E3 Ubiquitin Ligase Smurf1 Mediates Core-binding Factor α1/Runx2 Degradation and Plays A Specific Role in Osteoblast Differentiation

Osteoblast differentiation and bone formation is stimulated by bone morphogenetic protein (BMP)-2 and its downstream signaling molecules Smad1 and -5 and the osteoblast-specific transcription factor core-binding factor α1 (Cbfa1). Proteolytic degradation of Smad1 and Cbfa1 is proteasome-dependent, and intracellular concentrations of Smad1 and Cbfa1 are enhanced by inhibition of the 26 S proteasome. Smad1 degradation is mediated by the E3 ubiquitin ligase Smurf1 (Smad ubiquitin regulatory factor 1), but the specific E3 ligase responsible for Cbfa1 degradation has not been identified. Because Cbfa1 interacts with Smad1, whose degradation is mediated by Smurf1, we examined the effect of Smurf1 on Cbfa1 degradation in osteoblast precursor cells. Smurf1 interacts directly with Cbfa1 and mediates Cbfa1 degradation in a ubiquitin- and proteasome-dependent manner. Because Smurf1 controls the intracellular concentrations of several key molecules in the bone formation cascade, we examined the effect of a mutant form of Smurf1 in osteoblasts and found that expression of mutant Smurf1 markedly enhanced osteoblas tdifferentiation. Smurf1 therefore appears to be an important regulatory factor in osteoblast differentiation and a potential molecular target for identification of bone anabolic agents. Osteoblast differentiation and bone formation is stimulated by bone morphogenetic protein (BMP)-2 and its downstream signaling molecules Smad1 and -5 and the osteoblast-specific transcription factor core-binding factor α1 (Cbfa1). Proteolytic degradation of Smad1 and Cbfa1 is proteasome-dependent, and intracellular concentrations of Smad1 and Cbfa1 are enhanced by inhibition of the 26 S proteasome. Smad1 degradation is mediated by the E3 ubiquitin ligase Smurf1 (Smad ubiquitin regulatory factor 1), but the specific E3 ligase responsible for Cbfa1 degradation has not been identified. Because Cbfa1 interacts with Smad1, whose degradation is mediated by Smurf1, we examined the effect of Smurf1 on Cbfa1 degradation in osteoblast precursor cells. Smurf1 interacts directly with Cbfa1 and mediates Cbfa1 degradation in a ubiquitin- and proteasome-dependent manner. Because Smurf1 controls the intracellular concentrations of several key molecules in the bone formation cascade, we examined the effect of a mutant form of Smurf1 in osteoblasts and found that expression of mutant Smurf1 markedly enhanced osteoblas tdifferentiation. Smurf1 therefore appears to be an important regulatory factor in osteoblast differentiation and a potential molecular target for identification of bone anabolic agents. Bone formation is regulated by a number of growth regulatory factors, key signal transduction molecules, and critical transcription factors. Important among these are the bone morphogenetic proteins (BMPs), 1The abbreviations used are: BMP, bone morphogenetic protein; Cbfa1, Core-binding factor α1; Smad, Sma/Mother against decapentaplegic; Smurf1, Smad ubiquitin regulatory factor 1; PS1, proteasome inhibitor 1; SBE, Smad1 binding element; caBMPR-IB, constitutively active type IB BMP receptor; OC, osteocalcin; ALP, alkaline phosphatase; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin. which promote normal appositional bone growth and induce ectopic bone formation experimentally (1Wozney J.M. Mol. Reprod. Dev. 1992; 32: 160-167Crossref PubMed Scopus (613) Google Scholar). BMP-2 and -4 act through a complex serine-threonine kinase receptor mechanism to activate the signal transduction molecules Smad1 and -5, which in turn activate downstream target genes with other transcription factors. The mechanism of action and regulation of BMP signaling molecules during osteoblast differentiation are not fully defined. Cbfa1 2Other names for Cbfa1/Runx2 are PEBP2αA, AML-3, and Osf-2. is a transcription factor that belongs to the runt-domain gene family and plays an essential role in osteoblast differentiation, bone development, and postnatal bone formation (2Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3647) Google Scholar, 3Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y. Inada M. Sato M. Okamoto R. Kitamura R. