Characterization of Wise Protein and Its Molecular Mechanism to Interact with both Wnt and BMP Signals
2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês
10.1074/jbc.m109.025478
ISSN1083-351X
AutoresKatherine B. Lintern, Sonia Guidato, Alison Rowe, José W. Saldanha, Nobue Itasaki,
Tópico(s)Developmental Biology and Gene Regulation
ResumoCross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals. Cross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals. Wise is a secreted protein that was isolated from a functional screen of a chick cDNA library of embryonic tissues. It was identified as being able to alter the antero-posterior character of neuralized Xenopus animal caps by promoting activity of the Wnt pathway (1Itasaki N. Jones C.M. Mercurio S. Rowe A. Domingos P.M. Smith J.C. Krumlauf R. Development. 2003; 130: 4295-4305Crossref PubMed Scopus (265) Google Scholar). Independently, the homologous protein was isolated from a functional screen to detect genes that are preferentially expressed in the rat endometrium, which had been maximally sensitized to implantation, and named USAG-1 (uterine sensitization-associated gene-1) (2Simmons D.G. Kennedy T.G. Biol. Reprod. 2002; 67: 1638-1645Crossref PubMed Scopus (49) Google Scholar). The protein was identified a third time from the GenBankTM sequence data base of mouse as a putative secreted protein, shown to be a BMP antagonist, and named Ectodin (3Laurikkala J. Kassai Y. Pakkasjärvi L. Thesleff I. Itoh N. Dev. Biol. 2003; 264: 91-105Crossref PubMed Scopus (206) Google Scholar). The gene has also been called Sostdc1 (Sclerostin domain-containing 1) or Sostl (Sclerostin-like) due to the homology with Sclerostin-encoding gene Sost (4Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J. Am. J. Hum. Genet. 2001; 68: 577-589Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar, 5Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van Den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Crossref PubMed Scopus (890) Google Scholar). USAG-1/Wise/Ectodin/Sostdc1 is expressed in various tissues, such as the surface ectoderm of the posterior axis (1Itasaki N. Jones C.M. Mercurio S. Rowe A. Domingos P.M. Smith J.C. Krumlauf R. Development. 2003; 130: 4295-4305Crossref PubMed Scopus (265) Google Scholar, 6Shigetani Y. Itasaki N. Dev. Dyn. 2007; 236: 2277-2284Crossref PubMed Scopus (5) Google Scholar), branchial arches (3Laurikkala J. Kassai Y. Pakkasjärvi L. Thesleff I. Itoh N. Dev. Biol. 2003; 264: 91-105Crossref PubMed Scopus (206) Google Scholar, 6Shigetani Y. Itasaki N. Dev. Dyn. 2007; 236: 2277-2284Crossref PubMed Scopus (5) Google Scholar), the dermal papilla in hair follicles (7O'Shaughnessy R.F. Yeo W. Gautier J. Jahoda C.A. Christiano A.M. J. Invest. Dermatol. 2004; 123: 613-621Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), vibrissae (3Laurikkala J. Kassai Y. Pakkasjärvi L. Thesleff I. Itoh N. Dev. 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Stadler H.S. J. Biol. Chem. 2007; 282: 6843-6853Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), and embryonic and adult kidneys (13Yanagita M. Okuda T. Endo S. Tanaka M. Takahashi K. Sugiyama F. Kunita S. Takahashi S. Fukatsu A. Yanagisawa M. Kita T. Sakurai T. J. Clin. Invest. 2006; 116: 70-79Crossref PubMed Scopus (119) Google Scholar, 14Yanagita M. Oka M. Watabe T. Iguchi H. Niida A. Takahashi S. Akiyama T. Miyazono K. Yanagisawa M. Sakurai T. Biochem. Biophys. Res. Commun. 2004; 316: 490-500Crossref PubMed Scopus (115) Google Scholar). Wise appears to have a dual role in modulating the Wnt pathway. Injection of Wnt8 RNA into a ventral vegetal blastomere of Xenopus embryos at the four-cell stage induces a full secondary axis to form, and this is blocked by the addition of Wise RNA as well as other Wnt inhibitors (1Itasaki N. Jones C.M. Mercurio S. Rowe A. Domingos P.M. Smith J.C. Krumlauf R. Development. 