Tumor necrosis factor promotes Runx2 degradation through up-regulation of Smurf1 and Smurf2 in osteoblasts

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.

FIGURE 1. Decreased bone volume and osteoblast functions in TNF-Tg mice

A, femur from a 4-month-old TNF-Tg mouse and a wt littermate were fixed and processed. Paraffin-embedded sections were stained with hematoxylin and eosin. Lower bone volume in the metaphysis of the femur (green stars) is present in the TNF-Tg mouse (magnification, ×4). B, the distal femoral trabecular bone volume was measured as described previously (38). The values are the mean ± S.E. of eight or nine mice. *,p < 0.05 versus wt mice. C, bone marrow stromal cells were isolated from 4-month-old TNF-Tg mice and wt littermates and cultured in osteoblast differentiation medium containing 100 μg/ml l-ascorbic acid and 5 mm β-glycerophosphate in the presence or absence of 40 ng/ml BMP-2 for 2 weeks. The cells were fixed, and mineralized bone nodules were identified by vonKossa staining. The area of bone nodules was measured under light microscopy using point counting. The values are the mean ± S.E. of three wells. *,p < 0.05 versus wt cells. The same results were obtained from three pairs of TNF-Tg mice and wt littermates. D, mineralized bone nodules from one representative pair of TNF-Tg mice and wt littermates (magnification, ×2).

FIGURE 2. TNF increases Smurf1 expression and induces Runx2 degradation

A, C2C12 or 2T3 cells were treated with PBS or TNF (2.5–7.5 ng/ml) for 24, 48, and 72 h. Smurf1 and β-actin mRNA levels were measured by real time RT-PCR. The relative expression level of Smurf1 was normalized to β-actin in the same sample. The -fold increase was calculated as follows: (relative Smurf1 level in TNF-treated sample)/(relative Smurf1 level in PBS-treated sample). The values are the mean ± S.E. of three dishes. *, p < 0.05 versus the PBS-treated group. B, cells were treated with 2.5–7.5 ng/ml TNF for 72 h. Smurf1 and β-actin protein levels were examined by Western blot analysis. C, cells were treated with various doses of IL-1 and RANKL and with 7.5 ng/ml TNF for 72 h. The relative expression levels of Smurf1 mRNA were determined by real time RT-PCR, as described in A. The values are the mean ± S.E. of three dishes. *, p < 0.05 versus the PBS-treated group. D, cells were treated with 2.5–7.5 ng/ml TNF for 24, 48, and 72 h. Cell lysates were used for measuring caspase-3 activity or MTT assay. The values are the mean ± S.E. of three dishes. *, p<0.05 versus the PBS-treated group. E, cells were transfected with M-Smurf1 and/or F-Runx2 expression plasmids or empty vector for 24 h and cultured for 72 h in the presence of PBS or TNF (2.5–7.5 ng/ml). F-Runx2 expression was detected by Western blot using anti-FLAG antibody.

FIGURE 3. TNF induces ubiquitination of Runx2 protein

2T3 cells were treated with 7.5 ng/ml TNF for 72 h in the presence or absence of 0.1 mM MG132 for the last 12 h of TNF treatment, and endogenous Runx2 was immunoprecipitated (IP) by anti-Runx2 antibody. Ubiquitinated Runx2 (Ub-Runx2) protein ladders were detected by anti-ubiquitin antibody (upper panel). After stripping the antibody, total un-ubiquitinated Runx2 protein levels were determined by anti-Runx2 antibody (lower panel). MW, molecular mass.

FIGURE 4. Smurf1 or Smurf2 siRNA blocks TNF-induced Runx2 degradation

2T3 cells were infected with retroviral supernatant containing Smurf1 and/or Smurf2 siRNAs or empty vector, then cells were transfected with F-Runx2 expression plasmid (4 μg/dish) and treated with 7.5 ng/ml TNF for 72 h. Smurf1 (A) and Smurf2 (B) mRNA levels were measured by real time RT-PCR. The values are the mean ± S.E. of three dishes. The -fold increase was calculated as described in Fig. 2A. *, p<0.05 versus the empty vector-infected TNF group. C, the expression of F-Runx2 was determined by Western blot analysis using anti-FLAG antibody as described in Fig. 2E. D, ALP mRNA expression was assessed by real time RT-PCR. The values are the mean ± S.E. of three dishes. The -fold increase was calculated as described in Fig. 2A. *, p<0.05 versus empty vector-infected TNF group.

FIGURE 5. TNF targets both BMP and TGF-β signaling pathways in osteoblasts

A, 2T3 cells were infected with retroviral supernatant containing Smurf1 and/or Smurf2 siRNAs or empty vector, then cells were cotransfected with the Runx2 expression vector (Runx2), the empty expression vector (p3×FAG-CMV), and the Runx2 reporter vector (6×OSE2-OC-Luc) for 8 h and then treated with 7.5 ng/ml TNF for 48 h. B and C, 2T3 cells were infected with retroviral supernatant containing Smurf1 and/or Smurf2 siRNA or empty vector, then the BMP-2 and TGF-β signaling reporter constructs, 12×SBE-OC-Luc (B) or p3TP-Lux (C) were transfected into cells for 8 h. The cells were treated with 7.5 ng/ml TNF for 48 h and then stimulated with 50 ng/ml BMP-2 (B) or 2 ng/ml TGF-β (C) for 24 h in the presence of PBS or 10 ng/ml TNF. The values are the mean × S.E. of three dishes. *, p<0.05 versus empty vector-infected TNF group.

FIGURE 6. TNF increases Smurf1 expression in vivo

A, RNA and protein were extracted directly from the metaphyseal region of 4-month-old TNF-Tg mice and wt littermates. The relative expression levels of Smurf1, ALP, and OC were measured by real time RT-PCR (upper panels), as described in Fig. 2. The values are the mean ± S.E. of four pairs of TNF-Tg and wt mice. *, p < 0.05 versus wt mice. Smurf1 and β-actin protein levels were determined by Western blot analysis (lower panels). The data are representative of three pairs of TNF-Tg or wt mice. B, TNF (0.25 μg/injection, three times/day × 3 days, n = 2) or PBS was injected over the calvarial bones of wt mice. RNA and protein were extracted directly from calvarial bones. Expression levels of Smurf1, ALP, and OC mRNA and Smurf1 and β-actin protein were examined as described in A. The data show the values of individual mice.