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NLRP12，一种非炎性体形成的nod样受体（NLR），在骨中起着一种新的稳态调节剂的作用。越来越多的证据支持这样一种观点，即骨骼和免疫系统共享共同的信号分子来实现细胞内稳态。尽管我们已经做出了巨大的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并没有很好地表征。最近的研究表明，NLRP12以细胞类型和刺激特异性的方式，通过选择性下调NF-kB信号传导，作为先天免疫细胞激活的胞质负调节因子。鉴于替代NF-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通过交叉这一途径并对其激活施加负性控制，在骨中具有保护作用。我们在先天免疫细胞中进行的研究表明，NLRP12与nf -kappa- b诱导激酶（NIK）相关并促进其降解，NIK是促进转录因子RelB核易位所必需的中心上游激酶。在此之前，我们证明了NIK或RelB缺失的小鼠在体外不能产生破骨细胞，并且在体内炎症和骨转移模型中可以防止病理性骨丢失。我们的初步实验表明，破坏NLRP12在破骨细胞祖细胞中的功能会加速破骨细胞的形成，并显著增加RelB的核易位，这表明NLRP12在骨中具有负调控作用。本应用程序的主要目标是揭示NLRP12与骨重塑相关的功能，并阐明抑制破骨细胞发生的分子基础。具体来说，我们的目标是验证NLRP12作为破骨细胞发育的负调节因子的中心假设，通过靶向NF-kB替代途径的成分。目的1。确定NLRP12抑制破骨细胞谱系中NF-kB信号传导的分子机制及其对破骨细胞功能的潜在影响。我们将绘制NLRP12结构域(s)参与介导抑制替代性NFκB活化和破骨细胞形成。我们将通过检测NLRP12、TRAF3、NIK之间的直接蛋白-蛋白相互作用来确定NLRP12是否是破骨细胞前体中负调控E3泛素连接酶复合物的组成部分。此外，我们将研究NLRP12是否通过促进其泛素化来控制NIK的命运。目标2。探讨NLRP12在基础和病理条件下控制骨转换的作用。我们将使用rankl诱导的溶骨模型来确定NLRP12是否在预防病理性骨质流失中起作用。

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.