Kaneki Hiroyuki, Guo Ruolin, Chen Di, Yao Zhenqiang, Schwarz Edward M, Zhang Ying E, Boyce Brendan F, Xing Lianping
Department of Pathology and Laboratory Medicine, University of Rochester, School of Medicine and Dentistry, NY 14642, USA.
J Biol Chem. 2006 Feb 17;281(7):4326-33. doi: 10.1074/jbc.M509430200. Epub 2005 Dec 22.
Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Compared with the well established role of TNF in osteoclastogenesis, mechanisms by which TNF inhibits osteoblast function have not been fully determined. Runx2 is an osteoblast-specific transcription factor whose steady-state protein levels are regulated by proteasomal degradation, mediated by the E3 ubiquitin ligases, Smurf1 and Smurf2. We hypothesized that TNF inhibits osteoblast function through Smurf-mediated Runx2 degradation. We treated C2C12 and 2T3 osteoblast precursor cell lines and primary osteoblasts with TNF and found that TNF, but not interleukin-1, significantly increased Smurf1 and Smurf2 expression. TNF increased the degradation of endogenous or transfected Runx2 protein, which was blocked by treating cells with a proteasomal inhibitor or by infecting cells with small interfering (si)RNA against Smurf1 or Smurf2. TNF inhibited the expression of bone morphogenetic protein and transforming growth factor-beta signaling reporter constructs, and the inhibition of each was blocked by Smurf1 siRNA and Smurf2 siRNA, respectively. Overexpression of Smurf1 and/or Smurf2 siRNAs prevented the inhibitory effect of TNF on Runx2 reporter. Consistent with these in vitro findings, bones from TNF transgenic mice or TNF-injected wild type mice had increased Smurf1 and decreased Runx2 protein levels. We propose that one of the mechanisms by which TNF inhibits bone formation in inflammatory bone disorders is by promoting Runx2 proteasomal degradation through up-regulation of Smurf1 and Smurf2 expression.
肿瘤坏死因子（TNF）通过刺激破骨细胞骨吸收和抑制成骨细胞骨形成，在炎症性骨质流失的发病机制中发挥重要作用。与TNF在破骨细胞生成中已明确的作用相比，TNF抑制成骨细胞功能的机制尚未完全明确。Runx2是一种成骨细胞特异性转录因子，其稳态蛋白水平受E3泛素连接酶Smurf1和Smurf2介导的蛋白酶体降解调控。我们推测TNF通过Smurf介导的Runx2降解抑制成骨细胞功能。我们用TNF处理C2C12和2T3成骨细胞前体细胞系及原代成骨细胞，发现TNF而非白细胞介素-1显著增加Smurf1和Smurf2的表达。TNF增加内源性或转染的Runx2蛋白的降解，用蛋白酶体抑制剂处理细胞或用针对Smurf1或Smurf2的小干扰（si）RNA感染细胞可阻断这种降解。TNF抑制骨形态发生蛋白和转化生长因子-β信号报告构建体的表达，Smurf1 siRNA和Smurf2 siRNA分别阻断了对每种构建体的抑制。Smurf1和/或Smurf2 siRNA的过表达阻止了TNF对Runx2报告基因的抑制作用。与这些体外研究结果一致，TNF转基因小鼠或注射TNF的野生型小鼠的骨骼中Smurf1增加，Runx2蛋白水平降低。我们提出，TNF在炎症性骨病中抑制骨形成的机制之一是通过上调Smurf1和Smurf2的表达促进Runx2的蛋白酶体降解。
