Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins

Abstract

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-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 Res 2017;6:154-161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1.

Keywords: Bone formation; E3 ligase; Fracture; Itch; Osteoblasts.

Fig. 1

Increased expression of multiple Nedd4 subclass of E3 ligases in fracture callus. Three-month-old WT C57 BL/6J male mice underwent a surgical tibia fracture and were killed at different time points post-fracture. The expression levels of E3 ligases in callus tissue were examined by qPCR. Values are mean and standard deviation of three mice. The relative expression was calculated as fold change using the expression level at day 3 as 1. *p<0.05 versus samples from day three.

Fig. 2

Increased expression of NF-κB members in fracture callus. Samples were obtained from the callus tissue as in Figure 1. The expression levels of NF-κB members were determined by quantitative polymerase chain reaction (qPCR). Values are mean and standard deviation of three mice. The relative expression was calculated as fold change using the expression level at day 3 as 1. *p<0.05 versus samples from day 3.

Fig. 3

Increased total ubiquitinated proteins in fracture callus. WT mice received bone fracture surgery as in Figure 1. Callus tissue from fractured legs was harvested at day 7 after mice received MG132 for 24 hours. Total ubiquitinated (Ub) proteins were determined by Western blot analysis using anti-ubiquitin antibody. The cortical bone samples from the non-fractured legs were used as a control (Ctl).

Fig. 4

NF-κB upregulates Itch expression in osteoblasts. C2C12, an osteoblast/myoblast cell line, was infected with GFP control, NF-κB RelA or RelB virus for 24 hours. a) Expression levels of Itch protein were determined by Western blot analysis. b) Chromatin immunoprecipitation (ChIP) assays were performed on immunocomplexes that were pulled down with anti-RelA, anti-RelB antibody or Immunoglobulin G (IgG). Precipitated DNA was measured by polymerase chain reaction (PCR) and quantitative PCR using sequence-specific primers. Values are mean (sd) of determinates in triplicate. * p < 0.05 versus green fluorescent protein (GFP)-infected samples.

Altered expression patterns for genes associated with bone formation and remodelling in fracture callus of Itch KO mice. Itch KO and WT control mice underwent a surgical fracture and were killed at different time points post-fracture. The expression levels of genes related to osteoblast a) and osteoclast b) differentiation were examined by quantitative polymerase chain reaction (qPCR). Values are mean and standard deviation of triplicate experiments. The relative expression was calculated as fold change using the expression level at day 3 as 1.