Regulation of Type 2 NF-kappaB Activation and Inflammation

描述（由申请人提供）：本申请的长期目标是深入了解肿瘤坏死因子（TNF）受体相关因子2和3 （TRAF2和TRAF3）和NF-KB诱导激酶（NIK）在调节NF-KB激活和抑制炎症和自身免疫性疾病中的作用和作用机制。NF-kB转录因子，包括p50、p52、p65、c-Rel和Rel-B，是炎症、增殖和凋亡的关键调节因子。虽然NF-KB活性缺陷可导致细胞凋亡和免疫缺陷，但过度激活的NF-KB与许多疾病（如癌症和各种炎症综合征）有关。最近，NF- kb信号通路被分为1型（经典或典型）途径，其激活I:B1的降解并释放含有p50的活性NF-:B复合物，以及2型（替代或非典型）途径，其涉及诱导p100到p52的加工并形成含有p52的NF- kb复合物。我们的遗传学研究表明，TRAF3的缺失会导致构成型2 NF-KB活性的降低。traf3缺陷小鼠的早期产后死亡通过2型NF-KB p100基因的复合缺失得以挽救。此外，我们的初步研究表明，TRAF3也是响应TNF1和IL-12刺激的1型NF-KB激活的有效抑制剂。TRAF3-/-细胞中NF-KB通路缺乏抑制与B细胞体外不依赖刺激存活和体外和体内炎症细胞因子的深度超诱导相关。最近的研究表明，另一个TRAF家族成员TRAF2也是2型NF-KB通路的负调控因子。然而，TRAF2-和traf3介导的2型NF-KB激活负调控的分子机制仍有待阐明。我们最近的研究表明，NIK在未受刺激的细胞中以非常低的基础水平表达，但在CD40、b细胞活化因子（BAFF）受体（BAFF- r）和淋巴毒素2受体（LT2R）等受体激活后，以缓慢的动力学（8-12小时达到最大值）被高度诱导。有趣的是，traf2 -/-和TRAF3-/-细胞都具有高基础水平的NIK（相当于受体激活的野生型细胞），这与组成型激活的2型NF-KB相关。我们假设TRAF2和TRAF3通过招募NIK泛素复合物不断降解NIK并保持低基础水平的NF-KB激活来负性调节NF-KB。我们还假设受体激活诱导TRAF2和TRAF3从NIK泛素化复合体中分离，从而导致NIK积累和随后的2型NF-kB激活。在这项拨款申请中，我们将确定：1)TRAF2和TRAF3如何招募NIK泛素化复合物来控制基础水平的NIK泛素化和降解；2)受体激活如何导致这种泛素化复合物的解离，从而允许NIK积累；3)如何调节NIK激酶活性以诱导2型NF-kB活化；4) 2型NF-kB如何与1型NF-kB串扰并参与炎症。

DESCRIPTION (provided by applicant): 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.