Previously we discovered that Smurf1 interacts with bone-specific transcription factor Runx2 and induces Runx2 degradation in osteoblast precursor cells (24
). In the present studies we provide new evidence that the interaction of Smurf1 to the PY motif of Runx2 is not essential for Smurf1-induced Runx2 degradation. Here we demonstrate that Smurf1 can degrade Runx2 in a PY motif-independent manner through formation of a complex that includes Smad6. These findings provide new insights about the mechanism of Smurf1-induced Runx2 degradation.
In the present studies we found that Smad6 interacts with Runx2 in COS and 293 cells, and Smurf1 induces the degradation of PY-mutant Runx2 or Runx3 in COS and C2C12 cells. More importantly, we also demonstrate that endogenous Runx2 protein levels are increased by the shRNA of Smurf1 and Smad6. These results demonstrate that this Smad6-mediated Runx2 degradation is not simply due to the overexpression of Smurf1 or Smad6 in a specific cell line.
Recent studies show that Smad6 has a synergistic effect with Smurf1 on inhibition of BMP signaling. Smad6 binds type I BMP receptors and prevents binding and phosphorylation of Smads 1 and 5 (36
). Smad6 also facilitates Smurf1-induced type I BMP receptor degradation (23
). To determine the role of Smad6 and Smurf1 in chondrocyte maturation, Horiki et al.
) developed Col11-Smad6 and Col11-Smurf1 transgenic mice and demonstrate that Smurf1 enhances the effect of Smad6 on inhibition of chondrocyte differentiation and maturation (37
). Our results provide new evidence that Smad6 mediates Smurf1-induced Runx2 degradation. Because Smad6 and Smurf1 are co-localized in the nucleus as is Runx2, it is likely that Smad6-mediated Runx2 degradation occurs in the 26 S proteasome, which is located in the nucleus of cells.
Hect-domain E3 ligases frequently use adaptor proteins to mediate the degradation of substrate proteins. For example, Smurf2, another member of the Smurf family, induces the protein degradation of SnoN, a transcriptional repressor of transforming growth factor β
signaling, using Smad2 as an adaptor (30
). Similarly, Smurfs 1 and 2 target type I transforming growth factor β
and BMP receptors for degradation using Smads 6 and 7 as adaptor proteins (23
). The E6-AP is a Hect-domain E3 ubiquitin ligase. It binds E6 protein of human Papillomavirus
types 16 and 18 and targets the ubiquitin-proteasome degradation of tumor suppressor p53 (39
Our previous results demonstrated that transgenic mice overexpressing the Smurf1 transgene (Col1a1-Smurf1) in osteoblasts have impaired bone formation and osteoblast activity (25
). More recently, it has been reported that bone mass is increased in adult Smurf1 null mutant mice (40
). Surprisingly, Smurf1 null mutant mice had normal levels of Smads 1 and 5 and Runx2 proteins that have all previously been shown to be targeted by Smurf1 (22
). Because these proteins are also targeted by Smurf2 or other E3 ligases (31
), it is possible that Smurf2 and other E3 ligases may have played a redundant role in the degradation of these proteins in Smurf1 knock-out cells. In the present studies we found that the degradation of Runx2 is not only induced by Smurf1 but also induced by Smurf2 and WWP1. These findings demonstrate that Runx2 degradation involves multiple E3 ligases and is mediated through both PY motif-dependent and -independent mechanisms.
In summary, our findings indicate that Smad6 interacts with Runx2 and mediates Smurf1-induced Runx2 degradation. These results show that Smad6 and Smurf1 coordinately down-regulate Runx2 protein, which may serve as a negative regulatory mechanism for the BMP-Smad-Runx2 signaling pathway.