Wnt-3a and Dickkopf-1 stimulate neurite outgrowth in Ewing tumor cells via a Frizzled3- and c-Jun N-terminal kinase-dependent mechanism.
Recombinant Wnt-3a stimulated the rapid formation of elongated processes in Ewing sarcoma family tumor (ESFT) cells that were identified as neurites. The processes stained positively for polymerized actin and microtubules as well as synapsin I and growth-associated protein 43. Inhibition of the Wnt receptor, Frizzled3 (Fzd3), with antiserum or by short interfering RNA (siRNA) markedly reduced neurite extension. Knockdown of Dishevelled-2 (Dvl-2) and Dvl-3 also suppressed neurite outgrowth. Surprisingly, disruption of the Wnt/Fzd/lipoprotein receptor-related protein (LRP) complex and the associated beta-catenin signaling by treating cells either with the Wnt antagonist Dickkopf-1 (Dkk1) or LRP5/LRP6 siRNA enhanced neuritogenesis. Neurite outgrowth induced by Dkk1 or with LRP5/LRP6 siRNA was inhibited by secreted Fzd-related protein 1, a Wnt antagonist that binds directly to Wnt. Moreover, Dkk1 stimulation of neurite outgrowth was blocked by Fzd3 siRNA. These results suggested that Dkk1 shifted endogenous Wnt activity from the beta-catenin pathway to Fzd3-mediated, noncanonical signaling that is responsible for neurite formation. In particular, c-Jun amino-terminal kinase (JNK) was important for neurite outgrowth stimulated by both Wnt-3a and Dkk1. Our data demonstrate that Fzd3, Dvl, and JNK activity mediate Wnt-dependent neurite outgrowth and that ESFT cell lines will be useful experimental models for the study of Wnt-dependent neurite extension.
Endo
Yoshimi
Y
National Cancer Institute, Bldg. 37, Room 2042, 37 Convent Drive, MSC 4256, Bethesda, MD 20892-4256, USA.
Beauchamp
Elspeth
E
Woods
David
D
Taylor
William G
WG
Toretsky
Jeffrey A
JA
Uren
Aykut
A
Rubin
Jeffrey S
JS
D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology; D045744:Cell Line, Tumor / Q000187:drug effects / Q000648:ultrastructure; D000072261:Dishevelled Proteins; D051157:Frizzled Receptors / Q000032:analysis / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology*; D026502:LDL-Receptor Related Proteins / Q000037:antagonists & inhibitors / Q000502:physiology; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology*; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012512:Sarcoma, Ewing / Q000648:ultrastructure*; D051153:Wnt Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D051155:Wnt1 Protein / Q000502:physiology; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D000094742:Secreted Frizzled-Related Proteins
D048868:Adaptor Proteins, Signal Transducing; D000072261:Dishevelled Proteins; D051157:Frizzled Receptors; D036341:Intercellular Signaling Peptides and Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D026502:LDL-Receptor Related Proteins; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D051153:Wnt Proteins; D051155:Wnt1 Protein; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C000606581:DVL2 protein, human; C000606584:DVL3 protein, human; C105574:FZD3 protein, human; C402930:FZD4 protein, human; C115810:FZD7 protein, human; C555482:LRP5 protein, human; C555475:LRP6 protein, human; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins; C555510:WNT3A protein, human