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• W1979422179 abstract "Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 accumulation in a cell type-specific manner. The effect requires O-sulfation in heparin, and involves fibroblast growth factor-2 as well as fibroblast growth factor receptor-1. Interestingly, further investigation uncovers that heparin can also affect the post-translational modification of sFRP-1. We demonstrate that sFRP-1 is post-translationally modified by tyrosine sulfation at tyrosines 34 and 36, which is inhibited by the treatment of heparin. The results suggest that accumulation of sFRP-1 induced by heparin is in part due to the relative stabilization of unsulfated sFRP-1 and the direct stabilization by heparin. The study has revealed a multifaceted regulation on sFRP-1 protein by heparin. Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 accumulation in a cell type-specific manner. The effect requires O-sulfation in heparin, and involves fibroblast growth factor-2 as well as fibroblast growth factor receptor-1. Interestingly, further investigation uncovers that heparin can also affect the post-translational modification of sFRP-1. We demonstrate that sFRP-1 is post-translationally modified by tyrosine sulfation at tyrosines 34 and 36, which is inhibited by the treatment of heparin. The results suggest that accumulation of sFRP-1 induced by heparin is in part due to the relative stabilization of unsulfated sFRP-1 and the direct stabilization by heparin. The study has revealed a multifaceted regulation on sFRP-1 protein by heparin. Wnt proteins are a large family of structurally related secreted glycoproteins that mediate fundamental biological processes such as cell polarity and proliferation, tissue patterning, and tumorigenesis (1Dale T.C. Biochem. J. 1998; 329: 209-223Crossref PubMed Scopus (435) Google Scholar, 2Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar, 3Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1728) Google Scholar, 4Huelsken J. Birchmeier W. Curr. Opin. Genet. Dev. 2001; 11: 547-553Crossref PubMed Scopus (486) Google Scholar). The Wnt signaling event is initiated by the binding of Wnt proteins to a membrane receptor complex consisting of a seven-pass transmembrane molecule of the Frizzled (Fz) 2The abbreviations used are: Fz, Frizzled; sFRP-1, secreted Frizzled-related protein-1; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; CHO, Chinese hamster ovary; FBS, fetal bovine serum; MTX, methotrexate; Endo H, endoglycosidase H; PNGase, peptide:N-glycosidase; NTA, nitriloacetic acid; SEC, size exclusion column; MOPS, 4-morpholinepropanesulfonic acid; TCF, T-cell factor. 2The abbreviations used are: Fz, Frizzled; sFRP-1, secreted Frizzled-related protein-1; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; CHO, Chinese hamster ovary; FBS, fetal bovine serum; MTX, methotrexate; Endo H, endoglycosidase H; PNGase, peptide:N-glycosidase; NTA, nitriloacetic acid; SEC, size exclusion column; MOPS, 4-morpholinepropanesulfonic acid; TCF, T-cell factor. family (5Wang Y. Macke J.P. Abella B.S. Andreasson K. Worley P. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. J. Biol. Chem. 1996; 271: 4468-4476Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar) and members of the low density lipoprotein receptor-related family (LRP5 or LRP6) (6Wehrli M. Dougan S.T. Caldwell K. O'Keefe L. Schwartz S. Vaizel-Ohayon D. Schejter E. Tomlinson A. DiNardo S. Nature. 2000; 407: 527-530Crossref PubMed Scopus (715) Google Scholar, 7Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1078) Google Scholar, 8Pinson K.I. Brennan J. Monkley S. Avery B.J. Skarnes W.C. Nature. 