The focus of my lab has broadened to include both the study of secreted Frizzled-related proteins (sFRPs) and cellular responses to Wnt stimulation. We have embarked on the purification of Wnt proteins, not only to provide reagents to facilitate the analysis of sFRP function, but to foster investigation of Wnt activity and associated signaling. Defining the specificity of Wnt/Frizzled(Fz)/sFRP interactions is one of our primary goals. Currently, we have purified four recombinant sFRPs and have made progress in the isolation of three Wnt proteins. We also identified a cell line with a low background of Wnt/Fz expression, and separately introduced three Fzs into these cells to assist our study of Wnt/Fz/sFRP binding and signaling. Recently, we initiated a collaboration to explore the alleged role of Cripto as a novel Wnt co-receptor functioning in the canonical/?-catenin pathway. We developed a convenient model system to examine Wnt-dependent cell motility and associated changes in cell shape. We demonstrated that RhoA activation is important for cell migration, while ?-catenin signaling is not, and Dishevelled-2 (Dvl-2) had a predominant role as a mediator of Wnt activity in a transwell assay. This has led to an emphasis on aspects of Dvl regulation, particularly phosphorylation, thought to be important for non-canonical signaling and cell motility. We also have observed that Wnt-3a stimulates the assembly of fibronectin (FN) fibrils, a phenomenon that likely contributes to cell spreading and movement. Our longstanding study of sFRP-1 has continued in a variety of contexts. We have determined that methylation of specific sequences in the SFRP1 promoter disrupts binding of nuclear factors, which may contribute to epigenetic silencing of the gene that has been documented in several cancers. An Sfrp1 null mouse model has been developed, and we are sharing it with multiple labs to better understand the physiological activities of sFRP-1. In various experimental systems, we have been comparing and contrasting the effects of sFRP-1 and Dickkopf-1 (Dkk-1). While both inhibit the ?-catenin pathway, our results suggest that Dkk-1 might enhance non-canonical signaling in situations where it is blocked by sFRP-1. Besides the well-established role of sFRP-1 as a regulator of Wnt signaling, we have explored the possibility that it has novel binding partners and additional mechanisms of action. Using a phage-display peptide library, we identified a binding motif for sFRP-1 that is not present in Wnt proteins and might provide a clue to other protein-protein interactions. At one point, we thought that such interactions could be the basis for sFRP-1 inhibition of osteoclast formation, an effect which we demonstrated in collaboration with Dr. Matthew Gillespie. RANKL, a key mediator of osteoclastogenesis, contains a similar motif and bound to sFRP-1 in ELISA experiments. However, subsequent work suggests that sFRP-1 inhibitory activity does not require a direct interaction with RANKL. Taken together, our sFRP and Wnt studies will contribute to a better understanding of their roles in cancer, development and tissue homeostasis.
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