Regulation of NF-κB activity in astrocytes: effects of flavonoids at dietary-relevant concentrations.
ABSTRACT Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Sustained activation of nuclear transcription factor κB (NF-κB) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-κB signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNFα, 150ng/ml) increased NF-κB-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative IκBα construct. In addition, TNFα increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-κB activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((-)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1-1μM) for 18h. None of the flavonoids modulated constitutive or TNFα-induced NF-κB activity. Therefore, we conclude that NF-κB signalling in astrocytes is not a major target for flavonoids.
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ABSTRACT: The promoter of the murine gene encoding inducible nitric oxide synthase (iNOS) contains an NF-kappa B site beginning 55 base pairs upstream of the TATA box, designated NF-kappa Bd. Reporter constructs containing truncated promoter regions, when transfected into macrophages, revealed that NF-kappa Bd is necessary to confer inducibility by bacterial lipopolysaccharide (LPS). Oligonucleotide probes containing NF-kappa Bd plus the downstream 9 or 47 base pairs bound proteins that rapidly appeared in the nuclei of LPS-treated macrophages. The nuclear proteins bound to both probes in an NF-kappa Bd-dependent manner, but binding was resistant to cycloheximide only for the shorter probe. The proteins binding both probes reacted with antibodies against p50 and c-rel but not RelB; those binding the shorter probe also reacted with anti-RelA (p65). Pyrrolidine dithiocarbamate, which acts as a specific inhibitor of NF-kappa B, blocked both the activation of the NF-kappa Bd-binding proteins and the production of NO in LPS-treated macrophages. Thus, activation of NF-kappa B/Rel is critical in the induction of iNOS by LPS. However, additional, newly synthesized proteins contribute to the NF-kappa Bd-dependent transcription factor complex on the iNOS promoter in LPS-treated mouse macrophages.Journal of Biological Chemistry 03/1994; 269(7):4705-8. · 4.77 Impact Factor
Article: NF-kappaB-dependent production of nitric oxide by astrocytes mediates apoptosis in differentiated PC12 neurons following exposure to manganese and cytokines.[show abstract] [hide abstract]
ABSTRACT: Neuronal injury in manganism is accompanied by activation of astroglia within the basal ganglia that is thought to increase production of inflammatory mediators such as nitric oxide (NO). The present studies postulated that astroglial-derived NO mediates neuronal apoptosis induced by manganese (Mn) and pro-inflammatory cytokines. Pheochromocytoma (PC12) cells differentiated with nerve growth factor (NGF) were co-cultured with primary astrocytes and exposed to Mn and tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma). Mn enhanced cytokine-induced expression of inducible nitric oxide synthase (NOS2, EC 18.104.22.168) and production of NO in astrocytes that correlated with apoptosis in co-cultured neurons, as determined by caspase activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and nuclear morphology. Apoptosis in PC12 neurons required the presence of astrocytes and was blocked by overexpression of a phosphorylation-deficient mutant of IkappaBalpha (S32/36A) in astrocytes that prevented induction of NOS2. Pharmacologic inhibition of NOS2 with (+/-)-2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) significantly reduced neuronal apoptosis, and the addition of low concentrations of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), to neurons cultured without astrocytes was sufficient to recover the apoptotic phenotype following exposure to Mn and TNF-alpha/IFN-gamma. It is concluded that Mn- and cytokine-dependent apoptosis in PC12 neurons requires astroglial-derived NO and NF-kappaB-dependent expression of NOS2.Molecular Brain Research 12/2005; 141(1):39-47. · 2.00 Impact Factor
Article: Inactivation of astroglial NF-kappa B promotes survival of retinal neurons following ischemic injury.[show abstract] [hide abstract]
ABSTRACT: Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF-kappaB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia-reperfusion in wild-type (WT) and in GFAP-IkappaBalpha-dn transgenic mice, where the NF-kappaB classical pathway is suppressed specifically in astrocytes. The GFAP-IkappaBalpha-dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro-inflammatory genes, encoding Tnf-alpha, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO-synthase in the retinas of GFAP-IkappaBalpha-dn mice. These data suggest that certain NF-kappaB-regulated pro-inflammatory and redox-active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.European Journal of Neuroscience 08/2009; 30(2):175-85. · 3.63 Impact Factor