Editors' ChoiceNeuroscience

Delineating Wnt Pathways in Pain

Sci. Signal.  15 Jul 2014:
Vol. 7, Issue 334, pp. ec192
DOI: 10.1126/scisignal.2005684

Wnts are a family of ligands that stimulate signaling that is critical in embryonic development and contributes to some forms of cancer. A relatively newly identified function of Wnt signaling is implicated in pain sensation. Wnts are ligands for the Frizzled (Fzd) family of receptors, which stimulate pathways that stabilize β-catenin and stimulate β-catenin–dependent gene transcription, alter the cytoskeleton and contribute to planar cell polarity (the organization of epithelial sheets of cells), or stimulate the release of intracellular calcium stores. Simonetti et al. identified specific pathways through which Wnt signaling mediates peripheral nerve sensitivity to painful stimuli. Fzd3 mRNA and protein were detected in sensory neurons in the dorsal root ganglion (DRG) or the hindpaw of wild-type or transgenic mice expressing green fluorescent protein (GFP)–Fzd3. Intraplantar injection of recombinant Wnt3a increased the sensitivity of wild-type mice to mechanical and thermal stimuli. Intraplantar pretreatment with either an inhibitor of Dishevelled, which is downstream of Fzd in the β-catenin pathway, or a function-blocking antibody against Fzd1 and Fzd3 decreased the mechanical and thermal hypersensitivity induced by Wnt3a injection. Application of Wnt3a to an innervated skin slice from the hindpaw induced activity in some mechanical sensory nerve fibers. In cultured mouse DRG neurons, addition of Wnt3a increased the nuclear translocation of β-catenin and the expression of β-catenin transcriptional targets. Pharmacological inhibition of Ca2+/calmodulin kinase II (CaMKII) enabled stronger expression of Wnt3a-induced genes, consistent with the reported inhibition of β-catenin–mediated Wnt signaling by calcium-mediated Wnt signaling. However, compared with control mice, deleting or functionally blocking β-catenin transcriptional activity in all DRG neurons or only nociceptive (pain sensory) neurons embryonically or in adults abrogated the induction of a Wnt target gene but failed to prevent increased mechanical or thermal hypersensitivity induced by hindpaw injection of Wnt3a. This finding indicated that β-catenin–mediated Wnt signaling has only a minor role in enhancing pain sensitivity. In cultured DRG neurons, addition of Wnt3a increased intracellular calcium, the amount of the nociceptive ion channel TRPV1 at the cell surface, and the secretion of pain-stimulating cytokines. DRG neurons of mice injected with Wnt3a into the hindpaw had increased phosphorylation of CaMKII and the tyrosine kinase Src and TRPV1 at the cell surface. Pharmacologically inhibiting CaMKII or Src abolished thermal but not mechanical hypersensitivity. The planar cell polarity pathway of Wnt signaling is mediated in part by the guanosine triphosphatase Rac1 and its target c-Jun N-terminal kinase (JNK). Addition of Wnt3a to DRG cultures activated Rac1, and intraplantar injection of Wnt3a activated JNK in DRG neurons in mice. DRG neuron-specific knockdown of Rac1 or intraplantar injection of a JNK inhibitor abrogated Wnt3a-induced mechanical but not thermal hypersensitivity (the opposite of CaMKII or Src inhibition). Heterologous expression assays in HEK293 cells indicated that Wnt3a induced the membrane localization of the mechanical sensory ion channels TRPA1 and P2X3. Pharmacological inhibition of either channel in the hindpaw decreased Wnt3a-induced mechanical hypersensitivity in mice. The findings suggest that the Wnt-mediated calcium pathway and the Wnt pathway associated with the planar cell polarity mediate thermal and mechanical nociception, respectively.

Pain is commonly associated with cancer, particularly metastatic disease. Wnt3a was not present in DRG neurons or skin from the paws of wild-type mice, but Wnt5a was present in DRG neurons. When mice were injected in the heel with either fibrosarcoma cells or complete Freund’s adjuvant to model bone metastasis or inflammation, respectively, Wnt5a was unaffected, but lysates from the paws now had Wnt3a. Wnt5a-blocking antibodies injected into the heel had no effect on pain hypersensitivity of mice implanted in the heel with fibrosarcoma cells. In contrast, heel administration of Wnt3a-blocking antibodies, Fzd1/3-blocking antibodies, or secreted Fzd-related proteins, which sequester Wnt family ligands, reduced mechanical hypersensitivity in mice implanted in the heel with fibrosarcoma cells. The findings indicate that paracrine Wnt signaling might be therapeutically targeted to alleviate pain sensitivity in cancer patients.

M. Simonetti, N. Agarwal, S. Stösser, K. K. Bali, E. Karaulanov, R. Kamble, B. Pospisilova, M. Kurejova, W. Birchmeier, C. Niehrs, P. Heppenstall, R. Kuner, Wnt-Fzd signaling sensitizes peripheral sensory neurons via distinct noncanonical pathways. Neuron 83, 104–121 (2014). [Pubmed]