PAR2-dependent activation of GSK3ß regulates the survival of colon stem/progenitor cells.

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-3ß (GSK3ß) 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 GSK3ß. 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 GSK3ß activation through serine-9 dephosphorylation in normal and tumor cells. The PAR2-triggered GSK3ß activation implicates an arrestin/PP2A/GSK3ß complex that is dependent on the Rho kinase activity. Loss of PAR2 was associated with high levels of GSK3ß nonactive form, strengthening the role of PAR2 in GSK3ß activation. GSK3 pharmacological inhibition impaired the survival of PAR2-stimulated spheroids and serum-starved cells. Altogether our data identify PAR2/GSK3ß 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.