DRAGON, a Bone Morphogenetic Protein Co-receptor
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m410034200
ISSN1083-351X
AutoresTarek A. Samad, Anuradha Rebbapragada, Esther Bell, Ying E. Zhang, Yisrael Sidis, Sung‐Jin Jeong, Jason Campagna, Stephen Perusini, David Fabrizio, Alan L. Schneyer, Herbert Y. Lin, Ali H. Brivanlou, Liliana Attisano, Clifford J. Woolf,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoBone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)β superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFβ ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFβ signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFβ ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)β superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFβ ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFβ signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFβ ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling. Transforming growth factor beta (TGFβ) 1The abbreviations used are: TGFβ, transforming growth factor beta; GPI, glycosylphosphatidylinositol; RGM, repulsive guidance molecule; HEK, human embryonic kidney; PBS, phosphate-buffered saline; FBS, fetal bovine serum; E, embryonic day; HA, hemagglutinin; DN, dominant negative. superfamily ligands that include the TGFβ, bone morphogenetic protein (BMP), growth and differentiation factor, and nodal-related families play a pleiotropic role in vertebrate development by influencing cell specification, differentiation, proliferation, patterning, and migration (1.von Bubnoff A. Cho K.W. Dev. Biol. 2001; 239: 1-14Crossref PubMed Scopus (348) Google Scholar, 2.Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar). These functions require the tight control of ligand production, ensuring a highly ordered spatiotemporal distribution and specific activation, via receptor complexes, of particular intracellular signaling pathways. The TGFβ/activin/nodal ligand subfamily contributes to the specification of endoderm and mesoderm in pregastrula embryos and at gastrula stages, to dorsal mesoderm formation and anterior-posterior patterning (3.Stemple D.L. Curr. Biol. 2000; 10: 843-846Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 4.Kimelman D. Griffin K.J. Curr. Opin. Genet. Dev. 2000; 10: 350-356Crossref PubMed Scopus (137) Google Scholar). Later, TGFβ ligands influence the body axis and patterning of the nervous system (5.Altmann C.R. Brivanlou A.H. Int. Rev. Cytol. 2001; 203: 447-482Crossref PubMed Google Scholar). BMPs, a second major ligand subfamily, contribute to the ventralization of germ layers in the early embryo and suppress the default neural cell fate of the ectoderm (6.Munoz-Sanjuan I. Brivanlou A.H. Nat. Rev. Neurosci. 2002; 3: 271-280Crossref PubMed Scopus (486) Google Scholar). BMPs also participate later in development in the formation and patterning of the neural crest, heart, blood, kidney, limb, muscle, and skeletal system (7.Waite K.A. Eng C. Nat. Rev. Genet. 2003; 4: 763-773Crossref PubMed Scopus (233) Google Scholar). Signal transduction in the BMP subfamily is initiated by ligand binding to a receptor complex composed of two type I and two type II receptors. Three different BMP type I receptors (activin receptor-like kinase ALK2, ALK3, and ALK6) and three BMP type II receptors (BMP type II receptor (BMPRII), activin type IIA receptor (ActRIIA), activin type IIB receptor (ActRIIB)), each with intracellular serine/threonine kinase domains, have been identified (8.Attisano L. Wrana J.L. Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1148) Google Scholar). Ligand binding induces phosphorylation of the type I receptor by the type II receptor, which leads to phosphorylation of cytoplasmic receptor-activated Smads. The BMP subfamily signals through one set of receptor-activated Smads (Smad1, Smad5, and Smad8) whereas the TGFβ subfamily signals via another (Smad2, Smad3). The receptor-activated Smads form heteromeric complexes with a co-Smad, Smad4, which translocates from the cytoplasm to the nucleus to regulate gene expression. Multiple modulators enhance or reduce TGFβ and BMP signaling. The access of TGFβ ligands to receptors is inhibited by the soluble proteins LAP, decorin, and α2-macroglobulin that bind and sequester the ligands (2.Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar). Soluble BMP antagonists include Noggin, chordin, chordin-like, the DAN/Cerberus protein family, and sclerostin (2.Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar). TGFβ ligand access to receptors is also controlled by membrane-bound receptors. BAMBI acts as a decoy receptor, competing with the type I receptor (9.Onichtchouk D. Chen Y.G. Dosch R. Gawantka V. Delius H. Massague J. Niehrs C. Nature. 1999; 401: 480-485Crossref PubMed Scopus (579) Google Scholar), β-glycan (TGFβ type III receptor) enhances TGFβ binding to the type II receptor (10.Brown C.B. Boyer A.S. Runyan R.B. Barnett J.V. Science. 1999; 283: 2080-2082Crossref PubMed Scopus (340) Google Scholar, 11.Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (4040) Google Scholar, 12.del Re E. Babitt J.L. Pirani A. Schneyer A.L. Lin H.Y. J. Biol. Chem. 2004; 279: 22765-22772Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and endoglin enhances TGFβ binding to ALK1 in endothelial cells (13.Marchuk D.A. Curr. Opin. Hematol. 1998; 5: 332-338Crossref PubMed Scopus (65) Google Scholar, 14.Massague J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1671) Google Scholar, 15.Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4941) Google Scholar). Cripto, an EGF-CFC glycosylphosphatidylinositol (GPI)-anchored membrane protein, acts as a co-receptor, increasing the binding of the TGFβ ligands nodal, Vg1, and growth and differentiation factor 1 to activin receptors (16.Cheng S.K. Olale F. Bennett J.T. Brivanlou A.H. Schier A.F. Genes Dev. 2003; 17: 31-36Crossref PubMed Scopus (148) Google Scholar, 17.Shen M.M. Schier A.F. Trends Genet. 2000; 16: 303-309Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) while blocking activin signaling. Only co-receptors acting within the TGFβ/activin/nodal signal transduction pathway have been identified so far. We now find that DRAGON, a 436-amino-acid GPI-anchored protein identified by us and expressed early in the embryonic nervous system (18.Samad T.A. Srinivasan A. Karchewski L.A. Jeong S.J. Campagna J.A. Ji R.R. Fabrizio D.A. Zhang Y. Lin H.Y. Bell E. Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar), enhances BMP signaling in cells and developing embryos. DRAGON is a member of a gene family comprising two other GPI-linked proteins, repulsive guidance molecule (RGM) (19.Monnier P.P. Sierra A. Macchi P. Deitinghoff L. Andersen J.S. Mann M. Flad M. Hornberger M.R. Stahl B. Bonhoeffer F. Mueller B.K. Nature. 2002; 419: 392-395Crossref PubMed Scopus (257) Google Scholar) and HFE2 (20.Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (850) Google Scholar) that have diverse roles. DRAGON produces homophilic and heterophilic cell-cell neuronal adhesion (18.Samad T.A. Srinivasan A. Karchewski L.A. Jeong S.J. Campagna J.A. Ji R.R. Fabrizio D.A. Zhang Y. Lin H.Y. Bell E. Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar), RGM regulates retinotectal projections and neural tube closure (19.Monnier P.P. Sierra A. Macchi P. Deitinghoff L. Andersen J.S. Mann M. Flad M. Hornberger M.R. Stahl B. Bonhoeffer F. Mueller B.K. Nature. 2002; 419: 392-395Crossref PubMed Scopus (257) Google Scholar, 21.Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (152) Google Scholar), while mutations in the human HFE2 locus are linked to juvenile hemochromatosis (20.Papanikolaou G. Samuels M.E. Ludwig E.H. MacDonald M.L. Franchini P.L. Dube M.P. Andres L. MacFarlane J. Sakellaropoulos N. Politou M. Nemeth E. Thompson J. Risler J.K. Zaborowska C. Babakaiff R. Radomski C.C. Pape T.D. Davidas O. Christakis J. Brissot P. Lockitch G. Ganz T. Hayden M.R. Goldberg Y.P. Nat. Genet. 