PAR2-dependent activation of GSK3 beta regulates the survival of colon stem/progenitor cells

Protease-activated receptors PAR1 and PAR(2) 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-3 beta (GSK3 beta) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR(1) and PAR(2) effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3 beta. First, PAR(1) and PAR(2) were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR(2) activation decreased numbers and size of normal or cancerous spheroids, and PAR(2)-deficient spheroids showed increased proliferation, indicating that PAR(2) represses proliferation. PAR(2)-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR(2). Accordingly, active caspase-3 was strongly increased in PAR(2)-deficient normal spheroids. PAR(2) but not PAR(1) triggered GSK3 beta activation through serine-9 dephosphorylation in normal and tumor cells. The PAR(2)-triggered GSK3 beta activation implicates an arrestin/PP2A/GSK3 beta complex that is dependent on the Rho kinase activity. Loss of PAR(2) was associated with high levels of GSK3 beta nonactive form, strengthening the role of PAR2 in GSK3 beta activation. GSK3 pharmacological inhibition impaired the survival of PAR(2)-stimulated spheroids and serum-starved cells. Altogether our data identify PAR(2)/GSK3 beta 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