Trpc1-Mediated Ca2+ Signaling Enhances Intestinal Epithelial Restitution By Increasing Alpha 4 Association With Pp2ac After Wounding

Gut epithelial restitution after superficial wounding is an important repair modality regulated by numerous factors including Ca2+ signaling and cellular polyamines. Transient receptor potential canonical-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs) and its activation increases epithelial restitution by inducing Ca2+ influx after acute injury. alpha 4 is a multiple functional protein and implicated in many aspects of cell functions by modulating protein phosphatase 2A (PP2A) stability and activity. Here we show that the clonal populations of IECs stably expressing TRPC1 (IEC-TRPC1) exhibited increased levels of alpha 4 and PP2A catalytic subunit (PP2Ac) and that TRPC1 promoted intestinal epithelial restitution by increasing alpha 4/PP2Ac association. The levels of alpha 4 and PP2Ac proteins increased significantly in stable IEC-TRPC1 cells and this induction in alpha 4/PP2Ac complexes was accompanied by an increase in IEC migration after wounding. alpha 4 silencing by transfection with siRNA targeting alpha 4 (si alpha 4) or PP2Ac silencing destabilized alpha 4/PP2Ac complexes in stable IEC-TRPC1 cells and repressed cell migration over the wounded area. Increasing the levels of cellular polyamines by stable transfection with the Odc gene stimulated alpha 4 and PP2Ac expression and enhanced their association, thus also promoting epithelial restitution after wounding. In contrast, depletion of cellular polyamines by treatment with alpha-difluoromethylornithine reduced alpha 4/PP2Ac complexes and repressed cell migration. Ectopic overexpression of alpha 4 partially rescued rapid epithelial repair in polyamine-deficient cells. These results indicate that activation of TRPC1-mediated Ca2+ signaling enhances cell migration primarily by increasing alpha 4/PP2Ac associations after wounding and this pathway is tightly regulated by cellular polyamines.