NLRP12 is a Critical Negative Regulator of Alternative NF-kB Signaling in Bone

描述(由申请人提供)：本奖学金申请的总体目标是证明NLRP12，一种形成节点样受体(NLR)的非炎症性小体，在骨骼中发挥一种新型的体内平衡调节功能。越来越多的证据支持这样的观点，即骨骼和免疫系统共享共同的信号分子，以实现细胞动态平衡。尽管已经做出了重大努力来促进我们对促进破骨细胞发育的信号通路的理解，但对这些调节通路施加负面控制的因素还没有得到很好的表征。最近的研究表明，NLRP12以一种细胞类型和刺激特异性的方式，通过选择性下调替代的核因子-kB信号，发挥天然免疫细胞激活的胞浆负调节作用。鉴于替代的核因子-kB在驱动破骨细胞分化和功能方面的指导作用，我们假设NLRP12通过与这一途径交叉并对其激活施加负面控制而在骨中起到保护作用。我们提出这一假说是基于在先天性免疫细胞中进行的研究，该研究表明NLRP12与核因子-kappa-B诱导激酶(NIK)的降解有关并促进其降解，NIK是促进转录因子RelB核转位所必需的中央上游激酶。此前，我们在炎症模型和体内骨转移模型中证明，NIK或RelB全球缺陷的小鼠在体外无法生成破骨细胞，并受到保护，不受病理性骨丢失的影响。我们的初步实验表明，破骨细胞前体细胞中NLRP12功能的破坏加速了破骨细胞的形成，并显著增加了RelB的核转位，这表明NLRP12在骨中具有负面调节作用。这项应用的一个主要目标是揭示NLRP12与骨重建相关的功能，长期目标是阐明抑制破骨细胞生成的分子基础。具体地说，我们的目标是验证中心假设，即NLRP12通过靶向另一种核因子-kB途径的组件发挥破骨细胞发育的负面调节作用。目的：1.确定NLRP12抑制破骨细胞系细胞中选择性核因子-kB信号转导的分子机制(S)及其对破骨细胞功能的潜在影响。我们将绘制NLRP12结构域(S)，该结构域参与介导抑制替代的NFκB激活和破骨细胞的形成。我们将通过检测NLRP12、TRAF3、NIK之间的直接蛋白质-蛋白质相互作用来确定NLRP12是否是破骨细胞前体中负调控的E3泛素连接酶复合体的组成部分。此外，我们还将研究NLRP12是否通过促进其泛素化来控制NIK的命运。目的2.研究NLRP12在基础和病理条件下对体内骨转换的调控作用。我们将使用RANKL诱导的骨溶解模型来确定NLRP12是否具有防止病理性骨丢失的作用。

DESCRIPTION (provided by applicant): The overall goal of this fellowship application is to demonstrate that NLRP12, a non-inflammasome forming NOD-like receptor (NLR), functions a novel homeostatic regulator in bone. Accumulating evidence supports the notion that common signaling molecules are shared by the skeletal and immune systems to achieve cellular homeostasis. Although significant efforts have been made to advance our understanding of signaling pathways that promote osteoclast development, factors that exert negative control over these regulatory pathways are not well characterized. Recent studies have shown that NLRP12, in a cell type and stimuli-specific manner, functions as a cytosolic negative regulator of innate immune cell activation through select down-regulation of alternative NF-kB signaling. Given the instructive role of alternative NF-kB in driving osteoclast differentiation and function, we hypothesize that NLRP12 has a protective role in bone by intersecting this pathway and exerting negative control over its activation. We formulated this hypothesis based on studies performed in innate immune cells demonstrating that NLRP12 associates with and promotes the degradation of NF-kappa-B-inducing kinase (NIK), the central upstream kinase essential for promoting nuclear translocation of transcription factor RelB. Previously, we demonstrated that mice globally deficient in NIK or RelB fail to make osteoclasts in vitro and are protected from pathological bone loss in models of inflammation and bone metastasis in vivo. Our preliminary experiments demonstrate that disruption of NLRP12 function in osteoclast progenitors accelerates osteoclast formation and significantly augments the nuclear translocation of RelB, suggesting that NLRP12 has a negative regulatory role in bone. A major goal of this application is to uncover the function of NLRP12 as it relates to bone remodeling with the long-term goal of elucidating the molecular basis for suppression of osteoclastogenesis. Specifically, we aim to test the central hypothesis that NLRP12 functions as a negative regulator of osteoclast development by targeting components of the alternative NF-kB pathway. Aim 1. Determine the molecular mechanism (s) by which NLRP12 suppresses alternative NF-kB signaling in cells of the osteoclast lineage and its potential impact on osteoclast function. We will map the NLRP12 domain(s) involved in mediating suppression of alternative NFκB activation and osteoclast formation. We will determine whether NLRP12 is a constituent of the negative regulatory E3 ubiquitin ligase complex in osteoclast precursors by examining direct protein-protein interactions between NLRP12, TRAF3, NIK. Additionally, we will examine whether NLRP12 controls the fate of NIK by promoting its ubiquitylation. Aim 2. Investigate the role of NLRP12 in controlling bone turnover in vivo under basal and pathological conditions. We will use a RANKL-induced osteolytic model to determine whether NLRP12 has a role in preventing pathological bone loss.