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Docosahexaenoic acid potentiates interleukin-1beta induction of nitric oxide synthase through mechanism involving p44/42 MAPK activation in rat vascular smooth muscle cells.

ABSTRACT: The effect of docosahexaenoic acid (DHA) on nitric oxide (NO) production and inducible NO synthase (iNOS) expression induced by interleukin (IL)-1beta, and whether the effect of DHA is related to its effect on mitogen-activated protein kinase (MAPK) activation were investigated in cultured rat vascular smooth muscle cells (VSMCs). DHA and eicosapentaenoic acid (EPA), although less potent, increased the NO production induced by IL-1beta (3 ng ml(-1)) in a concentration-dependent manner (3 - 30 microM) Arachidonic acid had no significant effect. The stimulatory effect of DHA (30 microM) on the NO production was more obvious at lower concentrations of IL-1beta. IL-1beta induced iNOS protein and mRNA expressions, which were significantly potentiated by DHA. EPA (30 microM) had a tendency to increase the iNOS protein and mRNA expressions, but arachidonic acid had no effect. IL-1beta-induced iNOS protein expression was significantly inhibited by PD 98059 (10 microM), a selective inhibitor of p44/42 MAPK kinase, both in the absence and the presence of DHA. SB 203580 (10 microM), a selective inhibitor of p38 MAPK activity, had no significant effect, although had a tendency to inhibit slightly. IL-1beta increased the phosphorylation of p44/42 MAPK, while it did not apparently increase the phosphorylation of p38 MAPK. DHA significantly potentiated the IL-1beta-induced phosphorylation of p44/42 MAPK, while it had no significant effect on the phosphorylation of p38 MAPK. These results suggest that DHA increases NO production by potentiating iNOS expression induced by IL-1beta through mechanism involving p44/42 MAPK signalling cascade in rat VSMCs. The present study may contribute to the understanding of basic mechanisms underlying the beneficial effects of DHA on various cardiovascular disorders.

SUBMITTER: Hirafuji M

PROVIDER: S-EPMC1573387 | biostudies-other | 2002 Jun

REPOSITORIES: biostudies-other

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Project description:ObjectivesTo investigate the presence and functionality of oestrogen receptor alpha (ERalpha) in interleukin (IL)1beta-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17beta oestradiol (E2) effects on IL1beta-induced inducible nitric oxide synthase (iNOS) expression.MethodsThe presence and functionality of ERalpha were investigated by immunocytochemistry and transient expression of an E2-responsive reporter construct. iNOS expression and production were determined by transient expression of a chimeric iNOS promoter-luciferase construct and protein immunoblotting. Nitric oxide (NO) production was determined by the Griess reaction. DNA-binding activities of nuclear factor-kappaB (NF-kappaB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)-ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry.ResultsERalpha was identified in the nucleus of chondrocytes. ERalpha efficiently transactivated a transiently expressed E2-responsive construct. On IL1beta treatment, ERalpha partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1beta-induced NO production (-34%, -31% and -36%, respectively). E2 decreased IL1beta-induced iNOS protein expression (-40%). Transient expression of an iNOS promoter construct strongly suggested that iNOS expression was inhibited at the transcriptional level, and EMSA-ELISA assays showed that E2 reduced (-60%) the IL1beta-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1beta was also strongly decreased by E2.ConclusionsOur data support a reciprocal antagonism between oestrogens and IL1beta, ultimately resulting in the decrease of cytokine-dependent NO production through transcriptional inhibition of iNOS expression. This effect was associated with selective inhibition of p65 DNA binding and nuclear translocation.

Project description:Initial Ca2+-dependent contraction of the intestinal smooth muscle mediated by G(q)-coupled receptors is attenuated by RGS4 (regulator of G-protein signalling 4). Treatment of colonic muscle cells with IL-1beta (interleukin-1beta) inhibits acetylcholine-stimulated initial contraction through increasing the expression of RGS4. NF-kappaB (nuclear factor kappaB) signalling is the dominant pathway activated by IL-1beta. In the present study we show that RGS4 is a new target gene regulated by IL-1beta/NF-kappaB signalling. Exposure of cultured rabbit colonic muscle cells to IL-1beta induced a rapid increase in RGS4 mRNA expression, which was abolished by pretreatment with a transcription inhibitor, actinomycin D, implying a transcription-dependent mechanism. Existence of the canonical IKK2 [IkappaB (inhibitor of NF-kappaB) kinase 2]/IkappaBalpha pathway of NF-kappaB activation induced by IL-1beta in rabbit colonic muscle cells was validated with multiple approaches, including the induction of reporter luciferase activity and endogenous NF-kappaB-target gene expression, NF-kappaB-DNA binding activity, p65 nuclear translocation, IkappaBalpha degradation and the phosphorylation of IKK2 at Ser(177/181) and p65 at Ser(536). RGS4 up-regulation by IL-1beta was blocked by selective inhibitors of IKK2, IkappaBalpha or NF-kappaB activation, by effective siRNA (small interfering RNA) of IKK2, and in cells expressing either the kinase-inactive IKK2 mutant (K44A) or the phosphorylation-deficient IkappaBalpha mutant (S32A/S36A). An IKK2-specific inhibitor or effective siRNA prevented IL-1beta-induced inhibition of acetylcholine-stimulated PLC-beta (phopsholipase C-beta) activation. These results suggest that the canonical IKK2/IkappaBalpha pathway of NF-kappaB activation mediates the up-regulation of RGS4 expression in response to IL-1beta and contributes to the inhibitory effect of IL-1beta on acetylcholine-stimulated PLC-beta-dependent initial contraction in rabbit colonic smooth muscle.

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Docosahexaenoic acid potentiates interleukin-1beta induction of nitric oxide synthase through mechanism involving p44/42 MAPK activation in rat vascular smooth muscle cells.

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