2000; 407: 535-538Crossref PubMed Scopus (878) Google Scholar). The binding of Wnts to the receptors activates various intracellular signaling pathways according to the Wnt, Fz, and cell type involved (3Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1728) Google Scholar, 4Huelsken J. Birchmeier W. Curr. Opin. Genet. Dev. 2001; 11: 547-553Crossref PubMed Scopus (486) Google Scholar). In the canonical or Wnt/β-catenin pathway, Wnt binding activates disheveled protein and leads to the inhibition of glycogen synthase kinase-3β and subsequent stabilization of β-catenin. β-Catenin translocates to the nucleus, interacts with DNA-binding proteins of the T-cell factor/lymphoid enhancer-binding factor family, and activates transcription of target genes (1Dale T.C. Biochem. J. 1998; 329: 209-223Crossref PubMed Scopus (435) Google Scholar, 2Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar, 3Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1728) Google Scholar, 4Huelsken J. Birchmeier W. Curr. Opin. Genet. Dev. 2001; 11: 547-553Crossref PubMed Scopus (486) Google Scholar).Wnts bind to Fz proteins through a cysteine-rich domain, which contains 10 cysteines conserved in all members of the Fz family (5Wang Y. Macke J.P. Abella B.S. Andreasson K. Worley P. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. J. Biol. Chem. 1996; 271: 4468-4476Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 9Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. Nature. 1996; 382: 225-230Crossref PubMed Scopus (1214) Google Scholar). The cysteine-rich domain is not unique to Fz and is also the N-terminal domain of secreted Frizzled-related proteins (sFRPs), a family of glycoproteins that are ∼300 amino acids in length (10Leyns L. Bouwmeester T. Kim S.H. Piccolo S. De Robertis E.M. Cell. 1997; 88: 747-756Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 11Wang S. Krinks M. Lin K. Luyten F.P. Moos Jr., M. Cell. 1997; 88: 757-766Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 12Rattner A. Hsieh J.C. Smallwood P.M. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2859-2863Crossref PubMed Scopus (485) Google Scholar, 13Finch P.W. He X. Kelley M.J. Uren A. Schaudies R.P. Popescu N.C. Rudikoff S. Aaronson S.A. Varmus H.E. Rubin J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6770-6775Crossref PubMed Scopus (361) Google Scholar, 14Melkonyan H.S. Chang W.C. Shapiro J.P. Mahadevappa M. Fitzpatrick P.A. Kiefer M.C. Tomei L.D. Umansky S.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13636-13641Crossref PubMed Scopus (289) Google Scholar, 15Wolf V. Ke G. Dharmarajan A.M. Bielke W. Artuso L. Saurer S. Friis R. FEBS Lett. 1997; 417: 385-389Crossref PubMed Scopus (70) Google Scholar, 16Salic A.N. Kroll K.L. Evans L.M. Kirschner M.W. Development. 1997; 124: 4739-4748Crossref PubMed Google Scholar). sFRPs are capable of binding to Wnts and Fz receptors, and are also modulators of Wnt signaling (17Kawano Y. Kypta R. J. Cell Sci. 2003; 116: 2627-2634Crossref PubMed Scopus (1337) Google Scholar). sFRP-1 is a 35-kDa prototypical member of the sFRP family (14Melkonyan H.S. Chang W.C. Shapiro J.P. Mahadevappa M. Fitzpatrick P.A. Kiefer M.C. Tomei L.D. Umansky S.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13636-13641Crossref PubMed Scopus (289) Google Scholar, 18Uren A. Reichsman F. Anest V. Taylor W.G. Muraiso K. Bottaro D.P. Cumberledge S. Rubin J.S. J. Biol. Chem. 2000; 275: 4374-4382Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 19Bafico A. Gazit A. Pramila T. Finch P.W. Yaniv A. Aaronson S.A. J. Biol. Chem. 1999; 274: 16180-16187Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 20Chong J.M. Uren A. Rubin J.S. Speicher D.W. J. Biol. Chem. 2002; 277: 5134-5144Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). It has been shown that sFRP-1 acts as a biphasic modulator of Wnt signaling, counteracting Wnt-induced effects at high concentrations and promoting them at lower concentrations (18Uren A. Reichsman F. Anest V. Taylor W.G. Muraiso K. Bottaro D.P. Cumberledge S. Rubin J.S. J. Biol. Chem. 2000; 275: 4374-4382Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Deletion of sFRP-1 in mice leads to decreased osteoblast and osteocyte apoptosis and elevated bone mineral density (21Bodine P.V. Zhao W. Kharode Y.P. Bex F.J. Lambert A.J. Goad M.B. Gaur T. Stein G.S. Lian J.B. Komm B.S. Mol. Endocrinol. 