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.

Public Health Relevance

Osteoclasts are the bone resorbing cells and their normal function is crucial to maintain bone homeostasis. Differentiation and function of osteoclasts is regulated by the factors M-CSF and RANK-ligand which induce crucial signal transduction pathways including MAP kinases and NF-?B. These signaling pathways are activated by the upstream kinase TGF-?-activated kinase (Tak1). The direct role of Tak1 in osteoclasts is not fully clear. We provide evidence that myeloid-specific deletion of Tak1 leads to osteopetrosis in mice. We discovered a novel mechanism whereby deletion of Tak1 leads to accumulation of the protein NUMBL, previously described in neurons. NUMBL in turn mediates degradation of key proteins of the osteoclast machinery including TRAF6, NEMO, and Notch1-NICD resulting with arrest of osteoclastogenesis. We propose to decipher the molecular mechanism underlying the role of Tak1/NUMBL signal in osteoclasts and its implications to bone development. Our studies are expected to unravel an entirely new area in osteoclast biology.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Skeletal Biology Structure and Regeneration Study Section (SBSR)
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Chen, Faye H
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Washington University
Schools of Medicine
Saint Louis
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Swarnkar, Gaurav; Zhang, Kaihua; Mbalaviele, Gabriel et al. (2014) Constitutive activation of IKK2/NF-?B impairs osteogenesis and skeletal development. PLoS One 9:e91421
Yamanaka, Yasuhiro; Clohisy, John C F; Ito, Hiroshi et al. (2013) Blockade of JNK and NFAT pathways attenuates orthopedic particle-stimulated osteoclastogenesis of human osteoclast precursors and murine calvarial osteolysis. J Orthop Res 31:67-72
Abu-Amer, Y (2013) NF-*B signaling and bone resorption. Osteoporos Int 24:2377-86
Zhang, Yanhong; Otero, Jesse E; Abu-Amer, Yousef (2013) Ubiquitin-like domain of IKK* regulates osteoclastogenesis and osteolysis. Calcif Tissue Int 93:78-85
Dai, S; Abu-Amer, W; Karuppaiah, K et al. (2011) Evidence that the kinase-truncated c-Src regulates NF-ýýB signaling by targeting NEMO. J Cell Biochem 112:2463-70
Yamanaka, Yasuhiro; Karuppaiah, Kannan; Abu-Amer, Yousef (2011) Polyubiquitination events mediate polymethylmethacrylate (PMMA) particle activation of NF-kappaB pathway. J Biol Chem 286:23735-41
Otero, Jesse E; Dai, Simon; Alhawagri, Muhammad A et al. (2010) IKKbeta activation is sufficient for RANK-independent osteoclast differentiation and osteolysis. J Bone Miner Res 25:1282-94
Darwech, Isra; Otero, Jesse E; Alhawagri, Muhammad A et al. (2010) Tyrosine phosphorylation is required for IkappaB kinase-beta (IKKbeta) activation and function in osteoclastogenesis. J Biol Chem 285:25522-30
Abu-Amer, Yousef (2009) Inflammation, cancer, and bone loss. Curr Opin Pharmacol 9:427-33
Darwech, Isra; Otero, Jesse; Alhawagri, Muhammad et al. (2009) Impediment of NEMO oligomerization inhibits osteoclastogenesis and osteolysis. J Cell Biochem 108:1337-45

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