The function of Plexin-B1/PlexinB2 in intestinal stem cells

Plexins are a family of single-transmembrane proteins that serve as receptors for sem-aphorins and for mechanical forces. Nine plexins have been identified and classified into four groups, Plexin-A1-A4, Plexin-B1-B3, Plexin-C1, and Plexin-D1. In previous work, it has been observed that Plexin-B2 was highly expressed in intestinal epithelial cells. By generating knockout mice, Matkovic et al found that deletion of Plexin-B1 and Plexin-B2 in intestinal epithelial cells reduced the number of intestinal stem cells. In this thesis, it was found that deletion of Plexin-B1 and Plexin-B2 caused failure of intestinal regeneration after damage induced by the chemotherapeutic agent, 5-fluoruracil (5-FU). Intestinal organoids lacking Plexin-B1 and Plexin-B2 showed reduced budding structures and down-regulation of stem cell marker and Wnt target gene expression. However, the mRNA expression levels of Lgr5 and Olfm4 and TOPflash assay were not affected by ligands of B plexins (Sema4B, Sema4C, Sema4D, Sema4G, and Ang) indicating that Plexin-B1 and Plexin-B2 contributed to Wnt signaling independently of ligands. In HEK293T cells, the knockdown of Plexin-B2 decreased Wnt reporter activi-ty as induced by wnt3a and R-spondin but not Dvl1 and β-catenin. In addition, by con-ducting co-immunoprecipitation experiments, it was found that Plexin-B2 interacted with Wnt receptors, including Lrp6 and Fzd5/8. Finally, it was observed that Plexin-B2 exhibited co-localization with Fzd5 in the cytoplastic area of HEK-T cells after Wnt3a and R-spondin stimulation providing evidence that Plexin-B2 was involved in the endo-cytosis processes. Diminished internalization of Fzd5 was noted subsequent to Plexin-B2 knockdown, under conditions of Wnt3a and R-spondin stimulation, as well as Znrf3 overexpression. In conclusion, Plexin-B2 promotes canonical Wnt pathway signaling required to sustain the self-renewal of intestinal stem cells.