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Nutritional Immunology

Docosahexaenoic Acid Consumption Inhibits Deoxynivalenol-Induced CREB/ATF1 Activation and IL-6 Gene Transcription in Mouse Macrophages¹

Qunshan Jia^,**, Hui-Ren Zhou^*,,**, Yuhui Shi^,** and James J. Pestka^*,,**,2

^* Department of Microbiology and Molecular Genetics, Department of Food Science and Human Nutrition, and ^** Center for Integrative Toxicology, Michigan State University, East Lansing, MI

² To whom correspondence should be addressed. E-mail: pestka{at}pilot.msu.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The mycotoxin deoxynivalenol (DON) induces IgA nephropathy in mice by upregulating IL-6 expression, which is suppressed by (n-3) PUFA consumption. The purpose of this study was to test the hypothesis that consumption of the (n-3) PUFA docosahexaenoic acid (DHA) interferes with DON-induced transcriptional and post-transcriptional upregulation of IL-6 mRNA in murine macrophages. DON evoked expression of IL-6 mRNA and IL-6 heterogenous nuclear RNA (hnRNA), an indicator of ongoing IL-6 transcription, in macrophages elicited from mice fed control AIN-93G diet for 4 wk, whereas expression of both RNA species was suppressed in macrophages from mice fed AIN-93G modified to contain 30 g DHA/kg diet for the same time period. DON enhanced IL-6 mRNA stability similarly in macrophages from control and DHA-fed mice suggesting that (n-3) PUFA effects were not post-transcriptional. DON upregulated binding activity of cAMP response element binding protein (CREB) and activator protein (AP-1) to their respective consensus sequences in nuclear extracts from control-fed mice, whereas both activities were suppressed in nuclear extracts from DHA-fed mice. DON induced phosphorylation of CREB at Ser-133 and ATF1 at Ser-63 as well as intranuclear binding of phospho-CREB/ATF1 to the cis element of the IL-6 promoter in control macrophages, whereas both activities were inhibited in macrophages from DHA-fed mice. DHA consumption blocked DON-induced phosphorylation of the CREB kinase AKT. Inhibition of AKT suppressed both CREB/ATF1 phosphorylation and IL-6 transcription. These data suggest that DHA consumption suppresses DON-induced IL-6 transcription in macrophages in part by interfering with AKT-dependent phosphorylation and subsequent binding of CREB/ATF1 to the IL-6 promoter.

KEY WORDS: • (n-3) PUFA • deoxynivalenol • inflammation • mycotoxin • cytokine • macrophage

The increased ratio of (n-6) to (n-3) PUFA present in the diet in industrialized countries over the last 3 decades has been linked etiologically to an increased risk of inflammatory disease (1). Dietary supplementation with (n-3) PUFA found in fish oil, particularly docosahexaenoic acid (DHA)³ and eicosapentaenoic acid (EPA), can potentially reverse this trend (2). A recent CDC-National Health Interview Survey determined that 26 million U.S. adults consume (n-3) PUFA supplements (3). In animal models, (n-3) PUFA attenuate experimental induction of inflammation, proinflammatory cytokine production, delayed-type hypersensitivity, and graft vs. host responses in animal models (4). The anti-inflammatory properties of the (n-3) PUFA have been applied to prophylaxis and therapy of human inflammatory diseases including atherosclerosis (5), rheumatoid arthritis (6), psoriasis (7), and IgA nephropathy (IgAN) (8).

IgAN, the most common form of human primary glomerulonephritis, has marked kidney mesangial IgA deposition as its diagnostic hallmark (9). Between 15 and 40% of IgAN patients develop end-stage renal disease. Aberrant elevation of mucosal and systemic IgA contributes to IgA deposition in kidney mesangium as well as resultant inflammation and nephritis (10). Epidemiologic studies revealed a negative correlation between (n-3) PUFA tissue levels and IgAN (11), whereas there is a positive correlation with (n-6) PUFA and the disease (12). Several clinical trials demonstrated elegantly that consumption of (n-3) PUFA found in fish oil retarded renal disease progression in human IgAN patients by reducing inflammation and glomerulosclerosis (7,8).

