Regulation of Type 2 NF-kappaB Activation and Inflammation

描述(由申请人提供)：这项建议的长期目标是深入了解肿瘤坏死因子受体相关因子2和3(TRAF2和TRAF3)和核因子-kB诱导激酶(NIK)在调节核因子-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。核因子-kB转录因子包括p50、p52、p65、c-Rel和Rel-B，是炎症、增殖和细胞凋亡的重要调节因子。虽然核因子-kB活性缺陷会导致细胞凋亡和免疫缺陷，但核因子-kB的过度激活与癌症和各种炎症综合征等多种疾病有关。目前，核因子-KB信号通路分为第一类(经典或典型)途径和第二类(替代或非典型)途径，前者激活I：B1的降解和含有p50的活性核因子-B复合体的释放，后者涉及p100到p52的诱导加工和含有p52的核因子-KB复合体的形成。我们的遗传学研究表明，TRAF3的缺失导致了结构性的2型核因子-KB活性。在TRAF3缺陷小鼠中观察到的出生后早期死亡是通过2型NF-KBP100基因的复合缺失而挽救的。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激活的有效抑制因子。在TRAF3-/-细胞中，缺乏对这两个核因子-KB通路的抑制与B细胞在体外的成分非刺激性存活以及在体外和体内对炎性细胞因子的深刻超诱导有关。最近的研究表明，TRAF家族的另一个成员TRAF2也是2型NF-KB途径的负调控因子。然而，TRAF2和TRAF3介导的2型NF-KB激活负调控的分子机制仍未阐明。我们最近的研究表明，在未经刺激的细胞中，NIK在很低的基础水平上表达，但在CD40、B细胞激活因子受体(BAFF-R)和淋巴毒素2受体(LT2R)等受体激活后，高度诱导以缓慢的动力学(8-12小时达到最大值)。有趣的是，TRAF2-/-和TRAF3-/-细胞都有很高的基础水平的NIK(相当于受体激活的野生型细胞)，这与结构性激活的2型核因子-KB相关。我们假设TRAF2和TRAF3通过招募一个Nik泛素复合体来持续降解Nik并保持低水平的NF-kB激活，从而负向调节NF-kB。我们还假设，受体激活诱导TRAF2和TRAF3从Nik泛素复合体中解离，从而允许Nik积累和随后的2型NF-kB激活。在这项赠款提案中，我们将确定：1)TRAF2和TRAF3如何招募NIK泛素化复合体来控制基础水平的NIK泛素化和降解；2)受体激活如何导致这种泛素复合体的解离以允许NIK积累；3)Nik激酶活性如何被调节以诱导2型NF-kB激活；以及4)2型NF-kB如何与1型NF-kB串扰并参与炎症反应。 项目简介：许多疾病，如癌症和许多炎症性疾病，都与核因子-kB转录因子的过度激活有关。这项拟议的研究将聚焦于核因子-kB的一个关键的负调控因子，通过研究它如何作为门卫来防止核因子-kB的过度激活，以及失去这样的门卫将如何在动物模型中导致炎症反应和致死性表型。阐明抑制核因子-kB活性的分子机制无疑将有助于设计治疗与核因子-kB过度激活相关的人类疾病的新策略。

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.