Ubiquitin ligase Smurf1 mediates tumor necrosis factor-induced systemic bone loss by promoting proteasomal degradation of bone morphogenetic signaling proteins

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.

FIGURE 1.

TNF reduces expression of osteoblast marker genes in bone marrow-derived osteoblasts from adult mice. Bone marrow cells from 4–8-month-old WT mice were cultured in osteoblast differentiation medium ± TNF (7.5 ng/ml) for various times. Expression of ALP, osteocalcin, and actin mRNA was determined by real time reverse transcriptase-PCR. -Fold changes were calculated by dividing the value of a given time point by the value obtained from day 0 as 1. Values are the mean ± S.D. of 3 loadings.

FIGURE 2.

Chronic exposure to TNF decreases Smad1 and Runx2 protein levels in bone marrow-derived mature osteoblasts and in long bones from TNF-Tg mice. Murine (A and B) or human bone marrow cells (E and F) were cultured in osteoblast differentiation medium ± TNF (7.5 ng/ml) for 14 days, and total RNA and protein were harvested. Long bone samples (C and D) from 6-month-old TNF-Tg mice or WT littermates were harvested and pooled (n = 3/genotype). Expression of Smurf1, Smurf2, and ALP mRNA was determined by real time reverse transcriptase-PCR as in Fig. 1 (A, C, and E). Expression of phospho-Smad1/5, total Smad1/5, Runx2, and Smurf1 proteins was assessed by Western blot analysis (B, D, and F). The intensity of protein bands was measured in a densitometer. The -fold changes of a given protein over β-actin were calculated using PBS-treated samples as 1. ^*, p < 0.05 versus PBS-treated or WT samples.

FIGURE 3.

TNF increases Smad1 degradation by promoting its ubiquitination. A, 2T3 preosteoblasts were either transfected with M-Smad1 expression vector ± TNF or co-transfected with F-Smurf1 expression vectors for 72 h. The expression of M-Smad1 was examined by Western blot analysis with anti-c-Myc antibody. B, cells were transfected with M-Smad1 ± TNF for 72 h. MG132 (10 μm) was added for the last 4 h. M-Smad1 was immunoprecipitated (IP) by anti-c-Myc antibody, and ubiquitinated Smad1 protein ladders were detected by anti-ubiquitin antibody (upper panel). Total Smad1 and β-actin protein levels were determined by Western blot (WB) using anti-c-Myc or anti-actin antibody, respectively (lower panel).

FIGURE 4.

TNF-induced osteoblast inhibition is reduced in Smurf1^-/- mice. Long bone samples were harvested and pooled from 7.5-month-old WT, Smurf1^-/-, TNF-Tg, and TNF-Tg/Smurf1^-/- mice (n = 3/genotype). Total RNA and protein were extracted. The expression of ALP and osteocalcin mRNA (A), and Smad1/5 and Runx2 proteins (B) were determined by real time reverse transcriptase-PCR and Western blot (WB) analysis, respectively, as in Fig. 2. The intensity of protein bands was measured in a densitometer. The -fold changes of a given protein over β-actin were calculated using WT samples as 1. ^*, p < 0.05 versus TNF-Tg mice. Calvarial pre-osteoblasts were isolated from 3–5-day-old newborn pups of Smurf1^-/- and WT mice and treated with TNF for 72 h. MG132 (10 μm) was added for the last 4 h. Endogenous Smad1 was immunoprecipitated (IP) by anti-Smad1 antibody, and ubiquitinated (upper panel) and total Smad1 (lower panels) proteins were detected by anti-ubiquitin or anti-Smad1 antibody, respectively (C).

FIGURE 5.

TNF-induced systemic bone loss is prevented in TNF-Tg/Smurf1^-/- mice. Bodies of 4th and 2nd lumber vertebrae were isolated from 7.5-month-old WT, Smurf1^-/-, TNF-Tg, or TNF-Tg/Smurf1^-/- mice and subjected to micro-CT analysis and histology examination, respectively. A, representative three-dimensional reconstructed image shows significantly reduced trabecular bone structure in TNF-Tg mice, particularly in the middle portion of the vertebral bodies (box). Reduced bone volume was not observed in TNF-Tg/Smurf1^-/- mice. B, trabecular bone parameters were analyzed at the middle portion of the vertebral bodies. Values are the mean ± S.D. of 4–8 mice. ^*, p < 0.05 versus TNF-Tg mice; #, p < 0.05 versus WT mice.

FIGURE 6.

Chronic exposure to TNF reduces bone mechanical strength, which is partially restored in TNF-Tg/Smurf1^-/- mice. Lumbar 4 vertebral bodies from 7.5-month-old WT, Smurf1^-/-, TNF-Tg, and TNF-Tg/Smurf1^-/- mice were tested in compression at a rate of 1 mm/min until failure. The representative load-deformation curves from WT, Smurf1^-/-, TNF-Tg, and TNF-Tg/Smurf1^-/- mice show that TNF-Tg mice have reduced maximal load and work to failure but TNF-Tg/Smurf1^-/- mice have a similar compressive strength compared with WT mice (A). Biomechanical properties including maximum deformation, maximum compressive load to failure, energy to failure, and stiffness are shown in B. Values are the mean ± S.D. of 4–9 mice. ^*, p < 0.05 versus TNF-Tg mice; #, p < 0.05 versus WT mice.

FIGURE 7.

Smurf1 deletion does not affect osteoclast function. Knee sections from 7.5-month-old TNF-Tg and TNF-Tg/Smurf1^-/- mice were stained for TRAP activity for identifying osteoclasts (arrows). The number of TRAP⁺ osteoclasts and inflammatory area were assessed. Representative TRAP-stained sections indicate a similar degree of osteoclastogenesis, bone erosion, and inflammation between TNF-Tg and TNF-Tg/Smurf1^-/- mice (A). Histomorphometric analyses of the number of osteoclasts and percentage of erosion and inflammation are shown in B. Values are the mean ± S.D. of 6 mice per group. Spleen cells from WT and Smurf1^-/- mice were cultured with RANKL and macrophage colony-stimulating factors for 7 days to form osteoclasts. The cells were fixed in formalin and stained for TRAP activity. The number of TRAP⁺ cells was counted. Values are the mean ± S.D. of 4 wells (C). A similar result was repeated using another pair of WT and Smurf1^-/- mice.

FIGURE 8.

A model of Smurf1-induced BMP signal protein degradation in TNF-induced osteoblast inhibition in inflammatory arthritis. In inflamed joints, elevated TNF increases Smurf1 expression in osteoblasts locally and systemically. Increased Smurf1 constitutively increases proteasomal degradation of BMP signal proteins Runx2 and Smad1 and decreases steady-state levels of these osteoblast positive regulators, leading to osteoblast inhibition and bone loss.