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)

Interferongamma inducible protein-10 (IP10 or CXCL10), a Th-1 affiliated chemokine, is expressed by activated glial cells and may contribute to the trafficking of immune cells in the inflamed central nervous system. This study examines the regulation of the expression of this chemokine in cultured microglial cells focusing on the roles of mitogen-activated protein (MAP) kinase cascades. Exposure of a mouse microglial cell line, BV-2, to lipopolysaccharide (LPS) and IFNgamma led to an induction of IP10 mRNA and protein as determined by RT-PCR and ELISA, respectively. This induction was suppressed by pharmacological inhibitors of p38 MAPK (i.e., SB203580) and c-Jun N-terminal kinase (JNK, SP600125), suggesting the involvement of the two kinases in IP10 expression. LPS also induced the activity of an IP10 promoter reporter (luciferase) construct transfected into BV-2 cells in a MAP kinase- and NFkappaB-dependent manner. The use of deletion constructs revealed that the kinase-targeted sequences were within the region between -533 bp and -332 bp upstream of the transcriptional start site. Co-transfection of IP10 luciferase with the active forms of the upstream kinases in the MAP kinase cascades, i.e., MAPK kinase-3 (MKK3), MKK6 (the immediately upstream activators of p38 kinase) and a MAP3K, i.e., TGFbeta-activated kinase-1 (TAK1), produced a marked stimulation of the promoter activity. The results of this study indicate that the MAP kinase cascades prominently regulate IP10 gene expression in microglial cells.
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PMID:MAP kinase regulation of IP10/CXCL10 chemokine gene expression in microglial cells. 1663 81

Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IkappaB kinase promotes phosphorylation and degradation of NFkappaB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IkappaB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.
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PMID:Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages. 1698 Mar 1

Mitogen-activated protein/ERK kinase kinase 3 (MEKK3) is a Ser/Thr protein kinase belonging to the MEKK/STE11 subgroup of the MAP3K family. Recently, we found that MEKK3 plays a critical role in interleukin-1 (IL-1) receptor and Toll-like receptor 4 signalling using established primary mouse embryonic fibroblast (MEF) cell lines. However, the function of MEKK3 in immune cells has not been studied because germ-line MEKK3 knockout mice are embryonically lethal between embryonic days 10 and 11. In this study, we used small interference RNA to the mouse Mekk3 gene to specifically knock down MEKK3 expression in the macrophage line Raw264.7. We found that the lipopolysaccharide-induced IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production was dramatically decreased in MEKK3 knockdown cells whereas the tumour necrosis factor-alpha and IL-1beta production were not affected. We also observed that the ERK1/2, p38 and JNK MAPK induction in MEKK3 knockdown cells were moderately inhibited within the first 60 min of stimulation, while the ERK and p38 were more severely inhibited after 2-4 hr of stimulation. Degradation of IkappaBalpha was also partially blocked in MEKK3 knockdown cells. Notably, the impairment in IL-6 and GM-CSF production in the MEKK3 knockdown cells was restored by reintroducing a human Mekk3 cDNA that could not be targeted by mouse Mekk3-siRNAs. In conclusion, this study showed that MEKK3 is a crucial and specific regulator of the proinflammatory cytokines IL-6 and GM-CSF in macrophages and provided a novel method for investigating MEKK3 function in other immune cells.
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PMID:MEKK3 is essential for lipopolysaccharide-induced interleukin-6 and granulocyte-macrophage colony-stimulating factor production in macrophages. 1711 70

