Tumor Necrosis Factor Promotes Runx2 Degradation through Up-regulation of Smurf1 and Smurf2 in Osteoblasts

Hiroyuki Kaneki,Ruolin Guo,Brendan F Boyce,Edward M Schwarz,Lianping Xing,Zhenqiang Yao,Ying E Zhang,Di Chen

doi:10.1074/jbc.m509430200

Abstract

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.

Highlights

Tumor necrosis factor (TNF)2 is a major contributor to pathologic bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation
To examine whether osteoblast function is altered in TNF-Tg mice, bone marrow stromal cells were isolated from 4-monthold TNF-Tg mice and wt littermates
Osteoblast-mediated bone formation cannot compensate for accelerated osteoclastic bone resorption, suggesting a direct inhibitory effect of TNF on osteoblasts

Summary

Introduction

Tumor necrosis factor (TNF) is a major contributor to pathologic bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. In patients with rheumatoid arthritis, TNF and other cytokines are overproduced in inflamed joints by various cells infiltrating the synovial membrane This leads to severe local erosion of cartilage and bone, periarticular osteopenia, as well as systemic osteoporosis [1, 2]. The most important mechanistic finding of TNF-mediated osteoblast inhibition was the demonstration that TNF decreases Runtrelated gene 2 (Runx2) expression and its DNA binding activity in osteoblasts [10]. We and others have demonstrated that the E3 ubiquitin ligase, Smad ubiquitin regulatory factor (Smurf), regulates osteoblast differentiation by promoting proteasomal degradation of the BMP signaling protein, Smad and Smad, and of the osteoblast transcription factor, Runx2 [15,16,17,18]. There is little information on the regulation of Smurfs expression under physiological and pathological conditions

Methods

Results

Conclusion