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TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation.

ABSTRACT: Tumor necrosis factor receptor-associated factor 6 (TRAF6), the crucial adaptor molecule of receptor activator of NF-kappaB (RANK), plays an essential role in governing the formation of multi-nucleated osteoclasts. TRAF6 is a RING-dependent ubiquitin (Ub) ligase that in conjunction with Ubc13/Uev1A catalyzes its own auto-ubiquitination via Lys63-linked poly-Ub chains. While the receptor-adaptor function of TRAF6 in RANK signaling is well understood, the significance of its Ub ligase activity in this process remains largely unknown. In this study, we show that retroviral expression of TRAF6, but not a RING mutant of TRAF6 was able to rescue TRAF6-deficient monocytes for the activation of IKK and osteoclast differentiation by RANKL. Furthermore, a catalytically inactive Ubc13 or stable knockdown of Ubc13 significantly prevents RANK-mediated TRAF6 ubiquitination and NF-kappaB and JNK activation. These data establish a signaling cascade in which regulated Lys63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of osteoclast differentiation.

SUBMITTER: Lamothe B

PROVIDER: S-EPMC2732028 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation.

Lamothe Betty B Webster William K WK Gopinathan Ambily A Besse Arnaud A Campos Alejandro D AD Darnay Bryant G BG

Biochemical and biophysical research communications 20070611 4

Tumor necrosis factor receptor-associated factor 6 (TRAF6), the crucial adaptor molecule of receptor activator of NF-kappaB (RANK), plays an essential role in governing the formation of multi-nucleated osteoclasts. TRAF6 is a RING-dependent ubiquitin (Ub) ligase that in conjunction with Ubc13/Uev1A catalyzes its own auto-ubiquitination via Lys63-linked poly-Ub chains. While the receptor-adaptor function of TRAF6 in RANK signaling is well understood, the significance of its Ub ligase activity in ...[more]

PMID: 17572386

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Project description:We recently reported that oral administration of a (+)-vitisin A-enriched product prepared from Vitis thunbergii obviously ameliorated bone loss in ovariectomized mice and (+)-vitisin A was able to inhibit receptor activator of NF-?B ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells. Here we further clarified the mechanism(s) by which (+)-vitisin A targets osteoclastic differentiation and activity. Osteoclast-characteristic enzyme activity was determined using gel zymography or spectroflurometric-based assay. Expression of signal molecules was analyzed via Western blot or immunoprecipitation. Results showed that (+)-vitisin A suppressed RANKL-induced multinuclear cells (MNCs) formation and bone resorption which was accompanied with reduction in ?3 integrin, osteoclast stimulatory transmembrane protein (OC-STAMP), matrix metalloproteinase-9 (MMP-9) and cathepsin K proteins expression. (+)-Vitisin A also down-regulated the proteolytic activities of MMP-9 and cathepsin K via targeting at the late stage function. (+)-Vitisin A prominently abrogated RANKL-triggered nuclear translocations of NF-?B, AP-1 (c-Fos/c-Jun dimer) and associated induction and nuclear accumulation of nuclear factor of activated T cells c1 (NFATc1). The upstream I?B degradation as well as ERK and JNK phosphorylation were also substantially repressed. Transfection with siRNA targeting tumor necrosis factor receptor associated factor 6 (TRAF6) clearly restrained RANKL-induced MNCs formation and NFATc1 induction. Interesting, RANKL triggered poly-ubiquitination of TRAF6 and associated TRAF6-TAK1 (transforming growth factor ?-activated kinase 1) complex formation was prominently attenuated by (+)-vitisin A. Furthermore, the interaction between c-src tyrosine kinase (c-Src) and ?3 was markedly induced by RANKL stimulation. (+)-Vitisin A significantly attenuated this interaction when concomitant treated with RANKL in RAW264.7 cells, but failed to affect c-Src/?3 complex formation when post-cultured with MNCs. Taken together, (+)-vitisin A suppressed bone resorption possibly via interruption of RANKL-induced TRAF6 ubiquitination and associated downstream signaling pathways. Furthermore, action through negative regulation of the proteolytic activity of MMP-9 and cathepsin K might also contribute to the anti-resorption effect of (+)-vitisin A.

Dataset Information

TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation.

Publications

TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation.

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