Deoxynivalenol-induced mitogen-activated protein kinase phosphorylation and IL-6 expression in mice suppressed by fish oil

The trichothecene mycotoxin deoxynivalenol (DON) induces IgA hyperelevation and mesangial IgA deposition in mice that mimics the early stages of human IgA nephropathy (IgAN). Among potential mediators of this disease, interleukin-6 (IL-6) is likely to play a particularly critical role in IgA elevation and disease exacerbation. Based on previous findings that dietary fish oil (FO) suppresses DON-induced IgAN, we hypothesized that FO inhibits the induction of IL-6 expression by this mycotoxin in vivo and in vitro. Mice were fed modified AIN 93G diet amended with 7% corn oil (CO) or with 1% corn oil plus 6% menhaden fish oil (FO) for up to 8 weeks and then exposed acutely to DON by oral gavage. DON-induced plasma IL-6 and splenic mRNA elevation in FO-fed mice were significantly suppressed after 8 weeks when compared to the CO-fed group. The effects of FO on phosphorylation of mitogen-activated protein kinases (MAPKs), critical upstream transducers of IL-6 up-regulation, were also assessed. DON-induced phosphorylation of extracellular signal regulated protein kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases 1 and 2 (JNK1/2) was significantly suppressed in spleens of mice fed with FO, whereas p38 was not. Splenic COX-2 mRNA expression, which has been previously shown to enhance DON-induced IL-6, was also significantly decreased by FO, whereas plasma levels of the COX-2 metabolite, prostaglandin E_2, were not affected. To confirm in vivo findings, the effects of pretreatment with the two primary n-3 PUFAs in FO, eicosapentaenoic acid (20:5[n-3]; EPA) and docosahexaenoic acid, (22:6[n-3]; DHA), on DON-induced IL-6 expression were assessed in LPS-treated RAW 264.7 macrophage cells. Consistent with the in vivo findings, both EPA and DHA significantly suppressed IL-6 superinduction by DON, as well as impaired DON-induced ERK1/2 and JNK1/2 phosphorylation. In contrast, the n-6 PUFA arachidonic acid (20:4[n-3]) had markedly less effects on these MAPKs. Taken together, the capacity of FO and its component n-3 PUFAs to suppress IL-6 expression as well as ERK 1/2 and JNK 1/2 activation might explain, in part, the reported suppressive effects of these lipids on DON-induced IgA nephropathy.     

HCV-induced PKR activation is stimulated by the mitogen- and stress-activated protein kinase MSK2

The replication of viral nucleic acids triggers cellular antiviral responses. The double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays a key role in this antiviral response. We have recently reported that JFH-1 HCV replication in Huh-7 cells triggers PKR activation. Here we show that the HCV-induced PKR activation is further stimulated by the mitogen- and stress-activated protein kinase 2 (MSK2), a member of the 90 kDa ribosomal S6 kinase (RSK) family that has emerged as an important downstream effector of ERK and p38 MAPK signaling pathways. We show that MSK2 binds PKR and stimulates PKR phosphorylation, whereas the closely related MSK1 and RSK2 have no effect. Our data further indicate that MSK2 functions as an adaptor in mediating PKR activation, apparently independent of its catalytic activity. These results suggest that, in addition to viral dsRNA, stress signaling contributes to the regulation of cellular antiviral response.     

A phosphosite screen identifies autocrine TGF-beta-driven activation of protein kinase R as a survival-limiting factor for eosinophils

The differential usage of signaling pathways by chemokines and cytokines in eosinophils is largely unresolved. In this study, we investigate signaling similarities and differences between CCL11 (eotaxin) and IL-5 in a phosphosite screen of human eosinophils. We confirm many previously known pathways of cytokine and chemokine signaling and elucidate novel phosphoregulation in eosinophils. The signaling molecules that were stimulated by both agents were members of the ERK1/2 and p38 MAPK pathways and their downstream effectors such as RSK and MSK1/2. Both agents inhibited S6 kinase, protein kinase Cepsilon, and glycogen synthase kinase 3 alpha and beta. The molecules that were differentially regulated include STATs and protein kinase R (PKR). One of the chief findings in this investigation was that PKR and eukaryotic initiation factor 2alpha are phosphorylated under basal conditions in eosinophils and neutrophils. This basal phosphorylation was linked to autocrine secretion of TGF-beta in eosinophils. TGF-beta directly activates PKR in eosinophils. Basal phosphorylation of PKR was inhibited by incubation of eosinophils with a neutralizing anti-TGF-beta Ab suggesting its physiological importance. We show that inhibition of PKR activity prolongs eosinophil survival. The eosinophil survival factor IL-5 strongly suppresses phosphorylation of PKR. The biological relevance of IL-5 inhibition of phospho-PKR was established by the observation that ex vivo bone marrow-derived eosinophils from OVA-immunized mice had no PKR phosphorylation in contrast to the high level of phosphorylation in sham-immunized mice. Together, our findings suggest that survival of eosinophils is in part controlled by basal activation of PKR through autocrine TGF-beta and that this could be modulated by a Th2 microenvironment in vivo.     

