The transcription factor NF-(B is essential for osteoclastogenesis and is considered a key modulator of inflammatory responses. Activation of NF-(B entails induction of a large I(B kinase (IKK) complex that comprises IKK1, IKK2, and IKK(/NEMO. IKK2 and IKK1 activate the canonical (p50/p65) and non- canonical (p52/relB) NF-(B pathways, respectively. Recent studies implicate IKK2 and IKK1 as essential for osteoclastogenesis, yet the mechanisms underling this function, remain unclear. In addition, IKK2 is considered as a key mediator of inflammatory events induced by TNF, LPS, and other pro-inflammatory agents (through the classical NF-(B pathway), whereas less is known about the role of IKK1 is such responses. In the past few years we have investigated molecular pathways regulating osteoclast activity in osteolytic responses, such as inflammatory osteolysis and arthritic bone erosion, and unveiled critical regulatory steps modulating NF-(B activation in osteoclast precursors. In this regard, we find that disruption of NF-(B activation at multiple levels attenuates osteoclastogenesis and inflammatory bone erosion. Specifically, we have shown that administration of dominant-negative forms of I(B(, that resist phosphorylation by IKK2;and most recently inhibition of IKK assembly with NEMO, using a decoy NEMO binding domain (NBD) peptide, all are successful approaches to inhibit osteoclastogenesis and bone erosion, in vitro and in vivo. Most importantly, we have preliminary evidence that, 1) failure of RANKL-induced osteoclastogenesis by IKK1-null OCPs is rescued by re-introduction of IKK1 cDNA constructs, and that 2) tissue-specific deletion of IKK2 in OCPs (using CD11b-cre) results with skeletally deformed mice. It is evident that osteoclastogenesis is impaired in the absence of IKK1 or IKK2, however, the precise mechanism(s) underlying signaling of IKKs in basal and inflammatory osteoclastogenesis remain scarce. Moreover, the relative contribution of either protein to these responses is indefinite. It is also known that IKK1 and IKK2 share considerable sequence and domain similarities, albeit they maintain distinct and non-overlapping functions. Thus, we hypothesize that IKK1 and IKK2 differentially regulate basal and inflammatory osteoclastogenesis through the alternative and classical NF-(B activation pathways. Hence, clarifying the individual roles of IKKs in osteoclastogenesis by investigating the details of their molecular signaling in osteoclasts, utilizing germ-line and specific tissue deletions of the relevant genes, will enable us to design strategies and selective inhibitors directed against IKK1 and IKK2 that may be useful for regulating osteoclastogenesis and alleviating inflammatory osteolysis in various bone resorptive disorders. Thus, we propose to investigate the following specific aims: 1) Determine the molecular role of IKK1 and IKK2 in basal and inflammatory osteoclastogenesis. 2) Determine the mechanism by which tyrosine phosphorylation regulates the fate and activity of IKK2 in the osteoclast lineage and it's impact on osteoclastogenesis. 3) Determine the role of IKK1 and IKK2 in inflammatory osteolysis, in vivo.

Public Health Relevance

The transcription factor NF-(B is essential for osteoclastogenesis and modulates inflammatory responses. NF-(B is activated by the serine kinases, I(B kinase (IKK)-1, IKK-2, and IKK(/NEMO. Gene deletions of IKK1 or IKK2 led to defects in osteoclasts and inflammatory responses. Using IKK1 and IKK2-null mice, we propose to investigate the molecular domain contributions of IKK1 and IKK2 to osteoclastogenesis and inflammatory osteolysis, as is the case in inflammatory arthritis. Our proposal holds promise to identify novel selective anti-osteolytic therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR054326-05
Application #
8240429
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Chen, Faye H
Project Start
2008-07-07
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
5
Fiscal Year
2012
Total Cost
$317,814
Indirect Cost
$108,726
Name
Washington University
Department
Orthopedics
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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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