Abstract
1. Although accumulating studies have identified I kappa B kinase (IKK) to be essential for controlling NF-kappa B activity in response to several cytokines, the upstream kinases that control IKK activity are still not completely known. We have previously reported that G protein-coupled P2Y(6) receptor activation by UTP potentiates lipopolysaccharide (LPS)-induced I kappa B phosphorylation and degradation, and NF-kappa B activation in J774 macrophages. In this study, we investigated the upstream kinases for IKK activation by UTP and LPS. 2. In murine J774 macrophages, LPS-induced NF-kappa B activation was inhibited by the presence of PDTC, D609, Ro 31-8220, PD 098059 and SB 203580. 3. Accompanying NF-kappa B activation, LPS induced I kappa B degradation and IKK activation were reduced by PDTC, D609, Ro 31-8220 and PD 098059, but not by SB 203580. 4. Although UTP itself slightly induced IKK activation, this response was synergistic with LPS. BAPTA/AM and KN-93 (a calcium/calmodulin-dependent protein kinase (CaMK) inhibitor) attenuated UTP- but not LPS-stimulated IKK activity. Synergistic IKK activation between LPS and thapsigargin was further demonstrated in peritoneal macrophages. 5. LPS and UTP co-stimulation additively increased p65 NF-kappa B phosphorylation. In vitro kinase assays revealed that LPS and UTP induced extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase activation were respectively inhibited by PD098059 and SB 203580. 6. Taken together, we demonstration that Gq protein-coupled P2Y(6) receptor activation can potentiate LPS-stimulated IKK activity. While PKC and ERK participate in IKK activation by LPS and UTP, the phosphatidylinositide-phospholipase C-dependent activation of CaMK plays a major role in UTP potentiation of the LPS response.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Benzylamines / pharmacology
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Bridged-Ring Compounds / pharmacology
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Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Cell Line
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Enzyme Activation / drug effects
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Enzyme Inhibitors / pharmacology
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Flavonoids / pharmacology
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I-kappa B Kinase
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I-kappa B Proteins / metabolism
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Imidazoles / pharmacology
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Indoles / pharmacology
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Lipopolysaccharides / pharmacology*
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Macrophages / cytology
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Macrophages / drug effects*
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Macrophages / enzymology
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Macrophages, Peritoneal / cytology
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Macrophages, Peritoneal / drug effects
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Macrophages, Peritoneal / metabolism
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Mitogen-Activated Protein Kinases / drug effects
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Mitogen-Activated Protein Kinases / metabolism*
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NF-kappa B / drug effects
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NF-kappa B / metabolism
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Norbornanes
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Phosphorylation / drug effects
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Protein Kinase C / antagonists & inhibitors
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Protein Kinase C / metabolism*
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Protein Serine-Threonine Kinases / drug effects*
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Protein Serine-Threonine Kinases / metabolism
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Pyridines / pharmacology
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Receptors, Purinergic P2 / physiology
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Sulfonamides / pharmacology
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Thapsigargin / pharmacology
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Thiocarbamates
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Thiones / pharmacology
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Time Factors
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Transcription Factor RelA
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Uridine Triphosphate / pharmacology
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p38 Mitogen-Activated Protein Kinases
Substances
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Benzylamines
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Bridged-Ring Compounds
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Enzyme Inhibitors
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Flavonoids
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I-kappa B Proteins
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Imidazoles
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Indoles
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Lipopolysaccharides
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NF-kappa B
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Norbornanes
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Pyridines
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Receptors, Purinergic P2
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Sulfonamides
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Thiocarbamates
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Thiones
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Transcription Factor RelA
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KN 93
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tricyclodecane-9-yl-xanthogenate
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Thapsigargin
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Protein Serine-Threonine Kinases
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I-kappa B Kinase
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Protein Kinase C
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Calcium-Calmodulin-Dependent Protein Kinases
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Mitogen-Activated Protein Kinases
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p38 Mitogen-Activated Protein Kinases
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SB 203580
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2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
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Uridine Triphosphate
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Ro 31-8220