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Cell-surface Heparan Sulfate Proteoglycans Potentiate Chordin Antagonism of Bone Morphogenetic Protein Signaling and Are Necessary for Cellular Uptake of Chordin

2004; Elsevier BV; Volume: 279; Issue: 49 Linguagem: Inglês

10.1074/jbc.m408129200

ISSN

1083-351X

Autores

Reema Jasuja, Benjamin L. Allen, William N. Pappano, Alan C. Rapraeger, Daniel S. Greenspan,

Tópico(s)

Connective tissue disorders research

Resumo

Signaling by bone morphogenetic proteins (BMPs) plays a central role in early embryonic patterning, organogenesis, and homeostasis in a broad range of species. Chordin, an extracellular antagonist of BMP signaling, is thought to readily diffuse in tissues, thus forming gradients of BMP inhibition that result in reciprocal gradients of BMP signaling. The latter determine cell fates along the embryonic dorsoventral axis. The secreted protein Twisted Gastrulation (TSG) is thought to help shape BMP signaling gradients by acting as a cofactor that enhances Chordin inhibition of BMP signaling. Here, we demonstrate that mammalian Chordin binds heparin with an affinity similar to that of factors known to functionally interact with heparan sulfate proteoglycans (HSPGs) in tissues. We further demonstrate that Chordin binding in mouse embryonic tissues was dependent upon its interaction with cell-surface HSPGs and that Chordin bound to cell-surface HSPGs (e.g. syndecans), but not to basement membranes containing the HSPG perlecan. Surprisingly, mammalian TSG did not bind heparin unless prebound to Chordin and/or BMP-4, although Drosophila TSG has been reported to bind heparin on its own. Results are also presented that indicate that Chordin-HSPG interactions strongly potentiate the antagonism of BMP signaling by Chordin and are necessary for the retention and uptake of Chordin by cells. These data and others regarding Chordin diffusion have implications for the paradigm of how Chordin is thought to regulate BMP signaling in the extracellular space and how gradients of BMP signaling are formed. Signaling by bone morphogenetic proteins (BMPs) plays a central role in early embryonic patterning, organogenesis, and homeostasis in a broad range of species. Chordin, an extracellular antagonist of BMP signaling, is thought to readily diffuse in tissues, thus forming gradients of BMP inhibition that result in reciprocal gradients of BMP signaling. The latter determine cell fates along the embryonic dorsoventral axis. The secreted protein Twisted Gastrulation (TSG) is thought to help shape BMP signaling gradients by acting as a cofactor that enhances Chordin inhibition of BMP signaling. Here, we demonstrate that mammalian Chordin binds heparin with an affinity similar to that of factors known to functionally interact with heparan sulfate proteoglycans (HSPGs) in tissues. We further demonstrate that Chordin binding in mouse embryonic tissues was dependent upon its interaction with cell-surface HSPGs and that Chordin bound to cell-surface HSPGs (e.g. syndecans), but not to basement membranes containing the HSPG perlecan. Surprisingly, mammalian TSG did not bind heparin unless prebound to Chordin and/or BMP-4, although Drosophila TSG has been reported to bind heparin on its own. Results are also presented that indicate that Chordin-HSPG interactions strongly potentiate the antagonism of BMP signaling by Chordin and are necessary for the retention and uptake of Chordin by cells. These data and others regarding Chordin diffusion have implications for the paradigm of how Chordin is thought to regulate BMP signaling in the extracellular space and how gradients of BMP signaling are formed. Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-β, transforming growth factor-β; BMP, bone morphogenetic protein; DPP, Decapentaplegic; SOG, Short Gastrulation; TLD, Tolloid; TSG, Twisted Gastrulation; HSPG, heparan sulfate proteoglycan; HS, heparan sulfate; FGF, fibroblast growth factor; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; mAb, monoclonal antibody; AP, alkaline phosphatase; CR, cysteine-rich domain; dpc, day postcoitus.-like bone morphogenetic proteins (BMPs) were first isolated from osteogenic extracts of bone, and some BMPs are capable of inducing ectopic bone formation (1Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3352) Google Scholar, 2Celeste A.J. Iannazzi J.A. Taylor R.C. Hewick R.M. Rosen V. Wang E.A. Wozney J.M. Proc. Natl. Acad. Sci. U. S. 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Acad. Sci. U. S. A. 1994; 91: 10255-10259Crossref PubMed Scopus (416) Google Scholar, 7Hawley S.H. Wünnenberg-Stapleton K. Hashimoto C. Laurent M.N. Watabe T. Blumberg B.W. Cho K.W. Genes Dev. 1995; 9: 2923-2935Crossref PubMed Scopus (303) Google Scholar), whereas specification of dorsal mesoderm (Spemann's organizer) and neuroectoderm requires inhibition of BMP signaling. Dorsal inhibition of BMP signaling is achieved by secreted BMP antagonists such as Chordin and Noggin, which are expressed in Spemann's organizer (for a review, see Ref. 8Cho K.W.Y. Blitz I.L. Curr. Opin. Genet. Dev. 1998; 8: 443-449Crossref PubMed Scopus (70) Google Scholar). In Drosophila, dorsoventral patterning of the embryonic axis is mediated by the BMP-2/4 homolog Decapentaplegic (DPP) (9Padgett R.W. St. Johnston R.D. Gelbart W.M. Nature. 1987; 325: 81-84Crossref PubMed Scopus (575) Google Scholar) and the BMP-7 homolog Screw (10Arora K. Levine M.S. O'Connor M.B. Genes Dev. 1994; 8: 2588-2601Crossref PubMed Scopus (175) Google Scholar), whereas Chordin is represented by the homolog Short Gastrulation (SOG) (11Francois V. Sollway M. O'Neill J.W. Emery J. Bier E. Genes Dev. 1994; 8: 2602-2616Crossref PubMed Scopus (270) Google Scholar, 12Francois V. Bier E. Cell. 1995; 80: 19-20Abstract Full Text PDF PubMed Scopus (116) Google Scholar). Drosophila homologs do not exist for the other vertebrate extracellular antagonists of BMP signaling, such as Noggin and the Dan family of BMP inhibitors (13Massagué J. Chen Y.-G. Genes Dev. 2000; 14: 627-644PubMed Google Scholar). In Drosophila, DPP is expressed dorsally, whereas SOG is expressed ventrolaterally. Secreted SOG is thought to inhibit signaling by binding DPP in an extracellular latent complex (14Marqués G. Musacchio M. Shimell J.J. Wünnenberg-Stapleton K. Cho K.W.Y. O'Connor M.B. Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar), and it appears that SOG diffuses to form a concentration gradient, resulting in a reciprocal gradient of DPP signaling, which, in turn, determines cell fates along the dorsoventral axis (14Marqués G. Musacchio M. Shimell J.J. Wünnenberg-Stapleton K. Cho K.W.Y. O'Connor M.B. Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 15Srinivasan S. Rashka K.E. Bier E. Dev. Cell. 2002; 2: 91-101Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Although a similar model has been generally accepted for vertebrate systems (16Piccolo S. Sasai Y. Lu B. De Robertis E.M. Cell. 1996; 86: 589-598Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar), there are differences. First, in addition to the SOG ortholog Chordin, vertebrates possess various other extracellular BMP antagonists. Second, it remains unclear to what extent diffusion of Chordin over long distances is involved in vertebrate patterning (17Blitz I.L. Shimmi O. Wünnenberg-Stapleton K. O'Connor M.B. Cho K.W.Y. Dev. Biol. 2000; 223: 120-138Crossref PubMed Scopus (57) Google Scholar). Specifically, although high levels of overexpressed recombinant Chordin appear capable of diffusing to directly inhibit BMP over long distances, there is evidence suggesting that