PAR2-dependent activation of GSK3β regulates the survival of colon stem/progenitor cells

Abstract

Protease-activated receptors PAR₁ and PAR₂ play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor cells promoted by proinflammatory mediators may be critical in oncogenesis. The glycogen synthase kinase-3β (GSK3β) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR₁ and PAR₂ effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3β. First, PAR₁ and PAR₂ were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR₂ activation decreased numbers and size of normal or cancerous spheroids, and PAR₂-deficient spheroids showed increased proliferation, indicating that PAR₂ represses proliferation. PAR₂-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR₂. Accordingly, active caspase-3 was strongly increased in PAR₂-deficient normal spheroids. PAR₂ but not PAR₁ triggered GSK3β activation through serine-9 dephosphorylation in normal and tumor cells. The PAR₂-triggered GSK3β activation implicates an arrestin/PP2A/GSK3β complex that is dependent on the Rho kinase activity. Loss of PAR₂ was associated with high levels of GSK3β nonactive form, strengthening the role of PAR₂ in GSK3β activation. GSK3 pharmacological inhibition impaired the survival of PAR₂-stimulated spheroids and serum-starved cells. Altogether our data identify PAR₂/GSK3β as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer.