Regulation of Type 2 NF-kappaB Activation and Inflammation

描述（由申请人提供）：本提案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（TRAF 2和TRAF 3）和NF-κ B诱导激酶（NIK）在调节NF-κ B活化和抑制炎症性和自身免疫性疾病中的作用和作用机制。NF-κ B转录因子包括p50、p52、p65、c-Rel和Rel-B，它们是炎症、增殖和凋亡的关键调节因子。虽然缺陷的NF-κ B活性可导致细胞凋亡和免疫缺陷，但过度活化的NF-κ B已与许多疾病如癌症和多种炎性综合征相关。近来，NF-κ B信号通路已被分类为1型（经典或规范）通路和2型（替代或非规范）通路，1型通路激活I：B1的降解和含有p50的活性NF-：B复合物的释放，2型通路涉及p100到p52的诱导加工和含有p52的NF-κ B复合物的形成。我们的遗传学研究已经证明TRAF 3的缺失导致组成型2型NF-κ B活性。在TRAF 3缺陷型小鼠中观察到的出生后早期致死性通过2型NF-κ B p100基因的复合缺失来挽救。此外，我们的初步研究表明，TRAF 3也是一种有效的抑制剂，1型NF-κ B激活响应TNF-1和IL-12的刺激。TRAF 3-/-细胞中两种NF-κ B途径抑制的缺乏与离体B细胞的组成性刺激非依赖性存活以及体外和体内炎性细胞因子的显著超诱导相关。最近的研究表明，另一个TRAF家族成员TRAF 2也是2型NF-κ B通路的负调节因子。然而，负责TRAF 2和TRAF 3介导的2型NF-κ B激活的负调控的分子机制仍有待阐明。我们最近的研究表明，NIK在未刺激的细胞中以非常低的基础水平表达，但在受体如CD 40、B细胞活化因子（BAFF）受体（BAFF-R）和光敏素2受体（LT 2 R）活化后以缓慢的动力学（8-12小时达到其最大值）被高度诱导。有趣的是，TRAF 2-/-和TRAF 3-/-细胞都具有高基础水平的NIK（相当于受体活化的野生型细胞），其与组成性活化的2型NF-κ B相关。我们假设TRAF 2和TRAF 3通过募集NIK泛素化复合物来不断降解NIK并保持NF-κ B活化的基础水平较低，从而负调控NF-κ B。我们还假设受体激活诱导TRAF 2和TRAF 3从NIK泛素化复合物中解离，从而允许NIK积累和随后的2型NF-kB激活。在这项资助计划中，我们将确定：1）TRAF 2和TRAF 3如何募集NIK泛素化复合物以控制NIK的基础水平的泛素化和降解; 2）受体活化如何导致这样的泛素化复合物解离以允许NIK积累; 3）NIK激酶活性如何被调节以诱导2型NF-κ B活化;以及4）2型NF-κ B如何与1型NF-κ B相互作用并参与炎症反应。 项目叙述：许多疾病，如癌症和许多炎症性疾病与NF-κ B转录因子的过度激活有关。拟议的研究将集中在NF-κ B的关键负调节因子，通过检查它如何作为守门人发挥作用，以防止NF-κ B的过度激活，以及失去这样的守门人将如何导致动物模型中的炎症反应和致命表型。阐明负责抑制NF-κ B活性的分子机制无疑将有助于设计新的策略来治疗与NF-κ B过度激活相关的人类疾病。

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.