2003; 130: 4295-4305Crossref PubMed Scopus (265) Google Scholar). Activation of the Wnt/β-catenin pathway in hair follicles triggers regeneration of hair growth, and expression of Wise appears to have a defined role to inhibit this (15Beaudoin 3rd, G.M. Sisk J.M. Coulombe P.A. Thompson C.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14653-14658Crossref PubMed Scopus (119) Google Scholar). In this context, Wise expression is repressed by the nuclear receptor co-repressor, Hairless, which results in activation of the Wnt pathway; thus, a model of periodic regeneration of hair follicles has been proposed (15Beaudoin 3rd, G.M. Sisk J.M. Coulombe P.A. Thompson C.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14653-14658Crossref PubMed Scopus (119) Google Scholar, 16Thompson C.C. Sisk J.M. Beaudoin 3rd, G.M. Cell Cycle. 2006; 5: 1913-1917Crossref PubMed Scopus (61) Google Scholar). In addition, Wise and its homologue USAG-1 have been shown to block Wnt1, Wnt3a, and Wnt10b activities in reporter assays (14Yanagita M. Oka M. Watabe T. Iguchi H. Niida A. Takahashi S. Akiyama T. Miyazono K. Yanagisawa M. Sakurai T. Biochem. Biophys. Res. Commun. 2004; 316: 490-500Crossref PubMed Scopus (115) Google Scholar, 15Beaudoin 3rd, G.M. Sisk J.M. Coulombe P.A. Thompson C.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14653-14658Crossref PubMed Scopus (119) Google Scholar, 17Blish K.R. Wang W. Willingham M.C. Du W. Birse C.E. Krishnan S.R. Brown J.C. Hawkins G.A. Garvin A.J. D'Agostino Jr., R.B. Torti F.M. Torti S.V. Mol. Biol. Cell. 2008; 19: 457-464Crossref PubMed Scopus (55) Google Scholar). Wise was found to bind to the Wnt co-receptor, LRP6, sharing the binding domain with Wnt ligands. Importantly, Wise was found to compete with Wnt8 for binding to LRP6, therefore suggesting a mechanism for inhibition of the Wnt pathway whereby Wise blocks the binding of ligand and receptor (1Itasaki N. Jones C.M. Mercurio S. Rowe A. Domingos P.M. Smith J.C. Krumlauf R. Development. 2003; 130: 4295-4305Crossref PubMed Scopus (265) Google Scholar). Wise may also be retained in the endoplasmic reticulum and inhibit the trafficking of LRP6 to the cell surface (18Guidato S. Itasaki N. Dev. Biol. 2007; 310: 250-263Crossref PubMed Scopus (26) Google Scholar). Wise also binds LRP4 (19Ohazama A. Johnson E.B. Ota M.S. Choi H.Y. Choi H.J. Porntaveetus T. Oommen S. Itoh N. Eto K. Gritli-Linde A. Herz J. Sharpe P.T. PLoS ONE. 2008; 3: e4092Crossref PubMed Scopus (146) Google Scholar), a member of the LRP family functioning inhibitory to Wnt signals (20Johnson E.B. Hammer R.E. Herz J. Hum. Mol. Genet. 2005; 14: 3523-3538Crossref PubMed Scopus (108) Google Scholar). It is noteworthy that Wise was isolated from a screen designed to detect the activation of the Wnt/β-catenin pathway, not inhibition. The exact mechanism of how Wise exerts such a context-dependent modulation on the Wnt pathway is yet to be clarified. Osteoblast differentiation of MC3T3-E1 cells, as measured by alkaline phosphatase activity, can be induced by a wide range of BMP molecules. In this assay, Ectodin, the mouse ortholog of Wise, was shown to inhibit differentiation induced by BMP2, -4, -6, or -7 in a dose-dependent manner (3Laurikkala J. Kassai Y. Pakkasjärvi L. Thesleff I. Itoh N. Dev. Biol. 2003; 264: 91-105Crossref PubMed Scopus (206) Google Scholar). Similarly, Ectodin (also known as USAG-1) was also found to inhibit the bone differentiation induced by BMP2, -4, or -7 in C2C12 cells (14Yanagita M. Oka M. Watabe T. Iguchi H. Niida A. Takahashi S. Akiyama T. Miyazono K. Yanagisawa M. Sakurai T. Biochem. Biophys. Res. Commun. 