HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mandrekar, P. Articles by Szabo, G. Search for Related Content PubMed PubMed Citation Articles by Mandrekar, P. Articles by Szabo, G.

Acute Alcohol Exposure Exerts Anti-Inflammatory Effects by Inhibiting IB Kinase Activity and p65 Phosphorylation in Human Monocytes¹

Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Acute alcohol use is associated with impaired immune responses and decreased proinflammatory cytokine production. Our earlier studies have shown that acute alcohol intake inhibits NF-B DNA binding in an IB-independent manner. We report using human peripheral blood monocytes and Chinese hamster ovary cells transfected with CD14 cells that acute alcohol treatment in vitro exerts NF-B inhibition by disrupting phosphorylation of p65. Immunoprecipitation of p65 and IB revealed that acute alcohol exposure for 1 h decreased NF-B-IB complexes in the cytoplasm. Phosphorylation of p65 at Ser⁵³⁶ is mediated by IB kinase (IKK) and is required for NF-B-dependent cellular responses. We show that acute alcohol treatment decreased LPS-induced IKK and IKK activity resulting in decreased phosphorylation of p65 at Ser⁵³⁶. Furthermore, nuclear expression of IKK increased after alcohol treatment, which may contribute to inhibition of NF-B. Decreased phosphorylation of nuclear p65 at Ser²⁷⁶ was likely not due to alcohol-induced inhibition of protein kinase A and mitogen- and stress-activated protein kinase-1 activity. Although decreased IB phosphorylation after acute alcohol treatment was attributable to reduced IKK activity, degradation of IB during alcohol exposure was IKK-independent. Alcohol-induced degradation of IB in the presence of a 26S proteasome inhibitor suggested proteasome-independent IB degradation. Collectively, our studies suggest that acute alcohol exposure modulates IB-independent NF-B activity primarily by affecting phosphorylation of p65. These findings further implicate an important role for IKK in the acute effects of alcohol in immune cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Acute alcohol consumption is associated with immunosuppressive effects due to impairment in inflammatory mediator production and alterations in innate immune responses resulting in increased susceptibility to infections (1, 2, 3). Acute alcohol exposure decreases production of inflammatory cytokines such as TNF-, IL-1, and IL-6 both in vivo and in vitro in human and murine monocytic cells (4, 5, 6). The production of TNF- and proinflammatory cytokines is regulated at various levels of the signaling cascade including activation of NF-B, a transcription factor that binds to the promoter region of proinflammatory cytokine genes (7, 8).

NF-B, a pivotal transcription factor regulated by various stresses of bacterial or viral stimuli, serves as the central mediator of innate immune responses (9, 10). The NF-B transcription factor consists of two subunits of either homodimers or heterodimers of RelA/p65, c-Rel, and p50. The complexes are held in the cytoplasm and prevented from activation by a class of proteins referred to as inhibitors of NF-B or IB proteins. Upon stimulation, the IB proteins are phosphorylated by the IB kinases (IKK)³ IKK, IKK, and IKK, ubiquitinated, and degraded thereby releasing the NF-B complex for nuclear translocation (11). Recent studies have shown that in addition to this canonical NF-B activation, NF-B proteins are modified posttranslationally, and these changes can influence transcriptional activity. Examples of these modifications include the phosphorylation and acetylation of p65 (12) and S-nitrosylation of Cys⁶² of p50 (13). Phosphorylation of RelA/p65 and p50 are crucial in NF-B activation and target gene transcription (14, 15). LPS signaling promotes phosphorylation of the p65 subunit at Ser²⁷⁶ and Ser⁵³⁶ that is required for transactivation of gene expression (16). Ser²⁷⁶ of p65 is phosphorylated by protein kinase A (PKA) during IB degradation and this phosphorylation is necessary for the recruitment of CREB-binding protein (CBP)/p300 to p65 for active transcription (17, 18, 19). The phosphorylation of p65 at Ser⁵³⁶ by IKK following LPS stimulation increases NF-B-driven transcriptional activity (20). Recent studies have shown that p65 phosphorylation on Ser⁵³⁶ is not dependent on IB degradation and defines an IB-independent NF-B pathway (21). Thus, activation of NF-B could be regulated through distinct mechanisms in the signaling cascade.

Our previous studies have shown that in vitro acute ethanol treatment of monocytes in the absence of bacterial stimulus increases NF-B binding, specifically of the p50-p50 homodimer complex (22). However, in the presence of a bacterial stimulus, such as LPS, acute ethanol exposure decreased NF-B DNA binding of the p65-p50 heterodimer complex, which is responsible for transactivation of target genes (23). Previous studies have also shown that acute alcohol treatment affects NF-B nuclear translocation and DNA binding in human monocytes without affecting LPS-induced IB degradation, regardless of the stimuli, through various inflammatory receptors (24). Hence, it was postulated that acute alcohol exposure could inhibit NF-B in an IB-independent manner. Various IB degradation-independent mechanisms have been defined primarily including phosphorylation of p65 (21). In this study, we investigated the effect of acute alcohol exposure on IB-independent mechanisms of NF-B inhibition. We report the effect of alcohol on NF-B-mediated transactivation, phosphorylation of p65 at Ser⁵³⁶ and Ser²⁷⁶ positions, IKK activity, and IKK and IKK expression in human monocytes or Chinese hamster ovary (CHO) cells transfected with CD14 (CHO-CD14) used as a cell line model for monocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Cell culture and reagents

Monocytes from human peripheral blood were isolated by selective adherence from Ficoll-Hypaque purified mononuclear cell preparations as previously described (25). Healthy individuals age 18–60 years, females and males, who have no previous alcohol abuse history and consumed fewer than six drinks per week were recruited in the study.

