Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution

Jaladanki N. Rao, Xin Guo, Lan Liu, Tongtong Zou, Karnam S. Murthy, Jason X.J. Yuan, Jian Ying Wang

Research output: Contribution to journalArticle

46 Scopus citations

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 Ca2+ concentration ([Ca2+]cyt) through controlling voltage-gated K+ channel expression and membrane potential (Em) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca2+]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 [Ca2+]cyt, by either polyamine depletion or exposure to the Ca2+-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKα proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca2+]cyt also inhibited cell migration after wounding. Elevation of [Ca2+]cyt induced by the Ca2+ ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKα 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 [Ca2+]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.

Original languageEnglish (US)
Pages (from-to)C848-C859
JournalAmerican Journal of Physiology - Cell Physiology
Volume284
Issue number4 53-4
StatePublished - Apr 1 2003
Externally publishedYes

Keywords

  • Cdx2 gene
  • Cytoskeleton
  • Dominant negative mutant RhoA
  • Intracellular calcium
  • Mucosal injury

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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