TNFalpha activates c-Jun amino terminal kinase through p47(phox).
Reactive oxygen intermediates have been implicated in the transduction of TNFalpha signals, although the source of such oxidants has not been established. We found that activation of ECV-304 cells by TNFalpha 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 p47(phox), demonstrating that these nonphagocytic cells express this adapter protein known to specifically initiate assembly of the NADPH oxidase in professional phagocytes. A mutant p47(phox), defective in the first Src homology 3 (SH3) domain (p47W(193)R), diminished JNK activation by TNFalpha. Surprisingly, p47(phox) 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 TNFalpha. A p47-GFP fusion protein localized to the cortical cytoskeleton in living cells; further, stimulation of cells with TNFalpha 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 p47(phox), which appears to localize to the cytoskeleton and participate in TNFalpha signaling. We speculate that this physical targeting may prove important in conferring signal specificity and enhancing signaling efficiency of unstable oxidants.
Gu
Ying
Y
University of Texas Southwestern and Dallas VA Medical Center, Dallas, Texas 75216, USA.
Xu
You Cheng
YC
Wu
Ru Feng
RF
Souza
Rhonda F
RF
Nwariaku
Fiemu E
FE
Terada
Lance S
LS
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002460:Cell Line; D004730:Endothelium, Vascular / Q000502:physiology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D019255:NADPH Oxidases / Q000502:physiology; D016877:Oxidants / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology; D010750:Phosphoproteins / Q000502:physiology*; D017382:Reactive Oxygen Species; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000970:Antineoplastic Agents; D016877:Oxidants; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases