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)

A high-throughput screening (HTS) assay for inhibitors of nitric oxide (NO) production by activated microglia was developed and used to compare the relative activities of various anti-inflammatory compounds and cell-permeable protein kinase inhibitors. BV-2 cells, an immortalized line that retains phenotypic features of microglia and produces NO in response to lipopolysaccharide (LPS), were used in the activation paradigm for the HTS assay. A characteristic feature of the compounds that were the most potent dose-dependent inhibitors of NO production is their ability to modulate serine/threonine protein kinases. The anti-inflammatory compound K252a, an inhibitor of calmodulin (CaM)-regulated protein kinases, had one of the highest potencies in the assay. Other classes of kinase inhibitors, including the protein kinase A inhibitor H-89, the mitogen activated protein kinase inhibitors PD98059 and SB203580, and the tyrosine kinase inhibitor genistein, were less potent and efficacious than K252a or the general serine/threonine/tyrosine kinase inhibitor staurosporine. K252a suppresses production of the inducible nitric-oxide synthase (iNOS). The inhibitory effect of K252a is not due to cell toxicity and does not correlate with inhibition of NFkappaB nuclear translocation. The mechanism of action appears to involve inhibition of phosphorylation of the transcription factor CREB, a protein whose activity is modulated by phosphorylation by CaM-dependent protein kinases. These data suggest that signal transduction pathways mediated by CaM-dependent protein kinases warrant future study as potential drug discovery targets.
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PMID:Screening in a cell-based assay for inhibitors of microglial nitric oxide production reveals calmodulin-regulated protein kinases as potential drug discovery targets. 1053 68

The kinin B(1) receptor (B(1)R) gene is strongly upregulated following tissue injury and inflammation. In an attempt to define the regulatory elements that account for the control of B(1)R gene expression, we have conducted in vivo footprinting analysis of the B(1)R gene promoter region in three human cell types: embryonic lung fibroblast cells (IMR-90), embryonic kidney cells (HEK-293), and primary cultures of vascular umbilical smooth muscle cells. Initial in vitro delineation of the B(1)R gene promoter by transient transfection experiments with a reporter gene indicated that a 1.4-kb region, located just upstream of the transcription initiation site, bears all the characteristics of a core promoter with a functional TATA box and additional positive and negative control elements, as some of them could be tissue-specific. In vivo ultraviolet and dimethylsulfate footprinting analyses of the 1.4-kb region revealed no difference between the footprint patterns in the three cell types studied. We found that even in the noninduced state, the B(1)R gene promoter is possibly bound by several sequence-specific DNA binding proteins (GATA-1, PEA3, AP-1, CAAT, Sp1, Pit-1a, Oct-1, CREB). Some other footprints were detected on sequences that do not correspond to any known transcription factor binding site. No additional changes in protein-DNA complexes were observed upon treatment with interleukin-1 beta (IL-1beta) or bacterial lipopolysaccharide, shown previously to induce B(1)R gene expression. These results indicate that complex protein-DNA interactions exist at the B(1)R gene promoter prior to induction by external stimuli even in cells (HEK-293) that do not express a functional B(1)R.
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PMID:In vivo protein-DNA interactions at the kinin B(1) receptor gene promoter: no modification on interleukin-1 beta or lipopolysaccharide induction. 1084 22

Stimulation of macrophages by lipopolysaccharide (LPS) leads to the rapid activation of MAP kinases (MAPK) and the subsequent induction of cytokine gene expression. We sought to determine whether LPS-inducible cytokine genes were differentially regulated in macrophages derived from different tissues. Our studies revealed that PD98059, an inhibitor of the extracellular-regulated kinase (ERK) pathway, blocked LPS-induced activation of tumor necrosis factor alpha (TNF-alpha) gene expression in a murine cell line derived from alveolar macrophages but not in a nonpulmonary macrophage cell line. These findings were confirmed using primary murine alveolar and peritoneal macrophages. This suggests that the TNF-alpha promoter contains MAPK-dependent and -independent regulatory elements that are used in a cell type-specific manner. We also found that differences in MAPK-regulated signaling were not mediated by NF-KB, LITAF, Egr-1, CREB, or ATF2/ c-Jun. Together, these studies demonstrate that transcriptional activation of the TNF-alpha gene requires the ERK signaling cascade in selected macrophage populations.
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PMID:Activation of TNF-alpha transcription utilizes distinct MAP kinase pathways in different macrophage populations. 1085 63

