The long-term objective of this proposal is to gain insight into the role and the mechanisms of action of the tumor necrosis factor (TNF) receptor-associated factors 2 and 3 (TRAF2 and TRAF3) and NF-KB inducing kinase (NIK) in the regulation of NF-KB activation and in suppression of inflammatory and autoimmune diseases. The NF-kB transcription factors, including p50, p52, p65, c-Rel and Rel-B, are critical regulators of inflammation, proliferation and apoptosis. While defective NF-KB activity can lead to cell apoptosis and immunodeficiency, overactivated NF-KB has been associated with numerous diseases such as cancers and a variety of inflammatory syndromes. Recently, the NF-KB signaling pathways have been categorized into the type 1 (classical or canonical) pathway, which activates the degradation of I:B1 and the release of active NF- :B complexes containing p50, and the type 2 (alternative or noncanonical) pathway, which involves the induced processing of p100 to p52 and the formation of NF-KB complexes containing p52. Our genetic studies have demonstrated that loss of TRAF3 results in constitutive type 2 NF-KB activity. The early post-natal lethality observed in TRAF3-deficient mice is rescued by compound loss of the type 2 NF-KB p100 gene. In addition, our preliminary studies indicate that TRAF3 is also a potent inhibitor of type 1 NF-KB activation in response to TNF1 and IL-12 stimulation. Lack of inhibition of both NF-KB pathways in TRAF3-/- cells correlated with constitutive stimulus-independent survival of B cells ex vivo and profound super-induction of inflammatory cytokines in vitro and in vivo. Recent studies indicated that another TRAF family member, TRAF2, is also a negative regulator of the type 2 NF-KB pathway. However, the molecular mechanisms responsible for TRAF2- and TRAF3-mediated negative regulation of type 2 NF-KB activation remain to be elucidated. Our recent studies showed that NIK is expressed at very low basal levels in unstimulated cells but is highly induced with a slow kinetics (8-12 hours to reach its maximum) after activation of receptors such as CD40, B-cell activating factor (BAFF) receptor (BAFF-R) and lymphotoxin 2 receptor (LT2R). Interestingly, bothTRAF2-/- and TRAF3-/- cells have high basal levels of NIK (equivalent to receptor activated wild type cells), which correlate with constitutively activated type 2 NF-KB. We hypothesize that TRAF2 and TRAF3 negatively regulate NF-KB by recruiting a NIK ubiquination complex to constantly degrade NIK and to keep basal levels of NF-KB activation low. We also hypothesize that receptor activation induces dissociation of TRAF2 and TRAF3 from the NIK ubiquination complex allowing NIK accumulation and subsequent type 2 NF-kB activation. In this grant proposal, we will determine: 1) how TRAF2 and TRAF3 recruit a NIK ubiquination complex to control ubiquitination and degradation of NIK at basal levels;2) how receptor activation leads to dissociation of such a ubiquination complex to allow NIK accumulation;3) how NIK kinase activity is regulated in order to induce type 2 NF-kB activation;and 4) how type 2 NF-kB can crosstalk with type 1 NF-kB and involve in inflammation. Project Narrative: Many diseases such as cancers and numerous inflammatory diseases are associated with overactivation of NF-kB transcription factors. The proposed studies will focus a critical negative regulator of NF-KB by examining how it functions as a gate keeper to prevent overactivation of NF-kB and how loss of such a gate keeper would lead to inflammatory responses and lethal phenotypes in animal models. Elucidating the molecular mechanisms responsible for inhibiting NF-kB activity will undoubtedly help in designing novel strategies to treat human diseases associated overactivation of NF-kB.
|Feng, Feng; Yuan, Lingmin; Wang, Yao E et al. (2013) Crystal structure and nucleotide selectivity of human IFIT5/ISG58. Cell Res 23:1055-8|
|Zhang, Peng; Reichardt, Anna; Liang, Huanhuan et al. (2012) Single amino acid substitutions confer the antiviral activity of the TRAF3 adaptor protein onto TRAF5. Sci Signal 5:ra81|
|Razani, Bahram; Zarnegar, Brian; Ytterberg, A Jimmy et al. (2010) Negative feedback in noncanonical NF-kappaB signaling modulates NIK stability through IKKalpha-mediated phosphorylation. Sci Signal 3:ra41|
|Zarnegar, Brian J; Wang, Yaya; Mahoney, Douglas J et al. (2008) Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK. Nat Immunol 9:1371-8|