Abstract
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.
Publication types
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Research Support, N.I.H., Intramural
MeSH terms
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Adaptor Proteins, Signal Transducing / physiology
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Cell Line, Tumor / drug effects
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Cell Line, Tumor / ultrastructure
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Dishevelled Proteins
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Frizzled Receptors / analysis
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Frizzled Receptors / physiology*
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Humans
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Intercellular Signaling Peptides and Proteins / genetics
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Intercellular Signaling Peptides and Proteins / physiology*
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JNK Mitogen-Activated Protein Kinases / physiology*
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LDL-Receptor Related Proteins / antagonists & inhibitors
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LDL-Receptor Related Proteins / physiology
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Low Density Lipoprotein Receptor-Related Protein-5
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Low Density Lipoprotein Receptor-Related Protein-6
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Membrane Proteins / genetics
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Membrane Proteins / physiology
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Neoplasm Proteins / genetics
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Neoplasm Proteins / physiology*
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Neurites / drug effects
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Neurites / physiology*
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Phosphoproteins / physiology
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RNA, Small Interfering / pharmacology
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Receptors, G-Protein-Coupled / analysis
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Receptors, G-Protein-Coupled / physiology*
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Recombinant Fusion Proteins / physiology
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Sarcoma, Ewing / ultrastructure*
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Wnt Proteins / genetics
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Wnt Proteins / pharmacology
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Wnt Proteins / physiology*
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Wnt1 Protein / physiology
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Wnt3 Protein
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Wnt3A Protein
Substances
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Adaptor Proteins, Signal Transducing
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DVL2 protein, human
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DVL3 protein, human
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Dishevelled Proteins
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FZD3 protein, human
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FZD4 protein, human
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FZD7 protein, human
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Frizzled Receptors
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Intercellular Signaling Peptides and Proteins
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LDL-Receptor Related Proteins
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LRP5 protein, human
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LRP6 protein, human
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Low Density Lipoprotein Receptor-Related Protein-5
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Low Density Lipoprotein Receptor-Related Protein-6
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Membrane Proteins
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Neoplasm Proteins
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Phosphoproteins
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RNA, Small Interfering
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Receptors, G-Protein-Coupled
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Recombinant Fusion Proteins
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SFRP1 protein, human
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WNT3A protein, human
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Wnt Proteins
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Wnt1 Protein
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Wnt3 Protein
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Wnt3A Protein
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JNK Mitogen-Activated Protein Kinases