Abstract
Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of nuclear factor κ B (NF-κB)-driven proinflammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-κB activation and interleukin-8 (IL-8) production are not clear. Here, we examined participation of protein kinase C (PKC) in SP-induced IL-8 production in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells). SP (10-7 M) induced an early (1 min) phosphorylation of the PKC isoforms PKCδ, PKCθ, and PKCϵ, followed by I-κB kinase, IκBα, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 μM) blocked SP-induced IκBα and p65 phosphorylation and IL-8 production. The PKCδ inhibitor rottlerin at a low concentration (1 μM), but not pseudosubstrate PKCθ and PKCϵ inhibitors (10 μM), significantly reduced IL-8 secretion. PKCδ silencing by RNA interference reduced PKCδ protein expression and SP-induced PKCδ phosphorylation that was associated with diminished IL-8 promoter and NF-κB luciferase activities in response to SP. Moreover, overexpression of wild-type PKCδ increased SP-induced IL-8 promoter- and NF-κB-driven luciferase activities that were rottlerin-sensitive. We conclude that PKCδ plays an important role in SP-induced proinflammatory signaling in human colonocytes.
Footnotes
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This work was supported by National Institutes of Health Grant DK 47343 (to C.P.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.088013.
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ABBREVIATIONS: SP, substance P; NK-1R, neurokinin-1 receptor; NF, nuclear factor; IL-8, interleukin-8; TNFα, tumor necrosis factor α; MAPK, mitogen-activated protein kinase; EGFR, epidermal growth factor receptor; PKC, protein kinase C; DAG, diacylglycerol; CAPE, caffeic acid phenethyl ester; PMA, phorbol-12-myristate-13-acetate; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; IKK, IκB kinase complex; siRNA, small interfering RNA; Go6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; PD98059, 2′-amino-3′-methoxyflavone; DMSO, dimethyl sulfoxide.
- Received April 13, 2005.
- Accepted May 23, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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