Title: Repulsive Guidance Molecule (RGMa), a DRAGON Homologue, Is a Bone Morphogenetic Protein Co-receptor
Abstract: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF-β) superfamily of ligands, which regulate many mammalian physiologic and pathophysiologic processes. BMPs exert their effects through type I and type II serine/threonine kinase receptors and the Smad intracellular signaling pathway. Recently, the glycosylphosphatidylinositol (GPI)-anchored protein DRAGON was identified as a co-receptor for BMP signaling. Here, we investigate whether a homologue of DRAGON, repulsive guidance molecule (RGMa), is similarly involved in the BMP signaling pathway. We show that RGMa enhances BMP, but not TGF-β, signals in a ligand-dependent manner in cell culture. The soluble extracellular domain of RGMa fused to human Fc (RGMa.Fc) forms a complex with BMP type I receptors and binds directly and selectively to radiolabeled BMP-2 and BMP-4. RGMa mediates BMP signaling through the classical BMP signaling pathway involving Smad1, 5, and 8, and it up-regulates endogenous inhibitor of differentiation (Id1) protein, an important downstream target of BMP signals. Finally, we demonstrate that BMP signaling occurs in neurons that express RGMa in vivo. These data are consistent with a role for RGMa as a BMP co-receptor. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF-β) superfamily of ligands, which regulate many mammalian physiologic and pathophysiologic processes. BMPs exert their effects through type I and type II serine/threonine kinase receptors and the Smad intracellular signaling pathway. Recently, the glycosylphosphatidylinositol (GPI)-anchored protein DRAGON was identified as a co-receptor for BMP signaling. Here, we investigate whether a homologue of DRAGON, repulsive guidance molecule (RGMa), is similarly involved in the BMP signaling pathway. We show that RGMa enhances BMP, but not TGF-β, signals in a ligand-dependent manner in cell culture. The soluble extracellular domain of RGMa fused to human Fc (RGMa.Fc) forms a complex with BMP type I receptors and binds directly and selectively to radiolabeled BMP-2 and BMP-4. RGMa mediates BMP signaling through the classical BMP signaling pathway involving Smad1, 5, and 8, and it up-regulates endogenous inhibitor of differentiation (Id1) protein, an important downstream target of BMP signals. Finally, we demonstrate that BMP signaling occurs in neurons that express RGMa in vivo. These data are consistent with a role for RGMa as a BMP co-receptor. Bone morphogenetic proteins (BMPs) 1The abbreviations used are: BMP, bone morphogenetic protein; TGF-β, transforming growth factor β; p-Smad1/5/8, phosphorylated Smad1, 5, and 8; Id, inhibitor of differentiation; GPI, glycosylphosphatidylinositol; RGMa, repulsive guidance molecule; HJV, hemojuvelin; RGMa.Fc, soluble extracellular domain of RGMa fused to Fc portion of human immunoglobulin; DMEM, Dulbecco's modification of Eagle's medium; FBS, fetal bovine serum; CAGA-Luc or (CAGA)12MLP-Luc, TGF-β responsive firefly luciferase reporter; BRE-Luc, BMP responsive firefly luciferase reporter; α-RGMa, rabbit polyclonal RGMa antibody; DSS, disuccinimidyl suberate; PBS, phosphate-buffered saline; NeuN, neuron-specific nuclear protein; WT, wild type; DN, dominant negative; RLU, relative luciferase units. represent a large subfamily of the transforming growth factor β (TGF-β) superfamily of ligands, which also includes TGF-β1, -β2, -β3, activins, inhibins, growth and differentiation factors, nodal, Vg1, and Mullerian-inhibiting substance (1Shi Y. Massague J. Cell 2003. 2003; 113: 685-700Google Scholar). These cytokines play a key role in regulating cell proliferation, differentiation, apoptosis, migration, and patterning during development and in adult tissues (2Hogan B.L. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1725) Google Scholar, 3Zhao G.Q. Genesis. 