2004; 18: 1222-1237Crossref PubMed Scopus (401) Google Scholar).Heparin and heparan sulfate (HS) are mammalian glycosaminoglycans with the highest negative charge density of known biological macromolecules, and are known to bind by ionic interactions with a variety of proteins such as fibroblast growth factors (FGFs) (22Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar). The interactions of HS with heparin-binding proteins can have a direct effect on important cellular processes such as FGF cell signaling events (23Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1285) Google Scholar, 24Szebenyi G. Fallon J.F. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar). sFRP-1 as well as Wnts are heparin-binding proteins (13Finch P.W. He X. Kelley M.J. Uren A. Schaudies R.P. Popescu N.C. Rudikoff S. Aaronson S.A. Varmus H.E. Rubin J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6770-6775Crossref PubMed Scopus (361) Google Scholar, 16Salic A.N. Kroll K.L. Evans L.M. Kirschner M.W. Development. 1997; 124: 4739-4748Crossref PubMed Google Scholar, 18Uren A. Reichsman F. Anest V. Taylor W.G. Muraiso K. Bottaro D.P. Cumberledge S. Rubin J.S. J. Biol. Chem. 2000; 275: 4374-4382Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar) and, in fact, sFRP-1 was originally isolated and identified from the heparin-binding fraction of human embryonic lung fibroblast-conditioned medium (13Finch P.W. He X. Kelley M.J. Uren A. Schaudies R.P. Popescu N.C. Rudikoff S. Aaronson S.A. Varmus H.E. Rubin J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6770-6775Crossref PubMed Scopus (361) Google Scholar). The heparin-binding domain of sFRP-1 is in the C-terminal region of the protein and has weak homology with netrins (10Leyns L. Bouwmeester T. Kim S.H. Piccolo S. De Robertis E.M. Cell. 1997; 88: 747-756Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 11Wang S. Krinks M. Lin K. Luyten F.P. Moos Jr., M. Cell. 1997; 88: 757-766Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). The complex of sFRP-1 and Wg (Drosophila Wnt1 homologue) can be stabilized by heparin in vitro, suggesting that heparin or endogenous heparan-sulfate proteoglycan (HSPG) may promote sFRP-1/Wg binding by serving as a scaffold to facilitate interaction between sFRP-1 and Wg (18Uren A. Reichsman F. Anest V. Taylor W.G. Muraiso K. Bottaro D.P. Cumberledge S. Rubin J.S. J. Biol. Chem. 2000; 275: 4374-4382Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Lowering tissue HSPG levels has been shown to impair Wnt signaling in vivo, supporting the notion that HSPG plays an important role in the Wnt signaling regulation (25Kispert A. Vainio S. Shen L. Rowitch D.H. McMahon A.P. Development. 1996; 122: 3627-3637Crossref PubMed Google Scholar, 26Haerry T.E. Heslip T.R. Marsh J.L. O'Connor M.B. Development. 1997; 124: 3055-3064Crossref PubMed Google Scholar, 27Binari R.C. Staveley B.E. Johnson W.A. Godavarti R. Sasisekharan R. Manoukian A.S. Development. 1997; 124: 2623-2632Crossref PubMed Google Scholar, 28Hacker U. Lin X. Perrimon N. Development. 1997; 124: 3565-3573Crossref PubMed Google Scholar).In this study, we report two unexpected effects of heparin on sFRP-1. One is that heparin can stimulate sFRP-1 accumulation in a cell type-specific manner. The effect involves FGF-2 and FGF receptor-1. Surprisingly we also find that heparin can affect the post-translational modification of sFRP-1, which induces a mobility shift of the protein on SDS-PAGE. Further study shows that sFRP-1 is tyrosine-sulfated at tyrosines 34 and 36, and heparin treatment inhibits the modification. The data suggests that direct stabilization by heparin and the relative stabilization of unsulfated sFRP-1 contribute partly to the accumulation of sFRP-1 induced by heparin.EXPERIMENTAL PROCEDURESCell Lines and Cell Culture—All mammalian cell lines (HEK293, CHO, and Lec3.2.8.1) were grown and maintained in a humidified incubator with 5% CO2 at 37 °C. HEK293 cells were cultured in free-style 293 media (Invitrogen) supplemented with 