The CHO cells stably transfected with cDNA for human CD14 (CHO-CD14) were provided by Dr. D. Golenbock (University of Massachusetts Medical School, Worcester, MA). These cells were maintained in Ham’s F-12 medium containing 10% FBS, 400 U/ml hygromycin, and 0.5 mg/ml G418.

Cell stimulations

Cells (monocytes or CHO-CD14) were stimulated with Escherichia coli-derived LPS (100 ng/ml), 25 mM ethanol, and the combination of LPS and ethanol together at the times indicated in each experiment. The 25 mM in vitro ethanol concentration approximates a blood alcohol level of 0.1 g/dl, which is achieved in vivo after a dose of moderate drink and is slightly above the legal limit of blood alcohol concentration. Cell viability was not affected by ethanol or LPS treatment.

Preparation of nuclear and cytoplasmic extracts

Nuclear and cytoplasmic extracts from cells with or without stimulation at 37°C were performed by the method of Schatzle et al. (26). Briefly, at the end of the stimulation period, cells were scraped and washed in ice-cold PBS. Cells were then resuspended in cold hypotonic buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 10 µg/ml protease inhibitors such as aprotinin, antipain, and leupeptin (Sigma-Aldrich)) and incubated on ice for 30 min. Cells were then lysed in 0.6% Nonidet P-40 by vortexing for 20 s. The lysate was then centrifuged at 12,000 x g for 30 s to pellet the nuclei, and the supernatant was stored at –80°C as the cytoplasmic extract. The nuclear pellet was then resuspended in ice-cold buffer B (20 mM HEPES (pH 7.9), 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 20% glycerol). All tubes were kept on a shaker at 4°C for 30 min. The lysate was then centrifuged at 12,000 x g for 15 min and the supernatant was stored at –80°C as the nuclear extract. Protein content was determined in both the cytoplasmic and nuclear extract by the Bio-Rad Dye Reagent assay.

EMSA analysis

A consensus dsNF-B oligonucleotide (5'-AGTTGAGGGGACTTTCGC-3') was used for EMSA. End-labeling was accomplished by treatment with T4 polynucleotide kinase in the presence of [-³²P]ATP (DuPont-NEN). Labeled oligonucleotide was purified on a polyacrylamide copolymer column (Bio-Rad). Nuclear protein (5 µg) was added to a binding reaction mixture containing 20 mM HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 5% glycerol, 200 µg/ml BSA, 2 µg of polydeoxyinosinic-polydeoxycytidylic acid, and 50,000 cpm of -³²P-labeled NF-B oligonucleotide. Samples were incubated at room temperature for 30 min. All reactions were run on a 6% polyacrylamide gel, and the dried gel was exposed to an x-ray film at –80°C overnight. For the cold competition reaction, a 20-fold excess of specific unlabeled double-stranded probe was added to the reaction mixture before adding the labeled oligonucleotide. Supershift analysis was conducted by addition of 1 µl of Ab 30 min after addition of the labeled NF-B followed by 30 min of incubation at room temperature.

Immunoblotting and immunoprecipitation

Nuclear or cytoplasmic proteins (20 µg) were loaded onto each well, separated on 10% SDS-polyacrylamide gel and electroblotted onto nitrocellulose membranes. Nonspecific binding was blocked by incubation of the membranes in TBS/1% nonfat dried milk/0.1% Tween 20 followed by the Abs indicated in the experiment. The Abs against phospho-p65 Ser⁵³⁶ and phospho-p65 Ser²⁷⁶, phosphorylated mitogen- and stress-activated protein kinase-1 (MSK-1), and a PKA catalytic (PKAc) subunit were purchased from Cell Signaling Technology and p65 was purchased from Santa Cruz Biotechnology. The rabbit Abs against IB, phospho-IB, IKK, and IKK were from Santa Cruz Biotechnology. -actin and TATA-binding protein (TBP) Abs were from Abcam. The Abs were detected using HRP-conjugated secondary Abs (Santa Cruz Biotechnology) and chemiluminescence assay reagents from Amersham Biosciences.

For immunoprecipitations, samples were precleared with 50 µl of TrueBlot anti-rabbit IgG immunoprecipitation beads (eBioscience) for 1 h, and the precleared samples were incubated with 5 µg of anti-p65 Ab (BIOMOL) overnight at 4°C. An aliquot of the precleared sample (one-twentieth of the volume) before the immunoprecipitation reaction was designated as the input sample. Next day, 50 µl of the TrueBlot anti-rabbit IgG immunoprecipitation beads were added to each sample for 1 h. The beads were washed three times with lysing buffer and then eluted with sample buffer.

NF-B reporter assay

The NF-B-dependent reporter plasmid p(B)5-Luc, a gift from Dr. N. Mackman (The Scripps Research Institute, La Jolla, CA), contains five tandem copies of the NF-B site (5xNF-B) from the human TNF- gene. RAW 264.7 macrophages were transfected with the reporter plasmids 5xNF-B firefly luciferase and Renilla luciferase as control (Promega) using the transfection agent FuGENE 6 (Roche Applied Science). After 24 h of incubation, cells were treated with various concentrations of alcohol in the presence or absence of LPS (100 ng/ml) for 8 h, and luciferase activity was assessed with Dual Glo Luciferase assay reagent (Promega) according to the manufacturer’s instructions. NF-B transcriptional activity as detected by firefly luciferase activity was normalized with the Renilla luciferase activity. The relative light units represent an average of triplicate samples.