2003; 35: 43-56Crossref PubMed Scopus (307) Google Scholar, 4Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar). Like other members of the TGF-β superfamily, BMPs exert their effects through a common signal transduction pathway (1Shi Y. Massague J. Cell 2003. 2003; 113: 685-700Google Scholar). Signaling is initiated when ligand binds to combinations of two type I and two type II serine/threonine kinase receptors. Thus far, three type I receptors (BMPRIA/ALK3, BMPRIB/ALK6, and ALK2) and three type II receptors (BMP type II receptor (BMPRII), activin type IIA receptor (ActRIIA), and activin type IIB receptor (ActRIIB)) have been identified for BMP subfamily ligands. Ligand binding activates the receptor complex by inducing phosphorylation of the type I receptor by the type II receptor. Phosphorylated type I receptors then phosphorylate receptor-activated Smad1, 5, and 8 (Smad1/5/8). Phosphorylated Smad1, 5, and 8 (p-Smad1/5/8) then form heteromeric complexes with the common mediator Smad (Co-Smad), Smad4, and the activated Smad complexes move to the nucleus, where they act as transcriptional regulators to modulate gene expression. One important target of BMP signals includes the inhibitor of differentiation (Id) gene family, which serve as regulators of growth and differentiation in a variety of tissues (5Hollnagel A. Oehlmann V. Heymer J. Ruther U. Nordheim A. J. Biol. 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Cell. 1993; 73: 1435-1444Abstract Full Text PDF PubMed Scopus (778) Google Scholar). Glycosylphosphatidylinositol (GPI)-linked proteins from the epidermal growth factor-Cripto-Criptic-FRL-1 family are co-receptors necessary for nodal, Vg1, and growth and differentiation factor 1 signaling (11Shen M.M. Schier A.F. Trends Genet. 2000; 16: 303-309Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 12Cheng S.K. Olale F. Bennett J.T. Brivanlou A.H. Schier A.F. Genes Dev. 2003; 17: 31-36Crossref PubMed Scopus (147) Google Scholar). Cripto also inhibits activin signaling by preventing binding of the activin/type II receptor complex to type I receptors (13Gray P.C. Harrison C.A. Vale W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5193-5198Crossref PubMed Scopus (142) Google Scholar). We have recently identified the GPI-anchored protein DRAGON (RGMb) as the first co-receptor for BMP signaling (14Samad T.A. Rebbapragada A. Bell E. Zhang Y. Sidis Y. Jeong S.J. Campagna J.A. Perusini S. Fabrizio D.A. Schneyer A.L. Lin H.Y. Brivanlou A.H. Attisano L. Woolf C.J. J. Biol. Chem. 2005; 280: 14122-14129Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). DRAGON enhances cellular responses to BMP, but not TGF-β, signals in a ligand-dependent manner. DRAGON associates with BMP type I and type II receptors, and soluble DRAGON.Fc fusion protein binds selectively to BMP-2 and BMP-4, but not BMP-7 or other members of the TGF-β superfamily of ligands (14Samad T.A. Rebbapragada A. Bell E. Zhang Y. Sidis Y. Jeong S.J. Campagna J.A. Perusini S. Fabrizio D.A. Schneyer A.L. Lin H.Y. Brivanlou A.H. Attisano L. Woolf C.J. J. Biol. Chem. 2005; 280: 14122-14129Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). DRAGON is a member of the repulsive guidance molecule (RGM) family of genes, which also includes RGMa and hemojuvelin (HJV/RGMc/HFE2) (15Monnier P.P. Sierra A. Macchi P. Deitinghoff L. Andersen J.S. Mann M. Flad M. Hornberger M.R. 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Woolf C.J. J. Neurosci. 2004; 24: 2027-2036Crossref PubMed Scopus (93) Google Scholar, 18Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (151) Google Scholar, 19Schmidtmer J. Engelkamp D. Gene Expr. Patterns. 2004; 4: 105-110Crossref PubMed Scopus (81) Google Scholar, 20Oldekamp J. Kramer N. Alvarez-Bolado G. Skutella T. Gene Expr. Patterns. 2004; 4: 283-288Crossref PubMed Scopus (66) Google Scholar). Unlike DRAGON, RGMa and hemojuvelin also possess an RGD motif, which could be involved in cell attachment (15Monnier 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). RGMa and DRAGON are expressed in a complementary manner in the central nervous system (17Samad 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. 