5% fetal bovine serum (FBS). CHO-DUKX stable lines were grown in α media containing 10% FBS and 200 nm methotrexate (MTX). HEK293 stable lines were cultured in α media containing 10% FBS and 100 nm MTX. Lec3.2.8.1 stable lines were maintained in glutamine-free Dulbecco's modified Eagle's medium with 10% FBS and 25 μm methionine sulfoximine.DNA Constructs—For sFRP-1 DNA constructs, C-terminal His6 tags and the mutation V312L/F313E/ΔK314 were incorporated into PCR primers before the stop codon. The PCR products were digested with SalI and EcoRI. The gel-purified DNA fragments were subcloned into pSMED2 (resulted pWZ1028), pSMEDA (resulted pWZ1049), or pSMEG (resulted pWZ1097) behind a murine cytomegalovirus promoter. For expressing C-terminal His6-tagged Agg-2-His6, C-terminal FLAG-tagged Agg-1-E362Q-A520 (pWZ1071), or C-terminal FLAG-tagged Agg-1-A520 (pWZ1072), PCR fragments digested with SalI and XbaI were subcloned into pSMEDA vector. sFRP-1 mutants Y36F (pWZ1134), Y34F (pWZ1143), and Y34F/Y36F (pWZ1148), were generated with the Stratagene mutagenesis kit in pWZ1049. All constructs were confirmed by sequencing.Transient and Stable Expression—Transient expression was performed in either 50-ml spinners or 1-liter spinners. For the 50-ml culture volume, 25 μg of plasmid DNA was mixed with 400 μg of polyethylenimine (25 kDa, linear, neutralized to pH 7.0 by HCl, 1 mg/ml, Polysciences (Warrington, PA)) in 2.5 ml of serum-free 293 media. For the 1-liter volume, 500 μg of DNA was mixed with 4 mg of polyethylenimine in 50 ml of serum-free 293 media. Then the mixtures were mixed with either 50 ml or 1 liter of HEK293 cells in 293 media with 5% FBS at a cell density of 0.5 × 106 cells/ml. The spinners were incubated at 37 °C with a rotation rate of 170 rpm on a P2005 Stirrer (Bellco) for 72-144 h before harvest.For the establishment of CHO-DUKX sFRP-1 stable lines, construct pWZ1028 was transfected into CHO-DUKX cells and the transfectomas were selected against 50, 100, or 200 nm MTX for 3 weeks. After screening 72 colonies, three 200 nm MTX-resistant clones (200-10, -11, -12) with the highest expression of sFRP-1 were isolated. In this study, HEK293 cells were also found to be sensitive to MTX at concentrations of 100 nm and above, even though they have two copies of the dihydrofolate reductase gene. To construct a HEK293 stable line for sFRP-1, pWZ1028 was transfected into HEK293-EBNA and transfectomas were selected against 100 or 250 nm MTX for 3 weeks. The two best clones, 100-5 and 100-20, were then isolated. To establish Lec3.2.8.1 stable lines for sFRP-1, pWZ1097 was transfected into Lec3.2.8.1 and the transfectomas were selected against 10 or 25 μm methionine sulfoximine for 3 weeks. Several positive clones were isolated.Immunoblotting, Antibodies, and Chemicals—Immunoblotting was performed as described previously (29Zhong X. Kriz R. Seehra J. Kumar R. FEBS Lett. 2004; 562: 111-117Crossref PubMed Scopus (45) Google Scholar). Anti-His4 antibody (Qiagen) was used at 0.2 μg/ml. Rabbit polyclonal anti-fibroblast growth factor receptor (FGFR)-1 and anti-FGFR-2 were purchased from Sigma. Recombinant human FGF-1 (acidic) and FGF-2 (basic) were purchased from Sigma. Heparin, N-desulfated, N-acetylated heparin, and 2-O-desulfated heparin were purchased from Sigma. Endo H and PNGase F were purchased from New England Biolabs.Protein Purification—The conditioned media containing sFRP-1-(His6) from HEK293 cells or from Lec3.2.8.1 cells was supplemented with NaCl to 1 m final concentration and equilibrated with nickel-NTA (Qiagen) resin at 4 °C for about 1 h. The nickel-NTA resin was collected by centrifugation at 3,000 × g (Sorvall H-6000A/HBB-6), suspended in 1 m NaCl, 25 mm Tris·HCl, pH 7.5, and packed into a column (GE Healthcare). After extensive washing with 1 m NaCl, 25 mm Tris·HCl, pH 7.5 (buffer A), and buffer A plus 15 mm imidazole, sFRP-1-(His6) protein was eluted with buffer A plus 200 mm imidazole. The partially purified sFRP-1 was concentrated to about 2 ml using 10K