endogenous Chordin may employ a cell relay mechanism to effect long-range BMP inhibition (17Blitz I.L. Shimmi O. Wünnenberg-Stapleton K. O'Connor M.B. Cho K.W.Y. Dev. Biol. 2000; 223: 120-138Crossref PubMed Scopus (57) Google Scholar). In Drosophila, the gene products Tolloid (TLD) and Twisted Gastrulation (TSG) help shape the gradient of DPP signaling that forms the dorsoventral axis through interactions with SOG and DPP. Specifically, the metalloproteinase TLD cleaves SOG to release DPP from SOG·DPP complexes (14Marqués G. Musacchio M. Shimell J.J. Wünnenberg-Stapleton K. Cho K.W.Y. O'Connor M.B. Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar), whereas TSG increases the avidity with which SOG binds DPP, thus enhancing SOG antagonism of DPP signaling (18Ross J.J. Shimmi O. Vilmos P. Petryk A. Kim H. Gaudenz K. Hermanson S. Ekker S.C. O'Connor M.B. Marsh J.L. Nature. 2001; 410: 479-483Crossref PubMed Scopus (241) Google Scholar). In mammals, the TLD-related proteinases BMP-1 and mTLL-1 (mammalian Tolloid-like 1) process Chordin in vivo (19Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W.Y. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (231) Google Scholar, 20Pappano W.N. Steiglitz B.M. Scott I.C. Keene D.R. Greenspan D.S. Mol. Cell. Biol. 2003; 23: 4428-4438Crossref PubMed Scopus (105) Google Scholar), whereas overexpression of various TLD-related proteinases in Xenopus and zebrafish can counteract the dorsalizing effects of overexpressed Chordin (9Padgett R.W. St. Johnston R.D. Gelbart W.M. Nature. 1987; 325: 81-84Crossref PubMed Scopus (575) Google Scholar, 21Piccolo S. Agius E. Lu B. Goodman S. Dale L. De Robertis E.M. Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 22Blader P. Rastegar S. Fisher N. Strähle U. Science. 1997; 278: 1937-1940Crossref PubMed Scopus (168) Google Scholar, 23Wardle F.C. Welch J.V. Dale L. Mech. Dev. 1999; 86: 75-85Crossref PubMed Scopus (48) Google Scholar). Vertebrates also possess homologs of TSG (24Oelgeschläger M. Larrain J. Geissert De Robertis E.M. Nature. 2000; 405: 57-63Crossref Scopus (232) Google Scholar, 25Scott I.C. Blitz I.L. Pappano W.N. Maas S.A. Cho K.W.Y. Greenspan D.S. Nature. 2001; 410: 475-478Crossref PubMed Scopus (159) Google Scholar, 26Chang C. Holtzman D.A. Chau S. Chickering T. Woolf E.A. Holmgren L.M. Bodorova J. Gearing D.P. Holmes Brivanlou A.H. Nature. 2001; 410: 483-487Crossref PubMed Scopus (165) Google Scholar), which bind Chordin and the DPP ortholog BMP-4 in a ternary complex (24Oelgeschläger M. Larrain J. Geissert De Robertis E.M. Nature. 2000; 405: 57-63Crossref Scopus (232) Google Scholar, 25Scott I.C. Blitz I.L. Pappano W.N. Maas S.A. Cho K.W.Y. Greenspan D.S. Nature. 2001; 410: 475-478Crossref PubMed Scopus (159) Google Scholar, 26Chang C. Holtzman D.A. Chau S. Chickering T. Woolf E.A. Holmgren L.M. Bodorova J. Gearing D.P. Holmes Brivanlou A.H. Nature. 2001; 410: 483-487Crossref PubMed Scopus (165) Google Scholar), thereby enhancing antagonism of BMP signaling (25Scott I.C. Blitz I.L. Pappano W.N. Maas S.A. Cho K.W.Y. Greenspan D.S. Nature. 2001; 410: 475-478Crossref PubMed Scopus (159) Google Scholar, 26Chang C. Holtzman D.A. 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Horigan E. Sasaki M. Yamada Y. Hassell J.R. J. Biol. Chem. 1991; 266: 22939-22947Abstract Full Text PDF PubMed Google Scholar). Secreted growth factors that interact with HSPGs include, but are not limited to, fibroblast growth factors (FGFs), the epidermal growth factor and platelet-derived growth factor families, TGF-β1 and TGF-β2, and hepatocyte growth factor (34Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2320) Google Scholar). HSPGs have been shown to interact with various developmentally important ligands, including Wnt family members (34Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2320) Google Scholar) and TGF-β-like BMPs (35Ruppert R. Hoffmann E. Sebald W. Eur. J. Biochem. 