IKK assay

The IKK assay was performed by a method previously described (27). Briefly, IKK or IKK from the cytoplasmic extract (500 µg) was precipitated with Ab to IKK or IKK in the lysis buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1% Triton X-100) followed by treatment with 50 µl of anti-rabbit IgG beads (eBioscience). After 2 h, the beads were washed with lysis buffer and then assayed in kinase assay buffer containing 20 mM HEPES (pH 7.6), 10 mM MgCl₂, 1 mM DTT, 5 µCi of [-³²P]ATP (NEN Life science Products), 10 µM unlabeled ATP, and 0.5 µg of GST-IB (Santa Cruz Biotechnology). After incubation at 30°C for 30 min, the reaction was terminated by centrifugation and collecting supernatants. To quantitate kinase activity, 20 µl of supernatants containing the phosphorylated substrate were spotted onto p81 phosphocellulose paper (Upstate Biotechnology) and after repeated washing, cpm were determined using a scintillation counter. Furthermore, equal volumes (20 µl) of reaction mixture were boiled with 5x SDS sample buffer for 5 min, followed by resolution on a 10% polyacrylamide gel under reducing conditions. The gel was then dried, and radioactive bands visualized by PhosphoImager. To determine equal amounts of IKK and IKK in each sample, some of the immunoprecipitated sample was resolved on a 7.5% acrylamide gel, and Western blot analysis was performed as described.

PKA assay

PKA activity was assessed in monocytes using the PepTag nonradioactive protein assay kit (Promega), according to the manufacturer’s instructions. Briefly, each sample is mixed with the PKA reaction 5x buffer, PepTag A1 peptide, and PKA activator 5x solution and incubated at room temperature for 30 min. The reaction was terminated at 95°C for 10 min, after which 80% glycerol was added to each tube. The samples were then separated on 0.8% agarose gel at 100 V for 15 min. Phosphorylated peptide migrated toward the anode (positive), whereas nonphosphorylated peptide migrated toward the cathode (negative). The gel was photographed on a transilluminator. For positive control PKA assay, 10 ng of cAMP-dependent PKA and a PKAc subunit were used, and no PKAc subunit was added to the negative control.

Statistical analysis

All data are expressed as mean ± SE. Results between treatment groups were compared using the Wilcoxon signed rank, nonparametric data analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Acute alcohol consumption inhibits LPS-induced NF-B binding and transactivation potential

The mechanisms of the immunosuppressive effects of acute alcohol use have been linked to alterations in NF-B activation (1, 2, 3). Previous data suggested that universal inhibition of NF-B binding by alcohol via various receptor systems occurs independent of IB degradation (24). In this study, we investigated the effects of acute alcohol exposure on IB-independent mechanisms of NF-B activation. In human CHO-CD14 cells, used as a model cell line for human monocytes, LPS (100 ng/ml) induced a significant increase in NF-B DNA binding activity at 10 and 60 min as compared with nonstimulated cells (Fig. 1A). CHO cells expressing endogenous TLR4 and stably transfected with human CD14 provide an ideal system for investigation of TLR4-CD14-mediated NF-B activation (24). Alcohol was used at a physiologically relevant 25 mM dose that corresponds to blood alcohol levels (0.1 g/dl) slightly above the legal limit. Acute alcohol treatment (25 mM) decreased LPS-induced NF-B-binding activity significantly in CHO-CD14 cells both at 10 and 60 min (Fig. 1A). Previous work done by our group has shown that acute alcohol treatment alone does not affect the p65-p50 heterodimer but increases DNA binding of the p50-p50 homodimer (22). Supershift analysis confirmed that alcohol regulates dimers containing p65 or RelA protein in CHO-CD14 cells (Fig. 1B). Because acute alcohol consumption has been shown to inhibit the expression of TNF- mRNA, an NF-B-driven gene (23), we determined the effects of alcohol on the NF-B transcriptional activity in transient transfection experiments using a reporter gene construct containing five copies of the NF-B binding sites. LPS stimulation of cells transfected with the NF-B reporter construct resulted in a significant induction (4-fold) of NF-B-driven luciferase activity (Fig. 1C) compared with the unstimulated controls. Treatment of cells with LPS and alcohol together resulted in a significant inhibition (p < 0.01) of LPS-induced NF-B-driven luciferase activity in a dose-dependent manner (Fig. 1C). Alcohol (25, 50, 75, and 100 mM) added alone to the cells for 8 h did not have any effect on the NF-B-driven luciferase activity (data not shown). These results suggest that acute alcohol consumption not only reduces DNA binding activity of NF-B, but also significantly inhibits the transactivation potential of NF-B.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Acute alcohol decreases LPS-induced NF-B binding and transactivation in monocytic cells at indicated time points. A, CHO-CD14 cells were stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol (EtOH) at the times indicated. NF-B was detected in the nuclear extracts by EMSA using a 32P-labeled dsNF-B oligonucleotide. A 20-fold excess of unlabeled oligonucleotide was included as a cold competitor (Comp). Data show mean density ± SE of four experiments. B, Supershift analysis was conducted using anti-p65 Ab for 20 min. The gel is representative of three experiments that showed similar results. SS, supershifted complex; ns, nonspecific; Unst, unstimulated. C, RAW 264.7 macrophages were transiently transfected with NF-B reporter gene constructs carrying five tandem copies of the NF-B binding site in front of the firefly luciferase gene and the Renilla luciferase construct. At 24 h after transfection, cells were treated with either LPS alone or various concentrations of ethanol (Et) in the presence of LPS (100 ng/ml) for 8 h. Cells were then lysed to determine firefly luciferase activity and normalized to the Renilla luciferase activity. Data represent the fold induction of the luciferase gene as compared with the unstimulated control of a total of n = 3 experiments. *, p < 0.02; **, p < 0.04 compared with LPS).

