Stat1 as a Component of Tumor Necrosis Factor Alpha Receptor 1-TRADD Signaling Complex To Inhibit NF-κB Activation

Abstract

Activated tumor necrosis factor alpha (TNF-α) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-κB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-α treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-α-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-α-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-α-mediated I-κB degradation and NF-κB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-κB activation by TNF-α. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-κB activation.

ACKNOWLEDGMENTS

We thank D. V. Goeddel for the TNFR1, TNFR2, and TRADD expression vectors, V. Dixit for the FADD expression vector, X.-Y. Fu for the Stat1 expression vector, S. Ghosh for the NF-κB site luciferase reporter construct (pBIIx Luc), G. Stark for the Stat1-deficient human cell line U3A and its parental cell line 2fTGH, and David Levy for the Stat1 knockout mouse fibroblasts. We also thank Alicia Chung and Michelle Embree for their critical reading of the manuscript.

Support was provided by Brown University School of Medicine (Y.E.C.).

Y.W. and T.R.W. contributed equally to this work.