Deoxynivalenol (DON), a common contaminant of cereal grain–based foods(13), and other trichothecene mycotoxins activate multiple kinase signaling pathways via the ribotoxic stress response (14); as a consequence, they upregulate expression of multiple inflammation-related genes (15–18). DON induces aberrant elevation of IgA production and kidney mesangial IgA deposition in mice fed DON, which provides a unique preclinical model for studying IgAN in its early stages (19,20). Ex vivo reconstitution (21), antibody neutralization (22), and IL-6 knockout mice studies (23) indicate that induction of IL-6 gene expression in macrophages is crucial to DON-induced IgAN (20). Consumption of fish oil, DHA, or EPA retards serum and mesangial IgA elevation in DON-exposed mice and this retardation correlates with inhibition of IL-6 gene expression (24–27). However, the upstream mechanisms by which DHA downregulates DON-induced IL-6 gene expression in primary macrophages remain unknown.

Regulation of IL-6 gene expression involves multiple signal transduction pathways that affect gene transactivation, mRNA stability, and translational efficiency (28–30). Electrophoretic mobility shift assay (EMSA) and point mutation analyses showed that cAMP response element binding (CREB) protein, activator protein-1 (AP-1), CCAAT/enhancer binding protein ß (C/EBPß), and nuclear factor (NF)-B) all participate in IL-6 transcriptional upregulation (31,32). Acute oral exposure of mice to DON induces serum IL-6 as well as IL-6 mRNA expression in spleen and Peyer's patches (15,16), and DON induces AP-1, CREB, C/EBPß, and NF-B binding in both in vitro and in vivo models (15,33). In addition to its transcriptional effects, DON enhances mRNA stability for a number of cytokine genes including IL-6 (34,35).

The purpose of this study was to test the hypothesis that DHA consumption interferes with DON-induced transcriptional and post-transcriptional upregulation of IL-6 mRNA in murine macrophages.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Materials. All chemicals including DON were purchased from Sigma Chemical unless otherwise noted. Concentrated toxin solutions were handled in a fume hood. Labware contaminated with mycotoxin was detoxified by soaking for 1 h in 100 mL/L sodium hypochlorite.

    Animals and diets. Female B6C3F1 mice (7 wk old), weighing between 20 and 25 g, were obtained from Charles River. Housing, handling, and sample collection procedures conformed to the policies of the Michigan State University All University Committee on Animal Care in accordance with NIH guidelines. Mice were housed and diets prepared as described in a previous study (26). Briefly, corn oil, oleic acid (Dyets) and MEG-3^TM DHA-enriched oil (containing DHA 483 g/kg and 113 g/kg EPA; Ocean Nutrition) were added to AIN 93G basal diet (36) to yield a control diet (10 g corn oil and 60 g oleic acid/kg diet) and a diet containing 30 g DHA/kg diet (10 g corn oil and 60 g DHA enriched oil/kg diet). Mice were fed these diets for 4 wk before peritoneal macrophage harvest or spleen removal. The DHA concentration and time period were effective for increasing tissue (n-3) PUFA in previous studies (26).

    Peritoneal macrophage cultures. Mice were injected ip. with 1 mL of sterile thioglycollate (90 g/L). After 3 d, macrophages were collected from control and DHA-fed mice by peritoneal lavage with cold Hank's buffer (Invitrogen) and pelleted at 450 x g for 5 min (37). Cells were washed in Dulbecco's PBS (Sigma) and cultured in DMEM (Invitrogen) containing 100 mL/L heat-inactivated fetal bovine serum, 1 x 10⁵ U/L penicillin, and 100 mg/L streptomycin (Sigma) at 37°C under 7% CO₂ in a humidified incubator for 24 h before DON addition (38,39).