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Mueller B.K. Skutella T. J. Neurosci. 2004; 24: 3862-3869Crossref PubMed Scopus (80) Google Scholar) and neural tube closure (18Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (151) Google Scholar), while DRAGON contributes to neuronal cell adhesion through homophilic interactions (17Samad 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). RGMa also binds to the receptor neogenin (21Rajagopalan S. Deitinghoff L. Davis D. Conrad S. Skutella T. Chedotal A. Mueller B.K. Strittmatter S.M. Nat. Cell Biol. 2004; 6: 756-762Crossref PubMed Scopus (227) Google Scholar) and functions as a cell survival factor (23Matsunaga E. Tauszig-Delamasure S. Monnier P.P. Mueller B.K. Strittmatter S.M. Mehlen P. Chedotal A. Nat. Cell Biol. 2004; 6: 749-755Crossref PubMed Scopus (223) Google Scholar). Hemojuvelin is expressed most heavily in the liver, heart, and skeletal muscle, and is mutated in juvenile hemochromatosis, a disorder of iron overload (16Papanikolaou 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 (841) Google Scholar, 17Samad 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, 18Niederkofler V. Salie R. Sigrist M. Arber S. J. Neurosci. 2004; 24: 808-818Crossref PubMed Scopus (151) Google Scholar, 19Schmidtmer J. Engelkamp D. Gene Expr. Patterns. 2004; 4: 105-110Crossref PubMed Scopus (81) Google Scholar, 20Oldekamp J. Kramer N. Alvarez-Bolado G. Skutella T. Gene Expr. Patterns. 2004; 4: 283-288Crossref PubMed Scopus (66) Google Scholar, 24Rodriguez Martinez A. Niemela O. Parkkila S. Haematologica. 2004; 89: 1441-1445PubMed Google Scholar). Here, we investigate whether RGMa is involved in the BMP signaling pathway. Using a reporter assay, we show that transfection of RGMa cDNA into cells enhances BMP, but not TGF-β, signals in a ligand-dependent fashion. Binding and cross-linking studies in a cell-free system demonstrate that soluble RGMa.Fc fusion protein interacts with the BMP type I receptor ALK6 and binds directly to 125I-BMP-2 and 125I-BMP-4, but not other members of the TGF-β superfamily. Co-transfection of RGMa cDNA with dominant negative BMP type I receptors or with dominant negative Smad1 inhibits RGMa-mediated BMP signaling, suggesting that RGMa generates BMP signals via the classical BMP pathway. Transfection of RGMa cDNA into cells induces phosphorylation of endogenous Smad1/5/8 and up-regulates endogenous Id1. Finally, immunofluorescence microscopy of adult rat spinal cord sections, reveals that RGMa is expressed in vivo in neurons, which also show nuclear accumulation of p-Smad1/5/8. Taken together, these data suggest that RGMa functions as a BMP co-receptor. cDNA Subcloning—cDNA encoding murine RGMa was subcloned into the expression vector pCDNA4/HisB (Promega). cDNA encoding the extracellular domain of murine RGMa was amplified by polymerase chain reaction (PCR) and subcloned into the mammalian expression vector pIgplus (R & D Systems, Minneapolis, MN) into the restriction sites BamHI and HindIII in-frame with the Fc portion of human immunoglobulin (IgG) to generate soluble RGMa.Fc fusion protein. Cell Culture and Transfection—HEK 293 cells and LLC-PK1 cells were obtained from the American Type Culture Collection (ATCC CRL-1573 and CL-101, respectively) and cultured in Dulbecco's modification of Eagle's medium (DMEM; Cellgro Mediatech, Herndon, VA) containing 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA). All plasmid transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Stably transfected cells were selected and cultured in 2 mg/ml G418 (Cellgro Mediatech). Luciferase Assay—LLC-PK1 cells were transiently transfected with a TGF-β responsive firefly luciferase reporter, (CAGA)12MLP-Luc (CAGA-Luc, Ref. 25Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1588) Google