Alcohol decreases phosphorylation of IB and delays degradation of IB

Activation of NF-B is linked to phosphorylation and proteolytic degradation of IB (28, 29). Our previous data have shown that acute alcohol treatment inhibits LPS-induced NF-B activation concomitant to reduced levels of IB in human monocytes and CHO-CD14 cells (24). To determine the effect of alcohol treatment on early IB degradation induced by LPS, CHO-CD14 cells were treated with alcohol for 10 min and cytoplasmic extracts were analyzed for phosphorylated and total IB levels by Western blotting. LPS stimulation for 10 min increased phosphorylated IB levels and resulted in a substantial loss of total IB levels in the cytoplasm (Fig. 2A). In contrast, cells treated with acute alcohol (25 mM) showed no significant increase in phosphorylation and only a moderate decrease in IB in the cytoplasm after 10 min LPS stimulation (Fig. 2A). However, 60 min after LPS stimulation, both phosphorylated and total IB levels were low in alcohol-treated cells, whereas LPS induced an increase in phospho-IB levels in alcohol-naive cells. These results indicate that acute alcohol treatment affects IB phosphorylation but differentially regulates degradation of the IB protein at 10 and 60 min. These data also suggest that 60 min of alcohol treatment degrades IB regardless of decreased phosphorylation of IB. Taken together, alcohol-induced reduction of NF-B activity may be regulated via sequestration of NF-B proteins by IB at 10 min stimulation. However, down-regulation of NF-B binding by alcohol at 60 min appears to be IB-independent.

View larger version (47K): [in this window] [in a new window]   FIGURE 2. Alcohol decreases phosphorylation but delays degradation of IB. A, CHO-CD14 cells were unstimulated (Unst) or stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol (EtOH) at the times indicated. Cytoplasmic extracts were prepared and immunoblotted with phospho-IB, total IB, and -actin. B, CHO-CD14 cells were stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol at the times indicated. Cytoplasmic extracts were immunoprecipitated (IP) with anti-p65 Ab and analyzed by Western blot (WB) using the total IB Ab. C, CHO-CD14 cells were stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol at the times indicated after pretreatment with or without 10 µM MG-132. Cytoplasmic extracts were prepared and immunoblotted with the total IB Ab and -actin. NF-B binding in the nuclear extracts of the corresponding samples was determined by EMSA.

LPS induces NF-B-IB dissociation through a process initiated by inducible IB phosphorylation, a modification coupled to IB polyubiquitination and degradation via the proteasome pathway (11). To determine whether alcohol influences IB degradation by a proteasome-independent pathway, IB abundance was assayed in cells pretreated with a proteasome-selective inhibitor, MG-132, followed by alcohol and LPS treatment. Fig. 2C shows that LPS stimulation for 10 or 60 min induced proteolysis of IB that was inhibited in the presence of MG-132. Cells treated with alcohol for 10 min also showed increased accumulation of IB in the presence of MG-132 (Fig. 2C). However, in the presence of MG-132, alcohol treatment still resulted in decreased IB levels in LPS stimulated cells at 60 min. This was supported by higher nuclear NF-B binding activity, as indicated by EMSA (Fig. 2C, lower panel) and decreased IB levels in the cytoplasm in cells treated with MG-132 followed by LPS plus alcohol treatment. These data indicated that the alcohol-induced decrease in IB at 60 min involved a proteasome-independent process.

LPS-induced phosphorylation of p65 is inhibited by acute alcohol consumption

Recent studies demonstrate that posttranslational modification of NF-B proteins influences transcriptional activity (15, 16). Phosphorylation of p65 and p50 is associated with increased DNA binding capacity. Phosphorylation of p65 at Ser²⁷⁶ by PKA and phosphorylation at Ser⁵³⁶ position by IB kinases have been shown to play an important role in NF-B transcriptional activity (18, 19, 20) Because we found that acute alcohol exposure regulates NF-B inhibition in an IB-independent manner, we investigated whether alcohol-induced modulation of p65 phosphorylation played a role in NF-B inhibition. Using an Ab specific for phospho-Ser⁵³⁶ p65, we found that in the absence of alcohol, LPS stimulation for 10 min increased phosphorylation of p65 at Ser⁵³⁶, which was still present at 60 min in the nuclear extracts of human monocytes (Fig. 3A). However, acute alcohol treatment completely abolished early phosphorylation of p65 at Ser⁵³⁶ at 10 min and phospho-Ser⁵³⁶ p65 levels were significantly decreased at 60 min in the nucleus (Fig. 3A). In addition to reduced levels of phospho-Ser⁵³⁶ p65, there was a significant decrease in total p65 levels in the nucleus of alcohol-treated cells (Fig. 3A), indicating that alcohol inhibits both nuclear translocation and phosphorylation of p65.