The Fat-1 Mouse has Brain Docosahexaenoic Acid Levels Achievable Through Fish Oil Feeding

Fat-1 transgenic mice endogenously convert n-6 to n-3 polyunsaturated fatty acids (PUFA). The aims of this study were to test whether a) fish oil feeding can attain similar brain n-3 PUFA levels as the fat-1 mouse, and b) fat-1 mouse brain docosahexaenoic acid (22:6n-3; DHA) levels can be potentiated by fish oil feeding. Fat-1 mice and their wildtype littermates consumed either a 10% safflower oil (SO) or a 2% fish oil and 8% safflower oil chow (FO). Brain total lipid and phospholipid fraction fatty acids were analyzed using GC-FID. Wildtype mice fed FO chow had similar brain levels of DHA as fat-1 mice fed SO chow. Fat-1 mice fed FO chow had similar brain n-3 PUFA levels as fat-1 mice fed SO chow. In conclusion, brain levels of DHA in the fat-1 mouse can be obtained by and were not further augmented with fish oil feeding.     

Novel role for calcium-independent phospholipase A(2) in the macrophage antiviral response of inducible nitric-oxide synthase expression

The double-stranded (ds) RNA-dependent protein kinase (PKR) is a primary regulator of antiviral responses; however, the ability of dsRNA to activate nuclear factor-kappa B (NF-kappa B) and dsRNA + interferon gamma (IFN-gamma) to stimulate inducible nitric-oxide synthase (iNOS) expression by macrophages isolated from PKR(-/-) mice suggests that signaling pathways in addition to PKR participate in antiviral activities. We have identified a novel phospholipid-signaling cascade that mediates macrophage activation by dsRNA and viral infection. Bromoenol lactone (BEL), a selective inhibitor of the calcium-independent phospholipase A(2) (iPLA(2)), prevents dsRNA- and virus-induced iNOS expression by RAW 264.7 cells and mouse macrophages. BEL does not modulate dsRNA-induced interleukin 1 expression, nor does it affect dsRNA-induced NF-kappa B activation. Protein kinase A (PKA) and the cAMP response element binding protein (CREB) are downstream targets of iPLA(2), because selective PKA inhibition prevents dsRNA-induced iNOS expression, and the inhibitory actions of BEL on dsRNA-induced iNOS expression are overcome by the direct activation of PKA. In addition, BEL inhibits dsRNA-induced CREB phosphorylation and CRE reporter activation. PKR does not participate in iPLA(2) activation or iNOS expression, because dsRNA stimulates iPLA(2) activity and dsRNA + IFN-gamma induces iNOS expression and nitric oxide production to similar levels by macrophages isolated from PKR(+/+) and PKR(-/-) mice. These findings support a PKR-independent signaling role for iPLA(2) in the antiviral response of macrophages.     

Ultraviolet B-induced phosphorylation of histone H3 at serine 28 is mediated by MSK1.

