title: Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins.
abstract: OBJECTIVE
  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-κB 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-κB 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-κB 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-κB RelA and RelB proteins were found to bind to the NF-κB 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 Res2017;6:154-161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1.
authors.affiliation: Department of Orthopedics, China-Japan Union Hospital of Jilin University, 126 Xiantai Boulevard, Changchun, Jilin 130033, China.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Centre, 601 Elmwood Ave, Rochester, NY 14642, USA.
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Centre, 601 Elmwood Ave, Rochester, NY 14642, USA.
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Orthopedics, China-Japan Union Hospital of Jilin University, 126 Xiantai Boulevard, Changchun, Jilin 130033, China.
authors.forename: R
authors.initials: R
authors.lastname: Gu
authors.affiliation: Fourth Hospital of Hebei Medical University, 12 Jiankang Road, Shijiazhuang 050011, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Orthopedics, China-Japan Union Hospital of Jilin University, 126 Xiantai Boulevard, Changchun, Jilin 130033, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Gao
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Centre, 601 Elmwood Ave, Rochester, NY 14642, USA Lianping_xing@urmc.rochester.edu zhongligao123@outlook.com.
authors.forename: L
authors.initials: L
authors.lastname: Xing
keywords: Bone formation; E3 ligase; Fracture; Itch; Osteoblasts