2004; 316: 490-500Crossref PubMed Scopus (115) Google Scholar). Ectodin also inhibits BMP2- or BMP7-induced Msx2 expression in dissected mouse tooth buds in organ culture (3Laurikkala J. Kassai Y. Pakkasjärvi L. Thesleff I. Itoh N. Dev. Biol. 2003; 264: 91-105Crossref PubMed Scopus (206) Google Scholar). In tooth buds, Ectodin expression is detected in the dental ectoderm and mesenchymal cells excluding from the enamel knot (3Laurikkala J. Kassai Y. Pakkasjärvi L. Thesleff I. Itoh N. Dev. Biol. 2003; 264: 91-105Crossref PubMed Scopus (206) Google Scholar). Ectodin/USAG-1-deficient mice created by targeted-disruption show altered tooth morphology and extra teeth, indicating that Ectodin and BMP tightly control tooth development and patterning in mammals (8Kassai Y. Munne P. Hotta Y. Penttilä E. Kavanagh K. Ohbayashi N. Takada S. Thesleff I. Jernvall J. Itoh N. Science. 2005; 309: 2067-2070Crossref PubMed Scopus (241) Google Scholar, 21Murashima-Suginami A. Takahashi K. 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All of these findings indicate that USAG-1/Wise/Ectodin has a clear antagonistic effect on BMP signaling, where it binds BMP2, -4, -6, and -7 (3Laurikkala J. Kassai Y. Pakkasjärvi L. Thesleff I. Itoh N. Dev. Biol. 2003; 264: 91-105Crossref PubMed Scopus (206) Google Scholar, 14Yanagita M. Oka M. Watabe T. Iguchi H. Niida A. Takahashi S. Akiyama T. Miyazono K. Yanagisawa M. Sakurai T. Biochem. Biophys. Res. Commun. 2004; 316: 490-500Crossref PubMed Scopus (115) Google Scholar) and presumably prevents BMP binding to its receptors. Analysis of the sequence of Wise reveals that it has the C1XnC2XGXC3XnC4XnC5XC6 motif of a six-membered cystine knot, where C1 forms a disulfide bond with C4, C2 with C5, and C3 with C6 (for a review of the cystine knot, see Refs. 24Yanagita M. Cytokine Growth Factor Rev. 2005; 16: 309-317Crossref PubMed Scopus (122) Google Scholar, 25Vitt U.A. Hsu S.Y. Hsueh A.J. Mol. Endocrinol. 2001; 15: 681-694Crossref PubMed Scopus (211) Google Scholar, 26Balemans W. 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Thesleff I. Itoh N. J. Biol. Chem. 2003; 278: 24113-24117Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Sclerostin is involved in regulating bone mass (4Brunkow M.E. Gardner J.C. Van Ness J. Paeper B.W. Kovacevich B.R. Proll S. Skonier J.E. Zhao L. Sabo P.J. Fu Y. Alisch R.S. Gillett L. Colbert T. Tacconi P. Galas D. Hamersma H. Beighton P. Mulligan J. Am. J. Hum. Genet. 2001; 68: 577-589Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar, 5Balemans W. Ebeling M. Patel N. Van Hul E. Olson P. Dioszegi M. Lacza C. Wuyts W. Van Den Ende J. Willems P. Paes-Alves A.F. Hill S. Bueno M. Ramos F.J. Tacconi P. Dikkers F.G. Stratakis C. Lindpaintner K. Vickery B. Foernzler D. Van Hul W. Hum. Mol. Genet. 2001; 10: 537-543Crossref PubMed Scopus (890) Google Scholar) and also appears to antagonize both Wnt (29Li X. Zhang Y. Kang H. Liu W. Liu P. Zhang J. Harris S.E. Wu D. J. Biol. 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Chem. 2005; 280: 2498-2502Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 34Winkler D.G. Sutherland M.K. Geoghegan J.C. Yu C. Hayes T. Skonier J.E. Shpektor D. Jonas M. Kovacevich B.R. Staehling-Hampton K. Appleby M. Brunkow M.E. Latham J.A. EMBO J. 2003; 22: 6267-6276Crossref PubMed Scopus (837) Google Scholar) signals. This paper aims to analyze the dual role of Wise on Wnt and BMP pathways by probing the structural features of the protein and reconciling them to physiological properties. It also aims to reveal the molecular nature of the protein in view of possible glycosylation, secretion, and association with extracellular matrix. Secondary structure prediction was performed on the chick amino acid sequence and closely related sequences from zebrafish, Xenopus, mouse, and human using the program PHD (35Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar). The secondary structure prediction was used to guide a manual alignment of the five sequences. The result is shown in supplemental Fig. 1. This alignment was processed by the three-dimensional fold recognition server 3DPSSM (36Kelley L.A. MacCallum R.M. Sternberg M.J. J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1119) Google Scholar). The top fold was that of the cystine knot in human chorionic gonadotropin (37Wu H. Lustbader J.W. Liu Y. Canfield R.E. Hendrickson W.A. Structure. 