Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration

Expression of voltage-gated K+ (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E-m) and cytosolic free Ca2+ concentration ([ Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated [Ca2+](cyt) following activation of K+ channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha -difluoromethylornithine (DFMO) reduced whole cell K+ currents [I-K(v)] through Kv channels and caused membrane depolarization, which was associated with decreases in [Ca2+](cyt), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I-K(v), E-m, [Ca2+](cyt), and RhoA protein and restored cell migration to normal. Elevation of [Ca2+](cyt) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C-3 transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.