Smad6 interacts with Runx2 and mediates Smad ubiquitin regulatory factor 1-induced Runx2 degradation

Abstract

Runx2 is a bone-specific transcription factor that plays a critical role in bone development, postnatal bone formation, and chondrocyte maturation. The protein levels of Runx2 are regulated by the ubiquitin-proteasome pathway. In previous studies we discovered that E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1) induces Runx2 degradation in a ubiquitin-proteasome-dependent manner, and Smurf1 plays an important role in osteoblast function and bone formation. In the present studies we investigated the molecular mechanism of Smurf1-induced Runx2 degradation. Smurf1 interacts with the PY motif of substrate proteins, and a PY motif has been identified in the C terminus of the Runx2 protein. To determine whether Smurf1 induces Runx2 degradation through the interaction with the PY motif of Runx2, we created a mutant Runx2 with a PY motif deletion and found that Smurf1 retained some of its ability to induce the degradation of the mutant Runx2, suggesting that Smurf1 could induce Runx2 degradation through an indirect mechanism. Smurf1 has been shown to interact with Smads 1, 5, 6, and 7, and Smads 1 and 5 also interact with Runx2. In the present studies we found that Smads 1 and 5 had no effect on Smurf1-induced Runx2 degradation. Although Smads 6 and 7 bind Smurf1, it is not known if Smads 6 or 7 interacts with Runx2 and mediate Runx2 degradation. We performed immunoprecipitation assays and found that Smad6 but not Smad7 interacts with Runx2. Smad6 enhances Smurf1-induced Runx2 degradation in an ubiquitin-proteasome-dependent manner. These results demonstrate that in addition to its interaction with the PY motif of Runx2, Smurf1 induces Runx2 degradation in a Smad6-dependent manner. Smurf1-induced Runx2 degradation serves as a negative regulatory mechanism for the BMP-Smad-Runx2 signaling pathway.

FIGURE 1

a and b, Smurf1 induces Runx2 degradation in COS cells. a, FLAG-tagged Runx2 (F-Runx2) expression plasmid was co-transfected with different amounts of Smurf1 (0.1, 0.2, and 0.4 μg/dish, 6-cm culture dish) or mSmurf1(CA) (0.4 μg/dish) expression plasmid into COS cells. Smurf1 induced Runx2 degradation in a dose-dependent manner, and mSmurf1 had no effect on Runx2 degradation. WB, Western blot. b, Runx2 reporter construct, 6xOSE2-OC-Luc, was co-transfected with different amounts of Smurf1 (0.1, 0.2, and 0.4 μg/dish) or mSmurf1 expression plasmid (0.4 μg/dish) into COS cells. Smurf1 inhibited Runx2-induced luciferase activity of the Runx2 reporter in a dose-dependent manner, and mSmurf1 had no effect on Runx2-induced reporter activity. *, p < 0.05, one-way analysis of variance followed by Dunnett's test, compared with Runx2 alone (n = 4). c and d, the PY motif is not essential for Smurf1-induced Runx2 degradation. c, amino acid sequence analysis of Runx family members reveals that there is a conserved PY motif among Runx family members, and a mRunx2 expression plasmid with a PY motif deletion, mRunx2(–PY), has been generated. d, transfection of wild-type and mRunx2(–PY) expression plasmids with Smurf1 expression plasmid into COS cells resulted in a degradation of wild-type as well as mRunx2 proteins, although the intensity of the degradation is reduced for the mRunx2(–PY). e, transfection of wild-type and mRunx3(–PY) expression plasmids with Smurf1 expression plasmid into COS cells resulted in a similar result. Smurf1 induced wild-type and mRunx3(–PY) degradation with higher efficiency to wild-type Runx3. f, F-mRunx2 and F-mRunx3 plasmids were co-transfected with or without Smurf1 plasmid into C2C12 cells (0.4 μg/dish). Smurf1 induced mRunx2 and mRunx3 degradation in C2C12 cells.

FIGURE 2

a–d, Runx2 degradation rate is higher in C2C12 cells than in COS cells. a and b, a FLAG-tagged Runx2 expression plasmid was transfected into C2C12 and COS cells. Expressed Runx2 protein was quickly degraded within 48–72 h in C2C12 cells (a) but stable up to 72 h in COS cells (b). WB, Western blot. c and d, FLAG-tagged Runx2 (F-Runx2) expression plasmid was transfected into C2C12 and COS cells, and the pulse-chase assays were performed 24 h after transfection. The autoradiographic results from the pulse-chase experiments indicated that the degradation rates of Runx2 were much lower in COS cells than in C2C12 cells. e, Smad1 had no effect on Smurf1-induced Runx2 degradation. Smad1 expression plasmid was co-transfected with Runx2 and Smurf1 expression plasmids into COS cells. Smad1 had no effect on Runx2 protein levels or Smurf1-induced Runx2 degradation in COS cells. f, Smad6 protein is highly expressed in C2C12 cells. Expression of Smad6 protein was compared in C2C12 and COS cells. Smad6 expression is much higher in C2C12 cells than in COS cells.