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3653) Google Scholar, 4Otto F. Thornell A.P. Crompton T. Denze A. Gilmour K.C. Rosewell I.R. Stamp G.W.H. Beddington R.S.P. Mundlos S. Olsen B.R. Selby P. Owen M. Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2412) Google Scholar, 5Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty M. Gene Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (709) Google Scholar). Its expression and activity are regulated by many bone-derived growth factors, including BMPs (2Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3647) Google Scholar, 6Chen 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 (341) Google Scholar). BMPs may stimulate osteoblast differentiation by activating Cbfa1 through Smad1, as evidenced by reports that Smad1, the downstream effector of BMP signaling, directly interacts with Cbfa1 (7Hanai J.I. Chen L.F. Kanno T. Ohtani-Fujita N. Kim W.Y. Guo W.-H. Imamura T. Ishidou Y. Fukuchi M. Shi M.J. Stavnezer J. Kawabata M. Miyazono K. Ito Y. J. Biol. Chem. 1999; 274: 31577-31582Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar), but precise signaling mechanisms remain unclear. The activities of signaling proteins and transcription factors are regulated at both the transcriptional and post-translational levels. Recent reports (8Zhu H. Kavsak P. Abdollah S. Wrana J. Thomsen G.H.A. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar, 9Zhang Y. Chang C. Gehling D.J. Hemmati-Brivanlou A. Derynck R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 974-979Crossref PubMed Scopus (427) Google Scholar, 10Tintut Y. Parhami F. Le V. Karsenty G. Demer L.L. J. Biol. Chem. 1999; 274: 28875-28879Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) demonstrate that both Smad1 and Cbfa1 undergo ubiquitin-proteasome-mediated degradation. Protein ubiquitination involves a cascade of enzymatic reactions catalyzed by the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin ligases (11Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1459) Google Scholar), which play a crucial role in defining substrate specificity and subsequent protein degradation by the 26 S proteasomes. Smurf1 is a member of the Hect family of E3 ubiquitin ligases and has been found to interact with the BMP-activated Smad1 and -5, thereby triggering their ubiquitination and degradation (8Zhu H. Kavsak P. Abdollah S. Wrana J. Thomsen G.H.A. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar). Hect domain proteins represent a major subclass of E3 ligases and contain a conserved cysteine located at the carboxylterminal of the Hect domain that is capable of forming a thioester bond with ubiquitin (8Zhu H. Kavsak P. Abdollah S. Wrana J. Thomsen G.H.A. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar, 12Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar). Another motif often found in the Hect family of E3 ligase is the WW domain, which contains two highly conserved tryptophans and a conserved proline in an ∼30-amino acid region (8Zhu H. Kavsak P. Abdollah S. Wrana J. Thomsen G.H.A. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar, 13Rotin D. Curr. Top. Microbiol. Immunol. 