tetano
Editor, Senior Moderator
J Inflamm (Lond). 2010 Dec 7;7(1):59. [Epub ahead of print]
Tetra-O-methyl nordihydroguaiaretic acid (Terameprocol) inhibits the NF-kappaB-dependent transcription of TNF-alpha and MCP-1/CCL2 genes by preventing RelA from binding its cognate sites on DNA.
Oyegunwa AO, Sikes ML, Wilson JR, Scholle F, Laster SM.
Abstract
ABSTRACT:
BACKGROUND: Tetra-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-kappaB protein RelA, a transcription factor whose activity is critical to LPS-responsiveness.
METHODS: Reporter 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-kappaB response element. Cells were then treated with LPS, poly(I: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-kappaB 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-kappaB RelA and a fluorescein-conjugated secondary Ab. Western blots were performed using Abs to IkappaB-alpha and phospho-IkappaB-alpha. Bands were visualized by chemiluminescence.
RESULTS: In reporter assays with TLR-3, -4, and -8 over-expressing cells, TMP caused strong inhibition of NF-kappaB-dependent transcription. ChIP assays showed TMP caused virtually complete inhibition of RelA binding in vivo to promoters for the genes for TNF-alpha, MCP-1/CCL2, and RANTES/CCL5 although the LPS-dependent synthesis of IkappB-alpha 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-kappaB RelA nor the phosphorylation of IkappaB-alpha.
CONCLUSION: TMP acts indirectly as an inhibitor of NF-kappaB-dependent transcription by preventing RelA from binding the promoters of certain key cytokine and chemokine genes.
PMID: 21138578 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/21138578
Tetra-O-methyl nordihydroguaiaretic acid (Terameprocol) inhibits the NF-kappaB-dependent transcription of TNF-alpha and MCP-1/CCL2 genes by preventing RelA from binding its cognate sites on DNA.
Oyegunwa AO, Sikes ML, Wilson JR, Scholle F, Laster SM.
Abstract
ABSTRACT:
BACKGROUND: Tetra-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-kappaB protein RelA, a transcription factor whose activity is critical to LPS-responsiveness.
METHODS: Reporter 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-kappaB response element. Cells were then treated with LPS, poly(I: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-kappaB 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-kappaB RelA and a fluorescein-conjugated secondary Ab. Western blots were performed using Abs to IkappaB-alpha and phospho-IkappaB-alpha. Bands were visualized by chemiluminescence.
RESULTS: In reporter assays with TLR-3, -4, and -8 over-expressing cells, TMP caused strong inhibition of NF-kappaB-dependent transcription. ChIP assays showed TMP caused virtually complete inhibition of RelA binding in vivo to promoters for the genes for TNF-alpha, MCP-1/CCL2, and RANTES/CCL5 although the LPS-dependent synthesis of IkappB-alpha 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-kappaB RelA nor the phosphorylation of IkappaB-alpha.
CONCLUSION: TMP acts indirectly as an inhibitor of NF-kappaB-dependent transcription by preventing RelA from binding the promoters of certain key cytokine and chemokine genes.
PMID: 21138578 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/21138578