View larger version (65K): [in this window] [in a new window]   FIGURE 3. LPS-mediated phosphorylation of p65 is inhibited by acute alcohol exposure. A, Nuclear extracts from human monocytes unstimulated (Unst) or stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol (EtOH) at the times indicated were prepared and immunoblotted with phospho-p65 (Ser536, S536) initially and stripped and blotted with total p65. The membranes were re-stripped and blotted for TBP to demonstrate equal loading. A representative gel for phospho-p65 (Ser536-p65) and total p65 is shown from a total of six individuals. Data represent average mean density ± SE of phospho-p65 (Ser536) levels from a total of n = 6 individuals. *, p < 0.001; **, p < 0.01 compared with LPS. B, Nuclear and cytoplasmic extracts from human monocytes stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol for 60 min were prepared and immunoblotted with phospho-p65 (S536). The membranes were stripped and blotted for TBP for nuclear blots and -actin for cytoplasmic blots to demonstrate equal loading. Data represent average mean density ± SE of phospho-p65 (S536) levels of a total of n = 6 individuals. *, p < 0.001 compared with LPS. C, Nuclear and cytoplasmic extracts from human monocytes stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol (Et) for 60 min were prepared and immunoblotted with phospho-p65 (S276). The membranes were stripped and blotted for TBP for nuclear blots and -actin for cytoplasmic blots to demonstrate equal loading. Data represent average mean density ± SE of phospho-p65 (Ser276) levels of a total of n = 6 individuals. *, p < 0.01, compared with LPS. D, PKA assay. Nuclear (Nuc) and cytoplasmic (Cyto) extracts from human monocytes stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol (Et) for 60 min were incubated as described under standard PKA assay described in Materials and Methods. Phosphorylated peptide migrated toward the anode (+), whereas nonphosphorylated peptide migrated toward the cathode (–). The representative gel of a total of four experiments was photographed on a transilluminator. Positive control of 10 ng of cAMP-dependent PKAc subunit and negative control of no PKAc subunit are used. E, Human monocytes were stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol for 60 min. Whole cell extracts were immunoprecipitated (IP) with anti-p65 Ab and analyzed by Western blot (WB) using the total PKAc Ab (left) or the phospho-p65 (S276) Ab (right). Data represent average mean density ± SE of PKAc or phospho-p65 (S276) levels from a total n = 3 individuals. F, Whole cell extracts from human monocytes stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol for 10 and 60 min were prepared and immunoblotted with phospho-MSK-1 Ab. The membranes were stripped and blotted for -actin to demonstrate equal loading. Data represent average mean density ± SE of phospho-MSK-1 levels of a total of n = 3 individuals. *, p < 0.05 compared with LPS.

Additional studies indicate that p65 phosphorylation at Ser²⁷⁶ is also essential for NF-B-mediated cellular responses (17, 18, 30). We therefore, investigated whether acute alcohol treatment affects phosphorylation of p65 at Ser²⁷⁶, using an Ab specific for phospho-Ser²⁷⁶ p65 in cytoplasmic and nuclear extracts of alcohol-treated human monocytes. We found that LPS stimulation rapidly increased (60 min) the nuclear levels of phospho-Ser²⁷⁶ p65 (Fig. 3C). However, acute alcohol treatment prevented the LPS-induced increase in nuclear phospho-Ser²⁷⁶ p65 levels, whereas cytoplasmic phospho-Ser²⁷⁶ p65 remained at the same level in monocytic cells (Fig. 3C). PKA associated with both p65 and IB was shown to activate and phosphorylate the p65 subunit on Ser²⁷⁶ (17, 18). Because alcohol treatment decreased phospho-Ser²⁷⁶ p65 levels in the nucleus, we evaluated the effects of acute alcohol treatment on PKA enzyme activity both in the nucleus and cytoplasmic extracts of human monocytes stimulated with LPS in the presence or absence of alcohol. Fig. 3D shows that although PKA activity was induced in the cytoplasm by LPS stimulation, acute alcohol treatment did not affect PKA activity either in the nucleus or in the cytoplasm. Hence, decreases in nuclear levels of phospho-Ser²⁷⁶ p65 after alcohol treatment may be due to decreased nuclear translocation of total p65 as shown in Fig. 3A rather than changes in PKA activation. Because PKA remains associated in an inactive state with the NF-B-IB complex and signals causing degradation of IB result in a loss of PKAc from p65 immunoprecipitates as well as subsequent phosphorylation of p65 at Ser²⁷⁶ (18), we also determined whether PKAc specifically associated with the NF-B-IB complex is affected by alcohol. Human monocytes were treated with LPS in the presence or absence of acute alcohol for 60 min and then immunoprecipitation of p65 was conducted in total cellular extracts. This was followed by immunoblotting using the PKAc and phospho-Ser²⁷⁶ p65 Abs to determine the amount of PKA that remained associated with the NF-B-IB complex and the phosphorylation of p65 at Ser²⁷⁶. Fig. 3E shows that LPS treatment resulted in reduction in PKA in p65 immunoprecipitates and increased phospho-Ser²⁷⁶, whereas acute alcohol exposure plus LPS treatment retained association with p65 and resulted in no difference in phospho-Ser²⁷⁶ p65 compared with LPS alone. These results suggest that acute alcohol exposure does not affect PKA activity associated with the p65 and hence does not alter phospho-Ser²⁷⁶ p65 levels. In addition to the PKA, studies have shown that the MSK1 phosphorylates p65 at Ser²⁷⁶ (31). Fig. 3E shows that acute alcohol treatment increases LPS-induced phospho-MSK-1 levels at 10 and 60 min, whereas decreasing phospho-Ser²⁷⁶ p65 levels (Fig. 3F), indicating that it is less likely that the alcohol-activated MSK-1 has any effect on phosphorylation of p65 at Ser²⁷⁶. These data collectively suggest that acute alcohol exposure inhibits transcriptional activity of NF-B by the hindrance of p65 phosphorylation required for the functional activation of NF-B.