N-terminal tail phosphorylation of histone H3 plays an important role in gene expression, chromatin remodeling, and chromosome condensation. Phosphorylation of histone H3 at serine 10 was shown to be mediated by RSK2, mitogen- and stress-activated protein kinase-1 (MSK1), and mitogen-activated protein kinases depending on the specific stimulation or stress. Our previous study showed that mitogen-activated protein kinases MAP kinases are involved in ultraviolet B-induced phosphorylation of histone H3 at serine 28 (Zhong, S., Zhong, Z., Jansen, J., Goto, H., Inagaki, M., and Dong, Z., J. Biol. Chem. 276, 12932-12937). However, downstream effectors of MAP kinases remain to be identified. Here, we report that H89, a selective inhibitor of the nucleosomal response, totally inhibits ultraviolet B-induced phosphorylation of histone H3 at serine 28. H89 blocks MSK1 activity but does not inhibit ultraviolet B-induced activation of MAP kinases p70/85(S6K), p90(RSK), Akt, and protein kinase A. Furthermore, MSK1 markedly phosphorylated serine 28 of histone H3 and chromatin in vitro. Transfection experiments showed that an N-terminal mutant MSK1 or a C-terminal mutant MSK1 markedly blocked MSK1 activity. Compared with wild-type MSK1, cells transfected with N-terminal or C-terminal mutant MSK1 strongly blocked ultraviolet B-induced phosphorylation of histone H3 at serine 28 in vivo. These data illustrate that MSK1 mediates ultraviolet B-induced phosphorylation of histone H3 at serine 28.     

ERK activation is required for double-stranded RNA- and virus-induced interleukin-1 expression by macrophages

Double-stranded (ds) RNA, which accumulates during viral replication, activates the antiviral response of infected cells. In this study, we have identified a requirement for extracellular signal-regulated kinase (ERK) in the regulation of interleukin 1 (IL-1) expression by macrophages in response to dsRNA and viral infection. Treatment of RAW 264.7 cells or mouse macrophages with dsRNA stimulates ERK phosphorylation that is first apparent following a 15-min incubation and persists for up to 60 min, the accumulation of iNOS and IL-1 mRNA following a 6-h incubation, and the expression of iNOS and IL-1 at the protein level following a 24-h incubation. Inhibitors of ERK activation prevent dsRNA-induced ERK phosphorylation and IL-1 expression by macrophages. The regulation of macrophage activation by ERK appears to be selective for IL-1, as ERK inhibition does not attenuate dsRNA-induced iNOS expression by macrophages. dsRNA stimulates both ERK activation and IL-1 expression by macrophages isolated from dsRNA-dependent protein kinase (PKR)-deficient mice, indicating that PKR does not participate in this antiviral response. These findings support a novel PKR-independent role for ERK in the regulation of the antiviral response of IL-1 expression and release by macrophages.     

Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid

A number of studies have investigated the effects of fish oil on the production of pro-inflammatory cytokines using peripheral blood mononuclear cell models. The majority of these studies have employed heterogeneous blends of long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which preclude examination of the individual effects of LC n-3 PUFA. This study investigated the differential effects of pure EPA and DHA on cytokine expression and nuclear factor kappaB (NF-kappaB) activation in human THP-1 monocyte-derived macrophages. Pretreatment with 100 microM EPA and DHA significantly decreased lipopolysaccharide (LPS)-stimulated THP-1 macrophage tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and IL-6 production (P<.02), compared to control cells. Both EPA and DHA reduced TNF-alpha, IL-1beta and IL-6 mRNA expression. In all cases, the effect of DHA was significantly more potent than that of EPA (P<.01). Furthermore, a low dose (25 microM) of DHA had a greater inhibitory effect than that of EPA on macrophage IL-1beta (P<.01 and P<.04, respectively) and IL-6 (P<.003 and P<.003, respectively) production following 0.01 and 0.1 microg/ml LPS stimulation. Both EPA and DHA down-regulated LPS-induced NF-kappaB/DNA binding in THP-1 macrophages by approximately 13% (P< or =.03). DHA significantly decreased macrophage nuclear p65 expression (P< or =.05) and increased cytoplasmic IkappaBalpha expression (P< or =.05). Although similar trends were observed with EPA, they were not significant. Our findings suggest that DHA may be more effective than EPA in alleviating LPS-induced pro-inflammatory cytokine production in macrophages - an effect that may be partly mediated by NF-kappaB. Further work is required to elucidate additional divergent mechanisms to account for apparent differences between EPA and DHA.     