1998; 228: 115-133PubMed Google Scholar). The WW domains have a preference for binding to small proline-rich sequences, PPXY motifs, and different WW domains possess different substrate specificity. Although Cbfa1 has been reported to be degraded through the ubiquitin-proteasome pathway (10Tintut Y. Parhami F. Le V. Karsenty G. Demer L.L. J. Biol. Chem. 1999; 274: 28875-28879Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), the specific E3 ubiquitin ligase for Cbfa1 has not been identified. In the present studies, we examined the relationship between Smad1, Cbfa1, and Smurf1 in osteoblast precursor cells. Smad1 interacts directly with Cbfa1 in these cells, and both Smad1 and Cbfa1 are required for BMP-2 signaling. The intracellular concentrations of both Smad1 and Cbfa1 are dependent on osteoblast proteasomal function. Smurf1 mediates Cbfa1 degradation in a ubiquitin proteasome-dependent manner and therefore is a powerful regulator of osteoblast differentiation. Expression Plasmids—Myc-tagged Smad1 expression plasmid was obtained from Dr. R. Nishimura (Department of Biochemistry, Osaka University, Osaka, Japan), and FLAG-tagged Smurf1 and mutant Smurf1 (C710A) cDNA plasmids in SK vector were obtained from Dr. G. Thomsen and cloned into pcDNA3 expression vector. Cbfa1 cDNA was amplified by RT-PCR based on mouse Cbfa1 cDNA sequences (GenBank™ accession number AF010284) using RNA extracted from 2T3 osteoblast precursor cells and then cloned into p3×FLAG-CMV vector (Sigma). The 6×OSE2-OC-pGL3 reporter construct was generated by PCR as described by Ducy et al. (2Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3647) Google Scholar). The constitutively active type IB BMP receptor (caBMPR-IB) expression plasmid was obtained from Dr. J. Wrana (Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada). The HA-ubiquitin expression plasmid was obtained from Dr. X. Cao (Department of Pathology, University of Alabama at Birmingham, Alabama). Cell Culture and Transfections—C2C12 cells were cultured in Dulbecco's modified Eagle's medium, and 2T3 osteoblast precursor cells were cultured in α-minimal essential medium supplemented with 8% fetal calf serum. The cDNA expression plasmids were transiently transfected into these cells using LipofectAMINE Plus reagents (Invitrogen) in a 10-cm-diameter culture dish for immunoprecipitation assay, 6-well culture plates for Western blotting analysis, and 24-well plates for the luciferase assay. 5, 1, and 0.2 μg of expression plasmid were used for these transfections. After transfection (48 h), cells were lysed with lysis buffer. In proteasome inhibitor experiments, transfected cells were incubated with the proteasome inhibitors for 2, 6, or 24 h, the proteasome inhibitor was removed, and the cells were then cultured an additional 24 h. Immunoprecipitation and Immunoblotting—Expression plasmids were transfected into C2C12 cells separately or together for immunoprecipitation. In Western analysis, the total amounts of transfected plasmids in each group were equalized by addition of empty vector cDNA. 48 h after transfection, cells were washed three times with phosphate-buffered saline and solubilized in lysis buffer (150 mm NaCl, 1% Triton X-100, 0.5% doc, and 50 mm Tris buffer, pH 7.5). For Western blotting, 0.1% SDS was included in the lysis buffer. The protease inhibitors aprotinin (10 μg/ml), leupeptin (10 μg/ml), and phenylmethylsulfonyl fluoride (1 mm) were added to the lysis buffer. Cell lysates were centrifuged for 10 min at 4 °C at 10,000 × g and incubated with anti-Myc antibody for 4 h at 4 °C, followed by immunoprecipitation with protein G-agarose (Roche Applied Science) at 4 °C overnight. Immunoprecipitates were washed with lysis buffer five times, added to 1× reducing buffer containing 0.5 m β-mercaptoethanol, and boiled for 3 min. The immunoprecipitation and Western blotting samples were separated by SDS-PAGE, transferred to nitrocellulose membrane, immunoblotted with anti-FLAG or anti-Myc antibody, and visualized with horseradish peroxidase-coupled anti-mouse IgG antibody (Amersham Biosciences) with an enhancement by ECL detection kits (Amersham Biosciences). A monoclonal antibody against human Smad1 and a polyclonal antibody against human Smurf1 were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. A polyclonal antibody against human Cbfa1 was purchased from Oncogene Research Product, Cambridge, MA. Luciferase Assay—Oligonucleotides containing 6 copies of Cbfa1 response