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

描述（由申请人提供）：本研究金申请的总体目标是证明NLRP 12，一种非炎性小体形成NOD样受体（NLR），在骨中发挥新型稳态调节剂的作用。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内稳态。虽然已经作出了重大努力，以提高我们的理解信号通路，促进破骨细胞的发展，发挥负控制这些调控途径的因素没有得到很好的表征。最近的研究表明，NLRP 12，在一个细胞类型和刺激特异性的方式，作为一个先天性免疫细胞激活的胞质负调节通过选择性下调替代NF-κ B信号转导。考虑到替代性NF-κ B在驱动破骨细胞分化和功能中的指导作用， 我们假设NLRP 12通过与该途径交叉并对其激活施加负控制而在骨中具有保护作用。我们基于在先天免疫细胞中进行的研究制定了这一假设，这些研究表明NLRP 12与NF-κ B诱导激酶（NIK）相关并促进其降解，NIK是促进转录因子RelB核转位所必需的中心上游激酶。以前，我们证明了NIK或RelB全面缺陷的小鼠在体外不能制造破骨细胞，并且在体内炎症和骨转移模型中受到保护免于病理性骨丢失。我们的初步实验表明，破坏破骨细胞祖细胞中的NLRP 12功能会加速破骨细胞的形成，并显着增加RelB的核转位，这表明NLRP 12在骨中具有负调节作用。本申请的一个主要目标是揭示NLRP 12的功能，因为它与骨重建有关，长期目标是阐明抑制破骨细胞生成的分子基础。具体来说，我们的目标是测试的核心假设，即NLRP 12的功能作为破骨细胞的发展，通过靶向替代NF-κ B途径的组件的负调节。目标1。确定NLRP 12抑制破骨细胞谱系细胞中替代性NF-κ B信号传导的分子机制及其对破骨细胞功能的潜在影响。我们将映射NLRP 12域（S）参与介导抑制替代NF？B活化和破骨细胞形成。我们将通过检查NLRP 12、TRAF 3、NIK之间的直接蛋白质-蛋白质相互作用来确定NLRP 12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NLRP 12是否通过促进其泛素化来控制NIK的命运。目标二。研究NLRP 12在基础和病理条件下控制体内骨转换的作用。我们将使用RANKL诱导的溶骨性模型来确定NLRP 12是否在预防病理性骨丢失中发挥作用。

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 NF?B 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.