As a dendritic cell (DC) matures, it becomes more potent as an antigen-presenting cell. This functional change is accompanied by a change in DC immunophenotype. The signal transduction events underlying this process are poorly characterized. In this study, we have investigated the signal transduction pathways involved in the lipopolysaccharide (LPS)-induced maturation of human monocyte-derived DCs (MoDCs) in vitro. We show that exposure of immature MoDCs to LPS activates the p38 stress-activated protein kinase (p38SAPK), extracellular signal-regulated protein kinase (ERK), phosphoinositide 3-OH kinase (PI3 kinase)/Akt, and nuclear factor (NF)-kappaB pathways. Studies using inhibitors demonstrate that PI3 kinase/Akt but not the other pathways are important in maintaining survival of LPS-stimulated MoDCs. Inhibiting p38SAPK prevented activation of the transcription factors ATF-2 and CREB and significantly reduced the LPS-induced up-regulation of CD80, CD83, and CD86, but did not have any significant effect on the LPS-induced changes in macropinocytosis or HLA-DR, CD40, and CD1a expression. Inhibiting the NF-kappaB pathway significantly reduced the LPS-induced up-regulation of HLA-DR as well as CD80, CD83, and CD86. Inhibiting the p38SAPK and NF-kappaB pathways simultaneously had variable effects depending on the cell surface marker studied. It thus appears that different aspects of LPS-induced MoDC maturation are regulated by different and sometimes overlapping pathways.
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PMID:The PI3 kinase, p38 SAP kinase, and NF-kappaB signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells. 1091 Sep 20

The effect of lipopolysaccharide (LPS) on the expression of immediate early genes, such as c-fos and c-jun, was examined in C6 rat glioma cells. LPS (1 microg/ml) alone did not affect c-fos mRNA level. LPS, however, transiently increased c-jun mRNA level. Cycloheximide (CHX, 20 microM), a protein synthesis inhibitor, alone caused increases of c-fos and c-jun mRNA levels. LPS showed a potentiating effect in the regulation of c-fos mRNA level, whereas LPS showed an additive action for the regulation of CHX-induced c-jun mRNA expression. To determine if CREB and mitogen-activated protein kinases (MAPKs) are involved in the regulation of c-fos mRNA expression by LPS and CHX, Western blot was carried out using the phosphorylated form of antibodies against ERK, JNK, p38, and CREB. LPS transiently increased the phosphorylation of p38-MAPK and CREB. In addition, LPS alone elevated phosphorylation of ERK (p44/p42) MAPK in a time-dependent manner. Furthermore, LPS plus CHX enhanced phosphorylation of ERK, p38, and CREB in a synergistic manner. Our results suggest that the phosphorylation of ERK, p38, and CREB may be involved in the regulation of synergistic c-fos mRNA expression induced by LPS plus CHX in C6 rat glioma cells.
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PMID:Regulation of c-fos gene expression by lipopolysaccharide and cycloheximide in C6 rat glioma cells. 1092 99

In the present study, the mechanism by which dexamethasone (DEX) inhibited IL-1beta gene expression in bacterial lipopolysaccharide (LPS)-activated RAW 264.7 cells was investigated. The decrease in LPS-induced IL-1beta mRNA expression was demonstrated by quantitative reverse transcription polymerase chain reaction (RT-PCR). Since the promoter in IL-1beta gene contains binding motifs for NF-kappaB/Rel, AP-1, NF-IL6, and CREB/ATF, which appear to be important in LPS-mediated IL-1beta induction, the effects of DEX on the activation of these transcription factors were examined. Treatment of DEX to RAW 264.7 cells induced a dose-related inhibition of NF-kappaB/Rel and AP-1 in chloramphenicol acetyltransferase activity, while neither NF-IL6 nor CREB/ATF activation was affected by DEX. Treatment of RAW 264.7 cells with DEX inhibited DNA binding of NF-kappaB/Rel and AP-1 proteins to their cognate DNA sites as measured by electrophoretic mobility shift assay (EMSA). DEX treatment caused a significant reduction in nuclear c-rel, p65, and p50 protein contents, and these decreases were paralleled by the accumulation of cytoplasmic c-rel, p65, and p50. DEX treatment of RAW 264.7 cells did not inhibit the nuclear translocation of c-jun and c-fos. We found that the inhibition of IL-1beta production by DEX is not related to p38, which is important in the IL-1beta induction. These results suggest that DEX may inhibit IL-1beta gene expression by a mechanism involving the blocking of LPS-induced NF-kappaB/Rel and AP-1 activation.
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PMID:Dexamethasone inhibits IL-1 beta gene expression in LPS-stimulated RAW 264.7 cells by blocking NF-kappa B/Rel and AP-1 activation. 1093 15

