Exosomes derived from non-canonical WNT5A-expressing cells activate canonical WNT signaling in recipient cells

Exosomes derived from non-canonical WNT5A-expressing cells activate canonical WNT signaling in recipient cells

Purpose: WNT proteins regulate key developmental and physiological processes. Recent studies havefound that exosomes, which are small extracellular vesicles, are novel transport carriers of WNT signalingproteins. In the present study, we isolated exosomes from conditioned media of canonical ligand WNT3Aandnon-canonical ligand WNT5A-expressing HEK293T cells and analyzed their bioactivity.Methods: Exosomes were isolated by differential centrifugation followed by size exclusion chromatography.Isolated exosomes were subjected to Western blot analysis and nanoparticle tracking analysis for theircargo contents and size distribution, respectively.Results: Most enriched exosomes measured 120-140 nm and expressed canonical exosomal markers,including ALIX, CD63, and CD81. Expression of either WNT3A or WNT5A led to an increase in thetotal number of secreted exosomes. While WNT3A and activeβ-catenin were both recovered in exosomesderived from WNT3A-producing cells, only activeβ-catenin was detected in exosomes recovered fromWNT5A-producing cells. Intriguingly, both types of exosomes activatedβ-catenin-dependent response inrecipient cells, as evidenced by accumulation of cytosolic activeβ-catenin and transcriptional activation ofT-cell factor/lymphoid enhancer factor (TCF/LEF) responsive elements.Conclusion: These results suggest that WNT3A- and WNT5A-producing cells share a common mechanismfor packaging of activeβ-catenin in exosomes.