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Ubiquitin E3 ligase Itch negatively regulates osteoclast formation by promoting deubiquitination of tumor necrosis factor (TNF) receptor-associated factor 6.

ABSTRACT: Itch is a ubiquitin E3 ligase that regulates protein stability. Itch(-/-) mice develop an autoimmune disease phenotype characterized by itchy skin and multiorgan inflammation. The role of Itch in the regulation of osteoclast function has not been examined. We report that Itch(-/-) bone marrow and spleen cells formed more osteoclasts than cells from WT littermates in response to receptor activator of NF-?B ligand (RANKL) and was associated with increased expression of the osteoclastogenic transcription factors c-fos and Nfatc1. Overexpression of Itch in Itch(-/-) cells rescued increased osteoclastogenesis. RANKL increased Itch expression, which can be blocked by a NF-?B inhibitor. The murine Itch promoter contains NF-?B binding sites. Overexpression of NF-?B p65 increased Itch expression, and RANKL promoted the binding of p65 onto the NF-?B binding sites in the Itch promoter. Itch(-/-) osteoclast precursors had prolonged RANKL-induced NF-?B activation and delayed TNF receptor-associated factor 6 (TRAF6) deubiquitination. In WT osteoclast precursors, Itch bound to TRAF6 and the deubiquitinating enzyme cylindromatosis. Adult Itch(-/-) mice had normal bone volume, but they had significantly increased LPS-induced osteoclastogenesis and bone resorption. Thus, Itch is a new RANKL target gene that is induced during osteoclastogenesis. Itch interacts with the deubiquitinating enzyme and is required for deubiquitination of TRAF6, thus limiting RANKL-induced osteoclast formation.

SUBMITTER: Zhang H

PROVIDER: S-EPMC3829326 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Ubiquitin E3 ligase Itch negatively regulates osteoclast formation by promoting deubiquitination of tumor necrosis factor (TNF) receptor-associated factor 6.

Zhang Hengwei H Wu Chengwu C Matesic Lydia E LE Li Xing X Wang Zhiyu Z Boyce Brendan F BF Xing Lianping L

The Journal of biological chemistry 20130619 31

Itch is a ubiquitin E3 ligase that regulates protein stability. Itch(-/-) mice develop an autoimmune disease phenotype characterized by itchy skin and multiorgan inflammation. The role of Itch in the regulation of osteoclast function has not been examined. We report that Itch(-/-) bone marrow and spleen cells formed more osteoclasts than cells from WT littermates in response to receptor activator of NF-κB ligand (RANKL) and was associated with increased expression of the osteoclastogenic transcr ...[more]

PMID: 23782702

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Project description:OBJECTIVES:Ubiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown. METHODS:We examined the expression levels of E3 ligases and NF-?B members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in fracture callus from Itch knockout (KO) mice and their WT littermates were examined by qPCR. The effect of NF-?B on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay. RESULTS:The expression levels of WW Domain Containing E3 Ubiquitin Protein Ligase 1 (Wwp1), SMAD Specific E3 Ubiquitin Protein Ligase 1 (Smurf1), SMAD Specific E3 Ubiquitin Protein Ligase 2 (Smurf2) and Itch were all significantly increased in the fracture callus of WT mice, which was associated with elevated expression of NF-?B members and total ubiquitinated proteins. Callus tissue isolated from Itch KO mice expressed higher levels of osteoblast-associated genes, including Runx2, a positive regulator of osteoblast differentiation, but osteoclast-associated genes were not increased. Both NF-?B RelA and RelB proteins were found to bind to the NF-?B binding site in the mouse Itch promoter. CONCLUSIONS:Our findings indicate that Itch depletion may have a strong positive effect on osteoblast differentiation in fracture callus. Thus, ubiquitin E3 ligase Itch could be a potential target for enhancing bone fracture healing.Cite this article: J. Liu, X. Li, H. Zhang, R. Gu, Z. Wang, Z. Gao, L. Xing. Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins. Bone Joint Res 2017;6:154-161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1.

Dataset Information

Ubiquitin E3 ligase Itch negatively regulates osteoclast formation by promoting deubiquitination of tumor necrosis factor (TNF) receptor-associated factor 6.

Publications

Ubiquitin E3 ligase Itch negatively regulates osteoclast formation by promoting deubiquitination of tumor necrosis factor (TNF) receptor-associated factor 6.

Similar Datasets

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