modulation of Egr-1 expression has been best characterized in response to hypoxia/ischemia. Studies have shown that hypoxemia induces Egr-1 expression in the lung, leading to increased expression of tissue factor and fibrin accumulation (47). Although LPS has been previously reported to activate Egr-1 expression (36), this is the first report, to our knowledge, demonstrating that up-regulation of Egr-1 expression in response to an environmental stress, such as chronic ethanol exposure, can drive increased LPS-stimulated TNF␣ mRNA accumulation. Egr-1 has been characterized as a transcription factor that coordinates cellular responses to environmental stress, mediating increased expression of a number of target genes, including transforming growth factor ␤, platelet-derived growth factor, chemokines, and adhesion molecules (47). Because many of these same genes have been implicated in the development of alcoholic liver disease (39), it will be important to determine whether chronic ethanol-induced increases in Egr-1 activity also impact on the expression of other inflammatory and fibrogenic genes, in addition to the effect on TNF␣ mRNA reported here.