Science China Life Sciences

, Volume 57, Issue 5, pp 469–481 | Cite as

Bidirectional effect of Wnt signaling antagonist DKK1 on the modulation of anthrax toxin uptake

  • LiLi Qian
  • ChangZu Cai
  • PengFei Yuan
  • Sun-Young Jeong
  • XiaoZhou Yang
  • Venita DeAlmeida
  • James Ernst
  • Michael Costa
  • Stanley N. Cohen
  • WenSheng Wei
Open Access
Cover Article


LRP6, a co-receptor for the morphogen Wnt, aids endocytosis of anthrax complexes. Here we report that Dickkopf1 (DKK1) protein, a secreted LRP6 ligand and antagonist, is also a modulator of anthrax toxin sensitivity. shRNA-mediated gene silencing or TALEN-mediated gene knockout of DKK1 reduced sensitivity of cells to PA-dependent hybrid toxins. However, unlike the solely inhibitory effect on Wnt signaling, the effects of DKK1 overexpression on anthrax toxicity were bidirectional, depending on its endogenous expression and cell context. Fluorescence microscopy and biochemical analyses showed that DKK1 facilitates internalization of anthrax toxins and their receptors, an event mediated by DKK1-LRP6-Kremen2 complex. Monoclonal antibodies against DKK1 provided dose-dependent protection to macrophages from killing by anthrax lethal toxin (LT). Our discovery that DKK1 forms ternary structure with LRP6 and Kremen2 in promoting PA-mediated toxin internalization provides a paradigm for bacterial exploitation of mechanisms that host cells use to internalize signaling proteins.