The atheroprotective role of apolipoprotein E (apoE) is well established. During inflammation, expression of apoE in macrophages is reduced leading to enhanced atheromatous plaque development. In the present study, we investigated the signaling pathways involved in the repression of apoE gene expression in response to lipopolysaccharide (LPS) treatment, a condition that mimics the inflammatory stress, in mouse macrophages RAW 264.7. We identified Tpl-2 and MEKK1 as the kinases that are primarily responsible for the down-regulation of apoE promoter activity by LPS. Using a dominant negative form of IkappaB, we established that Tpl-2 and MEKK1 signaling pathways converge to NF-kappaB acting on the apoE core promoter -55/+73. In addition to NF-kappaB activation, LPS also activated c-Jun via its phosphorylation by JNK. The activity of the apoE promoter was repressed by c-Jun, whereas small interference RNA-mediated inhibition of endogenous c-Jun expression reversed the inhibitory effect of Tpl-2 on the apoE promoter. Transfection experiments and DNA binding assays showed that the binding site for c-Jun is in the -55/+73 region of the apoE promoter. Finally, we showed that LPS inhibited apoE gene expression via activation of the Tpl-2/MEK/ERK pathway acting on a different apoE promoter region. In summary, LPS represses apoE gene expression in macrophages via signaling pathways that involve the upstream kinases Tpl-2 and MEKK1, the intermediate mitogen-activated protein kinases ERK and JNK, and the downstream transcription factors AP-1 and NF-kappaB that inhibit the apoE promoter activity via distinct regions.
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PMID:Inflammatory signaling pathways regulating ApoE gene expression in macrophages. 1755 93

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-kappaB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1(-/-) macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1(-/-) macrophages. TPL-2(S400A) expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8(-/-) macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-kappaB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.
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PMID:Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in macrophages. 1770 78

Tumor progression loci-2 (Tpl2) (Cot/MAP3K8) is a serine/threonine kinase in the MAP3K family directly upstream of MEK. Recent studies using Tpl2 knockout mice have indicated an important role for Tpl2 in the lipopolysaccharide (LPS) induced production of tumor necrosis factor alpha (TNF-alpha) and other proinflammatory cytokines involved in diseases such as rheumatoid arthritis. Initial 4-anilino-6-aminoquinoline-3-carbonitrile leads showed poor selectivity for Tpl2 over epidermal growth factor receptor (EGFR) kinase. Using molecular modeling and crystallographic data of the EGFR kinase domain with and without an EGFR kinase-specific 4-anilinoquinazoline inhibitor (erlotinib, Tarceva), we hypothesized that we could diminish the inhibition of EGFR kinase by substitution at the C-8 position of our 4-anilino-6-aminoquinoline-3-carbonitrile leads. The 8-substituted-4-anilino-6-aminoquinoline-3-carbonitriles were prepared from the appropriate 2-substituted 4-nitroanilines. Modifications to the C-6 and C-8 positions led to the identification of compounds with increased inhibition of TNF-alpha release from LPS-stimulated rat and human blood, and these analogues were also highly selective for Tpl2 kinase over EGFR kinase. Further structure-activity based modifications led to the identification of 8-bromo-4-(3-chloro-4-fluorophenylamino)-6-[(1-methyl-1H-imidazol-4-yl)methylamino]quinoline-3-carbonitrile, which demonstrated in vitro as well as in vivo efficacy in inhibition of LPS-induced TNF-alpha production.
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PMID:Inhibitors of tumor progression loci-2 (Tpl2) kinase and tumor necrosis factor alpha (TNF-alpha) production: selectivity and in vivo antiinflammatory activity of novel 8-substituted-4-anilino-6-aminoquinoline-3-carbonitriles. 1771 8

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.
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PMID:Pharmacologic inhibition of tpl2 blocks inflammatory responses in primary human monocytes, synoviocytes, and blood. 1784 81

