Splicing Factor 3b Subunit 4 Binds BMPR-IA and Inhibits Osteochondral Cell Differentiation

Abstract

Bone morphogenetic protein (BMP)-2/4 play critical roles in early embryogenesis and skeletal development. BMP-2/4 signals conduct into cells via two types of serine/threonine kinase receptors, known as BMPR-I (IA and IB) and BMPR-II. Here we identified splicing factor 3b subunit 4 (SF3b4) as a molecule that interacts with BMPR-IA, using a yeast two-hybrid screening with a human fetal brain cDNA library. Co-immunoprecipitation/immunoblot analysis confirmed their interaction in mammalian cells. By separation of the cell components, SF3b4 was present in the cell membrane fraction with BMPR-IA as well as in the nucleus. Overexpression of SF3b4 inhibited BMP-2-mediated osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively, and the endogenous expression level of SF3b4 decreased during differentiation in ATDC5 cells. By reporter gene assay, SF3b4 suppressed Id reporter gene activity, specific to the Smad1/5/8 pathway, but not TGFβ-mediated reporter gene activity. Biotin labeling of the cell surface proteins followed by their immunoblot revealed that SF3b4 decreased the cell surface BMPRI-A levels. Further analysis by molecular modeling of the intracellular domain of BMPR-IA, coupled with binding studies of its several mutants, indicated that the site(s) for SF3b4 binding is not directly associated with the C-terminal lobe and the activation segment. Taken together, these results suggest that SF3b4, known to be localized in the nucleus and involved in RNA splicing, binds BMPR-IA and specifically inhibits BMP-mediated osteochondral cell differentiation. Bone morphogenetic protein (BMP)-2/4 play critical roles in early embryogenesis and skeletal development. BMP-2/4 signals conduct into cells via two types of serine/threonine kinase receptors, known as BMPR-I (IA and IB) and BMPR-II. Here we identified splicing factor 3b subunit 4 (SF3b4) as a molecule that interacts with BMPR-IA, using a yeast two-hybrid screening with a human fetal brain cDNA library. Co-immunoprecipitation/immunoblot analysis confirmed their interaction in mammalian cells. By separation of the cell components, SF3b4 was present in the cell membrane fraction with BMPR-IA as well as in the nucleus. Overexpression of SF3b4 inhibited BMP-2-mediated osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively, and the endogenous expression level of SF3b4 decreased during differentiation in ATDC5 cells. By reporter gene assay, SF3b4 suppressed Id reporter gene activity, specific to the Smad1/5/8 pathway, but not TGFβ-mediated reporter gene activity. Biotin labeling of the cell surface proteins followed by their immunoblot revealed that SF3b4 decreased the cell surface BMPRI-A levels. Further analysis by molecular modeling of the intracellular domain of BMPR-IA, coupled with binding studies of its several mutants, indicated that the site(s) for SF3b4 binding is not directly associated with the C-terminal lobe and the activation segment. Taken together, these results suggest that SF3b4, known to be localized in the nucleus and involved in RNA splicing, binds BMPR-IA and specifically inhibits BMP-mediated osteochondral cell differentiation. Bone morphogenetic proteins (BMPs) 2The abbreviations used are: BMP, bone morphogenetic protein; TGFβ, transforming growth factor-β; ICD, intracellular domain; SF3b4, splicing factor 3b subunit 4; 3AT, 3-amino-1,2,4-triazole; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; ALP, alkaline phosphatase; RRM, RNA recognition motif; TβR-I, TGFβ receptor I; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; WT, wild type; HA, hemagglutinin; SC, synthetic complete; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; RT, reverse transcription. 