Scholar), or a BMP responsive firefly luciferase reporter (BRE-Luc, Ref. 6Korchynskyi O. ten Dijke P. J. Biol. Chem. 2002; 277: 4883-4891Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar) (both kindly provided by Peter ten Dijke, Netherlands Cancer Institute), in combination with pRL-TK Renilla luciferase vector (Promega) in a ratio of 10:1 to control for transfection efficiency, with or without co-transfection with RGMa cDNA. Forty-eight hours after transfection, cells were serum-starved in DMEM supplemented with 1% FBS for 6 h and treated with varying amounts of TGF-β1, BMP-2, BMP-4, or BMP-7 ligands (R & D Systems) for 16 h, in the absence or presence of noggin (R & D Systems) or anti-BMP-2/4 antibody (R & D Systems). Cells were lysed, and luciferase activity was determined with the Dual Reporter Assay (Promega) according to the manufacturer's instructions. Experiments were performed in duplicate or triplicate wells. Relative luciferase units (RLU) were calculated as the ratio of firefly (reporter) and Renilla (transfection control) luciferase values. Reverse Transcription Polymerase Chain Reaction (RT-PCR)—LLC-PK1 cells were grown to confluence on 6-cm tissue culture plates. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) including DNase digestion with the RNase-Free DNase set (Qiagen) according to the manufacturer's instructions. First strand cDNA synthesis was performed using iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer's instructions. Transcripts of BMP-2 were amplified using the forward primer 5′-CGTGACCAGACTTTTGGACAC-3′ and reverse primer 5′-GGCATGATTAGTGGAGTTCAG-3′. Transcripts of BMP-4 were amplified using the forward primer 5′-AGCAGCCAAACTATGGGCTA-3′ and reverse primer 5′-TGGTTGAGTTGAGGTGGTCA-3′. Purification and Characterization of RGMa.Fc—HEK 293 cells stably expressing RGMa.Fc were cultured in DMEM supplemented with 5% FBS using 175-cm2 multifloor flasks (Denville Scientific, Southplainfield, NJ). RGMa.Fc was purified from the media of stably transfected cells via one-step Protein A affinity chromatography using Hi-Trap rProtein A FF columns (Amersham Biosciences) as previously described (26del 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). Purified protein was eluted with 100 mm glycine-HCl, pH 3.2 and neutralized with 0.3 m Tris-HCl, pH 9 as previously described (26del 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). Purified human RGMa.Fc was subjected to reducing SDS-PAGE using pre-cast NuPAGE Novex 4-12% Bis-Tris gels (Invitrogen), and gels were stained with Bio-safe Coomassie Blue (Bio-Rad) to determine the purity of RGMa.Fc and quantify protein concentration. For Western blot analysis, gels were electroblotted to polyvinylidene difluoride filter membranes (Bio-Rad). Membranes were blocked with TBS-T (Tris-buffered saline, 0.2% Tween 20) containing 6% milk powder for 1 h and washed three times with TBS-T for 10 min. Membranes were then probed with an affinity-purified rabbit polyclonal anti-mouse RGMa antibody (α-RGMa) raised against the peptide RMDEEVVNAVEDRDSQGLYLC (amino acids 296-316 in the C terminus of mouse RGMa upstream of its hydrophobic tail) 2GenBank™ accession number NM_177740. (1:2,000) at 4 °C overnight, or with anti-Fc antibody (Jackson ImmunoResearch, West Grove, PA) (1:2000) at room temperature for 1 h in blocking solution. Membranes were washed with TBS-T and incubated with donkey anti-rabbit or anti-goat horseradish peroxidase-linked secondary antibody (1:10,000) (Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was detected with chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA) and exposed to BioMax XAR film (Kodak, Rochester, NY). Ligand Iodination—2 μg of carrier-free human BMP-2 and BMP-4 ligand (R & D Systems) per reaction was iodinated with [125I] by the modified chloramine-T method as previously described (27Frolik C.A. Wakefield L.M. Smith D.M. Sporn M.B. J. Biol. Chem. 1984; 259: 