Liu J, Li X, Zhang H, Gu R, Wang Z, Gao Z, Xing L
Department of Orthopedics, China-Japan Union Hospital of Jilin University, 126 Xiantai Boulevard, Changchun, Jilin 130033, China.
Department of Pathology and Laboratory Medicine, University of Rochester Medical Centre, 601 Elmwood Ave, Rochester, NY 14642, USA.
Bone Joint Res. 2017 Mar;6(3):154-161. doi: 10.1302/2046-3758.63.BJR-2016-0237.R1.
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.
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.
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.
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. 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. 2017;6:154-161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1.
泛素E3连接酶介导的蛋白质降解调节成骨细胞功能。E3连接酶Itch通过调节相关蛋白质的泛素化和蛋白酶体降解来影响多种细胞功能。然而，在骨折修复过程中，成骨细胞分化和功能增强的与Itch相关的细胞和分子机制尚不清楚。
我们通过定量聚合酶链反应（qPCR）检测野生型（WT）小鼠骨折修复过程中骨痂样本中E3连接酶和NF-κB成员的表达水平，并通过蛋白质印迹分析检测泛素化蛋白质的总量。通过qPCR检测Itch基因敲除（KO）小鼠及其野生型同窝小鼠骨折骨痂中成骨细胞相关基因的表达水平。通过染色质免疫沉淀（ChIP）试验确定NF-κB对C2C12成骨细胞中Itch表达的影响。
野生型小鼠骨折骨痂中含WW结构域的E3泛素蛋白连接酶1（Wwp1）、SMAD特异性E3泛素蛋白连接酶1（Smurf1）、SMAD特异性E3泛素蛋白连接酶2（Smurf2）和Itch的表达水平均显著升高，这与NF-κB成员表达升高和泛素化蛋白质总量增加有关。从Itch基因敲除小鼠分离的骨痂组织中，成骨细胞相关基因的表达水平较高，包括成骨细胞分化的正向调节因子Runx2，但破骨细胞相关基因没有增加。发现NF-κB RelA和RelB蛋白均与小鼠Itch启动子中的NF-κB结合位点结合。
我们的研究结果表明，Itch缺失可能对骨折骨痂中的成骨细胞分化有强烈的正向作用。因此，泛素E3连接酶Itch可能是促进骨折愈合的潜在靶点。刘J，李X，张H，顾R，王Z，高Z，邢L。泛素E3连接酶Itch通过促进成骨蛋白的蛋白酶体降解负向调节成骨细胞功能。2017；6：154 - 161。DOI：10.1302/2046 - 3758.63.BJR - 2016 - 0237.R1。