FIGURE 3. Interaction of Smad6 with Runx2

a, Myc-tagged Runx2 (M-Runx2) expression plasmid was co-transfected with FLAG-tagged Smad6, Smad7, and mSmurf1(CA) expression plasmids into COS cells. IP assays were performed using an anti-Myc antibody followed by Western blot (WB) analysis using an anti-FLAG antibody. Smad6 and Smurf1 but not Smad7 were co-precipitated with Runx2 in COS cells. b, M-Runx2 was co-transfected with F-Smad6 and F-Smad7 in COS cells. IP assays were performed using an anti-FLAG antibody followed by Western blot analysis using an anti-Myc antibody. Smad6 but not Smad7 was co-precipitated with Runx2 into COS cells. c, Myc-tagged Runx2 and mRunx2(–PY) were co-transfected with F-Smad6 and F-Smad7 into COS cells. Smad6 but not Smad7 was detected by Western blot after Myc-Runx2 or Myc-mRunx2(–PY) proteins were immunoprecipitated by the anti-Myc antibody. d, Myc-tagged Runx2 was co-transfected with FLAG-tagged Smad6 or Smad7 into 293 cells. IP was performed using the anti-FLAG antibody followed by Western blot using the anti-Myc antibody. Only Smad6 interacts with Runx2 in this IP assay in 293 cells.

FIGURE 4

a–c, Smad6 mediates Smurf1-induced Runx2 ubiquitination and proteasomal degradation. a, different amounts of F-Smad6 expression plasmid (0.1, 0.2, and 0.4 μg/dish, 6-cm culture dish) was co-transfected with FLAG-tagged Runx2 (F-Runx2) and Smurf1 expression plasmids (0.5 μg/dish) into COS cells. Smad6 enhanced Smurf1-induced Runx2 degradation. Smad6 itself was also degraded with Runx2 by Smurf1 (sixth through eighth lanes). WB, Western blot. b, F-Runx2, Smurf1 and HA-ubiquitin expression plasmids were co-transfected into COS cells. IP was performed using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody. Smad6 enhanced Smurf1-induced Runx2 ubiquitination. Ub, ubiquitin. c, Runx2 expression plasmid was transfected into COS cells with Smad6 and Smurf1 expression plasmids and then treated with 5 μm proteasome inhibitor proteasome inhibitor 1 (PS-1; fifth and sixth lanes) for 4 h. Smurf1 induced Runx2 degradation which was enhanced by Smad6. In the fourth lane, Runx2 protein was totally degraded by Smad6/Smurf1. Proteasome inhibitor PS-1 significantly reversed the effects of Smurf1 alone or Smurf1/Smad6-induced Runx2 degradation (fifth and sixth lanes). d, Smad6 enhances the inhibitory activity of Smurf1 on Runx2-induced reporter activity. Runx2 reporter, the 6xOSE2-OC-Luc, was co-transfected with Runx2 expression plasmid into COS cells. Runx2 significantly stimulated the reporter activity. Co-transfection of Smurf1 significantly inhibited Runx2-induced reporter activity, and co-transfection of Smad6 slightly inhibited Runx2-induced reporter activity. Transfection of Smad6 significantly enhanced the inhibitory effect of Smurf1. *, p < 0.05, unpaired t test, compared with Runx2 alone (n = 4).

FIGURE 5. shRNA of Smurf1 and Smad6 enhance endogenous Runx2 protein levels

a and b, 2T3 cells were infected with shRNA of Smad6 (a) or Smurf1 (b), and expression of endogenous Smad6 and Smurf1 was examined by Western blot analysis. Infection of shRNA of Smad6 or Smurf1 significantly inhibited the expression of Smad6 (77%) and Smurf1 (81%) in 2T3 cells. c, 2T3 cells were infected separately or together with the retrovirus of shRNA of Smurf1 and Smad6, and changes in endogenous Runx2 protein levels were detected by Western blot analysis. Infection of Smurf1 shRNA increased Runx2 protein levels 1.9-fold, and infection of shRNA of Smurf1 and Smad6 together enhanced Runx2 protein levels 3.7-fold in 2T3 cells. d, retrovirus Smurf1 was infected into 2T3 cells and caused a complete degradation of endogenous Runx2 protein. Infection of shRNA of Smad6 reversed the effect of Smurf1 on Runx2 degradation.

FIGURE 6. Smad6 enhances Smurf2 and WWP1-induced Runx2 degradation

a and b, expression plasmids of Smurf2 (a) and WWP1 (b) were transfected into COS cells with Runx2 in the presence or absence of Smad6 expression plasmid. Smurf2 and WWP1 alone induced Runx2 degradation, and the effect of Smurf2 on Runx2 degradation was weaker compared with that of WWP1. Transfection of Smad6 significantly enhanced Smurf2 (a) and WWP1-induced (b) Runx2 degradation. F-Runx2, FLAG-tagged Runx2. WB, Western blot.