Vasoactive intestinal peptide (VIP) and the structurally related neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP), present in the microenvironment of lymphoid organs, modulate the function of inflammatory cells through specific receptors. VIP and PACAP inhibit the production of pro-inflammatory agents and stimulate the production of anti-inflammatory cytokines in activated macrophages. The effect is mediated through specific receptors and involves shedding of the CD14 lipopolysaccharide (LPS) receptor and the transcriptional regulation of cytokine genes through effects on de novo expression or nuclear translocation of NFkappaB, cAMP-element binding protein (CREB), c-Jun, and interferon regulatory factor 1 (IRF-1). The in vivo administration of VIP/PACAP results in a similar pattern of cytokine modulation which, presumably, mediates the protective effect of VIP/PACAP in a high-endotoxic murine model for septic shock. VIP/PACAP reduce the expression of the costimulatory B7.1/B7.2 molecules and the subsequent stimulatory activity for T helper (Th) cells in stimulated macrophages. In contrast, in unstimulated macrophages, VIP/PACAP induce specific B7.2 expression and promote Th2 cell differentiation. We propose that VIP/PACAP act as endogenous factors that regulate immune homeostasis and that the physiological consequences of VIP/PACAP presence in the immune microenvironment depend on the timing of the neuropeptide release and the activation stage of the neighboring immune cells.
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PMID:Neuropeptides as modulators of macrophage functions. Regulation of cytokine production and antigen presentation by VIP and PACAP. 1134 14

Antibody class switch recombination (CSR) occurs after antigen activation of B cells. CSR is directed to specific heavy chain isotypes by cytokines and B cell activators that induce transcription from the unrearranged, or germline (GL), C(H) region genes. Transforming growth factor (TGF)-beta1 is essential for switch recombination to IgA due to its ability to induce transcription from GL Ig alpha genes. It has been shown that the promoters which regulate transcription of mouse and human GL alpha RNAs contain a TGF-beta1-responsive element that binds Smad and core binding factor (CBFalpha)/AML/PEBPalpha/RUNX: They also contain other elements which bind the transcription factors CREB, BSAP and Ets family proteins. In this manuscript we demonstrate that two tandem Ets sites in the mouse GL alpha promoter bind the transcription factors Elf-1 and PU.1, and that the 3' site is essential for expression of a luciferase reporter gene driven by the GL alpha promoter. Binding of Elf-1 to the GL alpha promoter is inducible by lipopolysaccharide in nuclear extracts from splenic B cells. An NF-kappaB site is identified, although it does not contribute to expression of the promoter in reporter gene assays. Since CSR to IgA is greatly reduced in NF-kappaB/p50-deficient mice, these data support the hypothesis that NF-kappaB has roles in switching in addition to regulation of GL transcription. Finally, we demonstrate that nocodazole, which disrupts microtubules that sequester Smad proteins in the cytoplasm, stimulates transcription from the GL alpha promoter.
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PMID:Roles of Ets proteins, NF-kappa B and nocodazole in regulating induction of transcription of mouse germline Ig alpha RNA by transforming growth factor-beta 1. 1136

The expression of inducible nitric oxide synthase (NOS2) in glial cells is inhibited by neurotransmitters such as norepinephrine (NE) which elevate cAMP levels. We examined the molecular basis for this effect using a 2.2-kb fragment of the rat NOS2 promoter transfected into rat C6 glioma cells. Promoter activation (up to six-fold) by lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) was reduced by NE, which alone had no effect. However, a promoter construct extending to bp -130 and containing the proximal nuclear factor-kappa B (NF-kappaB) binding site was minimally activated by LPS and cytokines, but activated up to three-fold by NE. Deletion analysis identified a 27-bp region (bp -187 to -160) as critical for mediating this suppressive effect. This region also enhanced promoter activation by LPS and cytokines, and prevented activation by NE alone. Gel shift analysis revealed constitutive binding to this region, and induction by NE of additional complexes which could be blocked by an antibody against CREB. NE also increased levels of the IkappaBalpha protein which could contribute to its suppressive effects. These results identify a critical role for this 27-bp region in regulation of NOS2 promoter activation and suppression by cAMP.
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PMID:A 27-bp region of the inducible nitric oxide synthase promoter regulates expression in glial cells. 1143 80

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family play key roles in the regulation of genes implicated in the control of growth, differentiation, metabolism, and inflammation. The recent limited studies on the promoter regions of C/EBP genes, particularly C/EBPalpha, have indicated the potential existence of species-specific regulatory mechanisms. It is therefore essential that the promoter regions of different C/EBP genes from a wide range of species are investigated in detail. As an important step toward this goal, we report here the characterization of the Xenopus laevis C/EBPbeta gene promoter. Sequence analysis showed that the 1.6-kb promoter region contained putative binding sites for several transcription factors that have previously been implicated in the regulation of the C/EBPs, including C/EBP, CREB, Myb, STAT, and USF. The -288/+91 promoter region was capable of directing high levels of expression in the hepatoma Hep3B cell line. In addition, this minimal promoter could be autoregulated by both C/EBPalpha and C/EBPbeta and activated by lipopolysaccharide, interleukin-6 and CREB. These results therefore demonstrate that several aspects of C/EBPbeta regulation in mammals have been highly conserved in amphibians. However, a comparison of C/EBPbeta gene promoters characterized to date does indicate the existence of species-specific differences in autoregulation.
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PMID:Molecular characterization of the Xenopus CCAAT-enhancer binding protein beta gene promoter. 1144 61


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