2The abbreviations used are: BMP, bone morphogenetic protein; TGFβ, transforming growth factor-β; ICD, intracellular domain; SF3b4, splicing factor 3b subunit 4; 3AT, 3-amino-1,2,4-triazole; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; ALP, alkaline phosphatase; RRM, RNA recognition motif; TβR-I, TGFβ receptor I; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; WT, wild type; HA, hemagglutinin; SC, synthetic complete; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; RT, reverse transcription. are members of the transforming growth factor-β (TGFβ) superfamily, which is implicated in cellular functions, including proliferation, apoptosis, and adhesion (1Zhang J. Li L. Dev. Biol. 2005; 284: 1-11Crossref PubMed Scopus (174) Google Scholar, 2Chen D. Zhao M. Mundy G.R. Growth Factors. 2004; 22: 233-241Crossref PubMed Scopus (1676) Google Scholar). BMP-2 and -4 play critical roles in chondrogenesis and skeletal development (3Hogan B.L. Curr. Opin. Genet. Dev. 1996; 6: 432-438Crossref PubMed Scopus (655) Google Scholar, 4Wan M. Cao X. Biochem. Biophys. Res. Commun. 2005; 328: 651-657Crossref PubMed Scopus (310) Google Scholar, 5Tsumaki N. Yoshikawa H. Cytokine Growth Factor Rev. 2005; 16: 279-285Crossref PubMed Scopus (87) Google Scholar). In the early stages, BMP-2/4, expressed in specific areas of the limb bud, are essential for prechondrogenic condensations (6Pizette S. Niswander L. Dev. Biol. 2000; 219: 237-249Crossref PubMed Scopus (247) Google Scholar), and in the late stages, BMP-2, expressed in the growth plate, accelerates longitudinal bone growth by stimulating chondrocyte proliferation and hypertrophy (7De Luca F. Barnes K.M. Uyeda J.A. De-Levi S. Abad V. Palese T. Mericq V. Baron J. Endocrinology. 2001; 142: 430-436Crossref PubMed Scopus (123) Google Scholar). BMP-mediated signals conduct into the cell via two types of serine/threonine kinase receptors known as BMP receptor type I (IA and IB) and type II receptors (8Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (443) Google Scholar). With regard to BMP-2/4 signaling, three structurally related type I receptors, BMPR-IA (also known as ALK3), BMPR-IB (also known as ALK6), and ActRIA (also known as ALK2) and one type II receptor, BMPR-II, have been identified. When the ligand interacts with the receptor complex, BMPR-II transphosphorylates BMPR-I at the GS box to activate its kinase. BMPR-I activation induces the phosphorylation of transiently associated Smad1/5/8. These Smads then form heteromeric complexes with Smad4, which is then translocated into the nucleus. Once in the nucleus, they regulate the transcription of specific target genes in concert with various DNA-binding proteins, co-activators, or co-repressor proteins. Although the Smad pathway is widely represented in most of the cell types and tissues, additional pathways, including those of MAPKs (ERK1/2, p38 MAPK, SAPK/JNK) and phosphatidylinositol 3-kinase (4Wan M. Cao X. Biochem. Biophys. Res. Commun. 2005; 328: 651-657Crossref PubMed Scopus (310) Google Scholar) are activated following treatment with BMPs. Recently, a variety of molecules that modulate TGFβ-mediated signal transduction pathways have been identified, using yeast two-hybrid systems with Smads and the TGFβ receptor as bait proteins. These include NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) (9Kuratomi G. Komuro A. Goto K. Shinozaki M. Miyazawa K. Miyazono K. Imamura T. Biochem. J. 2005; 386: 461-470Crossref PubMed Scopus (164) Google Scholar), Tiul1 (TGIF-interacting ubiquitin ligase 1) (10Seo S.R. Lallemand F. Ferrand N. Pessah M. L'Hoste S. Camonis J. Atfi A. EMBO J. 2004; 23: 3780-3792Crossref PubMed Scopus (126) Google Scholar), WWP1 (WW domain-containing protein) (11Komuro A. Imamura T. Saitoh M. Yoshida Y. Yamori T. Miyazono K. Miyazawa K. Oncogene. 2004; 23: 6914-6923Crossref PubMed Scopus (157) Google Scholar), and PIASy (12Imoto S. Sugiyama K. Muromoto R. Sato N. Yamamoto T. Matsuda T. J. Biol. Chem. 2003; 278: 34253-34258Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) that bind Smad7; Dvl-1, Erbin, and Par-3 (13Warner D.R. Pisano M.M. Roberts E.A. Greene R.M. FEBS Lett. 2003; 539: 167-173Crossref PubMed Scopus (51) Google Scholar) that bind Smad3; and Daxx (14Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell Biol. 2001; 3: 708-714Crossref PubMed Scopus (299) Google Scholar), TRAP-1 (TGFβR-I associated protein-1) (15Charng M.J. Zhang D. Kinnunen P. Schneider M.D. J. Biol. Chem. 1998; 273: 9365-9368Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and STRAP (serine-threonine kinase receptor-associated protein) (16Datta P.K. Chytil A. Gorska A.E. Moses H.L. J. Biol. Chem. 1998; 273: 34671-34674Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) that bind TGFβ receptors. In contrast, only a few molecules, such as BRAM1 (BMP-receptor associated molecule 1) (17Kurozumi K. Nishita M. Yamaguchi K. Fujita T. Ueno N. Shibuya H. Genes Cells. 