Osteoclasts (OCs), the principal bone-resorbing cells, arise from hematopoietic precursors of monocyte-macrophage lineage upon stimulation of two key factors: M-CSF and RANKL (also known as OPGL/ODF/TRANCE). RANKL, identified as a member of TNF family, is a potent inducer of OC differentiation, activation and survival. RANKL exerts its effects by binding to its receptor RANK, identified as a member of TNF receptor superfamily, on OC precursors or mature OCs. Both RANKL-/- and RANK-/- mice develop osteopetrosis due to the failure to form OCs, indicating that RANK signaling is essential for differentiation. Furthermore, RANKL activates the resorptive function of the mature OCs and inhibits their apoptosis. Thus, RANKL is pivotal to generation, activation and survival of OCs. However, the specific motifs (residues) in the RANK cytoplasmic domain mediating OC differentiation, activation and survival have not been identified. We have developed a tool which permits us to identify the specific motifs mediating these three effects. Using a retroviral technology, we expressed a chimeric receptor containing the extracellular domain of TNFR1 linked to the transmembrane and cytoplasmic domains of RANK in authentic OC precursors isolated from TNFR1/R2 double knock-out mice. Treatment of the chimera-expressing OC precursors with TNF-alpha as a RANKL surrogate, plus M-CSF, generates OCs, indistinguishable from those induced by RANKL and M-CSF. Thus, using this chimeric protein approach, we are positioned to delineate the specific motifs in the RANK cytoplasmic domain mediating OC differentiation, activation and survival. Since members of TNF receptor superfamily transmit the intracellular signaling by binding intracellular adaptor proteins such as TRAFs, we hypothesize that RANK, as a member of the TNF receptor superfamily, initiates its signaling in OC differentiation, activation and survival by recruiting adaptor proteins, including TRAFs, to specific motifs in the RANK cytoplasmic domain.
Our Specific Aims are to identify: (1) specific motifs in the RANK cytoplasmic domain mediating OC differentiation; (2) specific motifs in the RANK cytoplasmic domain mediating OC activation; and (3) specific motifs in the RANK cytoplasmic domain mediating OC survival.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
1R01AR047830-01A1
Application #
6436676
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Program Officer
Sharrock, William J
Project Start
2001-09-28
Project End
2006-07-31
Budget Start
2001-09-28
Budget End
2002-07-31
Support Year
1
Fiscal Year
2001
Total Cost
$287,000
Indirect Cost
Name
University of Alabama Birmingham
Department
Pathology
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Jules, Joel; Chen, Wei; Feng, Xu et al. (2018) C/EBP? transcription factor is regulated by the RANK cytoplasmic 535IVVY538 motif and stimulates osteoclastogenesis more strongly than c-Fos. J Biol Chem 293:1480-1492
Jules, Joel; Wang, Shunqing; Shi, Zhenqi et al. (2015) The IVVY Motif and Tumor Necrosis Factor Receptor-associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor ?B (RANK) Cooperate to Induce Osteoclastogenesis. J Biol Chem 290:23738-50
Jules, Joel; Feng, Xu (2014) In vitro investigation of the roles of the proinflammatory cytokines tumor necrosis factor-? and interleukin-1 in murine osteoclastogenesis. Methods Mol Biol 1155:109-23
Zhao, Dongfeng; Shi, Zhenqi; Warriner, Amy H et al. (2014) Molecular mechanism of thiazolidinedione-mediated inhibitory effects on osteoclastogenesis. PLoS One 9:e102706
Hong, Huixian; Shi, Zhenqi; Qiao, Ping et al. (2013) Interleukin-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process. Biochem Biophys Res Commun 440:545-50
McCoy, Erin M; Hong, Huixian; Pruitt, Hawley C et al. (2013) IL-11 produced by breast cancer cells augments osteoclastogenesis by sustaining the pool of osteoclast progenitor cells. BMC Cancer 13:16
Izawa, Takashi; Zou, Wei; Chappel, Jean C et al. (2012) c-Src links a RANK/ýývýý3 integrin complex to the osteoclast cytoskeleton. Mol Cell Biol 32:2943-53
Jules, Joel; Zhang, Ping; Ashley, Jason W et al. (2012) Molecular basis of requirement of receptor activator of nuclear factor ?B signaling for interleukin 1-mediated osteoclastogenesis. J Biol Chem 287:15728-38
Cheng, Jing; Liu, Jianzhong; Shi, Zhenqi et al. (2011) Interleukin-4 inhibits RANKL-induced NFATc1 expression via STAT6: a novel mechanism mediating its blockade of osteoclastogenesis. J Cell Biochem 112:3385-92
Ashley, Jason W; Shi, Zhenqi; Zhao, Haibo et al. (2011) Genetic ablation of CD68 results in mice with increased bone and dysfunctional osteoclasts. PLoS One 6:e25838

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