10995-11000Abstract Full Text PDF PubMed Google Scholar). Binding Assay—25 ng of purified RGMa.Fc in 1× Tris-buffered saline/casein-blocking buffer (BioFX, Owings Mills, MD) or buffer alone was incubated with 125I-BMP-2 or 125I-BMP-4 in a total volume of 200 μl overnight at 4 °C, either alone or in the presence of 80 ng of cold BMP-2, BMP-4, BMP-7, or TGF-β1 for competition assays. For mixing studies, buffer alone, 10 ng of purified RGMa.Fc alone, 10 ng of ALK6.Fc alone (R & D Systems), or 10 ng each of RGMa.Fc and ALK6.Fc together were incubated in 1× Tris-buffered saline/casein-blocking buffer with 125I-BMP-2 in a total volume of 200 μl. The reaction mix was then incubated for 1.5 h at 4 °C on protein A-coated plates (Pierce), plates were washed with wash solution (KPL, Gaithersberg, MD), and individual wells were counted with a standard γ counter. DSS Cross-linking in Solution—100 μl of 125I-BMP-2 or 125I-BMP-4 (400,000 cpm) were incubated overnight at 4 °C with an equal volume of 20 mm HEPES (pH 7.8), 0.1% bovine serum albumin, and protease inhibitors (Roche Diagnostics, Mannheim, Germany) alone, or containing 25 ng of RGMa.Fc or ALK5.Fc (R & D Systems), in the absence or presence of 80 ng of cold BMP-2 or BMP-4. This mixture was incubated in the absence or presence of 2.5 mm disuccinimidyl suberate (DSS, Sigma) in dimethyl sulfoxide for 2 h on ice, followed by quenching of DSS activity with 40 mm Tris (pH 7.5) for 15 min. The mixture was then centrifuged and the supernatant incubated with protein A-Sepharose beads (Amersham Biosciences) at 4 °C for 2 h to precipitate hot BMP-2 or -4 bound to RGMa.Fc. Beads were washed with phosphate-buffered saline (PBS) and protein eluted by non-reducing Laemmli sample buffer (Bio-Rad). Eluted protein was separated by SDS-PAGE and analyzed by autoradiography. For receptor cross-linking studies, 200 ng of RGMa.Fc, 200 ng of ALK6.Fc, and/or 100 ng of BMP-2 were incubated in 100 μl of 20 mm HEPES (pH 7.8), 0.1% bovine serum albumin, and protease inhibitors at 4 °C overnight. The mixtures were then cross-linked with DSS, incubated with protein A beads, and eluted with non-reducing Laemmli sample buffer as described above. The protein complex was then separated by non-reducing SDS-PAGE, electroblotted to polyvinylidene difluoride membranes, and analyzed by Western blot using RGMa antibody (1:2000) as described above for RGMa.Fc. Measurement of Smad1/5/8 Phosphorylation and Id1 Expression—LLC-PK1 cells plated to 70% confluence were transiently transfected with 5 μg of RGMa cDNA or empty vector. Twenty-four hours after transfection cells were incubated in DMEM supplemented with 1% FBS in the absence or presence of 50 ng/ml BMP-2 for 2 h at 37 °C. Cells were sonicated and lysed in 200 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, and 10% glycerol containing a mixture of protease inhibitors (Roche Diagnostics) for twenty minutes on ice. After centrifugation for 20 min at 4 °C, the supernatant was assayed for protein concentration by colorimetric assay (BCA kit, Pierce). 30 μg of protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were probed with α-RGMa (1:2000) as described above for RGMa.Fc. Membranes were stripped in 0.2 m glycine, pH 2.5, 0.5% Tween 20 for 1 h, and re-probed in succession with rabbit polyclonal anti-p-Smad1/5/8 antibody (α-p-Smad1/5/8, Cell Signaling, Beverly, Ma) (1:1000) at 4 °C overnight according to the manufacturer's instructions, rabbit polyclonal anti-Smad1 antibody (α-Smad1, Upstate Biotechnology, Lake Placid, NY) (1:250) at 4 °C overnight, mouse monoclonal anti-β-actin antibody (α-β-actin, clone AC 15, Sigma) (1:5000) at room temperature for 1 h, and rabbit polyclonal anti-Id1 antibody (α-Id1, C 20, Santa Cruz Biotechnology) (1:200) at 4 °C overnight followed by the appropriate horseradish peroxidase-conjugated secondary antibody and chemiluminescence detection after each as described above. Chemiluminescence was quantitated using IPLab Spectrum software (Scanalytics, Vienna, VA). Northern Blot—Adult rat total RNA was separated on a 1.5% formaldehyde agarose gel and blotted onto GeneScreen Plus membrane (PerkinElmer Life Sciences) as previously described (28Costigan M. Mannion R.J. Kendall G. Lewis S.E. Campagna J.A. Coggeshall R.E. Meridith-Middleton J. Tate S. Woolf C.J. J. Neurosci. 