(A) The primary objective of this project has been to obtain a better understanding of the factors that account for the potentiation or inhibition of Wnt3a/beta-catenin signaling by secreted Frizzled-related proteins (sFRPs). Such knowledge would be beneficial in clinical applications that involve the use of sFRPs to control Wnt signaling. Cell context is a determinant of the effect sFRP1 has on Wnt3a/beta-catenin signaling. sFRP1 has biphasic activity in parental HEK293 cells and HEKSTF cells (a clonal line stably expressing a SuperTopFlash reporter construct), enhancing beta-catenin signaling at 1-10 nM and inhibiting it at 100-300 nM. In contrast, sFRP1 primarily stimulated Wnt3a activity in the mouse mammary epithelial line C57MG in this concentration range, but was a strict inhibitor in L929 fibroblasts (L cells) even at low concentrations. A similar pattern of cell-specific activity was observed with sFRP2. Receptor expression is a key factor in the response to sFRP1. Ectopic over-expression of Fzd5, but not Fzd2, in L cells enabled potentiation of Wnt3a activity. We had hypothesized that Fzd5 would contribute to a potentiating effect at low sFRP1 concentrations, because of its homology to DFz2, the Drosophila Fzd expressed in S2 cells that previously had shown a biphasic response to sFRP1 in the presence of Wingless. CRDsFRP1 mimicked the potentiating effect of sFRP1 in multiple settings, contradicting initial expectations that this domain would inhibit Wnt signaling. Moreover, CRDsFRP1 showed little avidity for Wnt3a compared with sFRP1, implying that the mechanism for potentiation by CRDsFRP1 probably does not require an interaction with Wnt protein. Together, these findings demonstrate that sFRPs can either promote or suppress Wnt/beta-catenin signaling, depending on cellular context, concentration and most likely the expression pattern of Fzd receptors. (B) A secondary objective of this project has been to develop the 32D myeloid progenitor cell line as a model system for the study of specificity in Wnt/Fzd interactions. 32D cells express little or no endogenous Fzd transcripts. We have stably transfected 32D cells with nine of the ten mammalian Fzds, all with N-terminal HA epitope tags. Treatment of 32D cells expressing individual Fzds with particular Wnts elicits responses in different Wnt signaling pathways, suggesting that this model would be useful in associating specific Wnt/Fzd combinations with distinct downstream signaling events.
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