Abstract 629 Secreted Frizzled Related Protein 2 Protects Cells from Apoptosis by Blocking the Effect of Canonical Wnt3a
We have demonstrated that mesenchymal stem cells (MSCs) overexpressing the survival gene Akt can confer paracrine protection to ischemic myocytes both in vivo and in vitro through the release of secreted frizzled related protein 2 (Sfrp2). However, the mechanisms mediating these effects of Sfrp2 have not been fully elucidated. In this study, we test the hypothesis that Sfrp2 exerts anti-apoptotic effect by antagonizing pro-apoptotic properties of specific Wnt ligands. To explore this possibility, we studied rat cardiomyoblasts subjected to hypoxia reoxygenation (HR) injury. We examined the effect of Wnt3a and Sfrp2 on HR-induced apoptosis. We choose to study Wnt3a because its expression is up-regulated in response to hypoxia. Wnt3a (3nM) significantly increased cellular caspase activities (35% increase, p<0.05, n=3) and TUNEL staining (38% increase) in response to HR. Sfrp2 attenuated significantly Wnt3a-induced caspase activities in a concentration dependent fashion (0nM, 8.50±0.47; 3nM, 7.28±1.03; 30nM, 7.45±0.49; 300nM, 5.65±0.35), achieving 36% inhibition at a concentration of 300nM. Using a solid phase binding assay, our data demonstrates that Sfrp2 physically binds to Wnt3a. In addition, we observed that 600nM Sfrp2 inhibits 47% of the beta-catenin / TCF transcriptional activities induced by Wnt3a(0.55±0.07 vs 1.03±0.04). Impressively, Dickkopf-1, a protein that binds to the Wnt coreceptor LRP, significantly inhibited the Wnt3a-activated caspase and transcriptional activities by 31% and 77% respectively (caspase activities: 8.62±0.05 vs12.54±0.49; transcriptional activities: 0.41±0.03 vs 1.77±0.01) Similarly, siRNA against beta-catenin inhibited the Wnt3a-activated caspase activities by 59% (6.44±0.92 vs 15.71±0.67). Consistent with this, significantly fewer TUNEL positive cells were observed in siRNA transfected cells than in control cells (43% decrease). Together, our data provide strong evidence to support the notion that Wnt3a is a canonical Wnt with pro-apoptotic action whose cellular activity is prevented by, at least in part, Sfrp2 through direct binding of these molecules. These results can explain the in vivo protective effect of Sfrp2 and highlight its therapeutic potential for the ischemic heart.