Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Toll-like receptor-4 (TLR4) can be activated by nonbacterial agonists, including saturated fatty acids. However, downstream signaling pathways activated by nonbacterial agonists are not known. Thus, we determined the downstream signaling pathways derived from saturated fatty acid-induced TLR4 activation. Saturated fatty acid (lauric acid)-induced NFkappaB activation was inhibited by a dominant-negative mutant of TLR4, MyD88, IRAK-1, TRAF6, or IkappaBalpha in macrophages (RAW264.7) and 293T cells transfected with TLR4 and MD2. Lauric acid induced the transient phosphorylation of AKT. LY294002, dominant-negative (DN) phosphatidylinositol 3-kinase (PI3K), or AKT(DN) inhibited NFkappaB activation, p65 transactivation, and cyclooxygenase-2 (COX-2) expression induced by lauric acid or constitutively active (CA) TLR4. AKT(DN) blocked MyD88-induced NFkappaB activation, suggesting that AKT is a MyD88-dependent downstream signaling component of TLR4. AKT(CA) was sufficient to induce NFkappaB activation and COX-2 expression. These results demonstrate that NFkappaB activation and COX-2 expression induced by lauric acid are at least partly mediated through the TLR4/PI3K/AKT signaling pathway. In contrast, docosahexaenoic acid (DHA) inhibited the phosphorylation of AKT induced by lipopolysaccharide or lauric acid. DHA also suppressed NFkappaB activation induced by TLR4(CA), but not MyD88(CA) or AKT(CA), suggesting that the molecular targets of DHA are signaling components upstream of MyD88 and AKT. Together, these results suggest that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4 and its downstream signaling pathways involving MyD88/IRAK/TRAF6 and PI3K/AKT and further suggest the possibility that TLR4-mediated target gene expression and cellular responses are also differentially modulated by saturated and unsaturated fatty acids.
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PMID:Reciprocal modulation of Toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids. 1286 24

Many of the proinflammatory effects of gram-negative bacteria are elicited by the interaction of bacterial lipopolysaccharide (LPS) with Toll-like receptor 4 (TLR4) expressed on host cells. TLR4 signaling leads to activation of NF-kappa B and transcription of many genes involved in the inflammatory response. In this study, we examined the signaling pathways involved in NF-kappa B activation by TLR4 signaling in human microvascular endothelial cells. Akt is a major downstream target of phosphoinositide 3 kinase (PI3-kinase), and PI3-kinase activation is necessary and sufficient for Akt phosphorylation. Consequently, Akt kinase activation was used as a measure of PI3-kinase activity. In a stable transfection system, dominant-negative mutants of myeloid differentiation factor 88 (MyD88) and interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK-1) (MyD88-TIR and IRAK-DD, respectively) blocked Akt kinase activity in response to LPS and IL-1 beta. A dominant-negative mutant (Mal-P/H) of MyD88 adapter-like protein (Mal), a protein with homology to MyD88, failed to inhibit LPS- or IL-1 beta-induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) inhibited the NF-kappa B luciferase activity, IL-6 production, and I kappa B alpha degradation elicited by LPS and IL-1 beta but not that stimulated by tumor necrosis factor alpha. The dominant-negative mutant of Akt partially inhibited the NF-kappa B luciferase activity evoked by LPS and IL-1 beta. However, expression of a constitutively activated Akt failed to induce NF-kappa B luciferase activity. These findings indicate that TLR4- and IL-1R-induced PI3-kinase activity is mediated by the adapter proteins MyD88 and IRAK-1 but not Mal. Further, these studies suggest that PI3-kinase is an important mediator of LPS and IL-1 beta signaling leading to NF-kappa B activation in endothelial cells and that Akt is necessary but not sufficient for NF-kappa B activation by TLR4.
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PMID:Phosphoinositide 3 kinase mediates Toll-like receptor 4-induced activation of NF-kappa B in endothelial cells. 1287 20

MyD88 is an adapter protein that is involved in Toll-like receptor (TLR)- and interleukin-1 receptor (IL-1R)-induced activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK). By directly binding IL-1R-associated kinase (IRAK)-1 and IRAK-4, MyD88 serves as a bridging protein, enabling IRAK-4-induced IRAK-1 phosphorylation. We previously identified a lipopolysaccharide-inducible splice variant of MyD88, MyD88(S), which specifically prevents the recruitment of IRAK-4 into the IL-1R complex and thus inhibits IRAK-4-mediated IRAK-1 phosphorylation. MyD88(S) is not able to activate NF-kappaB, and in contrast functions as a dominant negative inhibitor of TLR/IL-1R-induced NF-kappaB activation. Unexpectedly, we here demonstrate that MyD88(S) still allows JNK phosphorylation and activator protein (AP)-1-dependent reporter gene induction upon overexpression in HEK293T cells. These observations indicate that NF-kappaB and JNK activation pathways can already diverge at the level of MyD88. Moreover, the regulated expression of a MyD88 splice variant which specifically interferes with NF-kappaB- but not AP-1-dependent gene expression implies an important role for alternative splicing in the fine-tuning of TLR/IL-1R responses.
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PMID:MyD88S, a splice variant of MyD88, differentially modulates NF-kappaB- and AP-1-dependent gene expression. 1288 15

