Paget's disease is a metabolic bone disease that affects up to 3% of adults over 55 in which abnormal osteoclasts (OCL) both increase bone resorption and induce formation of poor quality bone leading to bone deformity, pain, pathological fractures, deafness, and nerve compression. Strong evidence supports a role for genetic mutations in the SQSTM1 gene (encoding p62) together with environmental agents such as measles virus nucleocapsid protein (MVNP) in the development of Paget's disease. This proposal is focused on understanding how MVNP alters OCL lineage cells and cooperates with p62P392L to generate pagetic lesions. Our group has recently found that MVNP complexes with and regulates the function of two IKK-family members TBK1 (a kinase) and optineurin (OPTN, an adaptor). TBK1 has a critical positive role in MVNP induction of aberrant pagetic-like OCL with increased OCL formation, nuclei/OCL and IL-6 production, while OPTN is a novel negative regulator of OCL formation and IL-6 production. MVNP interaction with the TBK1- OPTN complex results in increased TBK1 activity and decreased OPTN levels. TBK1 phosphorylates S177- OPTN, increasing OPTN activity as autophagy cargo receptor and its degradation. Intriguingly, we have found that MVNP increases autophagic flux. Further OPTN and p62, can both recruit CYLD, a deubiquitinase, to shut down TNFa and RANKL signaling, respectively, a function lost by pagetic p62 mutations. We hypothesize that MVNP interaction with the TBK1-OPTN complex is the key mechanism by which MVNP alters OCL biology and cooperates with mutant p62 to generate the pagetic OCL phenotype.
Aim 1 will determine the mechanisms by which MVNP regulates TBK1-OPTN activity. We will examine the nature of MVNP interactions with the TBK1-OPTN complex, the mechanism by which TBK1 is activated, if TBK1 phosphorylation of OPTN increases autophagy and TNFa/RANKL signaling, and if increased autophagy contributes to aspects of the pagetic OCL phenotype, in particular increased nuclei/OCL.
Aim 2 will test the hypothesis that increased expression of TBK1 alone or in combination with the p62P394L mutation are sufficient to induce pagetic OCL and bone lesions in mice. We will cross our recently made TRAP-TBK1 mice to our p62KI mice and will analyze both TRAP-TBK1 and TRAP-TBK1/p62KI mice for pagetic lesion formation. We will also conduct in vitro co-expression studies transducing TBK1 into MVNP-negative peripheral blood mononuclear cells (PBMC) from p62P392L-positive Paget's patients and normals to establish if increased TBK1 activity alone or plus p62P392L induces a pagetic phenotype.
Aim 3 will test the hypothesis that MVNP requires TBK1 in vivo to induce pagetic lesions. We will analyze formation of pagetic lesions in MVNP mice crossed with mice that have Tbk1 conditional knockout in OCL and evaluate if knockdown of TBK1 in MVNP+ PBMC from p62P392L-Paget's patients affects development of pagetic OCL. These studies will provide important insight into the pathogenesis of Paget's disease and may provide identification of novel targets for small molecule inhibitors for down-regulating OCL activity in Paget's disease and other pathological increases in OCL activity.

Public Health Relevance

Paget's disease is a metabolic bone disease that affects up to 3% of adults over 55 in which abnormal bone resorbing osteoclasts both increase bone resorption and induce formation of poor quality bone by osteoblasts in the areas of the lytic lesions leading to bone deformity, pain, pathological fractures, deafness, and nerve compression. The etiology of Paget's disease involves both environmental factors, such as measles virus (MV), and genetic predispositions, such as mutations in the sequestosome 1 gene encoding the p62 protein. This application proposes to increase our understanding of the mechanisms involved in the interaction of signals from the key MV protein involved in the disease etiology, MV nucleoprotein, and mutant p62 to generate aberrant osteoclasts and pagetic lesions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR057310-06A1
Application #
8962402
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Chen, Faye H
Project Start
2008-09-16
Project End
2020-06-30
Budget Start
2015-07-30
Budget End
2016-06-30
Support Year
6
Fiscal Year
2015
Total Cost
$429,219
Indirect Cost
$137,372
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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