element, OSE2, (6×OSE2) and 12 copies of Smad1 binding element (12×SBE) were synthesized in the DNA laboratory at The University of Texas Health Science Center at San Antonio. The oligonucleotides were cloned in front of the osteocalcin basal promoter (–155/+1), which was amplified by RT-PCR, and cloned in pGL3 vector. C2C12 and 2T3 cells were co-transfected with Cbfa1 expression plasmid and 6×OSE2-OC-pGL3 reporter plasmid in the presence or absence of Smurf1 or mutant Smurf1 expression plasmid. caBMPR-IB and Smad1 expression plasmids were co-transfected with 12×SBE-OC-luc or 12×SBE-SV40-luc in the presence or absence of mutant Cbfa1. Cell lysates were extracted 48 h later, and luciferase activity was measured and normalized by β-galactosidase activity. Alkaline Phosphatase (ALP) Activity and Osteocalcin Production— C2C12 and 2T3 cells were plated into 6-well culture plates grown to 60% confluency, transfected transiently with Smurf1 or mutant Smurf1 expression plasmids using LipofectAMINE Plus reagents (Invitrogen), or treated with proteasome inhibitors. 48 h after transfection, the medium was collected; the cells were washed twice with ice-cold phosphate-buffered saline, 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 osteocalcin in the medium was measured with a mouse osteocalcin immunoradiometric assay (IRMA) kit (Immutopics, Inc., San Clemente, CA). Bone inhibitor was the bone of the of D. Harris M.A. G. Feng Mundy G.R. Harris S.E. 1997; PubMed Scopus Google Scholar). of phosphate-buffered or 1, and for were after and were The were in between the and through and in were and with and bone was using an Inc., to a and Smurf1 Cbfa1 in Cbfa1 interacts with Smad1 protein (7Hanai J.I. Chen L.F. Kanno T. Ohtani-Fujita N. Kim W.Y. Guo W.-H. Imamura T. Ishidou Y. Fukuchi M. Shi M.J. Stavnezer J. Kawabata M. Miyazono K. Ito Y. J. Biol. Chem. 1999; 274: 31577-31582Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar) whose degradation is mediated by Smurf1 (8Zhu H. Kavsak P. Abdollah S. Wrana J. Thomsen G.H.A. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar), we examined the effect of Smurf1 on Cbfa1 degradation in precursor C2C12 cells and osteoblast precursor 2T3 cells. FLAG-tagged Cbfa1 expression plasmid was co-transfected with different amounts of Smurf1 expression plasmid into C2C12 and 2T3 cells. Expression of Smurf1 of Cbfa1 in a manner Expression of an Hect domain E3 with Cbfa1 effect on the degradation of Cbfa1 not that Cbfa1 degradation is a specific effect of the Smurf1 mediates Cbfa1 2T3 cells were transfected with Smurf1 expression plasmid. Expression of Cbfa1 protein was by Western using an antibody against human Cbfa1. of Smurf1 Cbfa1 protein in 2T3 cells that Smurf1 mediates degradation of Cbfa1 degradation of Cbfa1 is we co-transfected Smurf1, Cbfa1, and ubiquitin expression plasmids in C2C12 cells. After the Cbfa1 a Cbfa1 protein was that Cbfa1 degradation is degradation of Cbfa1 is proteasome-dependent, we treated transfected C2C12 cells with for h. Expression of Smurf1 in total degradation of Cbfa1, an effect by with were obtained different proteasome was used not demonstrate that Cbfa1 degradation is Smurf1 interacts with Cbfa1, Cbfa1, and mutant Smurf1 expression plasmids were co-transfected into C2C12 cells. Because is that Smurf1 mediates the degradation of proteins with which interacts (8Zhu H. Kavsak P. Abdollah S. Wrana J. Thomsen G.H.A. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar), we to the protein complex with Smurf1 using an expression plasmid of the mutant of Smurf1 as described by et al. (8Zhu H. Kavsak P. Abdollah S. Wrana J. Thomsen G.H.A. Nature. 1999; 400: 687-693Crossref PubMed Scopus (688) Google Scholar). proteins in and Western we found that Cbfa1 with A with the WW domain of Smurf1 was found in the of Cbfa1, that Smurf1 directly Cbfa1 and mediates its degradation. Cbfa1 degradation in Cbfa1, we examined the of Cbfa1 to activate a specific reporter P. Karsenty G. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar), in C2C12 cells. A Cbfa1 expression plasmid and reporter construct were co-transfected into C2C12 cells in the presence and absence of Smurf1 or Expression of Cbfa1 activity of of Smurf1 with Cbfa1 luciferase activity of reporter In of in effect on luciferase activity of the reporter gene demonstrate that Smurf1 mediates Cbfa1 degradation and to a in Cbfa1 activity. was effect of on luciferase activity of the reporter gene may be of the reporter gene by Cbfa1 Because has been reported that Cbfa1 degradation is in osteoblasts (10Tintut Y. Parhami F. Le V. Karsenty G. Demer L.L. J. Biol. Chem. 1999; 274: 28875-28879Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), in the present we treated C2C12 cells with an that and examined expression of Smurf1, Cbfa1 and the effect of on Cbfa1 degradation. Although Smurf1 Cbfa1 protein was by was in cells transfected with that Smurf1 may be in the Cbfa1 degradation. Smurf1 Smad1 in Smurf1 has been to Smad1 degradation in cells transfected with a Smad1 expression the of Smurf1 on Smad1 degradation in and in degradation of Smad1 protein by mechanism in have not been In the present studies, we the effect of Smurf1 on Smad1 degradation in osteoblast precursor cells and examined the regulatory of proteasome inhibitors on Smad1 examined the effect of Smurf1 on protein of Smad1 in C2C12 and 2T3 cells and found that Smurf1 mediates Smad1 degradation in a manner The effect of Smurf1 on Smad1 degradation is the mutant of Smurf1 (C710A) on Smad1 degradation and the proteasome inhibitor the effect of Smurf1 on Smad1 degradation in a manner were other different proteasome inhibitors as PS1, and were not the of proteasome inhibitors on the protein of Smad1, the C2C12 cells were treated with different proteasome inhibitors for 6 h. The with proteasome inhibitors Smad1 that Smad1 is ubiquitin-proteasome regulation in Cbfa1 for of BMP-2 has been reported to interact with Smad1, but of the mechanism of these two signaling molecules have not been defined. we examined Cbfa1 is required for BMP signaling by the of Cbfa1 with Smad1 in osteoblast precursor cells. Cbfa1 and Smad1 expression plasmids were co-transfected into C2C12 and 2T3 cells. of Cbfa1 and Smad1 was in both cells the role of Cbfa1 in BMP signaling, we generated copies of Smad1 response element (12×SBE) and cloned of an osteocalcin and a basal of BMP-2 or expression of caBMPR-IB stimulated luciferase activity of the reporter but not the reporter Because the osteocalcin basal promoter contains of the Cbfa1 response element we examined binding of Cbfa1 to response element in the osteocalcin basal promoter is required for BMP-2 signaling. A mutant Cbfa1 expression plasmid was generated as described by Ducy et al. P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty M. Gene Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (709) Google Scholar), and C2C12 cells were transfected with caBMPR-IB expression plasmid and the reporter construct with or Expression of the effect of caBMPR-IB on the reporter caBMPR-IB or BMP-2 on the osteocalcin basal promoter not the role of Cbfa1 in BMP signaling, the in the osteocalcin basal promoter was The reporter with its response to BMP-2 or caBMPR-IB that Cbfa1 is required for BMP-2 and Smad1 to activate downstream target genes in of Smurf1 in Osteoblast the of Smurf1 in osteoblast differentiation, a Smurf1 or expression plasmid was transfected into C2C12 cells, and ALP activity and osteocalcin were Expression of Smurf1 ALP activity as as osteocalcin in C2C12 cells not In transfection of basal as as ALP activity and osteocalcin in these cells 5, a and were obtained the expression plasmids were transfected into 2T3 cells not that Smurf1 mediates Cbfa1 and Smad1 degradation and of Cbfa1 and in a to the degradation of Cbfa1 and a between Smad1 degradation and osteoblast differentiation, we in Smad1 protein after C2C12 and 2T3 cells were transfected with mutant