Toll-like receptor (TLR)4 has recently been shown to reside in the Golgi apparatus of intestinal crypt epithelial m-ICcl2 cells, colocalizing with internalized lipopolysaccharide (LPS). Here we demonstrate that disruption of the integrity of the Golgi apparatus significantly reduced LPS-mediated nuclear factor kappaB activation. Also, the TLR4 adaptor protein MyD88 and the serine/threonine kinase IRAK-1 were rapidly recruited to the Golgi apparatus upon stimulation. LPS-mediated activation required lipid raft formation and intact clathrin-dependent internalization. In contrast to macrophages, prevention of ligand internalization by use of LPS-coated beads significantly impaired recognition by epithelial cells. The localization of TLR4 to the Golgi apparatus was abrogated by expression of a genetically modified form of the TLR4 binding chaperone gp96. Thus, our data provide evidence that in contrast to the situation in macrophages, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and require LPS internalization.
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PMID:Intracellular recognition of lipopolysaccharide by toll-like receptor 4 in intestinal epithelial cells. 1456 81

Pulmonary epithelial cells are continuously exposed to microbial challenges as a result of breathing. It is recognized that immune myeloid cells express Toll-like receptors (TLRs), which play a major role in detecting microbes and initiating innate immune responses. In contrast, little is known concerning the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function, and the signaling pathways involved. In this work, we demonstrated by reverse transcription-PCR and/or immunoblot that TLR4 and the accessory molecule MD-2 are constitutively expressed in distinct human alveolar and bronchial epithelial cells. We further characterized by flow cytometry, biotinylation/precipitation, and confocal microscopy the intracellular localization of TLR4 in these cells. Despite this intracellular compartmentalization of TLR4, pulmonary epithelial cells were responsive to the TLR4 activator lipopolysaccharide (LPS), a potent Gram-negative bacteria-associated molecular pattern. Using respiratory epithelial cells isolated from TLR4 knock-out and wild type mice, we demonstrated that TLR4 is the actual activating receptor for LPS in these cells. Furthermore we showed that this cell response to LPS involves a signaling complex including the kinases interleukin-1 receptor-associated kinase (IRAK), p38, Jnk, and ERK1/2. Moreover, using vectors expressing dominant-negative forms of MyD88 and TRAF6, we established that LPS-induced activation of respiratory epithelial cells is largely dependent on TLR4 signaling intermediates. Altogether these data demonstrate that TLR4 is a key element in the response of pulmonary epithelial cells to molecules derived from Gram-negative bacteria. The intracellular localization of TLR4 in lung epithelia is expected to play an important role in the prevention of the development of chronic inflammatory disease.
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PMID:Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4. 1460 Jan 54

Parenterally administered lipopolysaccharide (LPS) increases the concentration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) in the rat hippocampus and evidence suggests that this effect plays a significant role in inhibiting long-term potentiation (LTP). The anti-inflammatory cytokine IL-10, antagonizes certain effects of IL-1beta, so if the effects of LPS are mediated through an increase in IL-1beta, it might be predicted that IL-10 would also abrogate the effect of LPS. Here, we report that IL-10 reversed the inhibitory effect of LPS on LTP and the data couple this with an inhibitory effect on the LPS-induced increase in IL-1beta. LPS treatment increased hippocampal expression of IL-1 receptor Type I protein. Consistent with the LPS-induced increases in IL-1beta concentration and receptor expression, were downstream changes which included enhanced phosphorylation of IRAK and the stress-activated kinases, JNK and p38; these LPS-induced changes were reversed by IL-10, which concurs with the idea that these events are triggered by increased activation of IL-1RI by IL-1beta. We provide evidence which indicates that LPS treatment leads to evidence of cell death and this was reversed in hippocampus prepared from LPS-treated rats which received IL-10. The evidence is therefore consistent with the idea that IL-10 acts to protect neuronal tissue from the detrimental effects induced by LPS.
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PMID:Lipopolysaccharide-induced increase in signalling in hippocampus is abrogated by IL-10--a role for IL-1 beta? 1472 Feb 13