1994; 2: 545-558Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar) with 90% certainty. A three-dimensional structural model was built of the chick Wise sequence from residue 68 to 186 based on the human chorionic gonadotropin structure (Protein Data Bank code 1HCN) extracted from the Protein Data Bank (38Berman H. Henrick K. Nakamura H. Nat. Struct. Biol. 2003; 10: 980Crossref PubMed Scopus (1794) Google Scholar). The molecular modeling program QUAFNTA (Accelrys Inc.) was employed on a Silicon Graphics O2 computer running the IRIX operating system. An alignment from the chick, human, rat, and mouse amino acid sequences was made using the program ClustalX (39Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35075) Google Scholar) with default multiple alignment parameters. Secondary structure regions (β-strands) obtained from the structural model of chick Wise is shown in supplemental Fig. 2. HEK293 cells (ATCC) were propagated in Dulbecco's modified Eagle's medium with 10% fetal calf serum at 37 °C and 5% CO2. Cells were transfected with DNA constructs using Polyfect (Qiagen) according to the manufacturer's guidelines. FLAG-tagged chick Wise was subcloned into pCS2+, and deletions and point mutations were introduced into this clone using PCR with specific primers. All clones containing mutated constructs were sequenced throughout the Wise gene insert before being used in protein production and assays. The BMP4 construct is a fusion of a pro-region from the BMP2 and the mature region of BMP4 tagged with Myc (40Augsburger A. Schuchardt A. Hoskins S. Dodd J. Butler S. Neuron. 1999; 24: 127-141Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) subcloned into pCS2+. Recombinant BMP4 protein (R&D Systems) and anti-BMP4 antibody (R&D Systems) were also used. A clone containing a fusion of the extracellular domain of LRP6 and Fc domain of IgG (LRP6-IgG) was a gift from Xi He (41Tamai 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 (1071) Google Scholar). LRP6 extracellular domain (LRP6ECD, amino acids 1–1370) was subcloned into pCS2 after attaching a Myc tag and a stop codon. HEK293 cells were grown to 80% confluence in 80-cm2 flasks and transfected with expression plasmids. Aspirated medium was replaced with Opti-MEM (Invitrogen) after 24 h to provide a serum-free environment for protein collection. Condition medium was then collected after 3 and 5 days or after each further 24-h period for the next 4 days. They were clarified to remove debris and then applied to a 10-kDa cut-off centrifugal filter device (Millipore). The supernatant was concentrated 5 or 50 times, respectively, and stored in aliquots at −80 °C. HEK293 cells in T25 flasks were transfected with expression plasmids of FLAG-tagged Wise and Myc-tagged LRP6 extracellular domain (ECD) 3The abbreviations used are: ECDextracellular domainBREBMP response elementPNGase Fpeptide:N-glycosidase FHSPGheparan sulfate proteoglycan. or BMP4. After 24 h, the medium was replaced by OptiMEM (Invitrogen). After a further 24 h, medium was collected, added to anti-FLAG-agarose affinity gel beads (Sigma), and incubated at 4 °C for 6 h. In some experiments, Wise and LRP6ECD-IgG were individually transfected, and the conditioned media were mixed with bovine serum albumin or recombinant BMP4, followed by mixing with protein A beads (GE Healthcare). Beads were then washed five times with wash buffer (150 ml of NaCl, 50 mm Tris-HCl, pH 7.5, 0.1% Triton X-100). Protein