DKK1 anthrax toxin LRP6 TALENs internalization Kremen2 receptor Wnt 


  1. 1.
    Leppla SH. Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic amp concentrations of eukaryotic cells. Proc Natl Acad Sci USA, 1982, 79: 3162–3166PubMedPubMedCentralCrossRefGoogle Scholar
  2. 2.
    Young JA, Collier RJ. Anthrax toxin: receptor-binding, internalization, pore formation, and translocation. Annu Rev Biochem, 2007, 76: 243–265PubMedCrossRefGoogle Scholar
  3. 3.
    Baldari CT, Tonello F, Paccani SR, Montecucco C. Anthrax toxins: a paradigm of bacterial immune suppression. Trends Immunol, 2006, 27: 434–440PubMedCrossRefGoogle Scholar
  4. 4.
    Vitale G, Pellizzari R, Recchi C, Napolitani G, Mock M, Montecucco C. Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem Biophys Res Commun, 1998, 248: 706–711PubMedCrossRefGoogle Scholar
  5. 5.
    Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Copeland TD, Ahn NG, Oskarsson MK, Fukasawa K, Paull KD, Vande Woude GF. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science, 1998, 280: 734–737PubMedCrossRefGoogle Scholar
  6. 6.
    Vitale G, Bernardi L, Napolitani G, Mock M, Montecucco C. Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor. Biochem J, 2000, 352(Pt 3): 739–745PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Firoved AM, Miller GF, Moayeri M, Kakkar R, Shen Y, Wiggins JF, McNally EM, Tang WJ, Leppla SH. Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice. Am J Pathol, 2005, 167: 1309–1320PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Bradley KA, Mogridge J, Mourez M, Collier RJ, Young JA. Identification of the cellular receptor for anthrax toxin. Nature, 2001, 414: 225–229PubMedCrossRefGoogle Scholar
  9. 9.
    Scobie HM, Rainey GJ, Bradley KA, Young JA. Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc Natl Acad Sci USA, 2003, 100: 5170–5174PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Martchenko M, Jeong SY, Cohen SN. Heterodimeric integrin complexes containing beta1-integrin promote internalization and lethality of anthrax toxin. Proc Natl Acad Sci USA, 2010, 107: 15583–15588PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Liu S, Crown D, Miller-Randolph S, Moayeri M, Wang H, Hu H, Morley T, Leppla SH. Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo. Proc Natl Acad Sci USA, 2009, 106: 12424–12429PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Park JM, Greten FR, Li ZW, Karin M. Macrophage apoptosis by anthrax lethal factor through p38 map kinase inhibition. Science, 2002, 297: 2048–2051PubMedCrossRefGoogle Scholar
  13. 13.
    Arora N, Klimpel KR, Singh Y, Leppla SH. Fusions of anthrax toxin lethal factor to the ADP-ribosylation domain of pseudomonas exotoxin A are potent cytotoxins which are translocated to the cytosol of mammalian cells. J Biol Chem, 1992, 267: 15542–15548PubMedGoogle Scholar
  14. 14.
    Arora N, Leppla SH. Residues 1–254 of anthrax toxin lethal factor are sufficient to cause cellular uptake of fused polypeptides. J Biol Chem, 1993, 268: 3334–3341PubMedGoogle Scholar
  15. 15.
    Blanke SR, Milne JC, Benson EL, Collier RJ. Fused polycationic peptide mediates delivery of diphtheria toxin a chain to the cytosol in the presence of anthrax protective antigen. Proc Natl Acad Sci USA, 1996, 93: 8437–8442PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Wehrli M, Dougan ST, Caldwell K, O’Keefe L, Schwartz S, Vaizel-Ohayon D, Schejter E, Tomlinson A, DiNardo S. Arrow encodes an ldl-receptor-related protein essential for wingless signalling. Nature, 2000, 407: 527–530PubMedCrossRefGoogle Scholar
  17. 17.
    Tamai K, Semenov M, Kato Y, Spokony R, Liu C, Katsuyama Y, Hess F, Saint-Jeannet JP, He X. Ldl-receptor-related proteins in Wnt signal transduction. Nature, 2000, 407: 530–535PubMedCrossRefGoogle Scholar
  18. 18.
    Pinson KI, Brennan J, Monkley S, Avery BJ, Skarnes WC. An LDL-receptor-related protein mediates Wnt signalling in mice. Nature, 2000, 407: 535–538PubMedCrossRefGoogle Scholar
  19. 19.
    Lu Q, Wei W, Kowalski PE, Chang AC, Cohen SN. EST-based genome-wide gene inactivation identifies ARAP3 as a host protein affecting cellular susceptibility to anthrax toxin. Proc Natl Acad Sci USA, 2004, 101: 17246–17251PubMedPubMedCentralCrossRefGoogle Scholar
  20. 20.
    Wei W, Lu Q, Chaudry GJ, Leppla SH, Cohen SN. The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell, 2006, 124: 1141–1154PubMedCrossRefGoogle Scholar
  21. 21.
