RANKL coordinates multiple osteoclastogenic pathways by regulating expression of ubiquitin ligase RNF146
Yoshinori Matsumoto, … , Feng Cong, Robert Rottapel
Yoshinori Matsumoto, … , Feng Cong, Robert Rottapel
Published April 3, 2017; First published March 13, 2017
Citation Information: J Clin Invest. 2017;127(4):1303-1315. https://doi.org/10.1172/JCI90527.
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Research Article Bone biology Cell biology

RANKL coordinates multiple osteoclastogenic pathways by regulating expression of ubiquitin ligase RNF146

Abstract

Bone undergoes continuous remodeling due to balanced bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. Osteoclasts arise from the macrophage lineage, and their differentiation is dependent on RANKL, a member of the TNF family of cytokines. Here, we have provided evidence that RANKL controls the expression of 3BP2, an adapter protein that is required for activation of SRC tyrosine kinase and simultaneously coordinates the attenuation of β-catenin, both of which are required to execute the osteoclast developmental program. We found that RANKL represses the transcription of the E3 ubiquitin ligase RNF146 through an NF-κB–related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 results in the stabilization of its substrates, 3BP2 and AXIN1, which consequently triggers the activation of SRC and attenuates the expression of β-catenin, respectively. Depletion of RNF146 caused hypersensitivity to LPS-induced TNF-α production in vivo. RNF146 thus acts as an inhibitory switch to control osteoclastogenesis and cytokine production and may be a control point underlying the pathogenesis of chronic inflammatory diseases.

Authors

Yoshinori Matsumoto, Jose Larose, Oliver A. Kent, Melissa Lim, Adele Changoor, Lucia Zhang, Yaryna Storozhuk, Xiaohong Mao, Marc D. Grynpas, Feng Cong, Robert Rottapel

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Figure 1

RANKL stabilizes 3BP2 and AXIN1 protein levels through suppression of the E3 ubiquitin ligase RNF146.

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RANKL stabilizes 3BP2 and AXIN1 protein levels through suppression of th...
(A) Primary murine macrophages were cultured in the presence or absence of RANKL (50 ng/ml). Whole cell lysates were probed with the indicated antibodies for Western blot analysis. (B) Primary murine macrophages were cultured in the presence or absence of RANKL (50 ng/ml) and a WNT3a-conditioned medium. Whole cell lysates were probed with the indicated antibodies for Western blot analysis. (C) Whole cell lysates from cells in A were probed with the indicated antibodies for Western blot analysis. (D) Primary murine macrophages cultured in the presence or absence of RANKL (50 ng/ml) were treated with 10 μM MG132 for 4 hours prior to collection of cell lysates. 3BP2 immune complexes were probed with an anti–K48 linkage–specific polyubiquitin or anti-3BP2 antibody. (E) qPCR analysis of Rnf146 mRNA expression in primary murine macrophages cultured in the presence or absence of RANKL (50 ng/ml). n = 3. (F) Primary murine macrophages were cultured in the presence or absence of RANKL (50 ng/ml). RNF146 immune complexes were probed with an anti-RNF146 antibody. Whole cell lysates (WCLs) were probed with an anti-GAPDH antibody for Western blot analysis. (G) Primary murine macrophages infected with an empty vector control (mock) or RNF146-expressing retroviral vector were cultured in the presence or absence of RANKL (50 ng/ml). Whole cell lysates were probed with the indicated antibodies for Western blot analysis. (H) TRAP staining of osteoclasts infected with an empty vector control (mock) or RNF146-expressing retroviral vector and cultured in the presence or absence of RANKL for 7 days. Original magnification, ×40. n = 3. P values were determined by ANOVA with Tukey-Kramer’s post hoc test (B and G) or unpaired t test (A, C–F, and H). Data are presented as mean ± SEM. *P < 0.05.