Secreted Frizzled-Related Proteins and Wnt Signaling

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We established that cells from Ewing's sarcoma family of tumors (ESFT) form neurites in response to Wnt-3a and have begun to define the mechanisms that account for this cellular response. Fzd3 was identified as the primary Wnt receptor that mediates this process, which also involves the previously identified Wnt effector molecules, Dishevelled-2 and Dishevelled-3, and amino-terminal c-Jun kinase (JNK). Consistent with one of our general objectives (see above), we observed that Dickkopf-1 also promoted neurite outgrowth in ESFT cells, apparently by shifting the activity of endogenous Wnts to stimulate similar Wnt signaling pathways. Further analysis suggests that neurite outgrowth induced by Wnt-3a requires Dishevelled phosphorylation by casein kinase I delta (CKI delta). Moreover, preliminary experiments indicate that CKI delta and Dishevelleds may have critical roles in the formation of the primary cilium, implying that these proteins contribute to the formation of multiple cellular structures that require centrosomal function. For the past two years we have been investigating the role of R-spondins in the stimulation of Wnt/beta-catenin signaling and tumorigenesis. In collaboration with Dr. Robert Callahan's group, we demonstrated that Rspo2 potentiates Wnt/beta-catenin signaling in mammary epithelial cells and contributes to their oncogenic properties. However, some cellular responses appear to depend on other, yet to be determined, signaling pathways. Site-directed mutagenesis showed that specific point mutations dramatically reduced R-spondin2 activity in the beta-catenin pathway. Rspo2 activation of this pathway was associated with an unusually prolonged stimulation of Wnt co-receptor LRP6 phosphorylation and an accumulation of LRP6 at the cell surface. During the current fiscal year, two collaborative studies concerning the physiological or pathophysiological activities of sFRP-1 culminated in publications. In one study, we showed that increases in sFRP-1 expression and concomitant inhibition of Wnt/beta-catenin signaling could elevant intraocular pressure, predisposing individuals to glaucoma. The other report contained evidence that sFRP-1 regulates development of the prostate in mouse. In an ongoing examination of Wnt/Frizzled(Fzd)/sFRP interactions, members of my lab used a full set of ten epitope-tagged Fzds to define the specificity of sFRP-1/Fzd binding and confirmed that such interactions are mediated by the cysteine-rich domain of sFRP-1. Thus far, our efforts to demonstrate the functional significance of sFRP-1/Fzd binding are inconclusive.