TNFα activates c-Jun amino terminal kinase through p47phox

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68 Scopus citations

Abstract

Reactive oxygen intermediates have been implicated in the transduction of TNFα signals, although the source of such oxidants has not been established. We found that activation of ECV-304 cells by TNFα was accompanied by a transient burst of oxidants and activation of JNK, both of which were suppressed by two distinct inhibitors of the phagocyte NADPH oxidase and the thiol antioxidant N-acetyl cysteine (NAC). We cloned partial and full-length cDNA sequences from ECV-304 cells and human umbilical vein endothelial cells (HUVEC), respectively, for p47phox, demonstrating that these nonphagocytic cells express this adapter protein known to specifically initiate assembly of the NADPH oxidase in professional phagocytes. A mutant p47phox, defective in the first Src homology 3 (SH3) domain (p47W(193)R), diminished JNK activation by TNFα. Surprisingly, p47phox resided entirely in the particulate, not cytosolic, fraction of cells. Immunostaining suggested partial colocalization with cytoskeletal elements, and cytoskeletal disrupters decreased both oxidant production and JNK activation by TNFα. A p47-GFP fusion protein localized to the cortical cytoskeleton in living cells; further, stimulation of cells with TNFα caused a marked concentration of p47-GFP in membrane ruffles, actin-rich structures associated with intense respiratory burst activity in stimulated neutrophils. We conclude that nonphagocytic cells express p47phox, which appears to localize to the cytoskeleton and participate in TNFα signaling. We speculate that this physical targeting may prove important in conferring signal specificity and enhancing signaling efficiency of unstable oxidants.

Original languageEnglish (US)
Pages (from-to)62-74
Number of pages13
JournalExperimental Cell Research
Volume272
Issue number1
DOIs
StatePublished - Jan 1 2002

Keywords

  • Cytokines
  • Free radicals
  • Inflammation
  • Protein kinases

ASJC Scopus subject areas

  • Cell Biology

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