The Fas-associated death domain protein suppresses activation of NF-κB by LPS and IL-1β
Douglas D. Bannerman, Joan C. Tupper, James D. Kelly, Robert K. Winn, John M. Harlan
Douglas D. Bannerman, Joan C. Tupper, James D. Kelly, Robert K. Winn, John M. Harlan
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The Fas-associated death domain protein suppresses activation of NF-κB by LPS and IL-1β

Abstract

Activation of NF-κB by bacterial LPS promotes the upregulation of proinflammatory cytokines that contribute to the pathogenesis of Gram-negative septic shock. LPS activation of NF-κB is dependent upon the interaction of two death domain–containing (DD-containing) proteins, MyD88 and IL-1 receptor–associated kinase IRAK. Another DD-containing protein, Fas-associated death domain (FADD), also binds MyD88 through respective DD-DD interactions. Although FADD has been classically described as a proapoptotic signaling molecule, several reports have implicated a role for FADD in mediating NF-κB activation. In the present report, we investigated whether FADD could mediate LPS activation of NF-κB. Overexpression of FADD blocked LPS-induced NF-κB activation, whereas absence of FADD enhanced activation of NF-κB by LPS. Further, LPS-induced expression of two NF-κB–dependent gene products, IL-6 and KC, was enhanced in FADD–/– mouse embryo fibroblasts (MEFs) compared with wild-type. This increase in NF-κB activity correlated with enhanced IκB degradation. FADD–/– MEFs were also resistant to NF-κB activation induced by IL-1β. Finally, reconstitution of full-length FADD in the FADD–/– MEFs completely reversed the enhanced activation of NF-κB elicited by either LPS or IL-1β. Together, these data indicate that FADD negatively regulates LPS- and IL-1β–induced NF-κB activation and that this regulation occurs upstream of IκB degradation.

Authors

Douglas D. Bannerman, Joan C. Tupper, James D. Kelly, Robert K. Winn, John M. Harlan

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Reconstitution of FADD reverses the enhanced NF-κB activation and NF-κB–...
Reconstitution of FADD reverses the enhanced NF-κB activation and NF-κB–dependent gene expression in FADD–/– MEFs exposed to LPS or IL-1β. Lysates derived from FADD–/– MEFs stably transfected with either GFP vector alone or FADD were immunoblotted with anti-human FADD antibody to confirm expression (a). Molecular mass (in kDa) is indicated. In other experiments, FADD–/– MEFs expressing either GFP or FADD were treated for 4.5 hours with medium, LPS (100 ng/ml), or mIL-1β (10 ng/ml), lysed, and assayed for luciferase activity (b). Alternatively, MEFs were treated for 12 hours and the culture supernatants analyzed for IL-6 (c) or KC (d). Vertical bars represent mean (± SE) luciferase activity in arbitrary units (b) or pg/ml (c and d). *Significantly decreased compared with GFP-expressing cells exposed to the same treatment.