Bone development is tightly regulated by bone forming cells, osteoblasts, and bone resorbing cells, osteoclasts. Therefore, understanding the mechanisms governing osteoclastogenesis is crucial for addressing bone loss pathologies. Differentiation of osteoclasts is governed by RANK ligand which activates several signal transduction pathways, including MAP kinases and NF-?B pathways. Proximal activation entails recruitment of TRAF6 and other key proteins including TGF-?-activated kinase-1 (TAK1) to the receptor RANK. TRAF6, TAK1 and other signaling partners undergo extensive post-translational modifications aimed at stabilizing RANK signaling and enabling precise regulation and execution of proper down stream signals, primarily NF-?B activation. Precise regulation of NF-?B activity is crucial to maintain normal osteoclast activity and bone homeostasis. Conversely, abnormal activity of this transcription factor causes deleterious inflammatory osteolysis. In fact, we discovered recently that constitutive activation of IKK2 is sufficient to induce RANKL-independent osteoclastogenesis in vitro. More convincingly, we reported that knock-in of constitutively active IKK2 causes severe bone loss in mice. Given that IKK2 phosphorylation and activation is governed by TAK1, a MAP kinase heavily implicated in poly-ubiquitination and stabilization of RANK-TRAF6 complexes and down-stream signaling, we decided to investigate its molecular role in osteoclastogenesis. Thus, we generated mice harboring myeloid-specific deletion of TAK1. These mice displayed all hallmarks of osteopetrosis primarily defective osteoclastogenesis. Mechanistically, we observed that Tak1-null precursors fail to generate osteoclasts. More importantly, we discovered diminished expression of key osteoclastogenic proteins including TRAF6, NEMO and Notch-NICD. This phenomenon was associated with accumulation of NUMBL, a previously described neuron protein. Consistent with these observations, we established that exogenous expression of NUMBL induces degradation of TRAF6, NEMO, NOTCH1-NICD, and inhibits osteoclastogenesis in vitro. Inhibition of NUMBL using a dominant negative PTB-phosphotyrosine-binding of NUMBL and shRNAs knockdown of NUMBL enhanced expression of TRAF6 and NEMO and did not inhibit osteoclastogenesis in wild-type cells. In addition, inhibition of NUMBL using a dominant negative PTB of NUMBL and exogenous expression of NOTCH1-NICD restored osteoclastogenesis in TAK1-null cells. Based on these observations we hypothesize that """"""""NUMBL is a repressor of osteoclastogenesis and its expression is regulated by TAK1. Deletion of TAK1 leads to accumulation of NUMBL protein which induces degradation of TRAF6, NEMO and NICD proteins, and subsequently blocks osteoclastogenesis."""""""" To test this hypothesis, we propose to investigate the following specific aims: 1) Determine the mechanism by which TAK1 regulates NUMBL expression. 2) Determine the mechanism by which TAK1 deletion regulates and impedes expression of TRAF6, NEMO and NICD. 3) Determine the effect of genetic ablation of NUMBL on the osteopetrotic phenotype of TAK1-null mice.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR054326-07
Application #
8635282
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Chen, Faye H
Project Start
2006-12-01
Project End
2018-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
7
Fiscal Year
2014
Total Cost
$323,000
Indirect Cost
$110,500
Name
Washington University
Department
Orthopedics
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Wang, C; Qu, C; Alippe, Y et al. (2016) Poly-ADP-ribosylation-mediated degradation of ARTD1 by the NLRP3 inflammasome is a prerequisite for osteoclast maturation. Cell Death Dis 7:e2153
Swarnkar, Gaurav; Shim, Kyuhwan; Nasir, Amjad M et al. (2016) Myeloid Deletion of Nemo Causes Osteopetrosis in Mice Owing to Upregulation of Transcriptional Repressors. Sci Rep 6:29896
Zou, Wei; Rohatgi, Nidhi; Chen, Timothy Hung-Po et al. (2016) PPAR-γ regulates pharmacological but not physiological or pathological osteoclast formation. Nat Med 22:1203-1205
Qu, Chao; Bonar, Sheri L; Hickman-Brecks, Cynthia L et al. (2015) NLRP3 mediates osteolysis through inflammation-dependent and -independent mechanisms. FASEB J 29:1269-79
Chen, T H-P; Swarnkar, G; Mbalaviele, G et al. (2015) Myeloid lineage skewing due to exacerbated NF-κB signaling facilitates osteopenia in Scurfy mice. Cell Death Dis 6:e1723
Swarnkar, Gaurav; Karuppaiah, Kannan; Mbalaviele, Gabriel et al. (2015) Osteopetrosis in TAK1-deficient mice owing to defective NF-κB and NOTCH signaling. Proc Natl Acad Sci U S A 112:154-9
Swarnkar, Gaurav; Abu-Amer, Yousef (2015) Regulation of NF-κB signaling in osteoclasts and myeloid progenitors. Methods Mol Biol 1280:527-42
Swarnkar, Gaurav; Zhang, Kaihua; Mbalaviele, Gabriel et al. (2014) Constitutive activation of IKK2/NF-κB impairs osteogenesis and skeletal development. PLoS One 9:e91421
Abu-Amer, Y (2013) NF-κB signaling and bone resorption. Osteoporos Int 24:2377-86
Zhang, Yanhong; Otero, Jesse E; Abu-Amer, Yousef (2013) Ubiquitin-like domain of IKKβ regulates osteoclastogenesis and osteolysis. Calcif Tissue Int 93:78-85

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