Docosahexaenoic acid, a major constituent of fish oil diets, prevents activation of cyclin-dependent kinases and S-phase entry by serum stimulation in HT-29 cells

Cellular proliferation is regulated by cell cycle progression which, in turn, is controlled by sequential activation of various cyclin-dependent kinases (CDKs). To explore the mechanism(s) by which long chain polyunsaturated fatty acids (PUFAs) influence the growth of tumor cells, we compared the effects of different n-3 and n-6 fatty acids on the activity of CDKs. Docosahexaenoic acid (DHA), a major component of fish oil diets, is able to reduce serum-stimulated cyclin D1-, E-, and A- associated kinases activity in synchronized-HT-29 cells. The inhibitory effect of DHA on cyclin A-associated kinase activity is time-dependent, and is probably modulated by down-regulation of cyclin A protein expression. In addition, DHA inhibits the phosphorylation of pRb and DNA-binding activity of E2F-1 in response to serum stimulation, and prevents the serum-stimulated entry of S-phase in HT-29 cells. These results indicate that DHA may exert its negative effect on the growth of tumor cells by inhibiting the activation and expression of G1-associated cell cycle regulatory proteins. Since the synthetic antioxidant BHT is able to reverse the inhibition of serum-stimulated activation of cyclin A/CDK by DHA in a dose-dependent manner, endogenous oxidative stress produced by lipid peroxidation in HT-29 cells may be involved in the control of cell cycle progression.     

Activation of JNK1, RSK2, and MSK1 is involved in serine 112 phosphorylation of Bad by ultraviolet B radiation

The Bcl-2 family member Bad is a pro-apoptotic protein, and phosphorylation of Bad by cytokines and growth factors promotes cell survival in many cell types. Induction of apoptosis by UV radiation is well documented. However, little is known about UV activation of cell survival pathways. Here, we demonstrate that UVB induces Bad phosphorylation at serine 112 in JNK1, RSK2, and MSK1-dependent pathways. Inhibition of mitogen-activated protein (MAP) kinases including ERKs, JNKs, and p38 kinase by the use of their respective dominant negative mutant or a specific inhibitor for MEK1 or p38 kinase, PD98059 or SB202190, resulted in abrogation of UVB-induced phosphorylation of Bad at serine 112. Incubation of active MAP kinase members with Bad protein showed serine 112 phosphorylation of Bad by JNK1 only. However, activated RSK2 and MSK1, downstream kinases of ERKs and p38 kinase, respectively, also phosphorylated Bad at serine 112 in vitro. Cells from a Coffin-Lowry syndrome patient (deficient in RSK2) or expressing an N-terminal or C-terminal kinase-dead mutant of MSK1 were defective for UVB-induced serine 112 phosphorylation of Bad. Furthermore, MAP kinase pathway-dependent serine 112 phosphorylation was shown to be required for dissociation of Bad from Bcl-X(L). These data illustrated that UVB-induced phosphorylation of Bad at serine 112 was mediated through MAP kinase signaling pathways in which JNK1, RSK2, and MSK1 served as direct mediators.     

N-3 polyunsaturated fatty acid consumption produces neurobiological effects associated with prevention of depression in rats after the forced swimming test

Epidemiological data and clinical trials suggest that n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have preventive and therapeutic effects on depression; however, the underlying mechanism remains elusive. The present study aimed to examine the behavioral effects and antidepressant mechanism of n-3 PUFA using a forced swimming test. Eleven-week-old male Sprague-Dawley rats were fed an American Institute of Nutrition-93M diet containing 0%, 0.5% or 1% EPA and DHA relative to the total energy intake in their diet for 12 weeks (n=8 per group). Total dietary intake, body weight and hippocampus weights were not significantly different among groups. The groups administered 0.5% and 1% EPA+DHA diets had significantly higher levels of n-3 PUFA in their brain phospholipids compared to those in the control group. The immobility time was significantly decreased and the climbing time was significantly increased in the 0.5% and 1% EPA+DHA groups compared with those in the 0% EPA+DHA group. Plasma serotonin concentration and hippocampus c-AMP response element binding protein (CREB) expression were significantly increased in the 0.5% and 1% EPA+DHA groups compared with those in the 0% EPA+DHA group. Conversely, interleukin (IL)-6 expression was significantly reduced in the 0.5% and 1% EPA+DHA groups compared with that in the 0% EPA+DHA group. However, there were no dose-dependent effects of n-3 PUFA and no significant differences in expressions of IL-1β, tumor necrosis factor-α, brain-derived neurotrophic factor or phosphorylated CREB. In conclusion, long-term intake of EPA+DHA induced antidepressant-like effects in rats and overexpression of CREB via decreased IL-6 expression.