    Abrami L, Kunz B, Deuquet J, Bafico A, Davidson G, van der Goot FG. Functional interactions between anthrax toxin receptors and the Wnt signalling protein LRP6. Cell Microbiol, 2008, 10: 2509–2519PubMedCrossRefGoogle Scholar
  22. 22.
    Semenov MV, Tamai K, Brott BK, Kühl M, Sokol S, He X. Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Curr Biol, 2001, 11: 951–961PubMedCrossRefGoogle Scholar
  23. 23.
    Semenov MV, Zhang X, He X. Dkk1 antagonizes wnt signaling without promotion of LRP6 internalization and degradation. J Biol Chem, 2008, 283: 21427–21432PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Cselenyi CS, Lee E. Context-dependent activation or inhibition of Wnt-beta-catenin signaling by kremen. Sci Signal, 2008, 1: pe10PubMedCrossRefGoogle Scholar
  25. 25.
    Mao B, Wu W, Li Y, Hoppe D, Stannek P, Glinka A, Niehrs C. LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature, 2001, 411: 321–325PubMedCrossRefGoogle Scholar
  26. 26.
    Mao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM, Delius H, Hoppe D, Stannek P, Walter C, Glinka A, Niehrs C. Kremen proteins are dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature, 2002, 417: 66–667CrossRefGoogle Scholar
  27. 27.
    Bourhis E, Tam C, Franke Y, Bazan JF, Ernst J, Hwang J, Costa M, Cochran AG, Hannoush RN. Reconstitution of a frizzled8-Wnt3a-LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6. J Biol Chem, 2010, 285: 9172–9179PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    Bourhis E, Wang W, Tam C, Hwang J, Zhang Y, Spittler D, Huang OW, Gong Y, Estevez A, Zilberleyb I, Rouge L, Chiu C, Wu Y, Costa M, Hannoush RN, Franke Y, Cochran AG. Wnt antagonists bind through a short peptide to the first beta-propeller domain of LRP5/6. Structure, 2011, 19: 1433–1442PubMedCrossRefGoogle Scholar
  29. 29.
    Jackson-Cook C, Bae V, Edelman W, Brothman A, Ware J. Cytogenetic characterization of the human prostate cancer cell line P69SV40T and its novel tumorigenic sublines M2182 and M15. Cancer Genet Cytogenet, 1996, 87: 14–23PubMedCrossRefGoogle Scholar
  30. 30.
    Godbey WT, Barry MA, Saggau P, Wu KK, Mikos AG. Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery. J Biomed Mater Res, 2000, 51: 321–328PubMedCrossRefGoogle Scholar
  31. 31.
    Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet, 2000, 25: 217–222PubMedCrossRefGoogle Scholar
  32. 32.
    Zornetta I, Brandi L, Janowiak B, Dal Molin F, Tonello F, Collier RJ, Montecucco C. Imaging the cell entry of the anthrax oedema and lethal toxins with fluorescent protein chimeras. Cell Microbiol, 2010, 12: 1435–1445PubMedCrossRefGoogle Scholar
  33. 33.
    Harlow E, Lane D. Using Antibodies: A Laboratory Manual. Plainview: Cold Spring Harbor Lab Press, 1999Google Scholar
  34. 34.
    Leppla SH. Production and purification of anthrax toxin. Methods Enzymol, 1988, 165: 103–116PubMedCrossRefGoogle Scholar
  35. 35.
    Milne JC, Blanke SR, Hanna PC, Collier RJ. Protective antigen-binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus. Mol Microbiol, 1995, 15: 661–666PubMedCrossRefGoogle Scholar
  36. 36.
    Yang J, Yuan P, Wen D, Sheng Y, Zhu S, Yu Y, Gao X, Wei W. Ultimate system for rapid assembly of customized tal effectors. PLoS ONE, 2013, 8: e75649PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Veeman MT, Slusarski DC, Kaykas A, Louie SH, Moon RT. Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr Biol, 2003, 13: 680–685PubMedCrossRefGoogle Scholar
  38. 38.
    Willert K, Nusse R. Beta-catenin: a key mediator of Wnt signaling. Curr Opin Genet Dev, 1998, 8: 95–102PubMedCrossRefGoogle Scholar
  39. 39.
    Yang J, Zhang Y, Yuan P, Zhou Y, Cai C, Ren Q, Wen D, Chu C, Qi H, Wei W. Complete decoding of tal effectors for DNA recognition. Cell Res, 2014, doi: 10.1038/cr.2014.1019Google Scholar
  40. 40.
    Hanna PC, Acosta D, Collier RJ. On the role of macrophages in anthrax. Proc Natl Acad Sci USA, 1993, 90: 10198–10201PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C, Niehrs C. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature, 1998, 391: 357–362PubMedCrossRefGoogle Scholar
  42. 42.
    Jeong SY, Martchenko M, Cohen SN. Calpain-dependent cytoskeletal rearrangement exploited for anthrax toxin endocytosis. Proc Natl Acad Sci USA, 2013, 110: E4007–4015PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG. Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process. J Cell Biol, 2003, 160: 321–328PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Bann JG, Cegelski L, Hultgren SJ. LRP6 holds the key to the entry of anthrax toxin. Cell, 2006, 124: 1119–1121PubMedCrossRefGoogle Scholar