1998; 3: 257-264Crossref PubMed Scopus (43) Google Scholar) have been identified that bind BMP receptors. Since BMP transduces MAPKs as well as Smad1/5/8, there may be specific molecules that mediate those signaling pathways. In this study, we attempted to identify these intervening molecules specific to BMP-mediated signal transduction pathways, using a yeast two-hybrid system with a BMPR-IA intracellular domain (ICD) as a bait protein, and found that a molecule previously reported as splicing factor 3b subunit 4 (SF3b4) strongly binds the ICD but not its constitutively active form. In mammalian cells, a proportion of SF3b4 was present together with BMPR-IA in the cell membrane fraction, and co-immunoprecipitation/immunoblot analysis demonstrated their direct interaction. Overexpression of SF3b4 inhibited osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively. A reporter gene assay demonstrated that SF3b4 inhibits the Smad1/5/8-specific Id reporter gene activity. The levels of cell surface BMPR-IA in SF3b4-overexpressing cells were substantially decreased. We further performed molecular modeling of BMPR-IA, which provided insight into the sites of the interaction, although it was not determined. These results suggest that SF3b4 regulates osteochondral differentiation by interacting with BMPR-IA and down-regulating its cell surface levels. Yeast Two-hybrid Screening—To screen the molecules that bind the BMPR-IA ICD, a ProQuest™ two-hybrid system with Gateway™ technology and a ProQuest™ human fetal brain cDNA were bait cDNA BMPR-IA ICD and the BMPR-IA constitutively active form ICD were using the and into by Gateway™ with and reverse of BMPR-IA ICD and its constitutively active form with a at the T. were used as to cDNA of SF3b4 and its are in The were into yeast expression The bait and cDNA were into a yeast to the Yeast were and the synthetic and in were and for the following that were the and for were then further for the expression of three reporter genes and by growth and growth and of to in the of were and into cells using the the target cDNA were and their cDNA was using for of of SF3b4 for yeast two-hybrid assay using with Gateway™ for in mammalian for in mammalian in a Cell and and cells were in Dulbecco's modified Eagle's fetal bovine and C2C12 cells in ATDC5 cells were in the of chondrocyte and was to the cells. and was and MAPK, p38 MAPK, and were Cell The with were of SF3b4 and its were by the into H. N. K. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) using and or using the sites as indicated in cells were with the expression as using to the the cells were in and were and then by of the was to and the was for with and of protein were in the to a and to a The membrane was with in and were used as the in The signal was using was performed with to the immunoblot MAPKs and Smad1/5/8, following treatment with BMP-2 for the indicated at cells were in and of was performed as of the were to with was at three with the of SF3b4 the BMPR-IA levels the cell BMPRI-A were by these cells were with or as the cell were one for and the for direct immunoblot the cell surface proteins were with the cells were with phosphate-buffered and and for two at 4 with in and the cells were and for at 4 in and at for was for with at 4 The were to immunoblot analysis using of the were and to immunoblot analysis using and to SF3b4 and BMPR-IA, respectively. Cell by cells were with together with or as the were with in with Cell were with a The was then to and and then The were at 4 and the was The the was and at for at 4 the was and with the of the The at the of the was used as the The was a and and at for