For mRNA expression studies, macrophages (1 x 10⁹/L) were cultured with DON (250 µg/L) for 3 h. RNA was extracted and analyzed by real-time PCR for IL-6 mRNA or heterologous nuclear RNA (hnRNA). For mRNA stability studies, macrophages (1 x 10⁹/L) were incubated with 100 µg/L lipopolysaccharide (LPS) for 2 h to induce IL-6 transcription. The medium was removed and fresh medium containing transcriptional inhibitor DRB (100 µmol/L) and/or DON (250 µg/L). RNA was extracted at 0, 1, 3, and 6 h and analyzed by real-time PCR for IL-6 mRNA.

    Lipid extraction and analysis. Fatty acids were analyzed by a modification of the method of Hasler et al. (40) by GC utilizing a Shimadzu GC-2010 and standard FAME (Nu-Check-Prep).

    IL-6 gene expression. IL-6 mRNA and hnRNA were measured 3 h after DON stimulation by real-time PCR (26). 18S RNA was used to normalize target gene expression. Probe and primers for mouse IL-6 mRNA and endogenous control (18S RNA) were purchased as TaqMan assay reagents (PE Applied Biosystems). Real-time Polymerase Chain Reaction Primer Express software (PE Applied Biosystems) was employed to design primer pairs for mouse IL-6 hnRNA (forward primer: GTC CAA CTG TGC TAT CTG CTC ACT; backward primer: AGA AGG CAA CTGG ATG GAA GTC T). Target gene expression levels were calculated using the C_T method (PE Applied Biosystems User Bulletin 2).

    Nuclear extraction. For macrophage nuclear extracts, cells were cultured with DON (250 µg/L) for 30 min before extraction. For splenic nuclear extracts, mice were gavaged with DON (25 mg/kg body weight); 30 min later, spleen cells were isolated (15). Nuclear extracts were prepared by lysing cells in HEPES buffer (20 mmol/L, pH 7.9) containing 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, containing 1.5 mg/L each of aprotinin, pepstatin, leupeptin, and chymostatin for 15 min on ice. After the addition of Nonidet P-40 to a final concentration of 5 g/L, cell lysates were centrifuged at 1000 x g for 10 min at 4°C. The pelleted material was incubated with the aforementioned HEPES buffer containing 400 mmol/L KCl for 60 min on ice. Insoluble material was removed by centrifugation at 14000 x g for 15 min at 4°C. The protein concentration of the supernatant was determined using a Bio-Rad protein assay kit.

    Electrophoretic mobility shift assay (EMSA). EMSA were performed using consensus sequences for NF-B, 5' AGT TGA GGG GAC TTT CCC AGG C 3', mutant 5'AGT TGA GGC GAC TTT CCC AGG C 3'; C/EBP, 5' TGC AGA TTG CGC AAT CTG CA 3', mutant 5' TGC AGA GAC TAG TCT CTG CA 3'; AP-1, 5' CGC TTG ATG ACT CAG CCG GAA 3', mutant 5' CGC TTG ATG ACT TGG CCG GAA 3' and CRE, 5' AGA GAT TGC CTG ACG TCA GAC AGC TAG 3', mutant 5'-AGA GAT TGC CTG TGG TCA GAG AGC TAG-3' (Santa Cruz Biotechnology) (15). For competition experiments, synthetic wild-type and mutant oligonucleotides were used in 150-fold molar excess and incubated with nuclear extract for 15 min before the radiolabeled probe was loaded. Samples were then subjected to a nondenaturing PAGE at 30 mA using Tris borate-glycine buffer (45 mmol/L Tris borate, 1 mmol/L EDTA, pH 8.3). Blots were dried at 80°C for 3 h and analyzed by autoradiography.

