The IVVY Motif and Tumor Necrosis Factor Receptor-associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor κB (RANK) Cooperate to Induce Osteoclastogenesis.
Receptor activator of NF-κB (RANK) activation by RANK ligand (RANKL) mediates osteoclastogenesis by recruiting TNF receptor-associated factors (TRAFs) via three cytoplasmic motifs (motif 1, PFQEP(369-373); motif 2, PVQEET(559-564); and motif 3, PVQEQG(604-609)) to activate the NF-κB and MAPK signaling pathways. RANK also has a TRAF-independent motif (IVVY(535-538)), which is dispensable for the activation of TRAF-induced signaling pathways but essential for osteoclast lineage commitment by inducing the expression of nuclear factor of activated T-cells c1 (NFATc1) to regulate osteoclast gene expression. Notably, TNF/IL-1-mediated osteoclastogenesis requires RANK ligand assistance, and the IVVY motif is also critical for TNF/IL-1-mediated osteoclastogenesis by rendering osteoclast genes responsive to these two cytokines. Here we show that the two types of RANK cytoplasmic motifs have to be on the same RANK molecule to mediate osteoclastogenesis, suggesting a functional cooperation between them. Subsequent osteoclastogenesis assays with TNF or IL-1 revealed that, although all three TRAF motifs play roles in TNF/IL-1-mediated osteoclastogenesis, motifs 2 and 3 are more potent than motif 1. Accordingly, inactivation of motifs 2 and 3 blocksTNF/IL-1-mediated osteoclastogenesis. Mechanistically, double mutation of motifs 2 and 3, similar to inactivation of the IVVY motif, abrogates the expression of nuclear factor of activated T-cells c1 and osteoclast genes in assays reflecting RANK-initiated and TNF/IL-1-mediated osteoclastogenesis. In contrast, double inactivation of motifs 2 and 3 did not affect the ability of RANK to activate the NF-κB and MAPK signaling pathways. Collectively, these results indicate that the RANK IVVY motif cooperates with the TRAF-binding motifs to promote osteoclastogenesis, which provides novel insights into the molecular mechanism of RANK signaling in osteoclastogenesis.
From the Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and.
Joel
J
Jules
From the Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and the Department of Hematology, Guangzhou First People's Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180, China.
Shunqing
S
Wang
From the Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and.
Zhenqi
Z
Shi
From the Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and.
Jianzhong
J
Liu
From the Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and.
Shi
S
Wei
From the Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294 and xufeng@uab.edu.
Xu
X
Feng
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D020125:Mutation, Missense; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D010010:Osteoclasts / Q000378:metabolism*; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
D007375:Interleukin-1; D016328:NF-kappa B; D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; C494005:Nfatc1 protein, mouse; D053246:Receptor Activator of Nuclear Factor-kappa B; C439524:TNF protein, human; C505686:TNFRSF11A protein, human; C505687:Tnfrsf11a protein, mouse; D014409:Tumor Necrosis Factor-alpha
IL-1; IVVY motif; RANK; RANKL; TNF; TNF receptor-associated factor (TRAF); cell signaling; osteoclast