Inducible Tumor Necrosis Factor (TNF) Receptor-associated Factor-1 Expression Couples the Canonical to the Non-canonical NF-κB Pathway in TNF Stimulation

The NF-κB transcription factor mediates the inflammatory response through distinct (canonical and non-canonical) signaling pathways. The mechanisms controlling utilization of either of these pathways are largely unknown. Here we observe that TNF stimulation induces delayed NF-κB2/p100 processing and investigate the coupling mechanism. TNF stimulation induces TNF-associated factor-1 (TRAF-1) that directly binds NF-κB-inducing kinase (NIK) and stabilizes it from degradation by disrupting its interaction with TRAF2·cIAP2 ubiquitin ligase complex. We show that TRAF1 depletion prevents TNF-induced NIK stabilization and reduces p52 production. To further examine the interactions of TRAF1 and NIK with NF-κB2/p100 processing, we mathematically modeled TRAF1·NIK as a coupling signaling complex and validated computational inference by siRNA knockdown to show non-canonical pathway activation is dependent not only on TRAF1 induction but also NIK stabilization by forming TRAF1·NIK complex. Thus, these integrated computational-experimental studies of TNF-induced TRAF1 expression identified TRAF1·NIK as a central complex linking canonical and non-canonical pathways by disrupting the TRAF2-cIAP2 ubiquitin ligase complex. This feed-forward kinase pathway is essential for the activation of non-canonical pathway.

RelB/NF-κB2 Regulates Corticotropin-Releasing Hormone in the Human Placenta

Placental CRH may be part of a clock that governs the length of human gestation. The mechanism underlying differential regulation of CRH in the human placenta is poorly understood. We report here that constitutively activated RelB/nuclear factor-κB2 (NF-κB)-2 (p100/p52) acts as an endogenous stimulatory signal to regulate CRH by binding to an NF-κB enhancer of CRH gene promoter in the human placenta. Nuclear staining of NF-κB2 and RelB in villous syncytiotrophoblasts and cytotrophoblasts was coupled with cytoplasmic CRH in syncytial knots of cytotrophoblasts. Chromatin immunoprecipitation identified that CRH gene associated with both RelB and NF-κB2 (p52). Dexamethasone increased synthesis and nuclear translocation of RelB and NF-κB2 (p52) and their association with the CRH gene. In contrast, progesterone, a down-regulator of placental CRH, repressed NF-κB2 (p100) processing, nuclear translocation of RelB and NF-κB2 (p52), and their association with the CRH gene. Luciferase reporter assay determined that the NF-κB enhancer of CRH was sufficient to regulate transcriptional activity of a heterologous promoter in primary cytotrophoblasts. RNA interference-mediated repression of RelB or NF-κB2 resulted in significant inhibition of CRH at both transcriptional and translational levels and prevented the dexamethasone-mediated up-regulation of CRH transcription and translation. These results suggest that the noncanonical NF-κB pathway regulates CRH production in the human placenta and is responsible for the positive regulation of CRH by glucocorticoids.

TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells.

Abstract 3665 Poster Board III-601 Introduction Hodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear. Methods Electromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector. Results In this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells. Conclusions Not only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells. Disclosures: No relevant conflicts of interest to declare.

TRAF2 Ring Domain Plays An Essential Role in Inhibition of TNFα-Induced Cell Death Independent of NF-kB Activation.

Abstract 3621 Poster Board III-557 The proinflammatory cytokine TNFα regulates immune responses, inflammation and programmed cell death by activating the JNK/c-Jun and IKK/NF-κB signaling pathways. TRAF2 and RIP1 play critical roles in TNFα-induced activation of JNK and IKK, and inhibition of apoptosis. In the field, TRAF2 RING domain-mediated RIP1 ubiquitination is believed to play an essential role in TNFα-induced IKK activation, and the RING domain-deleted TRAF2 (TRAF2-ΔN) has been widely used as a dominant negative to block TNFα-induced JNK and IKK activation in transient overexpression systems. Here, we report that stable expression of TRAF2-Δ[N in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells at a physiological level almost completely restores TNFα-induced IKK activation, but does not restore RIP1 ubiquitination. In addition, stable expression of TRAF2-ΔN in T2/5 DKO cells efficiently inhibited TNFα-induced later-phase prolonged JNK activation, but failed to inhibit TNFα-induced cell death. The basal and inducible expression of antiapoptotic proteins in TRAF2-ΔN-expressing cells is totally normal, yet these cells are still sensitive to TNFα-induced cell death due to the inefficient recruitment of cIAP1 to the TNFR1 complex. Moreover, stable expression of TRAF2-WT, but not TRAF2-ΔN, in T2/5 DKO cells suppressed constitutive p100 processing. On the other hand, TRAF2-ΔN-expressing cells exhibited significant resistance to oxidative stress-induced cell death compared to TRAF2-WT-expressing cells. These data suggest that: i) the TRAF2 RING domain plays a critical role in the inhibition of cell death induced by TNFα, and is essential for suppression of p100 processing in unstimulated cells; ii) RIP1 polyubiquitination is not required for TNFα-induced IKK activation, but may play a role in inhibition of TNFα-induced cell death; and iii) prolonged JNK activation has no obligate role in TNFα-induced cell death. Disclosures: No relevant conflicts of interest to declare.