    Chromosome immunoprecipitation (ChIP) assay. The chromosome immunoprecipitation (ChIP) assay was conducted using a ChIP-IT Kit (Active Motif). After 30 min of exposure to DON (250 µg/L), peritoneal macrophage cells (2 x 10⁷), an amount sufficient for 4 ChIP reactions, were fixed using 1% (v:v) formaldehyde to cross-link protein and DNA on a shaking platform for 10 min at 25°C. Fixed chromatin DNA was sheared into 200- to 500-bp fragments using a Fisher Sonic Dismembranator 60 at 30% power with 6 pulses of 15 s each, and a 30-s rest on ice between each pulse. DNA/protein complexes were immunoprecipitated with rabbit polyclonal antibodies (3 mg/L antibody for each ChIP reaction) directed against the phospho-CREB (Cell signaling), c-jun, C/EBP, and p65 (Santa Cruz Biotechnology). After immunoprecipitation, the cross-linking was reversed, the proteins were removed by proteinase K treatment, and the DNA was purified on the column provided. The enriched DNA fragments were quantified by SYBR-Green real-time PCR (16) with primers to amplify a 82-bp segment CREB and C/EBP binding site (–149 to –231), a 97-bp segment AP-1 binding site (–240 to –332), and a 97-bp segment NF-B binding site (–240 to –332) upstream from the initiation site of the IL-6 gene. The specificity of the amplifications was confirmed both by dissociation curve and by generation of a single PCR product at the predicted size.

    Western analysis. For kinase phosphorylation studies, cells were cultured with DON (250 µg/L) for 30 min before protein extraction. Macrophages were washed with ice-cold phosphate buffer, lysed in 10 mmol/L Tris, pH 7.4, buffer containing 1.0 mmol/L sodium orthovanadate, and 10 g/L SDS and sonicated for 10 s. After centrifugation (1200 x g; 15 min), extracts were subjected to Western analysis (14) using specific antibodies for p38, phospho-p38, extracellular signal-regulated kinase (ERK), phospho-ERK, c-Jun N-terminal kinase (JNK), phospho-JNK antibodies, phospho-CREB, CREB (Cell Signaling), phospho c-jun, c-jun, phospho p65, p65, phospho C/EBPß and C/EBPß, phospho MSK1, phospho p90RSK, phospho AKT or AKT (Santa Cruz).

    Inhibitor studies. Inhibitors of p38 (SB203580, 20 µmol/L), ERK (PD98059, 100 µmol/L), JNK (SP600125, 10 µmol/L), AKT inhibitor IV (10 µmol/L), protein kinase A (KT5720, 1 µmol/L), CaMkII (KN62, 1 µmol/L), and adenyl cyclase (MDL13322, 1 µmol/L) (Calbiochem) were added to macrophage cultures 30 min before DON (250 µg/L) stimulation. Total RNA was extracted and IL-6 mRNA measured 3 h after DON treatment. Parallel cultures employed the MTT viability assay (41) to verify that these inhibitors were not cytotoxic at the concentrations employed.

    Statistics. Data were analyzed using Sigma Stat for Windows (Jandel Scientific). Data were subjected to 1-way ANOVA and pairwise comparisons were made by Student-Newman-Keuls methods. If data did not meet the normality assumption, they were subjected to Kruskal-Wallace ANOVA on Ranks and pairwise comparisons were made by Dunn's method. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
DHA consumption for 4 wk significantly increased DHA from 0.9 to 2.9%, increased EPA concentrations from 1.2 to 2.2%, and decreased the arachidonic acid level from 6.7 to 2.3% (P < 0.05) of total phospholipid fatty acids in thioglycollate-elicited peritoneal macrophages (Table 1). Treatment of the peritoneal macrophages with DON (250 µg/L) induced IL-6 mRNA (Fig. 1A) as well as IL-6 hnRNA an indicator of transcription (Fig. 1B), after 3 h (P < 0.05). These were suppressed in macrophages from DHA-fed mice by 90 and 80%, respectively, suggesting that DHA consumption inhibited IL-6 transcription ex vivo. To test whether DON and DHA affect IL-6 mRNA stability, peritoneal macrophages were stimulated with LPS for 2 h, treated with the transcriptional inhibitor DRB in the presence and absence of DON, and IL-6 mRNA was monitored over time. mRNA stability was markedly increased by DON in control macrophages, but this response was not altered in cultures from DHA-fed mice (Fig. 1C). Subsequent mechanistic studies therefore focused on transcription.

