Ubiquitin Ligase Smurf1 Mediates Tumor Necrosis Factor-induced Systemic Bone Loss by Promoting Proteasomal Degradation of Bone Morphogenetic Signaling Proteins

Ruolin Guo,Motozo Yamashita,Qian Zhang,Quan Zhou,Di Chen,David G Reynolds,Hani A Awad,Laura Yanoso,Lan Zhao,Edward M Schwarz,Ying E Zhang,Brendan F Boyce,Lianping Xing

doi:10.1074/jbc.m709848200

Abstract

Chronic inflammatory disorders, such as rheumatoid arthritis, are often accompanied by systemic bone loss, which is thought to occur through inflammatory cytokine-mediated stimulation of osteoclast resorption and inhibition of osteoblast function. However, the mechanisms involved in osteoblast inhibition remain poorly understood. Here we test the hypothesis that increased Smad ubiquitin regulatory factor 1 (Smurf1)-mediated degradation of the bone morphogenetic protein pathway signaling proteins mediates reduced bone formation in inflammatory disorders. Osteoblasts derived from bone marrow or long bone samples of adult tumor necrosis factor (TNF) transgenic (TNF-Tg) mice were used in this study. TNF decreased the steady-state levels of Smad1 and Runx2 protein similarly to those in long bones of TNF-Tg mice. In the presence of the proteasome inhibitor MG132, TNF increased accumulation of ubiquitinated Smad1 protein. TNF administration over calvarial bones caused decreases in Smad1 and Runx2 protein levels and mRNA expression of osteoblast marker genes in wild-type, but not in Smurf1(-/-) mice. Vertebral bone volume and strength of TNF-Tg/Smurf1(-/-) mice were examined by a combination of micro-CT, bone histomorphometry, and biomechanical testing and compared with those from TNF-Tg littermates. TNF-Tg mice had significantly decreased bone volume and biomechanical properties, which were partially rescued in TNF-Tg/Smurf1(-/-) mice. We conclude that in chronic inflammatory disorders where TNF is increased, TNF induces the expression of ubiquitin ligase Smurf1 and promotes ubiquitination and proteasomal degradation of Smad1 and Runx2, leading to systemic bone loss. Inhibition of ubiquitin-mediated Smad1 and Runx2 degradation in osteoblasts could help to treat inflammation-induced osteoporosis.

Highlights

Osteoporosis and fragility fractures are common and preventable complications of rheumatoid arthritis (RA).2 For example, one study has reported that 53.3% of RA patients had osteoporosis and 19.3% had vertebral fractures, rates that are much higher than those of the general population [1]
We observed a significant reduction in bone volume in long bones of tumor necrosis factor (TNF) transgenic (TNF-Tg) mice with established arthritis and a decrease in mineralized bone nodule formation from cells derived from bone marrow of these mice compared with wild-type (WT) littermates [14]
We found that treatment of 2T3 pre-osteoblasts with TNF for 3 days increases Smad ubiquitin regulatory factor 1 (Smurf1) expression and proteasomal degradation of Runx2 [14], leading to a hypothesis that elevated Smurf1-mediated bone morphogenetic protein (BMP)-signaling protein degradation may contribute to reduced osteoblast function in TNF-Tg mice

Summary

Introduction

Osteoporosis and fragility fractures are common and preventable complications of rheumatoid arthritis (RA). For example, one study has reported that 53.3% of RA patients had osteoporosis and 19.3% had vertebral fractures, rates that are much higher than those of the general population [1]. One study has reported that 53.3% of RA patients had osteoporosis and 19.3% had vertebral fractures, rates that are much higher than those of the general population [1] These complications are thought to occur through inflammatory stimulation of osteoclast bone resorption and inhibition of osteoblast function [2] mediated by cytokines, such as TNF [3,4,5]. We demonstrated that TNF increases the expression of Smad ubiquitin regulatory factor-1 (Smurf1) in osteoblasts and promotes the proteasomal degradation of Runx protein in osteoblast cell lines [14], suggesting that the proteasomal regulation of key transcription factors in osteoblasts may play an important role in inflammation-induced bone loss. Our findings provide a novel molecular mechanism of TNF-induced inhibition in osteoblast function and BMP signaling, which involves post-transcriptional regulation of protein levels through Smurf E3 ligase-mediated proteasomal degradation

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Results

Conclusion