1998; 18: 5891-5900Crossref PubMed Google Scholar) Immunohistochemistry—Freshly dissected adult rat lumbar spinal cord was embedded in OCT (Sakura, Tokyo, Japan), frozen on dry ice, cut by cryostat in 16-μm sections, and stored at -80 °C. Spinal sections were fixed in 4% paraformaldehyde, washed three times in PBS, and incubated for 1 h at room temperature in blocking buffer (1% bovine serum albumin, 0.5% Triton X in PBS). Fixed sections were incubated overnight at 4 °C in blocking buffer with rabbit polyclonal α-RGMa (1:500) or rabbit polyclonal α-p-Smad1/5/8 (1:100), in combination with mouse monoclonal anti-neuron-specific nuclear protein antibody (α-NeuN, 1:1000) (Chemicon, Temecula, CA) to visualize neuronal cell bodies. Sections were then washed three times in PBS and incubated with cyanin 3 (Cy3)-conjugated anti-rabbit and fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibodies (1:200 each, Jackson ImmunoResearch) for 1 h at room temperature. Finally, sections were washed three times in PBS and visualized by fluorescence microscopy. RGMa Mediates BMP, but Not TGF-β, Signaling—Because RGM family member DRAGON functions as a BMP co-receptor in LLC-PK1 porcine kidney epithelial cells (14Samad T.A. Rebbapragada A. Bell E. Zhang Y. Sidis Y. Jeong S.J. Campagna J.A. Perusini S. Fabrizio D.A. Schneyer A.L. Lin H.Y. Brivanlou A.H. Attisano L. Woolf C.J. J. Biol. Chem. 2005; 280: 14122-14129Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), we tested whether RGMa also mediated BMP signaling in these cells. LLC-PK1 cells were transfected with a BMP-responsive luciferase reporter (BRE-Luc, Ref. 6Korchynskyi O. ten Dijke P. J. Biol. Chem. 2002; 277: 4883-4891Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar) (Fig. 1, A and C) or a TGF-β responsive luciferase reporter (CAGA-Luc, Ref. 25Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1588) Google Scholar) (Fig. 1B) either alone or in combination with cDNA encoding RGMa. Transfected cells were then incubated with or without BMP-2 or TGF-β1 for 16 h followed by measurement of luciferase activity. In the absence of RGMa, stimulation with BMP-2 or TGF-β1 increased the relative luciferase activity for their respective reporters compared with unstimulated cells (Fig. 1, A and B, compare bars 2 to 1). Co-transfection with RGMa similarly increased BRE luciferase activity even in the absence of exogenous BMP ligand (Fig. 1A, bar 3). The RGMa-mediated BMP signaling was dose-dependent (Fig. 1C, white bars), reaching a peak at about 200 ng of cDNA per transfection. RGMa also augmented signaling produced by exogenous BMP-2 (Fig. 1C, black bars). In contrast, co-transfection with RGMa (up to 1 μg) did not increase the TGF-β responsive CAGA luciferase activity above baseline (Fig. 1B, bar 3). Similar results were seen in another cell line (HepG2 cells, data not shown). Taken together, these results demonstrate that like DRAGON, RGMa mediates BMP, but not TGF-β, signaling. RGMa-mediated BMP Signaling Is Ligand-dependent—The ability of RGMa to generate BMP signals even in the absence of exogenous BMP ligand raises the question of whether RGMa acts in a ligand-independent manner, or whether it augments signaling by endogenous BMP ligands. To investigate this question, we examined whether RGMa-mediated signaling was inhibited by noggin, a soluble BMP inhibitor that binds and sequesters BMP ligands, barring access to membrane receptors (4Balemans W. Van Hul W. Dev. Biol. 2002; 250: 231-250Crossref PubMed Google Scholar, 29Groppe 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: 636-642Cr