Smurf1 Inhibits Osteoblast Differentiation and Bone Formation in Vitro and in Vivo

Abstract

Bone morphogenetic proteins (BMPs) are required for normal postnatal bone formation and osteoblast differentiation. There is evidence from recent studies that BMP signaling in osteoblasts is controlled by an ubiquitin-proteasome regulatory mechanism involving a cascade of enzymatic reactions. The specificity of protein ubiquitination is determined by E3 ubiquitin ligases, which play a crucial role in defining substrate specificity and subsequent protein degradation by 26S proteasomes. We have examined the role of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1), a member of the Hect domain family of E3 ubiquitin ligases in osteoblast function. Smurf1 has been found to interact with BMP-activated Smad1 and -5 and to mediate degradation of these Smad proteins. Recently we have found that Smurf1 mediates the protein degradation of the osteoblast-specific transcription factor Runx2/Cbfa1. To determine the role of Smurf1 in osteoblast differentiation, in the present studies we transfected a Smurf1 expression plasmid into 2T3 osteoblast precursor cells and found that Smurf1 overexpression inhibits BMP signaling and osteoblast differentiation. To further investigate the role of Smurf1 in bone formation in vivo, we generated transgenic mice in which expression of the epitope-tagged Smurf1 transgene was targeted to osteoblasts using the murine 2.3-kb osteoblast-specific type I collagen promoter. In these transgenic mice, bone formation was significantly reduced during postnatal life. Our results demonstrate for the first time that Smurf1 plays a specific role in osteoblast differentiation and bone formation in vivo. Bone morphogenetic proteins (BMPs) are required for normal postnatal bone formation and osteoblast differentiation. There is evidence from recent studies that BMP signaling in osteoblasts is controlled by an ubiquitin-proteasome regulatory mechanism involving a cascade of enzymatic reactions. The specificity of protein ubiquitination is determined by E3 ubiquitin ligases, which play a crucial role in defining substrate specificity and subsequent protein degradation by 26S proteasomes. We have examined the role of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1), a member of the Hect domain family of E3 ubiquitin ligases in osteoblast function. Smurf1 has been found to interact with BMP-activated Smad1 and -5 and to mediate degradation of these Smad proteins. Recently we have found that Smurf1 mediates the protein degradation of the osteoblast-specific transcription factor Runx2/Cbfa1. To determine the role of Smurf1 in osteoblast differentiation, in the present studies we transfected a Smurf1 expression plasmid into 2T3 osteoblast precursor cells and found that Smurf1 overexpression inhibits BMP signaling and osteoblast differentiation. To further investigate the role of Smurf1 in bone formation in vivo, we generated transgenic mice in which expression of the epitope-tagged Smurf1 transgene was targeted to osteoblasts using the murine 2.3-kb osteoblast-specific type I collagen promoter. In these transgenic mice, bone formation was significantly reduced during postnatal life. Our results demonstrate for the first time that Smurf1 plays a specific role in osteoblast differentiation and bone formation in vivo. Bone morphogenetic proteins (BMPs) 1The abbreviations used are: BMP, bone morphogenetic protein; E3, ubiquitin ligase; Smurf1, Smad ubiquitin regulatory factor 1; Smad, Sma/Mother against dpp; Runx2, Runt domain transcription factor 2; Cbfa1, core-binding factor α1; TGF-β, transforming growth factor β; ALP, alkaline phosphatase; SBE, Smad1 binding element; OC, osteocalcin; dpp, decapentaplegic; BFR, bone formation rates; BrdUrd, 5-bromo-2′-deoxyuridine; αMEM, α minimal essential medium; FCS, fetal calf serum. play a specific role in osteoblast differentiation in vitro and bone formation in vivo (1Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R. Hewick R. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3423) Google Scholar, 2Wozney J.M. Rosen V. Clin. Orthop. 