at 4 The fraction in the was to and at 4 The was used as the membrane and membrane were to and and were used to these of the nucleus and cell respectively. for the assay, C2C12 cells were a at a of in growth the was with and the indicated the was and cells were with of and in was to and cells were for at of the cell was by of and then of as was The was well and in a at for followed by the at analysis of was performed as previously T. Yamaguchi A. M. N. T. V. A. T. J. Cell Biol. PubMed Scopus Google Scholar). cells were at in as the was with BMP-2 the cells were for with at with PBS, the cells were in and at cells were at in cells were with PBS, with for and with in was with and were levels were by the in for at The at was using a the was ATDC5 cells using a and cDNA was with the system The SF3b4 and their were using The a reporter at the and a at the The of the and are as reverse and for reverse and for and reverse and for was with The of and in were used as The reverse transcription was to the and analysis were performed to the were performed in to the of Smad4, and were T. T. M. T. T. N. R. Genes Cells. PubMed Scopus Google Scholar) and S. P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) were T. and P. respectively. C2C12 cells were in in the growth was with was performed using a In cells were with Cell were and for using a reporter assay system The reporter gene for BMP-2/4 and TGFβ were performed as previously using and R. M. K. J. M. A. Y. A. H. M. M. 2004; PubMed Scopus Google Scholar). of the cells with TGFβ not the of the reporter gene for were by analysis was performed using modeling of BMPR-IA of human BMPR-IA ICD was by A. R. F. A. Rev. Biophys. 2000; PubMed Scopus Google Scholar) using the of TGFβ receptor ICD as a The of BMPR-IA ICD was by G. 1996; PubMed Scopus Google Scholar) and R. D. PubMed Scopus Google Scholar). of of BMPR-IA of the BMPR-I of the was performed using the of human BMPR-IA ICD as the The were using and the of ICD of the BMPR-I was the to their were into as and and and 4 and and In the of human BMPR-IA and BMPR-IB we to were the of of the in BMPR-I (IA and IB) was we that the was and it was not used in the following The were the of BMPR-IA of a with for molecules that with BMPR-IA, we yeast a human fetal brain cDNA with BMPR-IA ICD and its constitutively active form as We by screening with BMPR-IA ICD, and of were identified as splicing factor 3b subunit 4 was identified as and the to be SF3b4 of the screening including the the plate, growth and and the plate, interaction. The interaction with BMPR-IA was further confirmed using a SF3b4 The the but not of the not The to bind to BMPR-IA ICD was confirmed using a With the BMPR-IA constitutively active form ICD as the screening the following SF3b4 is a protein involved in splicing of a of the in the P. R. Genes Dev. PubMed Scopus Google Scholar). molecule two well RNA recognition at the and a and at the the interaction BMPR-IA and SF3b4, SF3b4 were as and for their to with the BMPR-IA ICD in a yeast two-hybrid and BMPR-IA, but and that a of and the is for the interaction We further SF3b4 binds BMPR-IB and found that SF3b4 not bind the ICD of BMPR-IB in the yeast two-hybrid system not SF3b4 with BMPR-IA in we a in mammalian cells, using a SF3b4 and several with at the cells were transiently with expression of SF3b4 and and co-immunoprecipitation/immunoblot analysis was When was with SF3b4 was but and were not These results that SF3b4 binds BMPR-IA in the but its is for interaction, with the interaction in the yeast two-hybrid Since BMPR-IA to TβR-I, we SF3b4 interacts with or By co-immunoprecipitation/immunoblot SF3b4 was not with SF3b4 is a of the and is to be localized in the nucleus, BMPR-IA is the cell We these molecules are using and of and the cells were into the and cell membrane SF3b4 was in the cell membrane fraction as well as in the fraction, BMPR-IA was in the cell membrane fraction the of SF3b4 and BMPR-IA the cell SF3b4 of C2C12 we SF3b4 in C2C12 cells T. Yamaguchi A. M. N. T. V. A. T. J. Cell Biol. PubMed Scopus Google Scholar). cell SF3b4 and were they were with BMP-2 at