Tetra-O-methyl nordihydroguaiaretic acid Terameprocol inhibits the NF-κB-dependent transcription of TNF-α and MCP-1-CCL2 genes by preventing RelA from binding its cognate sites on DNAReportar como inadecuado




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Journal of Inflammation

, 7:59

First Online: 07 December 2010Received: 21 June 2010Accepted: 07 December 2010

Abstract

BackgroundTetra-O-methyl nordihydroguaiaretic acid, also known as terameprocol TMP, is a naturally occurring phenolic compound found in the resin of the creosote bush. We have shown previously that TMP will suppress production of certain inflammatory cytokines, chemokines and lipids from macrophages following stimulation with LPS or infection with H1N1 influenza virus. In this study our goal was to elucidate the mechanism underlying TMP-mediated suppression of cytokine and chemokine production. We focused our investigations on the response to LPS and the NF-κB protein RelA, a transcription factor whose activity is critical to LPS-responsiveness.

MethodsReporter assays were performed with HEK293 cells overexpressing either TLR-3 -4, or -8 and a plasmid containing the luciferase gene under control of an NF-κB response element. Cells were then treated with LPS, polyI:C, or resiquimod, and-or TMP, and lysates measured for luciferase activity.

RAW 264.7 cells treated with LPS and-or TMP were used in ChIP and EMSA assays. For ChIP assays, chromatin was prepared and complexes precipitated with anti-NF-κB RelA Ab. Cross-links were reversed, DNA purified, and sequence abundance determined by Q-PCR. For EMSA assays, nuclear extracts were incubated with radiolabeled probes, analyzed by non-denaturing PAGE and visualized by autoradiography.

RAW 264.7 cells treated with LPS and-or TMP were also used in fluorescence microscopy and western blot experiments. Translocation experiments were performed using a primary Ab to NF-κB RelA and a fluorescein-conjugated secondary Ab. Western blots were performed using Abs to IκB-α and phospho-IκB-α. Bands were visualized by chemiluminescence.

ResultsIn reporter assays with TLR-3 -4, and -8 over-expressing cells, TMP caused strong inhibition of NF-κB-dependent transcription.

ChIP assays showed TMP caused virtually complete inhibition of RelA binding in vivo to promoters for the genes for TNF-α, MCP-1-CCL2, and RANTES-CCL5 although the LPS-dependent synthesis of IκB-α was not inhibited. EMSA assays did not reveal an effect of TMP on the binding of RelA to naked DNA templates in vitro.

TMP did not inhibit the nuclear translocation of NF-κB RelA nor the phosphorylation of IκB-α.

ConclusionTMP acts indirectly as an inhibitor of NF-κB-dependent transcription by preventing RelA from binding the promoters of certain key cytokine and chemokine genes.

AbbreviationsTMPterameprocol

LPSlipopolysaccharide

TNF-αtumor necrosis factor-α

MCP-1monocyte chemotactic protein-1

NDGAnordihydroguaiaretic acid

NF-κBnuclear factor κB

q-PCRquantitative reverse transcriptase polymerase chain reaction

TLRtoll like receptor

ChIPchromatin immunoprecipitation

TTPtristetrapolin

EMSAelectrophoretic mobility shift assay.

Electronic supplementary materialThe online version of this article doi:10.1186-1476-9255-7-59 contains supplementary material, which is available to authorized users.

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Autor: Akinbolade O Oyegunwa - Michael L Sikes - Jason R Wilson - Frank Scholle - Scott M Laster

Fuente: https://link.springer.com/







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