MWCO concentrators (Vivascience) and applied to a Superdex™ 200 size exclusion column (SEC) (GE Healthcare) pre-equilibrated in Buffer A. The protein concentration of the SEC fractions containing sFRP-1 was determined by absorbance at 280 nm using the calculated extinction coefficient.Metabolic Labeling—Stable Lec3.2.8.1 cells for sFRP-1-(His6) (25-6) were grown to confluence and switched to a low sulfate RPMI medium (specialty medium), supplemented with 10% FBS. Before sulfate incorporation experiments, cells were pretreated or mock-treated with sodium chlorate for 3 h. 50 μCi/ml of [35S]sulfate (PerkinElmer Life Sciences) was incubated with cells in the presence or absence of heparin (50 μg/ml) for 18 h. For labeling in transient HEK293-EBNA expression, cells at 24 h post-transfection were resuspended into labeling medium with [35S]sulfate for 18 h as above. sFRP-1-(His6) in the conditioned media were affinity purified by passing through a small nickel-NTA column.Northern Blot Analysis—Total RNA was prepared from HEK293 cells using RNAqueous (Ambion). 10 μg of RNA was resolved by 1.1% agarose, 2% formaldehyde, MOPS gel electrophoresis, blotted onto Nytran Supercharge membranes (Schleicher and Schuell) with 8× SSC, and hybridized overnight at 50 °C with digoxigenin-labeled DNA probes in DIG easy Hyb solution (Roche). After washing at 60 °C (glyceraldehyde-3-phosphate dehydrogenase) with 0.5× SSC, 0.1% SDS and 0.2× SSC, 0.1% SDS, the membranes were blocked in Blocking reagent (Roche) for 30 min, and probed with alkaline phosphatase-labeled anti-digoxigenin antibody (Roche) for 30 min and with Tris saline buffer, 0.3% Tween 20. Signals were visualized with Supersignal (Pierce). Probes were generated by PCR using digoxigenin-labeled nucleotides (Roche).RESULTSHeparin Specifically Stimulates Recombinant sFRP-1 Accumulation in HEK293 Cells—A DNA construct expressing a C-terminal His6-tagged sFRP-1 was transiently transfected into HEK293 cells, and between 72 and 120 h, sFRP-1 was detected in the media, although at relatively low levels (Fig. 1A, lanes 1). As part of an effort to increase the expression of sFRP-1 for planned structure/function studies, heparin was tested as an additive to the cell culture media at various times during transfection. Adding heparin at the time of transfection reduced sFRP-1 expression to undetectable levels, most likely a result of the negatively charged heparin binding strongly to the polyanionic polyethylenimine thus interfering with the DNA uptake process (data not shown). Nevertheless, adding heparin 24 or 48 h after transfection significantly enhanced sFRP-1 accumulation in the media (Fig. 1A, lanes 2 and 3). When the cell pellets were analyzed, intracellular sFRP-1 was also accumulated with the treatment of heparin (lane 4 versus 5 and 6). We then tested various concentrations of heparin to determine the optimum concentration for sFRP-1 accumulation. As can be seen in Fig. 1B, heparin at concentrations above 5 μg/ml gave the highest sFRP-1 induction levels, whereas higher levels of heparin resulted in slightly lower sFRP-1 accumulation. Interestingly, under similar expression conditions, the unrelated secreted human proteins aggrecanase I and II (30Glasson S.S. Askew R. Sheppard B. Carito B. Blanchet T. Ma H.L. Flannery C.R. Peluso D. Kanki K. Yang Z. Majumdar M.K. Morris E.A. Nature. 2005; 434: 644-648Crossref PubMed Scopus (993) Google Scholar) did not show heparin inducible accumulation (Fig. 1C). Collectively, the results indicate that heparin specifically stimulates both extracellular and intracellular sFRP-1 accumulation in HEK293 cells.Purified sFRP-1 Is Biologically Active—To determine whether the enhancement of sFRP-1 by heparin affects biological activity, sFRP-1 produced by HEK293 cells was purified. As described under “Experimental Procedures,” 1 liter of conditioned medium from heparin-induced transient expression of sFRP-1 was prepared. NaCl was added to the conditioned medium at a final concentration of 1 m and sFRP-1 was