Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ]cyt) through controlling voltage-gated K ϩ channel expression and membrane potential (Em) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca 2ϩ ]cyt activates

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... nase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced [Ca 2ϩ ]cyt, by either polyamine depletion or exposure to the Ca 2ϩ -free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROK␣ proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca 2ϩ ]cyt also inhibited cell migration after wounding. Elevation of [Ca 2ϩ ]cyt induced by the Ca 2ϩ ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROK␣ proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via [Ca 2ϩ ]cyt can interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration. mucosal injury; intracellular calcium; Cdx2 gene; dominant negative mutant RhoA; cytoskeleton