was eluted from the beads in modified Laemmli buffer (2% SDS, 10% glycerol, 100 mm dithiothreitol, 60 mm Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 0.1% bromphenol blue) at 100 °C for 5 min before loading onto a denaturing SDS-polyacrylamide gel. For immunoprecipitation of LRP6ECD, a 4–15% gradient gel (Bio-Rad) was used to detect both Wise and LRP6ECD. For detecting Wise and BMP4, a 15% gel was used. Protein samples run on polyacrylamide gels were transferred onto polyvinylidene difluoride membrane. Membranes were then blocked in 10% milk protein in phosphate-buffered saline plus 0.1% Tween 20 before exposing to antibodies: anti-FLAG M2-horseradish peroxidase (Sigma), anti-Myc (Upstate Biotechnology, Inc.), or anti-Fc (Sigma) and anti-mouse horseradish peroxidase (Amersham Biosciences). Detection was by ECL (Amersham Biosciences; SuperSignal West Pico/Femto, Thermo) on x-ray film. extracellular domain BMP response element peptide:N-glycosidase F heparan sulfate proteoglycan. Deglycosylation of Wise protein was carried out by treating concentrated Wise-conditioned medium with peptide:N-glycosidase F (PNGase F; New England Biolabs), O-glycosidase (Roche Applied Science), endo-β-N-acetylglucosaminidase H (New England Biolabs), and endo-β-N-acetylglucosaminidase D (Merck) according to the manufacturer's guidelines. Typically, 50 times concentrated protein in conditioned medium was first denatured at 100 °C in 0.5% SDS, 1% 2-mercaptoethanol and then digested with the enzyme in manufacturer's buffer in a total of 50 μl at 37 °C for 60 min. For heparan sulfate treatment, HEK293 cells were transfected with Myc-tagged LRP6, and the medium was replaced on the following day with Wise conditioned medium together with 2 μg/ml heparan sulfate (Sigma). The cells were incubated for 1 h at 4 °C before the immunostaining procedure with anti-Myc and anti-FLAG antibodies. For the sodium chlorate experiment, HEK293 cells were transfected with Wise, split, and incubated with or without 20 nm of sodium chlorate for 2 days, after which the conditioned medium and cell extracts were collected for Western analyses. The Wnt pathway activity was detected by transfecting reporter constructs of 0.08 μg of TOPflash (Upstate Biotechnology) (42Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2896) Google Scholar) and 0.02 μg of Renilla luciferase reporter (pRL-TK; Promega) plasmids in each well of 24-well plates. For the BMP pathway, 0.15 μg of BMP response element (BRE) reporter, which contains Smad binding elements identified in the Id1 promoter (43Korchynskyi O. ten Dijke P. J. Biol. Chem. 2002; 277: 4883-4891Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar) or the Xvent2 promoter (44von Bubnoff A. Peiffer D.A. Blitz I.L. Hayata T. Ogata S. Zeng Q. Trunnell M. Cho K.W. Dev. Biol. 2005; 281: 210-226Crossref PubMed Scopus (50) Google Scholar), and 0.1–2 ng of Renilla reporter (pRL-CMV; Promega) were used. HEK293 cells were transfected with expression plasmids (Fig. 5C, BMP4 (0.05 μg), Wise (0.1 μg); Fig. 6C, Wnt1 (0.1 μg), Wise (0.1 μg), BMP4 (0.1 or 0.2 μg); Fig. 6D, BMP4 (0.05 μg), Wise (0.3 μg), LRP6 (0.005 or 0.01 μg)), each well receiving a total of 0.5 μg of DNA, using a control vector where necessary. After 24 h, medium was replaced with a serum-free medium, OptiMEM (Invitrogen), and cells were lysed 48 h post-transfection. The normalized luciferase activity was determined using the Stop and Glo Dual Luciferase System (Promega) on a Turner Luminometer. Bar graphs show the average of triplicate samples in one experiment, and the error bars display S.D. values. The same experiments were repeated at least three times.FIGURE 6Function of Wise on LRP6 and BMP4. A, immunoprecipitation (IP) assay. Conditioned media of HEK293 cells separately transfected with Wise or LRP6IgG were mixed together with