View larger version (17K): [in this window] [in a new window]   FIGURE 1  DON-induced IL-6 gene transcription but not IL-6 mRNA stability is suppressed in peritoneal macrophages from DHA-fed mice. For IL-6 mRNA (A) and hn/RNA (B) expression, macrophages were cultured with DON (250 µg/L) for 3 h and real-time PCR conducted. Data are means ± SEM, n = 3–6. Bars without a common letter differ, P < 0.05. For mRNA stability studies (C), LPS-stimulated macrophages were incubated with transcriptional inhibitor DRB (100 µmol/L) and/or DON (250 µg/L) and IL-6 mRNA was measured at intervals. Values are means ± SEM, n = 3. Data points without a common letter differ, P < 0.05. All data were normalized against 18S RNA and expressed relative to control value.

View larger version (26K): [in this window] [in a new window]   FIGURE 2  DON-induced transcription factor binding and phosphorylation are suppressed in peritoneal macrophages and spleens of DHA-fed mice. (A) Representative EMSA analysis conducted with labeled CREB, AP-1, C/EBP, and NF-B consensus sequences and nuclear extracts prepared from macrophages treated with and without DON. (B) EMSA competition experiments were used to verify specific binding using synthetic wild-type (W) and mutant (M) consensus sequences at 150-fold molar excess. (C) Representative EMSA analysis conducted with labeled consensus sequences and nuclear extracts prepared from spleens of mice treated with and without 25 mg DON/kg body weight.

View larger version (57K): [in this window] [in a new window]   FIGURE 3  DON-induced transcription factor phosphorylation is inhibited in macrophages from DHA-fed mice. Cells were treated with DON (250 µg/L) for 30 min and whole-cell lysates subjected to Western blotting using antibodies specific for phosphorylated and nonphosphorylated transcription experiments. Data are representative of 3 independent experiments.

View larger version (12K): [in this window] [in a new window]   FIGURE 4  DON-induced phospho-CREB intranuclear binding to IL-6 promoter is suppressed in macrophages from DHA-fed mice. Macrophages from mice fed control and DHA diets were incubated with DON (250 µg/L) for 30 min and subjected to ChIP using specific antibodies to phospho-CREB or nonspecific IgG control. DNA fragments were analyzed by real-time PCR using primers for phospho-CREB factor binding site in IL-6 promoter. Values are means ± SEM, n = 3–4. Bars without a common letter differ, P < 0.05.

View larger version (28K): [in this window] [in a new window]   FIGURE 5  MAPK does not have a role in attenuation of DON-induced CREB phosphorylation and IL-6 gene expression in macrophages of DHA-fed mice. (A) Peritoneal macrophages were cultured for 30 min with inhibitors for p38 (SB203580), ERK (PD98059), and JNK (SP600125) and then with DON (250 µg/L) for 3 h. CREB phosphorylation was measured by Western analysis. (B) IL-6 mRNA was measured by real-time PCR. Values are means ± SEM, n = 6. Bars without a common letter differ, P < 0.05. (C) Peritoneal macrophages were treated with DON (250 µg/L) for 30 min and cell lysates subjected to Western blotting using antibodies specific for phosphorylated and nonphosphorylated forms of p38, JNK, and ERK. Data are representative of 2 independent experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 6  DON-induced MSK1 and p90RSK phosphorylation is not altered in macrophages of DHA-fed mice. Peritoneal macrophages were treated with DON (250 µg/L) for 30 min and cell lysates subjected to Western blotting using antibodies specific for the phosphorylated form of MSK1 and p90RSK as well as acting as control for protein loading. Data are representative of 2 independent experiments.