2004; 36: 77-82Crossref PubMed Scopus (850) Google Scholar). Here, we show that DRAGON enhances BMP signaling, binds to specific BMP ligands, associates with BMP receptors, and potentiates BMP cellular signaling, indicating that DRAGON is a BMP co-receptor. The rabbit polyclonal DRAGON antibody was characterized and described previously (18.Samad T.A. Srinivasan A. Karchewski L.A. Jeong S.J. Campagna J.A. Ji R.R. Fabrizio D.A. Zhang Y. Lin H.Y. Bell E. Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar). Briefly, a rabbit polyclonal antibody was raised against the peptide sequence TAAAHSALEDVEALHPRKC (molecular weight, 2019.01), which is present in the C terminus of DRAGON up-stream of its hydrophobic tail and affinity-purified using the same peptide. The antibody binds with high affinity to recombinant DRAGON expressed in HEK293T-transfected cells, recognizing a band of 50-55 kDa in Western blots. Western blots of protein extracts from neonatal and adult dorsal root ganglion and dorsal root ganglion primary cultures show a similar band with an additional lower band of 35-40 kDa, indicating possible proteolytic cleavage of endogenous DRAGON, similar to that found for chick RGM (19.Monnier P.P. Sierra A. Macchi P. Deitinghoff L. Andersen J.S. Mann M. Flad M. Hornberger M.R. Stahl B. Bonhoeffer F. Mueller B.K. Nature. 2002; 419: 392-395Crossref PubMed Scopus (257) Google Scholar). Preincubation of DRAGON antibody with 1 μm of the above antigenic peptide (4 h at room temperature) results in a loss of both bands by Western blot and staining signals by immunohistochemistry. To obtain E2.5 embryos, 5-week-old female ICR mice were superovulated by injecting 5 IU of human chorionic gonadotropin (Sigma) 48 h after a prior administration of 5 IU of pregnant mare serum gonadotropin (Sigma). Treated females were mated with fertile male mice of the same strain and E2.5 embryos were flushed from oviducts. Embryos were washed with PBS containing 3% FBS (3% FBS-PBS) and fixed in 3.7% paraformaldehyde for 10 min at room temperature. They were permeabilized for 30min with 20 mm HEPES, 300 mm sucrose, 50 mm NaCl, 3 mm MgCl2, and 0.5% Triton X-100, pH7.4. After blocking with 3% FBS-PBS for 30min, embryos were incubated with the DRAGON antibody diluted 1:2000 in 3% FBS-PBS overnight at 4 °C, washed, and then incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories) for 2 h at room temperature. After washing with 3% FBS-PBS for 30min at room temperature, embryos were mounted with mounting medium containing propidium iodide (Vector Laboratories, Inc). Whole mount immunohistochemistry in mouse E10.5 embryos was carried out as described previously (22.Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar). Briefly, freshly dissected embryos were fixed with 4% paraformaldehyde overnight at 4 °C, washed in saline for 2 h, and then soaked in 80% methanol series. Endogenous peroxidase activity was quenched with 3% H2O2 in 80% methanol and 20% Me2SO for 3 h. After washing with Tris-buffered saline containing 1% Tween 20 (TBS-T) for 3 h, the embryos were incubated with DRAGON antibody (1:500) in TBS-T containing 5% skim milk and 5% Me2SO for 2 days at room temperature. The embryos were then washed 3 times and incubated with horseradish peroxidase-coupled anti-rabbit IgG (1:200 dilution in TBS-T containing 5% skim milk and 5% Me2SO). Horseradish peroxidase activity was detected with diaminobenzidine. The premade Northern blot containing ∼2 μg of poly(A)+ RNA/lane from mouse embryos at the indicated stages (Clontech, Inc.) was used. The membrane was hybridized with a 300-bp DRAGON cDNA probe as described previously (18.Samad T.A. Srinivasan A. Karchewski L.A. Jeong S.J. Campagna J.A. Ji R.R. Fabrizio D.A. Zhang Y. Lin H.Y. Bell E. Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar). Whole mount in situ hybridizations were carried out as described previously by Harland (23.Harland R.M. Methods Cell Biol. 1991; 36: 685-695Crossref PubMed Google Scholar). Embryos to be sectioned were post-fixed in 4% paraformaldehyde and embedded in 20% gelatin-PBS. Sections were cut between 50 and 100 μm. The BMP-inducible BRE-Luc (24.Korchynskyi O. ten Dijke P. J. Biol. Chem. 2002; 277: 4883-4891Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar), I-BRE-Luc (25.Benchabane H. Wrana J.L. Mol. Cell. Biol. 2003; 23: 6646-6661Crossref PubMed Scopus (76) Google Scholar), and Msx2-Luc (26.Sirard C. Kim S. Mirtsos C. Tadich P. Hoodless P.A. Itie A. Maxson R. Wrana J.L. Mak T.W. J. Biol. Chem. 2000; 275: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and the TGFβ-inducible 3TP-lux (27.Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1396) Google Scholar) have been described previously. HepG2, 10 T1/2, or LLC-PK1 cells were transiently transfected with calcium phosphate/DNA precipitation or Lipofectamine reagent (Invitrogen). The next day cells were washed with medium containing 0.2% FBS and treated overnight with BMP2, BMP4, or TGFβ as indicated. Cell lysates were harvested 48 h post-transfection, and luciferase activity was monitored by a plate reader luminometer. Relative light units were determined and corrected for transfection efficiency using β-galactosidase activity, as all plates were co-transfected with β-galactosidase expression construct to normalize transfection efficiency. Carrier-free human BMP2, -4, and -7, as well as TGFβ1, -2, and activin were purchased from R&D systems (Minneapolis, MN). Noggin was also purchased from R&D systems. TGFβ superfamily receptor constructs (28.Rebbapragada A. Benchabane H. Wrana J.L. Celeste A.J. Attisano L. Mol. Cell. Biol. 2003; 23: 7230-7242Crossref PubMed Scopus (469) Google Scholar) were transiently transfected into COS-1 cells with Lipofectamine reagent. Cells were affinity-labeled with 1.5 nm [125I]-BMP2 or 500 pm TGFβ for 4 h at 4 °C,andthe receptors were cross-linked to ligands with disuccinimidyl suberate (Pierce) as described previously (28.Rebbapragada A. Benchabane H. Wrana J.L. Celeste A.J. Attisano L. Mol. Cell. Biol. 2003; 23: 7230-7242Crossref PubMed Scopus (469) Google Scholar). Lysates were subjected to immunoprecipitation with anti-DRAGON polyclonal or anti-HA monoclonal antibodies (12CA5, Roche Diagnostics) and were separated by SDS-PAGE, and 125I-bound ligand was visualized by phosphorimaging. For co-immunoprecipitation assays, cells lysates were incubated with anti-DRAGON, anti-HA, or anti-FLAG (Sigma) antibodies (1:1000) for 4 h, and immune complexes were collected on protein G-Sepharose. Immunoprecipitates were transferred to nitrocellulose, and proteins were detected by immunoblotting with the indicated antibodies. DRAGON-Fc (18.Samad T.A. Srinivasan A. Karchewski L.A. Jeong S.J. Campagna J.A. Ji R.R. Fabrizio D.A. Zhang Y. Lin H.Y. Bell E. Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar) was generated by subcloning DRAGON cDNA without its GPI anchor into the mammalian expression vector pIgplus (R&D Systems, Minneapolis, MN) in-frame with the Fc portion of the human IgG. This allowed us to express a soluble DRAGON-Fc fusion protein. DRAGON-Fc collected in the medium of stably transfected HEK293T cells was purified using HiTrap protein A affinity columns (Amersham Biosciences) and eluted with 100 mm glycine-HCl, pH 3.0. The elution fraction was neutralized with 0.3 m Tris-HCl, pH 9. Purified DRAGON-Fc was run on SDS-polyacrylamide gel and immunoblotted with anti-Dragon antibody and anti-Fc antibody. For ligand binding assays, 2 μg of BMP2 ligand/reaction was iodinated with 125I by the modified chloramine-T method as described previously (29.Frolik C.A. Wakefield L.M. Smith D.M. Sporn M.B. J. Biol. Chem. 1984; 259: 10995-11000Abstract Full Text PDF PubMed Google Scholar). Purified soluble DRAGON-Fc (18.Samad T.A. Srinivasan A. Karchewski L.A. Jeong S.J. Campagna J.A. Ji R.R. Fabrizio D.A. Zhang Y. Lin H.Y. Bell E. Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar) was diluted in TBS/casein blocking buffer (BioFX, Owings Mills, MD) and incubated overnight in the presence or absence of 125I-labeled radioligands (50,000-100,000 counts). For competition binding assays, fixed amounts of radioligands (50,000-100,000 counts) were added to the samples together with increasing amounts (2 pm-500 nm) of non-radioactive TGFβ superfamily ligands or as indicated. Samples were then placed on protein A-coated 96-well plates (Pierce) for 90 min, washed 3 times with wash buffer (BioFX), and radioactivity was counted using a standard gamma counter. Reverse transcription-PCR was performed on isolated Xenopus animal cap explants or embryos, with orthidine decarboxylase as loading control (30.Munoz-Sanjuan I. Bell E. Altmann C.R. Vonica A. Brivanlou A.H. Development (Camb.). 2002; 129: 5529-5540Crossref PubMed Scopus (52) Google Scholar). DRAGON is expressed in mouse E2.5 pre-implantation embryos (Fig. 1A), and in postimplantation embryos (>E7) (Fig. 1B). In E10.5 embryos, DRAGON is found along the neural tube, in the dorsal root ganglia, and in the tips of the neural folds and the tail bud (Fig. 1C) as assessed by whole mount immunohistochemistry. Xenopus orthologs of both DRAGON and RGM are expressed maternally and throughout development and are detected at high levels by tadpole stages (Fig. 1D). Analysis by whole mount in situ hybridization of RGM expression at stage 12 reveals high levels in the ectoderm by the blastopore (Fig. 1E, i). More restricted expression is seen by stage 17 and stage 20 in the neural plate, specifically in the dorsal aspect of the neural tube (Fig, 1E, ii and iii). By stage 35, the expression of RGM is detected in the hindbrain, midbrain, and forebrain regions, as well as the branchial arches. In addition, expression is seen posteriorly in the somites and tail bud (Fig. 1E, iv). The expression pattern of DRAGON and RGM at different embryonic stages in both the developing mouse and Xenopus is comparable with the expression of the BMP type I receptors, ALK3 and ALK6 and BMP type II receptor (31.Dewulf N. Verschueren K. Lonnoy O. Moren A. Grimsby S. Vande S.K. Miyazono K. Huylebroeck D. ten Dijke P. Endocrinol. 1995; 136: 2652-2663Crossref PubMed Google Scholar, 32.Ebendal T. Bengtsson H. Soderstrom S. J. Neurosci. Res. 1998; 51: 139-146Crossref PubMed Scopus (163) Google Scholar) (Fig. 1). This observation prompted us to investigate whether DRAGON contributes to or modulates TGFβ superfamily signal transduction. TGFβ superfamily ligands are classified as TGFβ or BMP-like based on the particular Smad-dependent intracellular pathways they activate. We examined whether DRAGON activates these pathways using specific BMP- and TGFβ-responsive luciferase reporters in LLC-PK1 (kidney epithelial cells), as these cells are highly responsive to many TGFβ family members and thereby allow a parallel analysis of any action of DRAGON on TGFβ superfamily signaling. To determine whether DRAGON alters BMP signaling we used the BMP-inducible promoter BRE-Luc, an Id1 promoter-derived reporter construct (24.Korchynskyi O. ten Dijke P. J. Biol. Chem. 2002; 277: 4883-4891Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar). BMP2 stimulates BRE-Luc activity in LLC-PK1 cells (Fig. 2). Co-expression of DRAGON with BRE-Luc in the absence of exogenous ligand increases BRE-Luc activity to levels comparable with that achieved by BMP2 stimulation in the absence of DRAGON (Fig. 2A). To assess whether DRAGON regulates the signaling produced by BMP ligands, LLC-PK1 cells were co-transfected with the BRE-Luc