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.

Thrombin modifies growth, proliferation and apoptosis of human colon organoids: a protease‐activated receptor 1‐ and protease‐activated receptor 4‐dependent mechanism

“…Furthermore, we demonstrated that these detrimental effects of thrombin are mediated by the activation of both PAR1 and PAR4, the two functional thrombin receptors. We have previously demonstrated that PAR1 is expressed in human and mouse colon crypts and organoids (Nasri et al ., ). The presence of PAR4 in stem cells/progenitors has not been previously demonstrated, but our results revealed an active role for PAR4 in these cells.…”

“…This suggests that a compensatory mechanism might maintain the size of budding organoids in the presence of this high dose of thrombin, despite the anti‐proliferative effects induced by PAR1 activation. In a previous study, we observed that PAR1‐activating peptides increase the size of murine organoids in part by pushing differentiation processes (Nasri et al ., ). This could constitute the compensatory mechanism maintaining the size of budding organoids exposed to high doses of thrombin.…”

“…Although PAR2 is not considered as a thrombin receptor, recent work from the Hollenberg's laboratory has demonstrated that thrombin can signal through PAR2 (Mihara et al ., ). We have previously shown that activated PAR2 strongly reduced the proliferation of immature cells, although without inducing apoptosis or decreasing stem cell capacities in mouse cultured organoids (Nasri et al ., ). Thus, PAR2 could be another player, activated at high concentrations of thrombin (Mihara et al ., ), interfering with proliferation and metabolic activity.…”

G Protein–Coupled Receptor Signaling Through β-Arrestin–Dependent Mechanisms

“…83,84 On the other hand, β-arrestin2 does not associate with Akt downstream of PAR2, but forms a complex with PP2A/GSK3β in a Rho kinase dependent manner. 85 β-arrestin-PP2A-GSK3β signaling facilitates stem/progenitor cell survival in normal colon crypts and colon cancer. 85 …”

“…85 β-arrestin-PP2A-GSK3β signaling facilitates stem/progenitor cell survival in normal colon crypts and colon cancer. 85 …”