R-spondins, Secreted Frizzled-Related Proteins and the Regulation of Wnt Signali

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R-spondins (Rspos) are known to potentiate Wnt/beta-catenin signaling in various settings. Activation of this pathway is critical for many processes during embryonic development and tissue homeostasis in the adult. It is one of the mechanisms that supports the proliferation of embryonic stem cells and multi-potential progenitor cells. Constitutive activation of the beta-catenin pathway is common in many types of cancers, including colorectal, hepatocellular, mammary, prostate, renal, ovary and others. Understanding the properties of factors such as the Rspos that enhance signaling through this pathway and factors like sFRP-1 that block could lead to better ways to diagnose and treat cancer. In addition, because Wnt signaling has many functions during embryonic development and in the adult increased knowledge of its regulation by Rspos and sFRPs could have applications in the treatment of other diseases. Our site-directed mutagenesis of R-spondin2 demonstrated that specific amino acid substitutions 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. R-spondin2 co-immunoprecipitated with LRP6 and Kremens, but the lack of major differences between wild-type and mutant Rspo2 implied that additional interactions might be important for biological activity. Microarray analysis of C57MG mouse mammary epithelial cells treated with recombinant proteins showed that Rspo2 potentiated a large percentage of Wnt-3a-dependent changes in gene expression. Many novel Wnt target genes were identified and validated by quantitative RT-PCR and immunoblotting, including CXCR6, a chemokine receptor recently implicated in breast cancer metastasis. Several changes in gene expression induced by the combination of Rspo2 and Wnt-3a did not appear to be mediated by the canonical Wnt/beta-catenin/TCF pathway, suggesting that other mechanisms were associated with Rspo activity. Double transfection of C57MG cells with Rspo2 and Wnt-1 stimulated cell invasiveness in three-dimensional Matrigel cultures as well as transwell assays. Invasive properties were linked to JNK activation rather than beta-catenin signaling. Subcutaneous injection of mammary cells transfected either with Rspo2 or Wnt-1 expression vectors produced tumors in nude mice. Co-expression of Rspo2 and Wnt-1 altered the histologic phenotype of tumors relative to the pattern seen with the Wnt-1 transfectant. During the previous 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. We are continuing to work with Dr. Clark to identify genetic and/or epigenetic mechanisms that would predispose individuals to glaucoma by inhibiting the Wnt/beta-catenin pathway. The other article demonstrated that sFRP-1 regulates development of the prostate in mouse, using an Sfrp1 null mouse model created in my lab. We have provided these mice to several other labs interested in exploring the role of sFRP-1 in normal brain, breast and bone development as well as corresponding pathological conditions.