purified as described under “Experimental Procedures.” During the purification process, 1 m NaCl was included in all washing or elution buffers. As shown in Fig. 2, A and B, sFRP-1 was substantially pure after nickel-NTA (Fig. 2, A, left panel, and B, lanes 1 and 2), and was nearly homogeneous after the Superdex 200 SEC column (Fig. 2, A, right panel, and B, lanes 3 and 4). The sFRP-1 final yield was about 1 mg/liter and runs as an apparent monomer by analytical SEC analysis (Fig. 2A, right panel). N-terminal sequencing confirmed the identity of sFRP-1 and ESI-MS analysis revealed a heterogeneous and higher than expected mass that was assumed to be a result of N-linked glycosylation at two potential sites, Asn172 and Asn262 (data not shown).FIGURE 2Purified sFRP-1 is biologically active. A, protein elution profiling. Conditioned media from transfected 293 cells were passed through the nickel-NTA column as described under “Experimental Procedures.” The nickel-purified materials were further purified by the Superdex 200 size exclusion column as described under “Experimental Procedures.” B, Coomassie Blue-stained gel of purified sFRP-1-(His6). Protein samples after nickel-NTA or Superdex 200 were analyzed by SDS-PAGE under reduced (lanes 1 and 3) or non-reduced conditions (lanes 2 and 4). C, Wnt-3 antagonistic activity of purified sFRP-1. Wnt3 antagonistic assays with U2OS cells transfected with TCF-luciferase were performed as described in Ref. 31Bhat R.A. Stauffer B. Komm B.S. Bodine P.V. Protein Expression Purif. 2004; 37: 327-335Crossref PubMed Scopus (12) Google Scholar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the purified sFRP-1 protein was biologically active, the Wnt3 antagonistic activity of sFRP-1 was measured in a functional cell-based assay (31Bhat R.A. Stauffer B. Komm B.S. Bodine P.V. Protein Expression Purif. 2004; 37: 327-335Crossref PubMed Scopus (12) Google Scholar). In this assay, U2OS cells were co-transfected with a β-galactosidase-expressing plasmid, a Wnt3-expressing plasmid, as well as a reporter plasmid containing 16 copies of TCF DNA-binding sites placed in a reverse orientation upstream of a minimal thymidine kinase promoter and the luciferase gene. Then the cells were treated with various amounts of protein samples in the medium for 20 h. The cells were lysed and assayed for luciferase and β-galactosidase activity. The luciferase activity was normalized for transfection efficiency with β-galactosidase activity. As shown in Fig. 2C, Wnt3 increased the TCF luciferase reporter gene expression in the transfected U2OS cells. The addition of either nickel-NTA-purified sFRP-1 or SEC-purified sFRP-1 decreased the Wnt-mediated response in a dose-dependent manner, whereas the buffer had no effect on the Wnt-mediated TCF-luciferase reporter activation. These data clearly demonstrate that the purified sFRP-1 is functional.Heparin Induction of sFRP-1 Expression Is Cell-type Specific and Requires Heparin O-Sulfation—In addition to transient expression of sFRP-1 in HEK293 cells, stable CHO lines expressing sFRP-1 production were also established. Stable clones of CHO-Dukx cells were obtained by methotrexate selection and screened for sFRP-1 expression by immunoblotting, as described under “Experimental Procedures.” Three clones (200-10, 200-11, and 200-12) were isolated that expressed sFRP-1 at ∼50% lower levels than observed in HEK293 transient expression without heparin induction. sFRP-1 produced in CHO cells migrated at a higher apparent Mr than sFRP-1 from HEK293 cells (Fig. 3A, lanes 1-6 versus 7), which is likely due to more extensive N-linked carbohydrate modification in CHO cells. To determine whether heparin can also stimulate sFRP-1 accumulation in CHO cell lines, 90% confluent monolayers were treated with 50 μg/ml of heparin. Conditioned medium was harvested at 72 h post-treatment and analyzed for sFRP-1 expression by Western blot analysis. Interestingly, the expression of sFRP-1 in CHO cells was not induced by heparin (Fig. 3A, lane 1 