  45. 45.
    Liu S, Leppla SH. Cell surface tumor endothelium marker 8 cytoplasmic tail-independent anthrax toxin binding, proteolytic processing, oligomer formation, and internalization. J Biol Chem, 2003, 278: 5227–5234PubMedCrossRefGoogle Scholar
  46. 46.
    Zhang Y, Wang Y, Li X, Zhang J, Mao J, Li Z, Zheng J, Li L, Harris S, Wu D. The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd. Mol Cell Biol, 2004, 24: 4677–4684PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Binnerts ME, Tomasevic N, Bright JM, Leung J, Ahn VE, Kim KA, Zhan X, Liu S, Yonkovich S, Williams J, Zhou M, Gros D, Dixon M, Korver W, Weis WI, Abo A. The first propeller domain of LRP6 regulates sensitivity to Dkk1. Mol Biol Cell, 2009, 20: 3552–3560PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Cheng Z, Biechele T, Wei Z, Morrone S, Moon RT, Wang L, Xu W. Crystal structures of the extracellular domain of LRP6 and its complex with Dkk1. Nat Struct Mol Biol, 2011, 18: 1204–1210PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Bafico A, Liu G, Yaniv A, Gazit A, Aaronson SA. Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/arrow. Nat Cell Biol, 2001, 3: 683–686PubMedCrossRefGoogle Scholar
  50. 50.
    Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell, 2002, 110: 673–687PubMedCrossRefGoogle Scholar
  51. 51.
    Sakane H, Yamamoto H, Kikuchi A. LRP6 is internalized by Dkk1 to suppress its phosphorylation in the lipid raft and is recycled for reuse. J Cell Sci, 2010, 123: 360–368PubMedCrossRefGoogle Scholar
  52. 52.
    Yamamoto H, Sakane H, Yamamoto H, Michiue T, Kikuchi A. Wnt3a and Dkk1 regulate distinct internalization pathways of LRP6 to tune the activation of beta-catenin signaling. Dev Cell, 2008, 15: 37–48PubMedCrossRefGoogle Scholar
  53. 53.
    Krupnik VE, Sharp JD, Jiang C, Robison K, Chickering TW, Amaravadi L, Brown DE, Guyot D, Mays G, Leiby K, Chang B, Duong T, Goodearl AD, Gearing DP, Sokol SY, McCarthy SA. Functional and structural diversity of the human dickkopf gene family. Gene, 1999, 238: 301–313PubMedCrossRefGoogle Scholar
  54. 54.
    Mao B, Niehrs C. Kremen2 modulates Dickkopf2 activity during Wnt/LRP6 signaling. Gene, 2003, 302: 179–183PubMedCrossRefGoogle Scholar
  55. 55.
    Brott BK, Sokol SY. Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins. Mol Cell Biol, 2002, 22: 6100–6110PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Young JJ, Bromberg-White JL, Zylstra C, Church JT, Boguslawski E, Resau JH, Williams BO, Duesbery NS. LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin. PLoS Pathog, 2007, 3: e27PubMedPubMedCentralCrossRefGoogle Scholar
  57. 57.
    Ryan PL, Young JA. Evidence against a human cell-specific role for LRP6 in anthrax toxin entry. PLoS ONE, 2008, 3: e1817PubMedPubMedCentralCrossRefGoogle Scholar
  58. 58.
    Wigelsworth DJ, Krantz BA, Christensen KA, Lacy DB, Juris SJ, Collier RJ. Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen. J Biol Chem, 2004, 279: 23349–23356PubMedCrossRefGoogle Scholar
  59. 59.
    Scobie HM, Thomas D, Marlett JM, Destito G, Wigelsworth DJ, Collier RJ, Young JA, Manchester M. A soluble receptor decoy protects rats against anthrax lethal toxin challenge. J Infect Dis, 2005, 192: 1047–1051PubMedCrossRefGoogle Scholar
  60. 60.
    Zhou Y, Zhu S, Cai C, Yuan P, Li C, Huang Y, Wei W. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature, 2014, doi: 10.1038/nature13166Google Scholar
  61. 61.
    Liu S, Zhang Y, Moayeri M, Liu J, Crown D, Fattah RJ, Wein AN, Yu ZX, Finkel T, Leppla SH. Key tissue targets responsible for anthrax-toxin-induced lethality. Nature, 2013, 501: 63–68PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© The Author(s) 2014

Authors and Affiliations

  • LiLi Qian
    • 1
  • ChangZu Cai
    • 1
  • PengFei Yuan
    • 1
  • Sun-Young Jeong
    • 2
  • XiaoZhou Yang
    • 1
  • Venita DeAlmeida
    • 4
  • James Ernst
    • 5
  • Michael Costa
    • 4
  • Stanley N. Cohen
    • 2
    • 3
  • WenSheng Wei
    • 1
  1. 1.College of Life Sciences and State Key Laboratory of Protein and Plant Gene ResearchPeking UniversityBeijingChina
  2. 2.Department of GeneticsStanford University School of MedicineStanfordUSA
  3. 3.Department of MedicineStanford University School of MedicineStanfordUSA
  4. 4.Department of Cancer TargetsGenentech, Inc.South San FranciscoUSA
  5. 5.Department of Protein Chemistry and Protein EngineeringGenentech, Inc.South San FranciscoUSA

Personalised recommendations