Deubiquitinating enzyme CYLD negatively regulates RANK signaling and osteoclastogenesis in mice
Wei Jin, … , Jun You, Shao-Cong Sun
Wei Jin, … , Jun You, Shao-Cong Sun
Published April 1, 2008
Citation Information: J Clin Invest. 2008;118(5):1858-1866. https://doi.org/10.1172/JCI34257.
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Research Article Bone biology

Deubiquitinating enzyme CYLD negatively regulates RANK signaling and osteoclastogenesis in mice

Abstract

Osteoclastogenesis is a tightly regulated biological process, and deregulation can lead to severe bone disorders such as osteoporosis. The regulation of osteoclastic signaling is incompletely understood, but ubiquitination of TNF receptor–associated factor 6 (TRAF6) has recently been shown to be important in mediating this process. We therefore investigated the role of the recently identified deubiquitinating enzyme CYLD in osteoclastogenesis and found that mice with a genetic deficiency of CYLD had aberrant osteoclast differentiation and developed severe osteoporosis. Cultured osteoclast precursors derived from CYLD-deficient mice were hyperresponsive to RANKL-induced differentiation and produced more and larger osteoclasts than did controls upon stimulation. We assessed the expression pattern of CYLD and found that it was drastically upregulated during RANKL-induced differentiation of preosteoclasts. Furthermore, CYLD negatively regulated RANK signaling by inhibiting TRAF6 ubiquitination and activation of downstream signaling events. Interestingly, we found that CYLD interacted physically with the signaling adaptor p62 and thereby was recruited to TRAF6. These findings establish CYLD as a crucial negative regulator of osteoclastogenesis and suggest its involvement in the p62/TRAF6 signaling axis.

Authors

Wei Jin, Mikyoung Chang, Emmanuel M. Paul, Geetha Babu, Andrew J. Lee, William Reiley, Ato Wright, Minying Zhang, Jun You, Shao-Cong Sun

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Figure 6

CYLD targets TRAF6, which is promoted by p62.

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CYLD targets TRAF6, which is promoted by p62. (A) Wild-type and Cyld–/– ...
(A) Wild-type and Cyld–/– BMDMs were cultured for 2 days in M-CSF medium in either the absence (–) or presence (+) of GST-RANKL. TRAF6 was isolated by IP followed by IB to detect ubiquitin-conjugated TRAF6 (upper panel) or unmodified TRAF6 (lower panel). In lanes 5 and 6, the RANKL-treated cells were chased in RANKL-free media overnight before the TRAF6 ubiquitination assay. (B) Wild-type BMDMs were cultured for 2 days in M-CSF medium in the absence or presence of GST-RANKL. TRAF6 was isolated by IP followed by detection of the associated CYLD by IB (top panel). The lysates were subjected to IB to monitor the expression of CYLD and TRAF6 (middle and bottom panels). (C) Wild-type BMDMs were cultured for 2 days in M-CSF medium in the absence or presence of GST-RANKL. CYLD was isolated by IP followed by detection of the associated p62 by IB (top panel). The cell lysates were subjected to IB to monitor the expression of CYLD and p62 (middle and bottom panels). (D) 293 cells were transfected with CYLD along with vector control (V), wild-type p62, or p62ΔUBA. CYLD was isolated by IP, and its associated p62 was detected by IB (top panel). Protein expression in cell lysates was monitored by direct IB (middle and bottom panels). (E) 293T cells were transfected with either an empty vector or expression vectors encoding wild-type p62 or p62ΔUBA. Lanes 2–4 were also transfected with CYLD. Endogenous TRAF6 was isolated by IP followed by IB to detect the associated CYLD or the precipitated TRAF6 (top 2 panels). The cell lysates were subjected to IB to detect the expression of CYLD, EGFP-tagged p62 proteins, and TRAF6 (bottom 3 panels).