View larger version (26K): [in this window] [in a new window]   FIGURE 7  DON induces AKT-dependent CREB phosphorylation and IL-6 mRNA expression, which is suppressed in macrophages from DHA-fed mice. Peritoneal macrophages were cultured with DON (250 µg/L) and then analyzed for AKT or CREB/ATF1 phosphorylation by Western analysis and IL-6 mRNA by RT-PCR. (A) Concentration-dependent effects of DON on AKT were measured after 30 min. (B) Cells were pretreated with AKT inhibitor IV for 30 min, incubated with DON for 30 min, and CREB/ATF1 phosphorylation assessed. (C) Cells were pretreated with AKT inhibitor IV for 30 min, DON for 3 h, and IL-6 mRNA measured. Values are means ± SEM, n = 5. Bars without a common letter differ, P < 0.05. (D) Macrophages from mice fed the control or DHA diet were incubated with DON (250 µg/L) for 30 min and AKT phosphorylation assessed. Results are representative of at least 2 independent experiments.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The observation that DHA significantly abrogated IL-6 mRNA expression in macrophages from DHA-treated mice is highly consistent with previous in vivo studies of spleens of DHA-fed mice that were exposed acutely to DON (25,26). hnRNA concentrations can be used as a surrogate for the run-on assay to monitor ongoing gene transcriptional activity (44). Thus, DON-induced IL-6 hnRNA elevation and its suppression by DHA suggest that transcription is a target for this (n-3) PUFA. Although mRNA stabilization can also facilitate rapid post-transcriptional IL-6 mRNA levels (29,30), the inability of DHA to increase IL-6 mRNA degradation suggests that DHA acts primarily by impairing DON-induced IL-6 gene transcriptional activity.

Transcription factor activation occurs in RAW 264.7 murine macrophages incubated with DON (33) and in spleens of mice exposed acutely to the toxin (45). Here, DON induced CREB and AP-1 binding in peritoneal macrophages, and suppression by DHA is consistent with transcriptional modulation. The observed induction of these factors in spleen by DON and inhibition by dietary DHA suggest that the ex vivo peritoneal macrophage model is indeed predictive of events in the intact mouse.

A limitation of EMSA is that it might not always accurately predict interaction between transcriptional factors and DNA within a nucleus. This is because the condensed structure of chromatin blocks access by transcription factors and requires structural remodeling for factor binding and subsequent gene upregulation (46). ChIP enables correlation of in vitro binding data with actual intranuclear binding events (47). The observation that DHA blocked DON-induced phospho-CREB binding to the IL-6 promoter suggests that CREB might a key target of (n-3) PUFA in peritoneal macrophages. This contention was further supported by the finding that phosphorylation of CREB and CREB family member ATF1 was markedly activated by DON and impaired by DHA. CREB activation might have importance from the perspective of chromatin remodeling because subsequent binding to CREB-binding protein (CBP) can recruit and stabilize the RNA polymerase II transcription complex at the TATA box (48). CBP possesses an intrinsic histone acetyltransferase activity and contributes to chromatin structure modification, which can make the DNA template more accessible to transcriptional machinery. Because multiple stimuli and upstream signals can mediate activation of CREB and ATF1 (49), a key question relates to whether DHA can similarly target these pathways.

The potential effect of (n-3) PUFA on other transcription factors cannot be excluded because EMSA indeed showed suppression of AP-1 binding in macrophages from DHA-fed mice as well as AP-1, NF-B, and C/EBP in spleens of DHA-fed mice. Consistent with these findings, DHA feeding suppressed c-Jun and p65 phosphorylation in macrophages. DON-induced phosphorylation of CREB, c-Jun, and NF-B could indeed enhance their affinity for recruiting CBP to the IL-6 promoter, thus increasing transactivation potential (32,50). AP-1, NF-B, and C/EBP activation are reportedly suppressed by (n-3) PUFA in other models (51–53), further suggesting that these transcription factors could also be potential targets. The inability of ChIP to detect interactions between c-jun, p65, or C/EBP with the IL-6 promoter region might result from the masking of antigen epitopes that occurs when these transcription factors bind DNA or interact with other proteins (47). Additionally, formaldehyde fixation might modify the epitope of the factor and prevent antibody-binding. Thus, further exploration of the effects of DHA on the regulation of IL-6 transcription by factors other than CREB is warranted.

