Polyamines are required for phospholipase C-gamma(1) expression promoting intestinal epithelial restitution after wounding

Polyamines are required for phospholipase C-gamma(1) expression promoting intestinal epithelial restitution after wounding. Am J Physiol Gastrointest Liver Physiol 292: G335-G343, 2007. First published September 14, 2006; doi:10.1152/ajpgi.00282.2006.-Intestinal mucosal restitution occurs by epithelial cell migration, rather than by proliferation, to reseal superficial wounds after injury. Polyamines are essential for the stimulation of intestinal epithelial cell (IEC) migration during restitution in association with their ability to regulate Ca2+ homeostasis, but the exact mechanism by which polyamines induce cytosolic free Ca2+ concentration ([Ca2+](cyt)) remains unclear. Phospholipase C (PLC)-gamma(1) catalyzes the formation of inositol (1,4,5)-trisphosphate (IP3), which is implicated in the regulation of [Ca2+](cyt) by modulating Ca2+ store mobilization and Ca2+ influx. The present study tested the hypothesis that polyamines are involved in PLC-gamma(1) activity, regulating [Ca2+](cyt) and cell migration after wounding. Depletion of cellular polyamines by alpha-difluoromethylornithine inhibited PLC-gamma(1) expression in differentiated IECs (stable Cdx2-transfected IEC-6 cells), as indicated by substantial decreases in levels of PLC-gamma(1) mRNA and protein and its enzyme product IP3. Polyamine-deficient cells also displayed decreased [Ca2+](cyt) and inhibited cell migration. Decreased levels of PLC-gamma(1) by treatment with U-73122 or transfection with short interfering RNA specifically targeting PLC-gamma(1) also decreased IP3, reduced resting [Ca2+](cyt) and Ca2+ influx after store depletion, and suppressed cell migration in control cells. In contrast, stimulation of PLC-gamma(1) by 2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)-benzenesulfonamide induced IP3, increased [Ca2+](cyt), and promoted cell migration in polyamine-deficient cells. These results indicate that polyamines are absolutely required for PLC-gamma(1) expression in IECs and that polyamine-mediated PLC-gamma(1) signaling stimulates cell migration during restitution as a result of increased [Ca2+](cyt).