The protein kinase TAK1 (transforming growth factor-beta-activated kinase 1), which has been implicated in the activation of MAPK (mitogen-activated protein kinase) cascades and the production of inflammatory mediators by LPS (lipopolysaccharide), IL-1 (interleukin 1) and TNF (tumour necrosis factor), comprises the catalytic subunit complexed to the regulatory subunits, termed TAB (TAK1-binding subunit) 1 and either TAB2 or TAB3. We have previously identified a feedback-control mechanism by which p38alpha MAPK down-regulates TAK1 and showed that p38alpha MAPK phosphorylates TAB1 at Ser(423) and Thr(431). In the present study, we identified two IL-1-stimulated phosphorylation sites on TAB2 (Ser(372) and Ser(524)) and three on TAB3 (Ser(60), Thr(404) and Ser(506)) in human IL-1R cells [HEK-293 (human embryonic kidney) cells that stably express the IL-1 receptor] and MEFs (mouse embryonic fibroblasts). Ser(372) and Ser(524) of TAB2 are not phosphorylated by pathways dependent on p38alpha/beta MAPKs, ERK1/2 (extracellular-signal-regulated kinase 1/2) and JNK1/2 (c-Jun N-terminal kinase 1/2). In contrast, Ser(60) and Thr(404) of TAB3 appear to be phosphorylated directly by p38alpha MAPK, whereas Ser(506) is phosphorylated by MAPKAP-K2/MAPKAP-K3 (MAPK-activated protein kinase 2 and 3), which are protein kinases activated by p38alpha MAPK. Studies using TAB1(-/-) MEFs indicate important roles for TAB1 in recruiting p38alpha MAPK to the TAK1 complex for the phosphorylation of TAB3 at Ser(60) and Thr(404) and in inhibiting the dephosphorylation of TAB3 at Ser(506). TAB1 is also required to induce TAK1 catalytic activity, since neither IL-1 nor TNFalpha was able to stimulate detectable TAK1 activity in TAB1(-/-) MEFs. Surprisingly, the IL-1 and TNFalpha-stimulated activation of MAPK cascades and IkappaB (inhibitor of nuclear factor kappaB) kinases were similar in TAB1(-/-), MEKK3(-/-) [MAPK/ERK (extracellular-signal-regulated kinase) kinase kinase 3] and wild-type MEFs, suggesting that another MAP3K (MAPK kinase kinase) may mediate the IL-1/TNFalpha-induced activation of these signalling pathways in TAB1(-/-) and MEKK3(-/-) MEFs.
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PMID:Roles for TAB1 in regulating the IL-1-dependent phosphorylation of the TAB3 regulatory subunit and activity of the TAK1 complex. 1802 Oct 73

The protein kinase MEKK3 is essential for tumor necrosis factor alpha (TNFalpha)- and lipopolysaccharide-induced activation of nuclear factor kappaB, although the mechanism by which TNF receptor 1 and Toll-like receptors regulate MEKK3 is largely unknown. In this study we have identified MEKK3 Thr(294) as a novel site of phosphorylation that regulates MEKK3 binding with 14-3-3. Phosphorylation of MEKK3 at Thr(294) was observed for both endogenous and ectopically expressed MEKK3. Mutation of Thr(294) to alanine abolished 14-3-3-MEKK3 association and incubation with phosphorylated peptides mimicking Thr(P)(294) competed for 14-3-3 binding. Mutation of Thr(294) did not alter Ser(526) phosphorylation within the activation loop. However, expression of T294A MEKK3 elevated TNFalpha-stimulated NF-kappaB transcriptional activity, suggesting that Thr(294) phosphorylation and 14-3-3 binding negatively regulate MEKK3. Stimulation with TNFalpha or lipopolysaccharide caused a rapid decrease in Thr(294) phosphorylation of endogenous MEKK3 and subsequent loss of 14-3-3 association. Thus, this study identifies a potentially important regulatory step in MEKK3 signaling via dephosphorylation of Thr(294), which reduces 14-3-3 binding correlating with MEKK3 pathway activation.
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PMID:Phosphorylation of MEKK3 at threonine 294 promotes 14-3-3 association to inhibit nuclear factor kappaB activation. 1830 25

Cancer Osaka thyroid, also known as Tpl-2 (Cot) is a member of the MAP3K kinase family and plays a key role in the regulation of the immune response to pro-inflammatory stimuli such as lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha). A series of Cot constructs with an N-terminal 6xHis tag were transiently expressed in HEK293 cells: Cot(130-399) (kinase domain), Cot(1-388) (N-terminal and kinase domains), Cot(1-413), Cot(1-438) (containing a putative PEST sequence), Cot(1-457) (containing both PEST and degron sequences) and Cot(1-467) (full-length protein). These Cot proteins were pulled down using an anti-6xHis antibody and separated by 2D electrophoresis. The gels were silver-stained and 21 proteins were detected that did not appear, or had substantially reduced intensity, in the control sample. Three of these were identified by MS and MS/MS analysis as Hsp90, Hsp70 and Grp78. Hsp90 appeared to bind to the kinase domain of Cot and this interaction was further investigated using co-immuno-precipitation with both overexpressed Cot in HEK293 cells and endogenous Cot in Hela cells.
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PMID:Proteomics analysis of immunoprecipitated proteins associated with the oncogenic kinase cot. 1831 12


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