Several kinases can phosphorylate CREB and therefore might be subject to the effects of DHA (Fig. 8). p38 and ERK contribute to the phosphorylation of CREB at Ser¹³³ as well as CREB-mediated gene transcription (43,49). Although inhibition of both p38 and ERK 1/2 abrogated CREB/ATF1 phosphorylation and IL-6 gene expression in control macrophages, phosphorylation of these MAPK was not suppressed in macrophages from DHA-fed mice. DON induced phosphorylation of both MSK1 and p90RSK, which are crucial intracellular signaling transducers downstream of ERK and p38 that can phosphorylate CREB. However, DHA did not impair DON-induced MSK1 and p90RSK phosphorylation, suggesting that these downstream MAPK effectors were not targets for DHA inhibition of CREB/ATF1 phosphorylation. Thus, inhibition of p38 or ERK pathways could not be an explanation for suppression of CREB phosphorylation and, ultimately, IL-6 gene expression observed in macrophages from DHA-fed mice.

View larger version (19K): [in this window] [in a new window]   FIGURE 8  Summary of possible targets of DON-induced CREB activation and IL-6 expression. DHA suppresses AKT-mediated CREB activation by DON, whereas possible MAPK pathways involving MSK and RSK1 were not altered.

CREB phosphorylation and activation also occur via pathways involving adenylate cyclase and calmodulin (49). However, we could not demonstrate that DON upregulates intracellular cAMP or calcium in peritoneal macrophages or that inhibitors of these 2 pathways prevent DON-induced CREB phosphorylation (data not shown).

In addition to attenuating CREB kinases, it is possible that DHA consumption increases the activity of intranuclear protein phosphatases, which compete with CREB phosphorylation. Both Ser/Thr protein phosphatase type 1 (PP1) and type 2A (PP2A) were reported to dephosphorylate phospho-CREB (54,55). Furthermore, Siddiqui et al. (56,57) found that DHA induces PP1 and PP2A activity in Jurkat cells. Such a system could be quite complex because PP1 and PP2A activities can be regulated by intracellular inhibitors (58,59). Interestingly, phospho-AKT can be a target of these Ser/Thr phosphatases. Clearly, further study of competing CREB phosphorylation/dephosphorylation in the context of the DON/DHA model is warranted.

Taken together, DHA inhibition of DON-induced IL-6 gene transcription in macrophages is associated with impaired activation of several transcription factors, with the effects on CREB being the most robust. These results provide new insight into how (n-3) PUFA might suppress inflammatory diseases involving IL-6 and potentially other inflammatory genes (4–8). Further clarification of the upstream molecular mechanisms by which DHA alters phosphorylation of CREB and the role of AKT is warranted. CREB is essential to the regulation of many genes as evidenced by the 1300–1700 CRE motifs identified during stringent analyses of the mouse and human genomes (60). Thus, it will be necessary to clarify the specificity of the effects of DHA relative to other genes, different stimuli, and nonmacrophage phenotypes.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the advice of Drs. Dale Romsos, Maurice Bennink, Donald Jump, and Kate Claycombe. We acknowledge the kind donation of MEG-3^TM DHA oil from Dr. Colin Barrow (Ocean Nutrition Limited, Dartmouth, NS, Canada).

	FOOTNOTES

 
¹ Supported by Public Health Service Grants DK 58833 (JJP) from the National Institute for Diabetes Digestive and Kidney Diseases and ES 3358 (JJP) from the National Institute for Environmental Health Sciences and the Michigan State University Agricultural Experiment Station.

³ Abbreviations used: AP-1, activator protein 1; CBP, CREB-binding protein; C/EBPß, CCAAT/enhancer binding protein ß; ChIP, chromosome immunoprecipitation; CREB, cAMP response element binding protein; DON, deoxynivalenol; DHA, docosahexaenoic acid; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; hnRNA, heterologous nuclear RNA; IgAN, IgA nephropathy; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF, nuclear factor.

Manuscript received 19 July 2005. Initial review completed 24 August 2005. Revision accepted 31 October 2005.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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