Abstract 597: Negative regulation of TNFα-induced NFκB activation by XAF1 via inhibition of the TRADD/TRAF2/RIP1 signal complex

Abstract

Abstract Introduction: X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) was originally implicated as a tumor suppressor due to its inhibitory effect on anti-apoptotic XIAP. It contains an N-terminus tumor necrosis factor (TNF) receptor associated factor (TRAF) Zn finger domain that is highly conserved in TRAF family members. XAF1 expression can be induced by TNFα; however, its role in the signal cascade initiated by TNFα/TNF receptor (TNFR) 1 has not yet been clearly elucidated. Here we report that XAF1 can negatively regulate TNFα-induced nuclear factor kappa B (NFκB) activation via inhibition of signaling molecule TRAF2 and signal complex formation downstream of TNFα/TNFR1. Methods: To investigate the effects of XAF1 on TNFα signal transduction machinery, we assessed TNF-induced expression of the NFκB response gene IL-8 by real-time PCR, and phosphorylation of inhibitor of NFκB (IκB) by immunoblotting with an anti-phospho-IκB antibody, in HEK293 cells with an inducible XAF1 expression vector. Interaction between TRAF2 or RIP1 and XAF1 was investigated by immunoprecipitation. NFκB transcriptional activation potential, induced by ectopic TRAF2 or RIP1 with or without co-expressed XAF1, was quantified by luciferase assays. Inhibition of TRAF2 ubiquitination by XAF1 was also assessed by immunoblot. Transient transfection of plasmids expressing TRADD, TRAF2, RIP1 and/or XAF1 followed by immunoprecipition was performed to further investigate the role of XAF1 on the signal complex formation. Results: Luciferase assays and real-time PCR studies showed that XAF1 inhibited TNFα-induced NFκB activation and IL-8 expression. Both TRAF2 and RIP1 interacted with XAF1 according to our immunoprecipitation results but only TRAF2 activity was inhibited by XAF1. Poly-ubiquitination of TRAF2 was attenuated when XAF1 was present. In addition, immunoprecipitation and immunoblot data showed that XAF1 interfered with RIP1/TRADD association and regulated TRADD and RIP1 turnover. Discussion: Our data demonstrated an inhibitory effect of XAF1 on TNFα-induced NFκB activation and gene expression. A possible mechanism may be via inhibition of TRAF2, an E3 ubiquitin ligase responsible for signal transduction leading to NFκB activation. XAF1 inhibited both TRAF2-mediated NFκB activation and TRAF2 ubiquitination, which is essential for its signal delivery function. XAF1 also interacted with RIP1 yet did not block RIP1-induced NFκB promoter activity. However, the binding of RIP1 to TRADD, an adaptor protein of TNFR1-mediated signal pathway, was disrupted by XAF1. Furthermore, degradation of TRADD and RIP1 was accelerated when XAF1 was present. These data suggested that XAF1 may act as a TNF-induced negative regulator of NFκB activation by inhibiting TRAF2 ubiquitination and by regulating the integrity of the signal complex of TRADD/TRAF2/RIP1. Citation Format: Boren Lin, Da Xu, Douglas W. Leaman. Negative regulation of TNFα-induced NFκB activation by XAF1 via inhibition of the TRADD/TRAF2/RIP1 signal complex. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2014-597