Polyamines are required for phospholipase C-γ₁ expression promoting intestinal epithelial restitution after wounding

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)-γ₁ 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-γ₁ activity, regulating [Ca²⁺]_cyt and cell migration after wounding. Depletion of cellular polyamines by α-difluoromethylornithine inhibited PLC-γ₁ expression in differentiated IECs (stable Cdx2-transfected IEC-6 cells), as indicated by substantial decreases in levels of PLC-γ₁ mRNA and protein and its enzyme product IP ₃. Polyamine-deficient cells also displayed decreased [Ca ²⁺]_cyt and inhibited cell migration. Decreased levels of PLC-γ₁ by treatment with U-73122 or transfection with short interfering RNA specifically targeting PLC-γ₁ also decreased IP₃, reduced resting [Ca²⁺]_cyt and Ca ²⁺ influx after store depletion, and suppressed cell migration in control cells. In contrast, stimulation of PLC-γ₁ 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-γ₁ expression in IECs and that polyamine-mediated PLC-γ₁ signaling stimulates cell migration during restitution as a result of increased [Ca²⁺]_cyt.