1998; 346: 26-37Crossref PubMed Scopus (557) Google Scholar). 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 (3Zhao M. Harris S.E. Horn D. Geng Z. Nishimura R. Mundy G.R. Chen D. J. Cell Biol. 2002; 157: 1049-1060Crossref PubMed Scopus (156) Google Scholar). 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 (4Groppe J. Greenwald J. Wiater E. Rodriguez-Leon J. Economides A.N. Kwiatkowski W. Affolter M. Vale W.W. Belmonte J.C. Choe S. Nature. 2002; 420: 636-642Crossref PubMed Scopus (438) Google Scholar). Noggin has high affinity to bind BMP-2, -4, and -7 (5Smith W.C. Harland R.M. Cell. 1992; 70: 829-840Abstract Full Text PDF PubMed Scopus (956) Google Scholar) 2M. Zhao, M. Qiao, G. R. Mundy, and D. Chen, unpublished data. and sclerostin has high affinity to bind BMP-5, -6, and -7 (6Winkler D.G. Sutherland M.K. Geoghegan J.C. Yu C. Hayes T. Skonier J.E. Shpektor D. Jonas M. Kovacevich B.R. Staehling-Hampton K. Appleby M. Brunkow M.E. Latham J.A. EMBO J. 2003; 22: 6267-6276Crossref PubMed Scopus (903) Google Scholar, 7Kusu N. Laurikkala J. Imanishi M. Usui H. Konishi M. Miyake A. Thesleff I. Itoh N. J. Biol. Chem. 2003; 278: 24113-24117Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Recently it has been shown that transgenic mice overexpressing noggin or sclerostin transgene in osteoblasts develop an osteopenic/osteoporotic phenotype (8Devlin R.D. Du Z. Pereira R.C. Kimble R.B. Economides N. Jorgetti V. Canalis E. Endocrinology. 2003; 144: 1972-1978Crossref PubMed Scopus (162) Google Scholar, 6Winkler D.G. Sutherland M.K. Geoghegan J.C. Yu C. Hayes T. Skonier J.E. Shpektor D. Jonas M. Kovacevich B.R. Staehling-Hampton K. Appleby M. Brunkow M.E. Latham J.A. EMBO J. 2003; 22: 6267-6276Crossref PubMed Scopus (903) Google Scholar). 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 (9Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1269) Google Scholar, 10Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (7073) Google Scholar). 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 (11Hershko A. Cell. 1983; 34: 11-12Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 12Ciechanover A. Orian A. Schwartz A.L. BioEssays. 2000; 22: 442-451Crossref PubMed Scopus (717) Google Scholar). 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 (13Zhu H. Kavsak P. Abdollah S. Wrana J. Thomsen G.H.A. Nature. 1999; 400: 687-693Crossref PubMed Scopus (704) Google Scholar, 14Zhao M. Qiao M. Oyajobi B. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 15Murakami G. Watabe T. Takaoka K. Miyazono K. Imamura T. Mol. Biol. Cell. 2003; 14: 2809-2817Crossref PubMed Scopus (269) Google Scholar). Recent studies suggest that Smurf1 may also play an important role in osteoblast differentiation (14Zhao M. Qiao M. Oyajobi B. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 16Ying S.X. Hussain Z.J. Zhang Y.E. J. Biol. Chem. 2003; 278: 39029-39036Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). 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 (17Lin X. Liang M. Feng X.H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 18Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar, 19Bonni S. Wang H.R. Causing C.G. Kavsak P. Stroschein S.L. Luo K. Wrana J.L. Nat. Cell Biol. 2001; 3: 587-595Crossref PubMed Scopus (277) Google Scholar). It has also been reported that Smurf2 mediates Smad1 degradation (20Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (438) Google Scholar). 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 (21Rossert J.A. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar). 