versus 2; lane 3 versus 4; lane 5 versus 6). Similar results were also obtained when conditioned media were harvested at 96 h post-treatment with heparin (data not shown). Clearly the response to heparin in CHO cells is different from that in the HEK293 transient expression experiments and suggests a cell type-specific mechanism.FIGURE 3Heparin effect is cell-type specific and requires heparin O-sulfation. A, heparin cannot stimulate sFRP-1 accumulation in CHO cells. sFRP-1-(His6) expressing CHO stable lines were generated as described under “Experimental Procedures.” Cells were mock-treated or treated with 50 μg/ml of heparin for 72 h. Conditioned media (C.M.) were separated by SDS-PAGE and immunoblotted with anti-His4 antibody. Lane 7 shows non-heparin treated transient HEK293 expression and relates to Fig. 1A, lane 1. B, heparin can stimulate sFRP-1 accumulation in a HEK293 stable line. A stable line of sFRP-1-(His6) in HEK293 cell was generated as described under “Experimental Procedures.” Cells were treated or mock-treated with 50 μg/ml of heparin. Conditioned media were collected at different time points. Protein samples were separated by SDS-PAGE and immunoblotted with anti-His4 antibody. C, effects of modified heparins. sFRP-1 transfected HEK293 cells were left untreated (-) or incubated for 72 h with heparin, or N-desulfated, N-acetylated heparin (dN-heparin), or 2-O-desulfated heparin (dO-heparin), all at 50 μg/ml. Medium samples were collected and analyzed for immunoblotting with anti-His4 antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To rule out the possibility that heparin-induced sFRP-1 accumulation was unique to transient expression in HEK293 cells, a stable line of HEK293 expressing sFRP-1 was established as described under “Experimental Procedures.” The stable line was treated with heparin and conditioned media were collected at different time points and sFRP-1 levels were determined by Western blot analysis. As shown in Fig. 3B, the expression of sFRP-1 in this line responded to heparin at 48 h (lane 4 versus 3) and the effect increased with a longer incubation (lanes 6 and 8). The data clearly indicated that heparin induces sFRP-1 accumulation in both transient and stable HEK293 systems. The enhancement effect by heparin on sFRP-1 appears to be cell-type specific.The interaction of heparin-binding proteins with HS is determined by the sequence and sulfation level of the sugar moieties of HS (32Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (393) Google Scholar, 33Esko J.D. Lindahl U. J. Clin. Investig. 2001; 108: 169-173Crossref PubMed Scopus (782) Google Scholar, 35Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1226) Google Scholar). To test whether O-sulfation and N-sulfation are required for heparin activity in sFRP-1 accumulation, sFRP-1-transfected HEK293 cells were incubated with chemically modified heparin that was completely N-desulfated, followed by N-acetylation. Modified heparin lacking 2-O-sulfation was also examined for its ability to stimulate sFRP-1 accumulation. As shown in Fig. 3C, 2-O-desulfated heparin did not stimulate sFRP-1 accumulation (lane 4). In contrast, N-desulfated heparin (lane 3) and native heparin (lane 2) both induced sFRP-1 accumulation, suggesting that O-sulfation but not N-sulfation was necessary for the induction effect.Heparin Does Not Affect Protein Release from the Cell Surface Matrix or mRNA Level of sFRP-1—Heparin is known to release or extract extracellular proteins such as Wnts from the cell surface matrix (3Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1728) Google Scholar). To test whether the heparin effect is simply a competitive binding effect with cell surface proteoglycans, a whole cell sFRP-1 binding study was performed. As shown in Fig. 4A, purified His6-tagged sFRP-1 was incubated with HEK293 cells in cultured media in either the presence or absence of heparin. The concentration of sFRP-1 was at 1 μg/ml," @default.
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