title: Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution.
abstract: 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(2+) concentration ([Ca(2+)](cyt)) through controlling voltage-gated K(+) channel expression and membrane potential (E(m)) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca(2+)](cyt) activates Rho-kinase (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(2+)](cyt), by either polyamine depletion or exposure to the Ca(2+)-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca(2+)](cyt) also inhibited cell migration after wounding. Elevation of [Ca(2+)](cyt) induced by the Ca(2+) ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKalpha 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(2+)](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.
authors.affiliation: Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Jaladanki N
authors.initials: JN
authors.lastname: Rao
authors.forename: Xin
authors.initials: X
authors.lastname: Guo
authors.forename: Lan
authors.initials: L
authors.lastname: Liu
authors.forename: Tongtong
authors.initials: T
authors.lastname: Zou
authors.forename: Karnam S
authors.initials: KS
authors.lastname: Murthy
authors.forename: Jason X-J
authors.initials: JX
authors.lastname: Yuan
authors.forename: Jian-Ying
authors.initials: JY
authors.lastname: Wang
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D000071616:CDX2 Transcription Factor; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002465:Cell Movement; D003593:Cytoplasm / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism*; D024462:Myosin Type II / Q000378:metabolism; D009994:Osmolar Concentration; D010766:Phosphorylation; D011073:Polyamines / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D015534:Trans-Activators; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000577:Amides; D000071616:CDX2 Transcription Factor; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; D018994:Myosin Light Chains; D011073:Polyamines; D011725:Pyridines; D015534:Trans-Activators; C108830:Y 27632; D006160:Guanosine Triphosphate; D024462:Myosin Type II; D020742:rhoA GTP-Binding Protein; D002118:Calcium