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Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB
1Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.
Abstract:
Activated tumor necrosis factor alpha (TNF-alpha) 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-kappaB 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-alpha treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-alpha-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-alpha-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-alpha-mediated I-kappaB degradation and NF-kappaB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kappaB activation by TNF-alpha. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kappaB activation.
Insights
Signal transducer and activator of transcription 1 (Stat1) regulates tumor necrosis factor alpha receptor 1 (TNFR1) signaling. Stat1 suppresses NF-kappaB activation, impacting apoptosis and anti-apoptosis gene expression.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Immunology
Background:
- Tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) signaling initiates opposing pathways: apoptosis induction via caspase activation and anti-apoptosis via NF-kappaB activation.
- The precise molecular mechanisms regulating the balance between these pathways are not fully understood.
Purpose of the Study:
- To investigate the role of Signal transducer and activator of transcription 1 (Stat1) in TNFR1 signaling complex formation and its impact on downstream pathways.
- To elucidate the interaction of Stat1 with key TNFR1 signaling components.
Main Methods:
- Novel antibody array screening to identify protein interactions within the TNFR1-TRADD signaling complex.
- In vitro recombinant protein-protein interaction assays.
- Analysis of Stat1-deficient and Stat1-overexpressing cells to assess functional consequences.
Main Results:
- Stat1 was identified as a component of the TNFR1-TRADD signaling complex, with its association increasing upon TNF-alpha treatment.
- Stat1 directly interacts with TNFR1 and TNFR1-associated death domain protein (TRADD) but not with Fas-associated death domain protein (FADD).
- Stat1 deficiency enhanced TNF-alpha-induced TRADD-RIP and TRADD-TRAF2 complex formation, leading to increased NF-kappaB activation, while Stat1 overexpression inhibited this activation.
Conclusions:
- Stat1 acts as a negative regulator in the TNFR1 signaling pathway, specifically suppressing NF-kappaB activation.
- Stat1's interaction with TNFR1 and TRADD modulates the balance between pro-apoptotic and anti-apoptotic signaling outcomes.
- These findings reveal a novel function of Stat1 in controlling TNF-alpha-mediated inflammatory responses.