Smurf1 or treated with proteasome inhibitors. found that proteasome inhibitors Smad1 of a in protein of of Smad1 enhanced ALP activity and osteocalcin in C2C12 cells 5, a and that enhanced Smad1 by or proteasome inhibitors promote osteoblast differentiation. Osteoblast and Bone the role of proteasome inhibitors in osteoblast and bone we examined the of PS1, a proteasome on ALP activity and the BMP signaling reporter in C2C12 cells and on bone formation in in found that in C2C12 cells, with ALP activity and luciferase activity of the reporter in a manner. concentrations of 1, and were for into the of The were after the and were for bone formation 6, and were obtained a proteasome was in not these proteasome inhibitors stimulated bone formation in and I.R. Chen D. G. M. Harris S.E. G. A. Kim S. Mundy G.R. J. PubMed Scopus Google Scholar). demonstrate that inhibition of Smurf1 and proteasome degradation to osteoblast and that of the mechanism of proteasome inhibitor on bone formation may be to regulation of intracellular protein of Cbfa1 and have for the that E3 ubiquitin ligase Smurf1 mediates Cbfa1 degradation through the ubiquitin-proteasome Cbfa1 is required for BMP-2 signaling, and expression of mutant Smurf1 osteoblast differentiation. that Smurf1 plays an important role in osteoblast differentiation by Cbfa1 and Smad1 function. Cbfa1 is a critical transcription factor in osteoblast differentiation and (2Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3647) Google Scholar). BMPs have been to stimulate osteoblast differentiation in and in (1Wozney J.M. Mol. Reprod. Dev. 1992; 32: 160-167Crossref PubMed Scopus (613) Google Scholar), in by Cbfa1 expression (2Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3647) Google Scholar, 6Chen 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 (341) Google Scholar) through with Smad1 and Cbfa1 (7Hanai J.I. Chen L.F. Kanno T. Ohtani-Fujita N. Kim W.Y. Guo W.-H. Imamura T. Ishidou Y. Fukuchi M. Shi M.J. Stavnezer J. Kawabata M. Miyazono K. Ito Y. J. Biol. Chem. 1999; 274: 31577-31582Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). Smad1 is a downstream of BMP and plays a role in BMP receptor signaling T. Abdollah S. M. Wrana Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In the present studies, we found that Cbfa1 is required to activate BMP-2 signaling. has been reported P. G. M. H. J. Bone 2001; Google Scholar, D. X. H. Y. D.J. M. K. V. J. Bone PubMed Scopus Google Scholar) that Smad1 and Cbfa1 binding in the of several together with that Smad1 and Cbfa1 downstream target genes in Expression of mutant Smurf1 Cbfa1 degradation in C2C12 cells, that Smurf1 may be in Cbfa1 degradation by a protein kinase proteasomal degradation of protein in osteoblasts Cell 1998; PubMed Scopus Google Scholar), and has been reported that the anabolic effect of signaling V. Ducy P. Mol. PubMed Scopus Google Scholar). that may induce Cbfa1 degradation signaling and that Smurf1 is in to be signal transduction and gene cells of signaling proteins and transcription factors during Smurf1 may degradation of the protein but is required for Because Smurf1 mediates degradation of two critical proteins in the BMP signaling pathway and of Smurf1 osteoblast differentiation and we that Smurf1 plays an important role in bone formation in has been that in to enhanced of signaling and downstream target gene expression Y. Dev. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that Smurf1 may be a critical protein Cbfa1 and Smad1 osteoblast differentiation and bone Dr. Wrana (Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, for the constitutively active and Smad1 expression Dr. Thomsen (Department of and Cell Biology and for Cell and University of for the and mutant expression Dr. Cao (Department of Pathology, University of Alabama at Birmingham, for the HA-ubiquitin expression and Dr. (Department of Research for the expression vector.

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.