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TNFalpha activates c-Jun amino terminal kinase through p47(phox)
Ying Gu1, You Cheng Xu, Ru Feng Wu
1University of Texas Southwestern and Dallas VA Medical Center, Dallas, Texas 75216, USA.
Experimental Cell Research
|December 13, 2001
Summary
Nonphagocytic cells utilize p47(phox) protein, typically found in immune cells, to generate reactive oxygen species and activate JNK signaling in response to TNFalpha. This protein localizes to the cytoskeleton, influencing signal specificity.
Area of Science:
- Cellular Biology
- Immunology
- Biochemistry
Background:
- Reactive oxygen intermediates (ROIs) are implicated in tumor necrosis factor-alpha (TNFalpha) signal transduction.
- The specific cellular source of these oxidants in nonphagocytic cells remains unidentified.
Purpose of the Study:
- To investigate the source of oxidants involved in TNFalpha signaling in nonphagocytic cells.
- To determine the role of p47(phox) and its localization in TNFalpha-induced signaling pathways.
Main Methods:
- Activation of ECV-304 cells with TNFalpha and assessment of oxidant burst and JNK activation.
- Inhibition studies using NADPH oxidase inhibitors and N-acetyl cysteine (NAC).
- Cloning and expression of p47(phox) cDNA, generation of a mutant p47(phox), and localization studies using immunostaining and GFP fusion proteins.
- Treatment with cytoskeletal disrupters.
Main Results:
- TNFalpha stimulation induced a transient oxidant burst and JNK activation, suppressed by NADPH oxidase inhibitors and NAC.
- ECV-304 cells and HUVECs express p47(phox), an adapter protein crucial for NADPH oxidase assembly.
- A mutant p47(phox) impaired JNK activation; the protein localized to the particulate fraction and cytoskeleton, concentrating in membrane ruffles upon TNFalpha stimulation.
- Cytoskeletal disruption reduced oxidant production and JNK activation.
Conclusions:
- Nonphagocytic cells express p47(phox), which localizes to the cytoskeleton and participates in TNFalpha signaling.
- Cytoskeletal localization of p47(phox) may enhance signaling specificity and efficiency of reactive oxygen intermediates.