Proteolytic degradation of IB by acute alcohol treatment is not modulated by IKK complex inhibition

It has been shown that IKK complex is required not only for LPS-induced phosphorylation of IB, but also for phosphorylation of p65 (32). The observation that acute alcohol treatment inhibited phosphorylation of both IB and p65 prompted us to test the effect of acute alcohol treatment on LPS-induced IKK activation. Data in Fig. 4A demonstrate that treatment of human monocytes with LPS for 15 min significantly increased IKK and IKK activity leading to augmented phosphorylation of GST-IB substrate. However, addition of 25 mM alcohol abolished the LPS-induced IKK and IKK activity in human monocytes (Fig. 4A) and CHO-CD14 cells (similar results observed). Thus, taken together with the previously shown decreased phospho-IB, it appears that the decreased IKK and IKK activity may result in the inhibition of IB phosphorylation in alcohol-treated monocytic cells. However, reduced total IB levels in the cytoplasm after alcohol treatment may be indicative of an IKK-independent polyubiquitination and degradation.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Acute alcohol exposure inhibits IKK and IKK activity and protein expression. A, Human monocytes were stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol for 15 min. Immunoprecipitation (IP) of the cytoplasmic extracts with either IKK or IKK Abs in lysis buffer was performed as described in Materials and Methods. The kinase assays with the GST-IB as substrate were also performed as described. The proteins were separated on SDS-PAGE and the gels depict the 32P-phosphorylated GST-IB. Data represent the cpm incorporated in the substrate as measured by scintillation counter. *, p < 0.01 compared with LPS; n = 4 individuals). Equal protein in the immunoprecipitation samples was determined by immunoblotting with IKK or IKK Abs. B, Nuclear (Nuc) and cytoplasmic (Cyto) extracts from human monocytes pretreated with leptomycin B (LMB) for IKK and then stimulated with LPS (100 ng/ml) in the presence or absence of 25 mM ethanol (Et) for 60 min were prepared and immunoblotted with IKK (left) or IKK (right) Abs. The membranes were stripped and blotted for TBP for nuclear blots and -actin for cytoplasmic blots to demonstrate equal loading. The gels are representative of a total of six individuals.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In this study, we demonstrate that acute alcohol treatment regulates NF-B activation by a dual action involving p65 phosphorylation and IKK activity. We found that alcohol affects NF-B nuclear translocation, DNA binding activity, and transactivation potential and involves inhibition of IKK activity and decreased phosphorylation of p65 independent of IB degradation (Fig. 5). Our previous studies have shown that acute alcohol exposure potently inhibits transcription of inflammatory cytokines such as TNF- and IL-1 (24). Because these cytokines are specific target genes of NF-B, our study reveals how acute alcohol treatment may inhibit inflammatory cytokine gene activation. In this study, we show that acute alcohol consumption also suppresses NF-B-dependent reporter gene expression and IB phosphorylation.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Illustrating the effects of acute alcohol exposure on NF-B regulation. Acute alcohol treatment inhibits IKK or IKK activity and p65 phosphorylation to modulate NF-B mediated transcriptional activity independent of IB degradation.

Our previous studies have shown cytoplasmic retention of total p65 (23) 1 h after alcohol exposure, and in this study we observed decreased levels of total p65 in the nucleus. Furthermore, although p65-IB complexes were retained in the cytoplasm after 10 min of alcohol exposure, alcohol exposure for 1 h decreased p65-IB complexes in the cytoplasm as seen in the immunoprecipitation experiments. Thus, it appears that the p65 in the cytoplasm of alcohol-exposed cells is not in complex with IB, and the precise subcellular distribution of total p65 after alcohol exposure remains to be elucidated. Furthermore, recent studies have shown that after target-gene activation, p65 is degraded by the proteasome in the nucleus and in a DNA binding-dependent manner (36). Whether acute alcohol influences nuclear proteasomal activity and hence induces degradation of p65 in the nucleus is yet to be determined. Our experiments using MG-132, a proteasome inhibitor, showed increased NF-B binding 60 min after alcohol treatment, indicating a role for proteasome-mediated p65 degradation in the nucleus by alcohol.

In addition to inhibition of NF-B DNA binding activity, acute alcohol exposure promoted delayed IB degradation (Fig. 5) and inhibition of its resynthesis (37), which could be attributed to decreased NF-B binding in the promoter of the IB gene. Interestingly, although acute alcohol exposure decreases IKK and IKK activity as well as phosphorylation of IB, it appears that there was a delayed disappearance of the complexes of p65 and IB from the cytoplasm, as seen in the immunoprecipitation experiments. In addition to the ubiquitin-proteasome pathway, multiple proteolytic enzymes such as casein kinase II (38, 39), 90-kDa ribosomal S6 kinase (40), and m-calpain (41) have been implicated in IB degradation. Our studies cannot rule out the possibility that acute alcohol exposure may influence other proteolytic systems in monocytic cells and induce IB degradation independent of NF-B inhibition.

Our results show that acute alcohol treatment affects levels of IKK but not IKK in the nucleus and the cytoplasm. The catalytic activities of both IKK and IKK make essential contributions to IB phosphorylation and NF-B activation (42). Although IKK was demonstrated to function in the nucleus to regulate cytokine-induced NF-B activation by modifying histone function (43), a distinct role for nuclear IKK is not clearly defined. Furthermore, studies using IKK knockout mice demonstrate a role for IKK in the negative regulation of macrophage activation and inflammation (44). IKK has been shown to suppress NF-B activity by accelerating both the turnover of p65/RelA and c-Rel and their removal from proinflammatory gene promoters (44). Thus, increased IKK levels after acute alcohol exposure are likely to contribute to suppression of NF-B.

In conclusion, we propose that acute alcohol consumption has dual effects on NF-B regulation. One is an inhibitory effect of NF-B binding via decreased IKK activity and p65 phosphorylation and transcriptional activity and another is promotion of proteolytic degradation of IB. Acute alcohol exposure may thus affect inflammatory cytokine gene expression and exert its immunomodulatory, anti-inflammatory effects.

	Acknowledgment

 
We thank Karen Kodys for helping with preparation of the manuscript figures.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grant RO1 AA11576 from the Public Health Service, National Institute of Alcohol Abuse and Alcoholism and its contents are solely the responsibility of the authors and do not necessarily represent the views of the National Institute of Alcohol Abuse and Alcoholism.

² Address correspondence and reprint requests to Dr. Gyongyi Szabo, Department of Medicine, Lazare Research Building Room 215, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605. E-mail address: gyongyi.szabo{at}umassmed.edu

³ Abbreviations used in this paper: PKA, protein kinase A; PKAc, PKA catalytic subunit; CHO, Chinese hamster ovary; IKK, IB kinase; TBP, TATA-binding protein; MSK, mitogen- and stress-activated protein kinase; CBP, CREB-binding protein.

Received for publication June 7, 2006. Accepted for publication March 21, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Cook, R. J.. 1998. Alcohol abuse, alcoholism and damage to the immune system: a review. Alcohol. Clin. Exp. Res. 22: 1927-1942. [Medline]
2. Szabo, G.. 1999. Consequences of alcohol consumption on host defence. Alcohol Alcohol. 34: 830-841. [Abstract/Free Full Text]
3. Saad, J., R. Domati-Saad, T. R. Jerrells. 1993. Ethanol ingestion increases susceptibility of mice to Listeria monocytogenes. Alcohol. Clin. Exp. Res. 17: 75-85. [Medline]
4. Nelson, S., G. J. Bagby, G. Bainton, W. R. Warren. 1989. The effects of acute and chronic alcoholism on tumor necrosis factor and inflammatory response. J. Infect. Dis. 160: 422-429. [Medline]
5. Szabo, G., P. Mandrekar, D. Catalano. 1995. Inhibition of superantigen-induced T cell proliferation and monocytes IL-1, TNF and IL-6 production by acute ethanol treatment. J. Leukocyte Biol. 58: 342-350. [Abstract]
6. Verma, B. K., M. Fogarasi, G. Szabo. 1993. Down-regulation of tumor necrosis factor activity by acute ethanol treatment in human peripheral blood monocytes. J. Clin. Immunol. 13: 8-22. [Medline]
7. Collart, M. A., P. Bauerle, P. Vassali. 1990. Regulation of tumor necrosis factor transcription in macrophages: involvement of four B-like motifs and of constitutive and inducible forms of NF-B. Mol. Cell. Biol. 10: 1498-1506. [Abstract/Free Full Text]
8. Hiscott, J., J. Marios, J. Garoufalis, M. D’Addario, A. Roulston, I. Kwan, N. Pepin, J. Lacoste, H. Nguyen, G. Bensi. 1993. Characterization of a functional NF-B site in the human interleukin 1 promoter: evidence for a positive autoregulatory loop. Mol. Cell. Biol. 13: 6231-6240. [Abstract/Free Full Text]
9. Xiao, C., S. Ghosh. 2005. NF-B an evolutionarily conserved mediator of immune and inflammatory responses. Adv. Exp. Med. Biol. 560: 41-45. [Medline]
10. Ruland, J., T. W. Mak. 2003. From antigen to activation: specific signal transduction pathways linking antigen receptors to NF-B. Semin. Immunol. 15: 177-183. [Medline]
11. Hacker, H., and M. Karin. 2006. Regulation and function of IKK and IKK-related kinases. Sci. STKE 2006: re13.
12. Schmitz, M. L., I. Mattioli, H. Buss, M. Kracht. 2004. NF-B: a multifaceted transcription factor regulated at several levels. Chembiochem. 5: 1348-1358. [Medline]
13. Marshall, H. E., D. T. Hess, J. S. Stamler. 2004. S-Nitrosylation: physiological regulation of NF-B. Proc. Natl. Acad. Sci. USA 101: 8841-8842. [Free Full Text]
14. Li, C. C., R. M. Dai, E. Chen, D. L. Longo. 1994. Phosphorylation of NF-B1–p50 is involved in NF-B activation and stable DNA binding. J. Biol. Chem. 269: 30089-30092. [Abstract/Free Full Text]
15. Vermeulen, L., G. DeWilde, S. Notebaert, W. Vanden Berghe, G. Haegeman. 2002. Regulation of transcriptional activity of the nuclear factor-B p65 subunit. Biochem. Pharmacol. 64: 963-970. [Medline]
16. Viatour, P., M.-P. Merville, V. Bours, A. Chariot. 2005. Phosphorylation of NF-B and IB proteins: implications in cancer and inflammation. Trends Biochem. Sci. 30: 43-52. [Medline]
17. Zhong, H., M. J. May, E. Jimi, S. Ghosh. 2002. The phosphorylation status of nuclear NF-B determines its association with CBP/p300 or HDAC-1. Mol. Cell 9: 625-636. [Medline]
18. Zhong, H., R. E. Voll, S. Ghosh. 1998. Phosphorylation of NF-B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol. Cell 1: 661-671. [Medline]
19. Zhong, H., H. SuYang, H. Erdjument-Bromage, P. Tempst, S. Ghosh. 1997. The transcriptional activity of NF-B is regulated by the IB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89: 413-424. [Medline]
20. Sakurai, H., H. Chiba, H. Miyoshi, T. Sugita, W. Toriumi. 1999. IB kinases phosphorylate NF-B p65 subunit on serine 536 in the transactivation domain. J. Biol. Chem. 274: 30353-30356. [Abstract/Free Full Text]
21. Sasaki, C. Y., T. J. Barberi, P. Ghosh, D. L. Longo. 2005. Phosphorylation of RelA/p65 on serine 536 defines an IB-independent NF-B pathway. J. Biol. Chem. 280: 34538-34547. [Abstract/Free Full Text]
22. Mandrekar, P., D. Catalano, G. Szabo. 1997. Alcohol-induced regulation of nuclear regulatory factor- in human monocytes. Alcohol. Clin. Exp. Res. 21: 988-994. [Medline]
