Bone morphogenetic proteins (BMPs)1
play a specific role in osteoblast differentiation in vitro
and bone formation in vivo
). The role of BMPs in postnatal bone formation and maintenance of bone mass has recently been demonstrated in several animal models. In previous studies, we have shown that selective blockage of BMP receptor signaling by over-expression of a dominant-negative type I BMP receptor in osteoblasts inhibits postnatal bone growth and bone formation (3
). Noggin and sclerostin, two glycoproteins, which are produced in bone cells, demonstrate high affinity binding to BMP ligands and prevent the interaction between BMP ligands and their cognate receptors (4
). Noggin has high affinity to bind BMP-2, -4, and -7 (5
and sclerostin has high affinity to bind BMP-5, -6, and -7 (6
). Recently it has been shown that transgenic mice overexpressing noggin or sclerostin transgene in osteoblasts develop an osteopenic/osteoporotic phenotype (8
). However, the mechanism(s), that regulate BMP signaling during osteoblast differentiation are not fully understood.
One of the important regulatory mechanisms by which the activity of BMP signaling proteins is modulated involves ubquitin-mediated proteasomal degradation. The ubiquitin-proteasome proteolytic pathway is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction (9
). The formation of ubiquitin-protein conjugates requires three enzymes that participate in a cascade of ubiquitin transfer reactions: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). The specificity of protein ubiquitination is determined by E3 enzymes, and proteins polyubiquitinated by these enzymes are targeted to undergo degradation primarily by the 26S proteasome (11
Smurf1 is a member of the Hect domain family of E3 ubiquitin ligases and has been found to interact not only with BMP signaling proteins Smad1 and 5 but also the bone-specific transcription factor Runx2/Cbfa1 and type I BMP receptors and to mediate the degradation of these proteins (13
). Recent studies suggest that Smurf1 may also play an important role in osteoblast differentiation (14
). The related E3 ubiquitin ligase Smurf2 interacts with Smad2 and mediates degradation of Smad2 and proteins interacting with Smad2, such as type I TGF-β
receptor and SnoN (17
). It has also been reported that Smurf2 mediates Smad1 degradation (20
). The role of Smurf2 in osteoblast differentiation is currently unknown.
To determine the role of Smurf1 in osteoblast differentiation and bone formation, in the present studies we have utilized a Smurf1 expression plasmid stably transfected into osteoblast precursor 2T3 cells. We also generated transgenic mice in which expression of a Smurf1 transgene is targeted to osteoblasts using the osteoblast-specific type I collagen promoter (21
). We found that expression of Smurf1 inhibits osteoblast differentiation in 2T3 cells. In addition, overexpression of Smurf1 in osteoblasts in vivo
causes a significant reduction in trabecular bone volume and bone formation rates. Moreover, both osteoblast proliferation and differentiation are inhibited in Smurf1 transgenic mice. Our results demonstrate that Smurf1 plays a specific role in regulating BMP signaling, osteoblast differentiation, and bone formation.