Local activation of the macrophage by endotoxin is essential for the eradication of invasive gram-negative infections. Circulating endotoxin at lower concentrations results in immune cell activation at distant sites leading to tissue injury. Although the cellular mechanisms involved in these potentially dissimilar events are incomplete, it appears that the proximal kinase IRAK-1 plays a role. Thus, sense and antisense IRAK-1 oligonucleotides were used to determine the role IRAK-1 plays in macrophage activation by systemic (1-100 ng/mL) and local (1000 ng/mL) concentration of lipopolysaccharide (LPS) within THP-1 cells. Within the sense group, 1-1000 ng/mL of LPS within the sense group resulted in cellular activation of ERK-1/2, p38, and JNK/SAPK and the nuclear activation of NF-kappaB and AP-1. This activation was associated with proinflammatory cytokine production and cellular spreading. Systemic concentrations of LPS within the antisense group were associated with significant attenuation of intracellular signaling, cytokine production, and cellular spreading compared with the sense group. Local concentrations of LPS within the antisense group, however, were associated only with a delay in intracellular signaling, with no effect on cytokine production or cell spreading compared with the sense group. Based on these results, it appears that IRAK-1 is essential to macrophage activation at systemic, but not local, concentrations of LPS. These data suggest that redundant pathways exist that are functional at higher concentrations of LPS. Therefore, IRAK-1 appears to be the central kinase involved in the activation of the macrophage at distant sites during septic shock but is not necessary for activation in areas of local infection.
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PMID:Modulation of macrophage responsiveness to lipopolysaccharide by IRAK-1 manipulation. 1475 94

The interleukin-1 receptor-associated kinases (IRAKs) are important downstream signaling components of Toll-like receptors (TLRs). To date, four mammalian IRAKs have been found, namely IRAK-1, IRAK-2, IRAK-4, and IRAK-M. Herein, we show a detailed analysis of the genomic region encompassing the murine Irak2 gene and the molecular cloning of four isoforms of Irak2 (designated Irak2a, Irak2b, Irak2c, and Irak2d) generated by alternative splicing at the 5'-end of the gene. This alternative splicing has direct effects on the expression of the N-terminal death domain and/or inter-domain. No evidence of similar alternative splicing was found for the human IRAK2 gene. When overexpressed, Irak2a and Irak2b potentiated NF-kappaB activation by lipopolysaccharide. Importantly, Irak2c and Irak2d were inhibitory. The promoter for Irak2c differed from that of the other Irak2 isoforms in that it contained putative NF-kappaB binding sites. Lipopolysaccharide induced the expression of Irak2c, indicating a possible negative feedback effect on the signaling pathway. Alternative splicing of the Irak2 gene in mice will therefore generate agonistic or antagonistic Irak2 isoforms, which is likely to have consequences for the regulation of TLR signaling. These observations identify another distinguishing feature between mice and humans in the TLR system that is likely to be due to differences in the selective pressure imposed by pathogens on each species during evolution.
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PMID:The murine IRAK2 gene encodes four alternatively spliced isoforms, two of which are inhibitory. 1508 13

The phenomenon of endotoxin tolerance has been widely investigated, but to date, the molecular mechanisms of endotoxin tolerance remain to be resolved clearly. The discovery of the Toll-like receptor (TLR) family as the major receptors for lipopolysaccharide (LPS) and other bacterial products has prompted a resurgence of interest in endotoxin tolerance mechanisms. Changes of cell surface molecules, signaling proteins, pro-inflammatory and anti-inflammatory cytokines and other mediators have been examined. During tolerance expression of LPS-binding protein (LBP), CD14, myeloid differentiation protein-2 (MD-2) and TLR2 are unchanged or up-regulated, whereas TLR4 is transiently suppressed or unchanged. Proximal post-receptor signaling proteins that are altered in tolerance include augmented degradation of interleukin-1 receptor-associated kinase (IRAK), and decreased TLR4-myeloid differentiation factor 88 (MyD88) and IRAK-MyD88 association. Tolerance has also been shown to be associated with decreased Gi protein content and activity, decreased protein kinase C (PKC) activity, reduction in mitogen-activated protein kinase (MAP kinase) activity, and reduced activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB) induced gene transactivation. However, not all signaling proteins and pathways are suppressed in tolerance and induction of specific anti-inflammatory proteins and signaling pathways may serve important counter inflammatory functions. The latter include induction of IRAK-M and suppressor of cytokine-signaling-1 (SOCS-1), phosphoinositide-3-kinase (PI3K) signaling, and increased or maintained expression of inhibitor-kappaB (IkappaB) isoforms. Also at the nuclear level, increase in the NF-kappaB subunit p50 homodimer expression and increased activation of peroxisome-proliferator-activated receptors-gamma (PPARgamma) have been linked to tolerance phenotype. Although there are species and cellular variations in manifestation of the LPS tolerant phenotype, it is clear that the tolerance phenomena have evolved as a complex orchestrated counter regulatory response to inflammation.
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PMID:Molecular mechanisms of endotoxin tolerance. 1511 98

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.
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PMID:Implications of proteasome inhibition: an enhanced macrophage phenotype. 1513 96


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