1996; 237: 295-302Crossref PubMed Scopus (469) Google Scholar, 36Jackson S.M. Nakato H. Sugiura M. Jannuzi A. Oakes R. Kaluza V. Golden C. Selleck S.B. Development (Camb.). 1997; 124: 4113-4120Crossref PubMed Google Scholar). With regard to the latter, the Drosophila glypican dally is required for normal DPP signaling (36Jackson S.M. Nakato H. Sugiura M. Jannuzi A. Oakes R. Kaluza V. Golden C. Selleck S.B. Development (Camb.). 1997; 124: 4113-4120Crossref PubMed Google Scholar), whereas HS may be capable of modulating BMP-2 activities in cell culture and embryonic chick limb bud assays (35Ruppert R. Hoffmann E. Sebald W. Eur. J. Biochem. 1996; 237: 295-302Crossref PubMed Scopus (469) Google Scholar, 37Irie A. Habuchi H. Kimata K. Sanai Y. Biochem. Biophys. Res. Commun. 2003; 308: 858-865Crossref PubMed Scopus (107) Google Scholar, 38Takada T. Katagiri T. Ifuku M. Morimura N. Kobayashi M. Hasegawa K. Ogamo A. Kamijo R. J. Biol. Chem. 2003; 278: 43229-43235Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Recently, it has been shown that Noggin binds to HSPGs (39Paine-Saunders S. Viviano B.L. Economides A.N. Saunders S. J. Biol. Chem. 2002; 277: 2089-2096Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Here, we demonstrate that Chordin binds heparin-Sepharose with elution profiles similar to those of proteins, such as FGFs, that are known to functionally interact with HSPGs in tissues. We show that, surprisingly, mammalian TSG did not bind heparin, in contrast to Drosophila TSG (40Mason E.D. Williams S. Grotendorst G.R. Marsh J.L. Mech. Dev. 1997; 64: 61-75Crossref PubMed Scopus (34) Google Scholar), but that it would bind heparin if complexed to Chordin and/or TSG. We show that Chordin bound embryonic tissues in a manner that was dependent upon its interactions with tissue HSPGs and that Chordin bound cell-surface HSPGs and specifically cell-surface syndecans, but that it did not detectably bind basement membranes containing the major HSPG perlecan. Importantly, the results presented herein indicate that cell-surface HSPGs strongly potentiate Chordin antagonism of BMP signaling, effect the retention of Chordin at cell surfaces, and are necessary for the cellular uptake of Chordin. Implications of the various data for the modulation of BMP signaling and the formation of BMP signaling gradients are discussed. Production of Recombinant Proteins—FLAG-tagged mouse Chordin, FLAG-tagged Chordin fragments, and protein C-tagged mouse TSG were expressed and purified as described previously (19Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W.Y. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (231) Google Scholar, 25Scott I.C. Blitz I.L. Pappano W.N. Maas S.A. Cho K.W.Y. Greenspan D.S. Nature. 2001; 410: 475-478Crossref PubMed Scopus (159) Google Scholar). Concentrations of BMP-4 (R&D Systems), FLAG-tagged mouse Chordin, FLAG-tagged Chordin fragments, and protein C-tagged mouse TSG were calculated by comparing the intensities of Coomassie Blue-stained bands with protein standards of known concentrations. Heparin-Sepharose Chromatography—A heparin-Sepharose CL-6B slurry (Amersham Biosciences) was pelleted and resuspended in a double volume of phosphate-buffered saline (PBS) containing 1 mg/ml bovine serum albumin (BSA), and 100 μl were added to separate equimolar amounts of BMP-4, Chordin, or mouse TSG or to equimolar combinations of these proteins that had been preincubated together for 30 min at 37 °C. Heparin-Sepharose/protein mixtures were incubated for 2 h at 4 °C and then loaded onto a 0.8 × 4-cm column, and the flow-through was collected. The column was washed with 500 μl of PBS containing 1 mg/ml BSA, and stepwise elution was performed using 75-μl