a of and was By a of by BMP-2 but cell not BMP-2 was during the in and SF3b4 not By the assay, the in cell was BMP-2 treatment These results that SF3b4 inhibits the osteogenic differentiation of C2C12 cells. SF3b4 signaling is involved in chondrocytic differentiation. We SF3b4 using ATDC5 chondrocytic cells. These cells in a to they to to form We cell and of ATDC5 cells. to the ATDC5 cells, by cell not When with the in but that of SF3b4 during We further performed to a of the transcription levels of and of type II the level of expression in to and that of at and the level in SF3b4 to and at and The level of expression in to and at and but that in SF3b4 to only and at and Since the of SF3b4 inhibited chondrocyte we that the endogenous level of SF3b4 is down-regulated during the differentiation. By its expression level to that at expression of SF3b4 strongly that chondrocyte differentiation by down-regulating the expression of SF3b4 SF3b4 the Smad1/5/8 but the the of the of SF3b4 we a of pathways known to be activated by we the of SF3b4 Smad1/5/8 phosphorylation in ATDC5 and C2C12 cells. By immunoblot for the phosphorylation was as early as BMP-2 at to and in the and the cells were of the Smad1/5/8 phosphorylation to the two cell the of of SF3b4 Smad1/5/8 in the following not be by immunoblot we attempted to the using a reporter gene When a reporter known to be specific to the Smad1/5/8 pathway T. M. T. T. N. R. Genes Cells. PubMed Scopus Google Scholar), was the of and in C2C12 cells reporter gene to The of SF3b4 together with inhibited this in a When a of SF3b4 that the and not bind BMPR-IA was expressed in of the SF3b4, of the reporter gene was The of SF3b4 signaling were using the reporter gene assay S. P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The expression of in the of activated the reporter gene but of SF3b4 not These results that SF3b4 specifically inhibits the Smad1/5/8 pathway the phosphorylation of Smad1/5/8, by interacting with SF3b4 pathways play critical roles in chondrocyte proliferation and differentiation J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, K. T. S. S. K. F. Cell Res. PubMed Scopus Google Scholar, H. Y. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), we the of SF3b4 p38 MAPK, and in ATDC5 cells, using not and immunoblot analysis BMP-2 the phosphorylation of p38 with a at in and SF3b4-overexpressing cell with the phosphorylation BMP-2 with the at in cell and immunoblot analysis of phosphorylation the two BMP-2 not in cell types SF3b4 Cell BMPR-IA SF3b4 down-regulated the osteochondral it phosphorylation of Smad1/5/8 and We that SF3b4 regulate the cell surface levels of When levels of SF3b4 were expressed in cells, the levels of the receptor the cell surface decreased in with the expression levels of SF3b4 that SF3b4 may the BMP-mediated signal transduction by BMPR-IA the cell of the that SF3b4 not bind BMPR-IB ICD, we for a to the specific binding of SF3b4 with BMPR-IA using the in BMPR-IA ICD and BMPR-IB ICD and the of BMPR-IA of the in human BMPR-IA and revealed were the These were in the BMPR-IA or of these were in the C-terminal lobe and and two were in the activation to of the by modeling using as a and are and respectively. in a BMPR-IA and BMPR-IB are and the are In the of the for were the protein surface and and were to The surface by and was Although this the of the S. S. A. 1996; PubMed Scopus Google Scholar), it be the interaction surface the were involved in the interaction. we this surface is involved in the interaction using several SF3b4 to the ICD of BMPR-IA of the were with the in BMPR-IB not The of SF3b4 to bind the constitutively active form that the of the ICD by phosphorylation the we the activation is for the interaction by of and These were with the in SF3b4 the direct of the activation in the interaction with In this study, we have found that SF3b4 interacts with the intracellular domain of BMPR-IA, using a yeast two-hybrid SF3b4, identified as a of the to the recognition of the S. Genes Dev. 2004; PubMed Scopus Google Scholar). Although known to be localized in the nucleus, SF3b4 is into the cell membrane together with BMPR-IA, and they with in the Overexpression of SF3b4 inhibits the BMP-mediated Smad1/5/8 pathway but not the TGFβ-mediated pathway as by reporter gene SF3b4 substantially the osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively. Although SF3b4 phosphorylation of Smads and it the cell surface levels of a of SF3b4 to regulate BMP-mediated osteochondral cell differentiation by binding SF3b4 was the only molecule that the ICD of BMPR-IA of in the yeast two-hybrid a and T. Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar) identified the molecule that binds the ICD of BMPR-IA, which Since molecules, such as BRAM1 (17Kurozumi K. Nishita M. Yamaguchi K. Fujita T. Ueno N. Shibuya H. Genes Cells. 1998; 3: 257-264Crossref PubMed Scopus (43) Google Scholar), were SF3b4 to bind BMPR-IA strongly molecules previously identified. When the constitutively active form of BMPR-IA was used as molecule was These results suggest that SF3b4 interacts with BMPR-IA or that the of the form is the BMPR-IA, although it BMP analysis with various in the yeast two-hybrid system indicated that the and are for the interaction. domain analysis by and T. Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar) demonstrated that of the the substantially the interaction, that the N-terminal GS domain interacts with BMPR-IA, which to When analysis using the R. V. Res. 2003; PubMed Scopus Google Scholar) was the was identified as a domain that a not the SF3b4 is for interaction in mammalian cells. Taken together, the studies of the be to identify the interacting We have that BMP-2 transduces p38 and pathways in ATDC5 cells but that SF3b4 not their In contrast, SF3b4 substantially inhibits Smad1/5/8-specific Id reporter gene activity, SF3b4 not to the phosphorylation of Smad1/5/8 in the BMP-2 as by immunoblot may be a in the phosphorylation levels that be by the or the may in the following the phosphorylation is by the may be of Smad1/5/8 the in the BMPR-IA that interacts with SF3b4 to be of the Smad phosphorylation that SF3b4 cell surface levels of BMPR-IA that SF3b4 of the The of SF3b4 the which binds the domain R. P. S. A. 1998; PubMed Scopus Google Scholar). A of molecules the including SF3b4, have been to bind the domain K. S. J. K. J. G. G. L. G. M. F. L. G. R. P. A. P. T. Cell Biol. 2005; PubMed Scopus Google Scholar). the binds at its and the of T. M. G. T. K. Imamura T. Miyazono K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Although we have not the direct interaction of SF3b4 with SF3b4 may with molecules the domain and a specific The of a the of target and A. R. F. A. Rev. Biophys. 2000; PubMed Scopus Google Scholar). We the of the ICD of BMPR-IA and that of which the of a Since in the activation not the interaction, SF3b4 to with sites to Although we not the binding we The the interaction of these receptors with Smads the interaction of with been demonstrated by a in which is by the of or by of the C-terminal phosphorylation sites of Smads M. S. R. L. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Imamura T. S. M. A. K. J. Miyazono K. P. EMBO J. 16: PubMed Scopus Google Scholar). binding of BMPR-I with Smad1/5/8 not been reported The of BMPR-IA in this a the specific of these type I receptors. We have demonstrated that SF3b4 inhibits BMP-mediated osteogenic and chondrocytic differentiation in C2C12 and ATDC5 cell respectively. SF3b4 expression during chondrocytic differentiation this have various is by intracellular The of SF3b4 cell differentiation may be to cell although SF3b4 directly binds BMPR-IA, its not been in cell types T. Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar). By in SF3b4 is expressed at a level in and at P. P. A. Dev. PubMed Scopus Google Scholar), its in skeletal development. studies have that proteins in cellular the splicing of subunit various skeletal the with expression of and of subunit skeletal K. Y. T. H. Genes Dev. 2005; PubMed Scopus Google Scholar). Although these studies the by which SF3b4 regulates the cell surface BMPRI-A to be We T. Imamura and T. for expression and P. for the reporter gene for and G. for