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. Cell Culture and Transfection—2T3 osteoblast precursor cells were cultured in α minimal essential medium (αMEM) supplemented with 10% fetal calf serum (FCS) (22Chen D. Ji X. Harris M.A. Feng J.Q. Karsenty G. Celeste A.J. Rosen V. Mundy G.R. Harris S.E. J. Cell Biol. 1998; 142: 295-305Crossref PubMed Scopus (350) Google Scholar). FLAG-tagged Smurf1 expression plasmid and pcDNA3 empty vector (1 μg/well) were stably transfected into 2T3 cells in 6-well culture plates using LipofectAMINE plus reagents (Invitrogen). After transfection (48 h), cells were treated with G418 (300 μg/ml) for 3 weeks. Pooled G418-resistant cells were then cloned using a limiting dilution method and expression of Smurf1 mRNA and protein in selected subclones screened. Four Smurf1-positive subclones (2T3/Smurf1) were analyzed for alkaline phosphatase activity (ALP), expression of osteoblast marker genes and mineralized bone nodule formation, and compared with control subclones (2T3/vector). Generation of Col1a1-Smurf1 Transgenic Mice—To generate the Col1a1(2.3)-Smurf1 transgene, the truncated type IB BMP receptor (trBMPR-IB) cDNA at the BamHI site of a Col1a1-trBMPR-IB vector (3Zhao M. Harris S.E. Horn D. Geng Z. Nishimura R. Mundy G.R. Chen D. J. Cell Biol. 2002; 157: 1049-1060Crossref PubMed Scopus (156) Google Scholar) was replaced with a FLAG-tagged full-length Smurf1 cDNA. The transgene was released by KpnI/NarI digestion. Approximately 1 to 2 μl of a solution of purified DNA at a concentration of 2 μg/ml was microinjected into the pronuclei of fertilized one-cell mouse embryos derived from C57BL/6 mice. The injected embryos were reimplanted into B6D2F1 (C57BL/6 x DBA/2) pseudopregnant females. The presence of the transgene in the resulting pups was determined by preparing genomic DNA from the tail tissue and assaying by Southern blot analysis and PCR. Alkaline Phosphatase Activity Assay—Empty vector or Smurf1 expressing-2T3 subclones or primary osteoblasts isolated from wild-type or Col1a1-Smurf1 transgenic mice were plated into 96-well culture plates and grown to ∼60% confluence. The medium was then changed into αMEM with 2% FCS. 48 h after incubation, cells were washed with phosphate-buffered saline twice, and cell lysates were extracted with 0.05% Triton X-100. ALP activity in cell lysates was measured using a Sigma ALP assay kit (Sigma) and normalized by the protein content. Mineralized Bone Nodule Formation Assay—2T3/vector and 2T3/Smurf1 cells and primary osteoblasts isolated from the calvariae of neonatal wild-type and Smurf1 transgenic mice were used for mineralized bone nodule formation assays as described previously (3Zhao M. Harris S.E. Horn D. Geng Z. Nishimura R. Mundy G.R. Chen D. J. Cell Biol. 2002; 157: 1049-1060Crossref PubMed Scopus (156) Google Scholar, 22Chen D. Ji X. Harris M.A. Feng J.Q. Karsenty G. Celeste A.J. Rosen V. Mundy G.R. Harris S.E. J. Cell Biol. 1998; 142: 295-305Crossref PubMed Scopus (350) Google Scholar). The cells were plated in 24-well culture plates at a density of 2 × 104 cells/well and cultured with αMEM supplemented with 10% FCS. When the cells reached confluence (day 0), the medium was changed to αMEM containing 5% FCS, 100 μg/ml ascorbic acid, and 5 mm β-glycerol phosphate with or without 50 ng/ml BMP-2. The medium was changed every other day and fresh reagents were added. After 10-day incubation, von Kossa staining was performed to analyze the formation of mineralized bone nodules. Luciferase Assay—The BMP and TGF-β signaling reporter constructs, 12×SBE-OC-Luc (14Zhao M. Qiao M. Oyajobi B. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar) and p3TP-Lux (23Carcamo J. Zentella A. Massague J. Mol. Cell. Biol. 1995; 15: 1573-1581Crossref PubMed Google Scholar), were transfected into 2T3/vector and 2T3/Smurf1 cells. Cells were incubated for 24 h in the presence or absence of 50 ng/ml BMP-2 or 2 ng/ml TGF-β (R & D Systems, Minneapolis, MN). Cell lysates were extracted 48 h after transfection, and luciferase activity was measured using a Promega luciferase assay kit and normalized by β-galactosidase activity. RNA Analysis—Total RNA was isolated from 2T3/vector and 2T3/Smurf1 cells using RNAzol B solution (Tel-Test Inc., Friendswood, TX). Expression of osteoblast-specific genes was analyzed by Northern analysis (22Chen D. Ji X. Harris M.A. Feng J.Q. Karsenty G. Celeste A.J. Rosen V. Mundy G.R. Harris S.E. J. Cell Biol. 1998; 142: 295-305Crossref PubMed Scopus (350) Google Scholar). Expression of Smurf1 transgene in 2T3/Smurf1 cells and primary osteoblasts isolated from Col1a1-Smurf1 transgenic mice was analyzed by reverse transcriptase-PCR. For reverse transcriptase-PCR