reporter and DRAGON (2 and 20 ng) and incubated with BMP2. BMP2 (50 ng/ml) induces a 6-fold increase in luciferase activity in the absence of DRAGON and a 14-fold increase in DRAGON co-transfected cells (Fig. 2A). DRAGON expression had no effect on TGFβ signaling, assessed by using a TGFβ-responsive promoter (TGFβ-responsive CAGA reporter (12.del Re E. Babitt J.L. Pirani A. Schneyer A.L. Lin H.Y. J. Biol. Chem. 2004; 279: 22765-22772Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar)) in LLC-PK1 cells (data not shown). DRAGON expression activates BMP signaling in the absence of any added ligand (Fig. 2A). To determine whether this represents a ligand-independent action or is mediated by endogenous BMP ligands either produced by the cell lines or present in the medium, we investigated whether DRAGON activation of BMP signaling is blocked by administration of the soluble BMP antagonist Noggin (2.Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar). When added to the medium Noggin (50-1000 ng/ml) inhibits both the DRAGON- and BMP2-induced activation of the BRE-Luc reporter in LLC-PK1 cells (Fig. 2B). Noggin has no effect on TGFβ-induced stimulation of the TGFβ-responsive CAGA reporter (Fig. 2B). DRAGON-mediated activation of BMP signaling is, therefore, ligand-dependent. DRAGON significantly enhances BMP signaling and increases cellular sensitivity to BMP2 in LLC-PK1 cells (Fig. 2, A and B). To rule out a potential cell-type or reporter-specific effect of DRAGON expression, we examined the generality of this effect, i.e. whether it is restricted to one cell line or to one BMP-inducible reporter construct. We tested the effect of DRAGON expression in 10 T1/2 cells (mouse mesenchymal stem cells), another BMP-responsive cell line (28.Rebbapragada A. Benchabane H. Wrana J.L. Celeste A.J. Attisano L. Mol. Cell. Biol. 2003; 23: 7230-7242Crossref PubMed Scopus (469) Google Scholar), using another BMP-inducible promoter reporter construct, I-BRE-Luc, which contains a portion of intron 1 of the Smad7 gene (25.Benchabane H. Wrana J.L. Mol. Cell. Biol. 2003; 23: 6646-6661Crossref PubMed Scopus (76) Google Scholar). DRAGON significantly enhances BMP2-dependent reporter activity in this cell line at BMP2 concentrations of 25-300 pm (Fig. 2C). This effect is lost at 300 pm or higher, where maximal signaling is achieved. Similar results were obtained using LLC-PK1, 10 T1/2, and HepG2 cell lines and different BMP-responsive promoters (I-BRE-Luc, BRE-Luc, and Msx2-Luc) (Fig. 2, and data not shown). DRAGON significantly increases cellular sensitivity, therefore, to low doses of BMP2 in a manner that is neither reporternor cell-specific. These results suggest that the regulatory role of DRAGON in BMP signaling is a generalized phenomenon. This finding prompted us to study the mechanism of the enhancement of BMP signaling by DRAGON and where and how it interacts with BMP signaling components. DRAGON could potentially enhance BMP signaling through a direct modulation of the BMP signaling complex or through an independent receptor pathway that converges on the intracellular Smads. We therefore investigated whether DRAGON binds to BMP ligands, interacts directly with BMP receptors, and whether a dominant negative BMP receptor or Smad mutants reduce the DRAGON-induced enhancement of BMP signaling. DRAGON Binds to BMP but Not TGFβ Ligands—To determine whether cell surface-localized DRAGON binds directly to BMP ligands, COS-1 cells expressing DRAGON were incubated with [125I]-BMP2 or [125I]-TGFβ and receptor-bound ligand cross-linked. HA-tagged BMP type I receptor (ALK6) or TGFβ type II receptor (TβRII) served as positive controls for BMP2 or TGFβ binding, respectively. Immunoprecipitation of DRAGON from [125I]-BMP2-but not [125I]-TGFβ-labeled cells revealed the presence of two [125I]-BMP2-bound proteins of ∼55-65 kDa (Fig. 3A), consistent with the expected