aliquots of PBS containing increasing concentrations of NaCl. Elution was followed by addition of 4× SDS-PAGE sample buffer with 5% β-mercaptoethanol to fractions and electrophoresis on 4–15% acrylamide gradient gels (Bio-Rad). Immunoblotting by electrotransfer to polyvinylidene difluoride membranes, incubations of blots with antibodies, and washes were performed as described previously (41Lee S. Solow-Cordero D.E. Kessler E. Takahara K. Greenspan D.S. J. Biol. Chem. 1997; 272: 19059-19066Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Rabbit polyclonal antibody raised against peptide EPPALPIRSEKEPLPVRGA (20Pappano W.N. Steiglitz B.M. Scott I.C. Keene D.R. Greenspan D.S. Mol. Cell. Biol. 2003; 23: 4428-4438Crossref PubMed Scopus (105) Google Scholar), corresponding to residues 30–48 at the N terminus of the published mouse Chordin sequence (42Pappano W.N. Scott I.C. Clark T.G. Eddy R.L. Shows T.B. Greenspan D.S. Genomics. 1998; 52: 236-239Crossref PubMed Scopus (27) Google Scholar); anti-BMP-4 monoclonal antibody (R&D Systems); peroxidase-conjugated anti-protein C monoclonal antibody (Roche Applied Science), for detection of protein C epitope-tagged mouse TSG; biotinylated anti-FLAG monoclonal antibody (Sigma); and streptavidin-horseradish peroxidase conjugate and secondary antibodies (Amersham Biosciences) were all used at 1:5000. Cell Binding Assays and Immunofluorescence—Raji cells stably transfected with syndecan-1 (Raji-S1) (43Lebakken C.S. Rapraeger A.C. J. Cell Biol. 1996; 132: 1209-1221Crossref PubMed Scopus (66) Google Scholar), Raji cells similarly transfected with syndecan-4 (Raji-S4), and parental Raji cells cultured as described previously (44Allen B.L. Filla M.S. Rapraeger A.C. J. Cell Biol. 2001; 155: 845-857Crossref PubMed Scopus (126) Google Scholar), and 10t1/2 cells cultured in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS) (Hyclone Laboratories) were fixed by incubation with 4% paraformaldehyde in PBS for 1 h. Fixed cells were then incubated with or without 60 nm Chordin in RPMI 1640 medium and 10% FBS for 1 h at room temperature. Three 5-min washes with PBS were followed by incubation with anti-Chordin antibody (described above) at 1:200, with rat anti-syndecan-1 monoclonal antibody 281.2 at a concentration of 1 μg/ml, or with rabbit anti-syndecan-4 polyclonal antibody (see below) at 1:300 for 1 h. This was followed by three 5-min washes with PBS and then incubation with Alexa 546-conjugated goat anti-rabbit antibody or Alexa 488-conjugated donkey anti-rat antibody (Molecular Probes, Inc.) at 1:1000 for 30 min, followed by a final three 5-min washes with PBS. Rabbit anti-syndecan-4 polyclonal antibody was made using a syndecan-4 exodomain-glutathione S-transferase fusion protein. The antibody was passed over a glutathione S-transferase affinity column, leaving only syndecan-4-specific antibody. Transfected M2-10B4 cells were fixed as described above, but were not incubated for 1 h at room temperature prior to addition of anti-Chordin antibody. For experiments in which non-transfected and Chordin-transfected cells were co-cultured, transfected M2-10B4 cells were labeled with CellTracker™ Green 5-chloromethylfluorescein diacetate (Molecular Probes, Inc.) following the manufacturer's protocol. The labeled/transfected cells were plated in droplets of medium and allowed to adhere and spread for 5 h such that they formed colonies. After 5 h, nontransfected cells in sufficient medium to cover the bottom of the dish were plated such that they adhered and spread in areas surrounding the colonies of Chordin-transfected cells. Cells were co-cultured for an additional 24 h and then fixed and immunostained as described above. In Situ HS Binding Assays—In situ binding to HS was performed essentially as described previously (45Friedl A. Chang Z. Tierney A. Rapraeger A.C. Am. J. Pathol. 1997; 150: 1443-1455PubMed Google Scholar). Frozen tissue sections (cut to a thickness of 5 μm) were air-dried, fixed in 4% paraformaldehyde on ice, treated with 2 m NaCl to remove exogenous HS-binding molecules, and then blocked overnight in RPMI 1640 medium and 10% FBS at 4 °C. The following day, sections were incubated with 60 nm Chordin for 1 h, and after three washes with PBS, bound Chordin was detected using rabbit anti-Chordin antibody (described above) at 1:200 and Alexa 546-conjugated goat anti-rabbit antibody. Serial sections were incubated with rabbit anti-perlecan antibody (kind gift of Dr. J. R. Hassell, University of South Florida) at 1:1000 and detected with Alexa 546-conjugated goat anti-rabbit antibody at 1:1000. Removal of endogenous HS was accomplished by treatment of tissue sections or cells with 0.006 IU/ml heparinases I and III (heparin lyases; Seikagaku America, Inc.) for 2 h at 37 °C, followed by addition of fresh enzyme for an additional 2 h. Characterization of the distribution of total HS in tissues was performed with monoclonal antibody (mAb) 3G10 (Seikagaku America, Inc.), which detects unsaturated glucuronate residues remaining on core proteins following heparinase III treatment (46David G. Bai X.M. Van Der Schueren B. Cassiman J.-J. Van Den Berghe H. J. Cell Biol. 1992; 119: 961-975Crossref PubMed Scopus (407) Google Scholar). Sections were incubated with mAb 3G10 at 1:200 in RPMI 1640 medium and 10% fetal calf serum, followed by Cy3-conjugated donkey anti-mouse secondary antibody (Molecular Probes, Inc.) at 1:300. Staining of sections without prior heparinase III treatment showed no mAb 3G10 staining (data not shown). Western Blot Analysis—Cell lysate collection and subsequent Western blot analysis were performed as described previously (47Burbach B.J. Friedl A. Mundhenke C. Rapraeger A.C. Matrix Biol. 2003; 22: 163-177Crossref PubMed Scopus (51) Google Scholar). Briefly, 10t1/2 cells were lysed in cold PBS containing 1% Triton X-100, 0.1% SDS, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 2 μg/ml aprotinin, and the cell layer was then scraped into this solution. Following removal of the insoluble fraction by centrifugation, total protein levels were determined by BCA assay. 100 μg of total protein were then precipitated overnight in 2.5 volumes of methanol at -20 °C. Pellets were resuspended in heparinase buffer (50 mm HEPES (pH 6.5), 50 mm NaOAc, 150 mm NaCl, and 5 mm CaCl2) and incubated for 2 h at 37 °C with 0.0001 unit of heparinase I (IBEX), 0.0001 unit of heparinase III (IBEX), and 0.005 units of chondroitin ABC lyase (Sigma). Samples were resolved by SDS-PAGE, transferred to Immobilon-P (polyvinylidene difluoride; Millipore Corp.), fixed with 0.05% glutaraldehyde for 30 min, and blocked overnight at 4 °C in Tris-buffered saline containing 3% BSA. HSPGs were detected with mAb 3G10, followed by incubation with alkaline phosphatase (AP)-conjugated secondary antibody. Proteins were visualized with ECF detection reagent (Amersham Biosciences) and scanned on a Storm PhosphorImager (Amersham Biosciences). AP and BMP Signaling Assay—M2-10B4 cells were transfected either with the empty pcDNA3.1 vector (Invitrogen) or with the same vector containing cDNA coding for sequences of murine Chordin with a C-terminal FLAG epitope using LipofectAMINE (Invitrogen). The Chordin expression vector was constructed by excising the Chordin-FLAG insert from a previously described pCEP-based vector (19Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W.Y. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (231) Google Scholar) and inserting it between the pcDNA3.1 AflII and NotI sites. Transfected M2-10B4 cells were selected with G418; resistant colonies were ring-cloned; and one clonal line derived from empty vector-transfected cells and one of the clonal lines expressing the highest levels of recombinant Chordin were used for the AP assays. AP assays were performed essentially as described by Zebboudj et al. (48Zebboudj A.F. Imura M. Boström K. J. Biol. Chem. 2002; 277: 4388-4394Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Briefly, cells were washed twice with PBS and grown for 24 h either in DMEM and 10% FBS or in low sulfate DMEM (49Rapraeger A.C. Guimond S. Krufka A. Olwin B.B. Methods Enzymol. 1994; 245: 219-240Crossref PubMed Scopus (96) Google Scholar) containing 10% dialyzed FBS and 50 mm chlorate. Cells were then trypsinized and plated in triplicate on 96-well plates at 10,000 cells/well in DMEM and 10% FBS or in low sulfate DMEM containing 10% dialyzed FBS and 50 mm chlorate. After 16 h, cells were washed once with PBS and placed in 200 μl of DMEM and 10% FBS or in 200 μl of low sulfate DMEM containing 10% dialyzed FBS and 50 mm chlorate either with 6 nm BMP-4 (dissolved in 4 mm HCl and 0.1% BSA) or with 4 mm HCl and 0.1% BSA. AP activity was determined 48 h later. Cells were washed once with PBS and lysed by freeze-thawing twice in 50 μl of 0.2% Nonidet P-40 and 1 mm MgCl2. 150 μl of AP buffer (5 mg of p-nitrophenyl phosphate dissolved in 2.5 ml of Sigma 221 alkaline buffer solution diluted 1:2 with water) were added to each well, and plates were incubated for 1 h at 37 °C. AP activity was then measured by absorbance readings taken at 405 nm using a Universal Microplate Reader (Bio-Tek Instruments, Winooski, VT). Chordin and BMP-4, but Not Mouse TSG, Bind Heparin In Vitro—To gain insight into the potential of Chordin to bind HS in vivo, purified recombinant murine Chordin was incubated with heparin-Sepharose, and the mixture was placed in a column and eluted with a step gradient of 0.15–2.0 m NaCl. As shown in Fig. 1A, Chordin bound heparin, with the majority of bound Chordin eluting at 0.55–1.60 m NaCl and some requiring elution with 2.0 m NaCl or boiling in SDS. It has previously been shown that TGF-β-like BMPs (e.g. BMP-2) (35Ruppert R. Hoffmann E. Sebald W. Eur. J. Biochem. 1996; 237: 295-302Crossref PubMed Scopus (469) Google Scholar) bind heparin with high affinity. Here, BMP-4 was shown to bind heparin with an elution profile similar to that of Chordin (Fig. 1A). Murine TSG did not bind heparin under these conditions, as the vast majority was found in the flow-through and wash (Fig. 1A). This result was surprising, as Drosophila TSG has been reported to be a heparin-binding protein (40Mason E.D. Williams S. Grotendorst G.R. Marsh J.L. Mech. Dev. 1997; 64: 61-75Crossref PubMed Scopus (34) Google Scholar). Chordin binds BMP-4 and murine TSG and can bind the two proteins simultaneously in a ternary complex (24Oelgeschläger M. Larrain J. Geissert De Robertis E.M. Nature. 2000; 405: 57-63Crossref Scopus (232) Google Scholar, 25Scott I.C. Blitz I.L. Pappano W.N. Maas S.A. Cho K.W.Y. Greenspan D.S. Nature. 2001; 410: 475-478Crossref PubMed Scopus (159) Google Scholar, 26Chang C. Holtzman D.A. Chau S. Chickering T. Woolf E.A. Holmgren L.M. Bodorova J. Gearing D.P. Holmes Brivanlou A.H. Nature. 2001; 410: 483-487Crossref PubMed Scopus (165) Google Scholar). To determine whether binding to mouse TSG might decrease the affinity of Chordin and/or BMP-4 for heparin or whether binding of TSG to the other two proteins might increase its retention on heparin, the three proteins were preincubated together prior to incubation with heparin-Sepharose. As shown in Fig. 1B, in the presence of Chordin and BMP-4, some portion of the input murine TSG was retained on heparin and was eluted at fairly high concentrations of NaCl, similar to those at which Chordin and BMP-4 were eluted. As only som

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