analysis, DNase I-treated total RNA was reverse transcribed using oligo(dT). cDNA were amplified using primers specific for the FLAG-Smurf1 transgene. Western Blot Analysis—To determine expression of Smurf1 and to examine Smad1 and Runx2 protein levels in 2T3/Smurf1 cells, cell lysates were extracted from 2T3/vector and 2T3/Smurf1 cells or from osteoblasts isolated from wild-type and transgenic mice, mixed with sample buffer, and run on SDS-PAGE gels (Mini-PROTEIN II Ready gels, Bio-Rad). The proteins were transblotted onto a polyvinylidene difluoride membrane (Bio-Rad) in transblotting buffer (20 mm Tris, 150 mm glycine, 20% methanol, pH 8.0; TBS-T) at 4 °C, 100v for 1 h. The membrane was blocked with 5% dry milk in TBS-T for 1 h at room temperature and incubated with anti-FLAG monoclonal antibody in TBS-T for 2 h at room temperature. Incubation with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Biosciences) was performed at room temperature for 1 h. The membrane was then washed five times with TBS-T for 5 min. Immunostaining was detected using an enhanced chemiluminescence (ECL) system (Amersham Biosciences, and were performed on in transgenic and wild-type mice that been injected with and 2 were (3Zhao M. Harris S.E. Horn D. Geng Z. Nishimura R. Mundy G.R. Chen D. J. Cell Biol. 2002; 157: 1049-1060Crossref PubMed Scopus (156) Google Scholar). of the bone from were in in and with and or for in trabecular were analyzed and in osteoblast and as described previously (3Zhao M. Harris S.E. Horn D. Geng Z. Nishimura R. Mundy G.R. Chen D. J. Cell Biol. 2002; 157: 1049-1060Crossref PubMed Scopus (156) Google Scholar). The other of the bone were in and used for of bone formation or with von Kossa to determine in the bone and in bone bone volume was in the bone mm from the growth plates and as a of total tissue were as the S.E. from three mouse from of transgenic mice and wild-type The bone or and the between two was and and bone formation were (3Zhao M. Harris S.E. Horn D. Geng Z. Nishimura R. Mundy G.R. Chen D. J. Cell Biol. 2002; 157: 1049-1060Crossref PubMed Scopus (156) Google Scholar). was performed using Inc., and staining kit to the was injected into mice 24 h Bone were in 10% and in were and on were in a in two of for 5 and then in a of were for to was used as a signal for the and in the presence of was used as a staining J.Q. Zhang J. M. Horn D. J. Harris S.E. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Smurf1 in determine the of Smurf1 on osteoblast differentiation, subclones of the osteoblast precursor 2T3 cells stably overexpressing wild-type Smurf1 were Four high levels of Smurf1 mRNA and Smurf1 protein were selected and further Four transfected with empty vector were used as The of Smurf1 on ALP expression of osteoblast-specific and mineralized bone nodule formation in these cells were In 2T3/Smurf1 was an reduction in ALP activity compared with 2T3/vector Expression of several osteoblast-specific Runx2, type I and were also in Smurf1 cells To determine the of Smurf1 on osteoblast differentiation, we examined the of Smurf1 on formation of mineralized bone nodules. day in the presence of ascorbic acid, β-glycerol phosphate and BMP-2 2T3/vector subclones bone nodules. In in of 2T3/Smurf1 cells, were bone in the presence of the of BMP-2 that Smurf1 plays a specific role in BMP signaling and osteoblast differentiation. Smurf1 BMP then determined the of Smurf1 on levels of Smad1 and In 2T3/Smurf1 cells, Smad1 and Runx2 levels were significantly reduced compared with 2T3/vector cells a and Runx2 expression is by the signaling the in Runx2 protein to a of reduced mRNA expression and protein degradation. It has been reported that Smurf1 a with which is then from the and targeted to the type I TGF-β receptor for degradation T. M. G. T. K. Imamura T. Miyazono K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, C. G. M. T. Y. Imamura T. Miyazono K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). To determine the of Smurf1 on BMP and TGF-β signaling, we transfected a BMP signaling reporter 12×SBE-OC-Luc (14Zhao M. Qiao M. Oyajobi B. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar) and the TGF-β signaling reporter p3TP-Lux (23Carcamo J. Zentella A. Massague J. Mol. Cell. Biol. 1995; 15: 1573-1581Crossref PubMed Google Scholar) into 2T3/vector and 2T3/Smurf1 cells and treated cells with BMP-2 or In 2T3/Smurf1 cells, BMP signaling was significantly reduced In TGF-β signaling in 2T3/Smurf1 cells that Smurf1 inhibits BMP signaling in osteoblast precursor cells. of into 2T3/Smurf1 cells further inhibited BMP signaling that and Smurf1 BMP signaling by Smurf1 Bone Formation in further determine Smurf1 plays an essential role in bone formation in vivo, we generated transgenic mice in which expression of a FLAG-tagged Smurf1 transgene was targeted to osteoblasts using the 2.3-kb type I collagen promoter (21Rossert J.A. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (214) Google Scholar). The Col1a1-Smurf1 transgenic mice were and and into and expression of the FLAG-Smurf1 transgene in osteoblasts isolated from the transgenic mice was of two of transgenic mice a reduction of in trabecular bone volume and a significant of in bone formation in transgenic mice compared with their wild-type and In addition, ALP staining in trabecular bone was also significantly in Smurf1 transgenic mice There was a significant in osteoblast on bone but not not in Smurf1 transgenic mice. To determine osteoblast proliferation was in Smurf1 transgenic mice, we performed and found a significant in cells on of the calvariae of transgenic mice and In osteoblast as detected by staining was not in Smurf1 transgenic mice not We isolated primary osteoblasts from neonatal transgenic mice and wild-type and examined ALP activity and mineralized bone nodule formation. to in vitro ALP activity was significantly reduced and mineralized bone nodule formation was inhibited in of osteoblasts isolated from transgenic mice compared with from wild-type these results suggest that bone formation found in Smurf1 transgenic mice are to osteoblast proliferation and differentiation. We have demonstrated for the first time that the E3 ubiquitin ligase Smurf1 plays a specific role in bone formation in vivo and that expression of Smurf1 inhibits osteoblast proliferation and differentiation in vitro and in vivo. that these of Smurf1 on osteoblast proliferation and differentiation are to specific of BMP signaling in are with previous that expression of a dominant-negative and Smurf1 enhanced osteoblast differentiation in osteoblast precursor and 2T3 cells (14Zhao M. Qiao M. Oyajobi B. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). is also with a recent that expression of Smurf1 in cells osteoblast differentiation S.X. Hussain Z.J. Zhang Y.E. J. Biol. Chem. 2003; 278: 39029-39036Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). in in also to enhanced of signaling and expression of genes of Wang Y. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). these suggest that Smurf1 plays a specific role in regulating BMP signaling and osteoblast differentiation and bone formation. Our studies were performed using an overexpression The role of Smurf1 in bone formation in vivo to further by overexpressing a dominant-negative Smurf1 in osteoblasts in transgenic mice or by Smurf1 mice, specific to the osteoblast results that Smurf1 plays a specific and role in bone formation in vivo. a in the and the Western and and of for the of of the currently used in the of are bone including as as and related these of bone mass and prevent further bone have not been shown to or bone is a with may have of bone mass at in the at time of a that formation of a of bone is Recently we have that selective of the osteoblast proteasome bone formation in vivo and in vitro Chen D. G. G. M. A. S. Mundy G.R. J. Clin. 2003; PubMed Scopus Google Scholar), that selective of proteolytic degradation have on proteasome may not to with to their and Our demonstrate that the E3 ligase Smurf1 plays specific in several protein for BMP signaling and mediates subsequent proteasomal degradation of these proteins. the E3 ligase Smurf1 as a for the of We Thomsen of and Cell and for Cell and of for the FLAG-Smurf1