NF-κB- and C/EBPβ-driven Interleukin-1β Gene Expression and PAK1-mediated Caspase-1 Activation Play Essential Roles in Interleukin-1β Release from Helicobacter pylori Lipopolysaccharide-stimulated Macrophages
ABSTRACT Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1beta (IL-1beta) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1beta release and dissected the signaling pathways leading to LPS-induced IL-1beta secretion. We demonstrate that both the NF-kappaB and the C/EBPbeta-binding elements of the IL-1beta promoter drive LPS-induced IL-1beta gene expression. NF-kappaB activation requires the classical TLR4-initiated signaling cascade leading to IkappaB phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBPbeta activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBPbeta, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBPbeta. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1beta. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1beta gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1beta gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer.
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- "ti - vate both signals , increase the level of the precursor IL - 1b protein , and activate the IPAF inflammasome , leading to the maturation of IL - 1b in primary rat astrocytes . The regulation of proeIL - 1b expression by transcription factor , nuclear factor kappa - light - chain - enhancer of activated B cells ( NFkB ) , is well established ( Basak et al . , 2005 ) , and NFkB is known to be activated by fatty acids , such as PA or a high - fat diet ( Wang et al . , 2012 ) . In support of this , we observed that NFkB expression in the astrocytes is increased on PA treatment ( Supplementary Fig . 6 ) and inhibiting NFkB significantly reduced the mRNA level of proeIL - 1b ( Supplementary Fig . 7 ) "
ABSTRACT: Inflammatory response has been strongly implicated in the pathogenesis of numerous diseases, including Alzheimer's disease (AD). However, little is known about the molecular mechanisms initiating the generation of inflammatory molecules in the central nervous system, such as interleukin-1β (IL-1β). Previously we identified that palmitate can induce primary astrocytes to produce cytokines, causing AD-like changes in primary neurons. Here we investigated and identified that palmitate induced the activation of ice protease-activating factor (IPAF)-apoptosis-associated speck-like protein containing a caspase activation and recruitment domains (CARD) (ASC) inflammasome in astrocytes leading to the maturation of IL-1β, thereby implicating that not only pathogen-related factors can activate the IPAF-ASC inflammasome. Moreover, downregulating IPAF (which was found to be regulated by cAMP response element-binding protein) in astrocytes through silencing to decrease IL-1β secretion from the astrocytes reduced the generation of amyloid-β42 by primary neurons. Furthermore, the expression levels of IPAF and ASC were found significantly elevated in a subgroup of sporadic AD patients, suggesting an involvement of the IPAF-ASC inflammasome in the inflammatory response associated with AD, and thus could be a potential therapeutic target for AD.Neurobiology of aging 09/2013; 35(2). DOI:10.1016/j.neurobiolaging.2013.08.016 · 4.85 Impact Factor
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- "Pathogen-induced TLR signaling quickly phosphorylates and activates C/EBP␤ (Cloutier et al., 2009) via the mitogen-activated protein (MAP) kinase p38 (Basak et al., 2005). Activated C/EBP␤ was found to synergize with NF-B p65 (RelA) in the proinflammatory activation of a variety of cytokine encoding genes, including IL1␤ (Basak et al., 2005), IL-6, IL-8 (John et al., 2009; Matsusaka et al., 1993; Stein and Baldwin, 1993; Zhu et al., 2003), and IL12p40 (Plevy et al., 1997) but also the SAA2 encoding gene (Betts et al., 1993). C/EBP␤ was found to directly enhance NF-B signaling through reducing the levels of IB␣ (Cappello et al., 2009). "
ABSTRACT: Pathogen contact induces quickly in Mammary Epithelial Cells (MEC) the expression of the proinflammatory cytokine IL-8 and delayed that of the bactericidal β-defensin LAP. Both genes encoding these factors feature on their proximal promoter a composite NF-κB/CEBP binding site. We compare here in MEC the role of NF-κB and C/EBP factors in regulating basal and pathogen-induced expression of both genes from cattle. Abrogating NF-κB binding to that site by introduction of a single point mutation blocks promoter activity of both genes in reporter gene assays. Chromatin accessibility PCR and Chromatin immunoprecipitation reveal that the chromatin of the resting LAP promoter is tightly packed and NF-κB p50 homodimer binding prevails. Infection results in chromatin decompaction accompanied by predominant recruitment of NF-κB p65 for promoter activation. Overexpression of transcription factors confirms a stimulatory role of NF-κB p65 but also a repressive function of C/EBPβ for LAP promoter activity. These factors reverse roles to control IL-8 expression. NF-κB p65 homodimers already reside on the resting IL-8 promoter and induction recruits NF-κB p50. Overexpression of both NF-κB factors represses the promoter in MEC, but not in HEK293 cells. Inhibitors of NF-κB activation and nuclear recruitment both tremendously increase basal and pathogen stimulated IL-8 mRNA concentrations in MEC. Mutation of the C/EBP-binding site blocks and overexpression of C/EBPβ stimulates IL-8-promoter activity. Thus, the pathogen-induced fast activation of diverse transcription factors acting through a common promoter binding site is gene specifically differentiated into opposite functional significance for swiftly (IL-8) or slowly (LAP) induced genes in MEC.Molecular Immunology 03/2011; 48(6-7):895-908. DOI:10.1016/j.molimm.2010.12.018 · 3.00 Impact Factor