23. Mandrekar, P., D. Catalano, G. Szabo. 1999. Inhibition of lipopolysaccharide-mediated NFB activation by ethanol in human monocytes. Int. Immunol. 11: 1781-1790. [Abstract/Free Full Text]
24. Mandrekar, P., A. Dolganiuc, G. Bellerose, K. Kodys, L. Romics, R. Nizamani, G. Szabo. 2002. Acute alcohol inhibits the induction of nuclear regulatory factor B activation through CD14/Toll-like receptor 4, interleukin-1, and tumor necrosis factor receptors: a common mechanism independent of inhibitory B degradation?. Alcohol. Clin. Exp. Res. 26: 1609-1614. [Medline]
25. Szabo, G., P. Mandrekar, L. Girouard, D. Catalano. 1996. Regulation of human monocyte functions by acute ethanol treatment: decreased TNF, IL-1 and elevated IL-10, TGF production. Alcohol. Clin. Exp. Res. 20: 900-907. [Medline]
26. Schatzle, J. D., J. Kralova, H. R. Bose. 1995. Avian IB is transcriptionally induced by c-Rel and v-Rel with different kinetics. J. Virol. 69: 5383-5390. [Abstract]
27. Manna, S. K., A. Mukhopadhyay, B. B. Aggarwal. 2000. IFN- suppresses activation of nuclear transcription factors NF-B and activator protein 1 and potentiates TNF-induced apoptosis. J. Immunol. 165: 4927-4934. [Abstract/Free Full Text]
28. Ghosh, S., D. Baltimore. 1990. Activation in vitro of NF-B by phosphorylation of its inhibitor IB. Nature 344: 678-682. [Medline]
29. Beg, A. A., A. S. Baldwin. 1993. The IB proteins: multifunctional regulators of Rel/NF-B transcription factors. Genes Dev. 7: 2064-2070. [Free Full Text]
30. Okazaki, T., S. Sakon, T. Sasazuki, H. Sakurai, T. Doi, H. Yagita, K. Okumura, H. Nakano. 2003. Phosphorylation of serine 276 is essential for p65 NF-B subunit-dependent cellular responses. Biochem. Biophys. Res. Commun. 300: 807-812. [Medline]
31. Vermeulen, L., G. DeWilde, P. VanDamme, W. Vanden Berghe, G. Haegeman. 2003. Transcriptional activation of the NF-B p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J. 22: 1313-1324. [Medline]
32. Zandi, E., D. M. Rothwarf, M. Delhase, M. Hayakawa, M. Karin. 1997. The IB kinase complex (IKK) contains two kinase subunits, IKK and IKK, necessary for IB phosphorylation and NF-B activation. Cell 91: 243-252. [Medline]
33. Ear, T., A. Cloutier, P. P. McDonald. 2005. Constitutive nuclear expression of the IB kinase complex and its activation in human neutrophils. J. Immunol. 175: 1834-1842. [Abstract/Free Full Text]
34. Yamamoto, Y., R. Gaynor. 2004. IB kinases: key regulators of the NF-B pathway. Trends Biochem. Sci. 29: 72-79. [Medline]
35. Yang, F., E. Tang, K. Guan, C. Wang. 2003. IKK plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. J. Immunol. 170: 5630-5635. [Abstract/Free Full Text]
36. Saccani, S., I. Marazzi, A. A. Beg, G. Natoli. 2004. Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor B response. J. Exp. Med. 200: 107-113. [Abstract/Free Full Text]
37. Mandrekar, P., G. Bellerose, G. Szabo. 2002. Inhibition of NF-B binding correlates with increased nuclear glucocorticoid receptor levels in acute alcohol-treated human monocytes. Alcohol. Clin. Exp. Res. 26: 1872-1879. [Medline]
38. Lin, R., P. Beauparlant, C. Makris, S. Meloche, J. Hiscott. 1996. Phosphorylation of IB in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. Mol. Cell. Biol. 16: 1401-1409. [Abstract]
39. Barroga, C. F., J. K. Stevenson, E. M. Schwarz, I. M. Verma. 1995. Constitutive phosphorylation of IB by casein kinase II. Proc. Natl. Acad. Sci. USA 92: 7637-7641. [Abstract/Free Full Text]
40. Ghoda, L., X. Lin, W. C. Greene. 1997. The 90-kDa ribosomal S6 kinase (pp90^rsk) phosphorylates the N-terminal regulatory domain of IB and stimulates its degradation in vitro. J. Biol. Chem. 272: 21281-21288. [Abstract/Free Full Text]
41. Schaecher, K., J. M. Goust, N. L. Banik. 2004. The effects of calpain inhibition on IB degradation after activation of PBMCs: identification of the calpain cleavage sites. Neurochem. Res. 29: 1443-1451. [Medline]
42. Sizemore, N., N. Lerner, N. Dombrowski, H. Sakurai, G. R. Stark. 2002. Distinct roles of the IB kinase and subunits in liberating nuclear factor B (NF-B) from IB and in phosphorylating the p65 subunit of NF-B. J. Biol. Chem. 277: 3863-3869. [Abstract/Free Full Text]
43. Yamamoto, Y., U. N. Verma, S. Prajapati, Y.-T. Kwak, R. B. Gaynor. 2003. Histone H3 phosphorylation by IKK- is critical for cytokine-induced gene expression. Nature 423: 655-659. [Medline]
44. Lawrence, T., M. Bebein, G. Y. Liu, V. Nizet, M. Karin. 2005. IKK limits macrophage NF-B activation and contributes to the resolution of inflammation. Nature 434: 1138-1143. [Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted