Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events

Lipopolysaccharide (LPS), a highly conserved component of the outer membrane of gram-negative bacteria, stimulates macrophages to release various cytokine and eicosanoid mediators of the immune response. The mechanism by which LPS stimulates these cells is poorly characterized. One of the most rapid...

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Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1995
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2192101/
https://www.ncbi.nlm.nih.gov/pubmed/7790814
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description Lipopolysaccharide (LPS), a highly conserved component of the outer membrane of gram-negative bacteria, stimulates macrophages to release various cytokine and eicosanoid mediators of the immune response. The mechanism by which LPS stimulates these cells is poorly characterized. One of the most rapid LPS-stimulated events is the phosphorylation and activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase. We wished to examine the role of MAP kinase in LPS- induced signaling in murine macrophages by activating MAP kinase independently of LPS. An expression vector encoding a Raf-1:estrogen receptor (ER) chimeric protein was transfected into the murine macrophage cell line RAW 264.7. Activation of this chimeric protein (delta Raf-1:ER) by estradiol resulted in rapid and prolonged activation of MAP kinase, as expected from previous results implicating Raf-1 as an upstream activator of this signaling cascade. LPS stimulation induced accumulation of MAP kinase phosphatase 1 messenger RNA, whereas delta Raf-1:ER activation did not, perhaps accounting for the more prolonged activation of MAP kinase seen in response to delta Raf-1:ER activation. Similarly, activation of DNA binding by the transcription factor, nuclear factor (NF) kappa B, as assessed by electrophoretic mobility shift assay, occurred in response to LPS stimulation but not in response to delta Raf-1:ER activation or phorbol myristate acetate (PMA) stimulation. Using an enzyme-linked immunosorbent assay for murine tumor necrosis factor alpha (TNF-alpha), we found that LPS and PMA stimulation and delta Raf-1:ER activation induced secretion of TNF-alpha, although the amount of TNF-alpha secreted in response to delta Raf-1:ER activation and PMA stimulation was approximately 20-fold less than that secreted in response to LPS. Correspondingly, accumulation of TNF-alpha messenger RNA was weakly induced by delta Raf-1:ER activation or PMA stimulation, whereas strong induction was noted in response to LPS. These results suggest that Raf- 1 or PMA activation of MAP kinase in murine macrophages is sufficient for a small amount of TNF-alpha production and secretion in the absence of NF-kappa B activation, but LPS stimulation involves additional signaling events, such as NF-kappa B activation, that augment the response seen with activation of MAP kinase alone.
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spelling pubmed-21921012008-04-16 Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events J Exp Med Articles Lipopolysaccharide (LPS), a highly conserved component of the outer membrane of gram-negative bacteria, stimulates macrophages to release various cytokine and eicosanoid mediators of the immune response. The mechanism by which LPS stimulates these cells is poorly characterized. One of the most rapid LPS-stimulated events is the phosphorylation and activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase. We wished to examine the role of MAP kinase in LPS- induced signaling in murine macrophages by activating MAP kinase independently of LPS. An expression vector encoding a Raf-1:estrogen receptor (ER) chimeric protein was transfected into the murine macrophage cell line RAW 264.7. Activation of this chimeric protein (delta Raf-1:ER) by estradiol resulted in rapid and prolonged activation of MAP kinase, as expected from previous results implicating Raf-1 as an upstream activator of this signaling cascade. LPS stimulation induced accumulation of MAP kinase phosphatase 1 messenger RNA, whereas delta Raf-1:ER activation did not, perhaps accounting for the more prolonged activation of MAP kinase seen in response to delta Raf-1:ER activation. Similarly, activation of DNA binding by the transcription factor, nuclear factor (NF) kappa B, as assessed by electrophoretic mobility shift assay, occurred in response to LPS stimulation but not in response to delta Raf-1:ER activation or phorbol myristate acetate (PMA) stimulation. Using an enzyme-linked immunosorbent assay for murine tumor necrosis factor alpha (TNF-alpha), we found that LPS and PMA stimulation and delta Raf-1:ER activation induced secretion of TNF-alpha, although the amount of TNF-alpha secreted in response to delta Raf-1:ER activation and PMA stimulation was approximately 20-fold less than that secreted in response to LPS. Correspondingly, accumulation of TNF-alpha messenger RNA was weakly induced by delta Raf-1:ER activation or PMA stimulation, whereas strong induction was noted in response to LPS. These results suggest that Raf- 1 or PMA activation of MAP kinase in murine macrophages is sufficient for a small amount of TNF-alpha production and secretion in the absence of NF-kappa B activation, but LPS stimulation involves additional signaling events, such as NF-kappa B activation, that augment the response seen with activation of MAP kinase alone. The Rockefeller University Press 1995-07-01 /pmc/articles/PMC2192101/ /pubmed/7790814 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events
title Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events
title_full Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events
title_fullStr Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events
title_full_unstemmed Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events
title_short Activation of Raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events
title_sort activation of raf-1 and mitogen-activated protein kinase in murine macrophages partially mimics lipopolysaccharide-induced signaling events
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2192101/
https://www.ncbi.nlm.nih.gov/pubmed/7790814