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.
The exclusive ability of osteoclasts to resorb bone makes them critical for both normal and pathological bone loss. Noting their inherent resorptive properties, osteoclasts, when dysregulated, are often implicated in many systemic and local skeletal diseases, including osteoporosis, rheumatoid arthritis, periodontal disease and cancer metastasis to bone. Understanding the molecular mechanisms that control their activity is crucial for the prevention and treatment of unwarranted bone erosion.