bovine serum albumin or recombinant BMP4, as indicated (+, 100 ng; ++, 200 ng/700 μl) and immunoprecipitated with LRP6IgG, and the precipitated samples were analyzed on Western blots using anti-FLAG (Wise), anti-BMP4, or anti-Fc antibodies. Inputs of BMP4 and LRP6 indicate the presence of these proteins in the mix of relevant conditioned media and proteins. Input Wise is not shown because it was undetectable after diluting with other conditioned media. Concentrated Wise medium was checked on a separate blot prior to use, and an equal volume of the medium from the same batch was used for each of the immunoprecipitation samples. Immunoprecipitation of Wise (upper panel) is not compromised by the presence of BMP4. *, nonspecific bands. B, immunostaining of HEK293 cells stably expressing Myc-tagged LRP6 (green), treated with conditioned medium of FLAG-tagged Wise (red), without or with recombinant BMP4 (100 ng/ml). Additional BMP4 does not interfere with the binding of Wise to LRP6. C, TOPflash reporter assay. HEK293 cells were transfected with Wnt1 (0.1 μg), Wise (0.1 μg), and/or BMP4 (+, 0.1 μg; ++, 0.2 μg) constructs, as indicated, together with TOPflash and control Renilla reporters. Activation of TOPflash reporter by Wnt1 is suppressed by Wise. The addition of BMP4 does not affect the function of Wise in Wnt1 inhibition. D, BRE reporter assay. HEK293 cells were transfected with BMP4 (0.05 μg), Wise (0.3 μg), and/or LRP6 (+, 0.005 μg; ++, 0.01 μg) constructs, as indicated, together with BRE and control Renilla reporters. Suppression of BMP4 activity by Wise is largely prevented by additional LRP6. BSA, bovine serum albumin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To detect phosphorylation of Smad1/5/8 proteins, HEK293 cells were plated in 35-mm dishes 24 h prior to experimentation. Recombinant BMP4 (5 ng/ml) was premixed with 5 times concentrated Wise conditioned medium or similarly concentrated control conditioned medium and incubated for 2 h at 4 °C before applying to the cells. The cells were incubated at 37 °C for 1 h and then collected on ice with 200 μl of modified Laemmli buffer by scraping. The samples were boiled for 5 min and sonicated before loading onto polyacrylamide gels. Anti-phospho-Smad1/5/8 antibody (Cell Signaling) was used on Western blots together with anti-β-tubulin antibody, which serves as a loading control. The signals were detected by the fluorescent Western system for quantification (LI-COR). Cells transfected with Myc-tagged LRP6 were treated with conditioned medium of FLAG-tagged Wise- or Wise(Δheel)-expressing cells overnight. Cells were then fixed with 3% paraformaldehyde in phosphate-buffered saline, and immunostaining was carried out with anti-FLAG and anti-Myc antibodies. The predicted three-dimensional structural model of the core part of the Wise protein revealed that Wise is very likely to form a cystine knot (Fig. 1A). In addition, the two other cysteines at positions 89 and 147 are located very close to the tip of two "fingers" and are likely to anchor the fingers by a disulfide bond, analogous to other cystine knot proteins (27Avsian-Kretchmer O. Hsueh A.J. Mol. Endocrinol. 2004; 18: 1-12Crossref PubMed Scopus (186) Google Scholar). The two fingers curve in the same direction in parallel (toward the back of the plane in Fig. 1A). The domain between the C terminus and the 165th cysteine residue turns around and comes close to the first finger. Inspection of the amino acid sequence of chick Wise revealed two putative N-glycosylation sites, at positions 47 and 173. To investigate glycosylation of Wise, concentrated Wise conditioned medium was treated either with PNGase F, which cleaves most N-glycosylations between the innermost GlcNAc and the asparagine residue, or with O-glycosidase, wh
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