size of a BMP-bound DRAGON. The presence of two bands suggests that cell surface-localized DRAGON may be subject to post-translational modification. These data, indicating that cell surface-expressed DRAGON can bind BMP2 but not TGFβ, prompted us to assess whether DRAGON can directly bind BMP ligands. We examined whether DRAGON directly binds to BMP ligands using a soluble DRAGON-Fc fusion protein in a cell-free binding system (12.del Re E. Babitt J.L. Pirani A. Schneyer A.L. Lin H.Y. J. Biol. Chem. 2004; 279: 22765-22772Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). DRAGON-Fc binds [125I]-BMP2 with high affinity and an apparent dissociation rate constant (Kd) of 1.5 nm (Fig. 3B). This binding is competed with an excess of unlabeled BMP2 as well as by BMP4 (4 nm) (Fig. 3, B and C). In contrast, BMP7 (4 nm), activin A, TGFβ1, -2, or -3 (4 nm each) do not competitively inhibit the binding of DRAGON-Fc to BMP2 (Fig. 3C). Pretreatment of cells with DRAGON-Fc decreases BMP2-but not BMP7-mediated activation of the BRE-Luc promoter in a dose-dependent manner (60 and 300 ng/ml) (Fig. 3D) but had no effect on TGF-β1-dependent activation of the CAGA-Luc promoter (Fig. 3E). DRAGON directly interacts with members of the BMP but not the TGFβ ligand family, with a preference for BMP2 and BMP4. DRAGON Enhances BMP Signaling Only when Expressed on the Cell Surface—To assess whether the GPI anchor is required for the action of DRAGON on BMP signaling, we used the DRAGON-Fc fusion protein where the C-terminal GPI anchor is deleted and replaced by human Fc (18.Samad T.A. Srinivasan A. Karchewski L.A. Jeong S.J. Campagna J.A. Ji R.R. Fabrizio D.A. Zhang Y. Lin H.Y. Bell E. Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar). When cotransfected with the BRE-Luc reporter construct this DRAGON-Fc, which is secreted into the medium, fails to increase BMP signaling (Fig. 4A). Western blot confirms the expression of both DRAGON and DRAGON-Fc in the transfected cells (Fig. 4B). These results demonstrate that the expression of DRAGON on the cell surface is required for its action on BMP signaling. DRAGON Binds to BMP Type I and Type II Receptors—BMP type I and type II receptors are essential for mediating BMP responses. To test whether DRAGON directly interacts with BMP type I and type II receptors, HEK293T cells were transiently transfected either with DRAGON alone or with HA epitope-tagged type I and type II BMP receptors. Anti-Dragon immunoprecipitation followed by anti-HA immunoblotting reveals that DRAGON associates directly with all BMP type I receptors, (ALK2, ALK3, and ALK6) (Fig. 5A) and with the BMP type II receptors ActRII and ActRIIB (Fig. 5B). DRAGON Signaling Is Blocked by Dominant Negative ALK3, -6, and Smad1—We used dominant negative type I receptors (ALK1-DN, ALK3-DN, and ALK6-DN), which are deficient in kinase activity and unable to phosphorylate Smads (33.Clarke T.R. Hoshiya Y. Yi S.E. Liu X. Lyons K.M. Donahoe P.K. Mol. Endocrinol. 2001; 15: 946-959Crossref PubMed Scopus (157) Google Scholar), to confirm that DRAGON acts through modulation of BMP receptors and subsequent activation of the downstream Smad1 pathway (Fig. 5, C and D). Co-expression of DRAGON with ALK3-DN and ALK6-DN in LLC-PK1 cells decreases DRAG-ON-mediated induction of I-BRE luciferase activity to base line, whereas co-expression of ALK1-DN does not affect DRAG-ON-mediated BMP signaling (Fig. 5C). Co-expression of DRAGON in LLC-PK1 cells with a Smad1 dominant negative mutant that lacks the C-terminal phosphoacceptor domain (15.Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4941) Google Scholar, 34.Lo R.S. Chen Y.G. Shi Y. Pavletich N.P. Massague J. EMBO J. 1998; 17: 996-1005Crossref PubMed Scopus (210) Google Scholar) also dose dependently reduces DRAGON-induced signaling. In contrast, co-expression wit
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