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

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

Research output: Contribution to journalArticle

44 Citations (Scopus)

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)
JournalAmerican Journal of Physiology - Cell Physiology
Volume284
Issue number4 53-4
StatePublished - Apr 1 2003
Externally publishedYes

Fingerprint

rho-Associated Kinases
Phosphorylation
Polyamines
Myosins
Myosin Light Chains
Cell Movement
Guanosine Triphosphate
Protein Kinases
rhoA GTP-Binding Protein
Epithelial Cells
Voltage-Gated Potassium Channels
Stress Fibers
Ionomycin
Ionophores
Membrane Potentials
Proteins
Chemical activation
Membranes
Phenotype
Fibers

Keywords

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

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution. / Rao, Jaladanki N.; Guo, Xin; Liu, Lan; Zou, Tongtong; Murthy, Karnam S.; Yuan, Jason; Wang, Jian Ying.

In: American Journal of Physiology - Cell Physiology, Vol. 284, No. 4 53-4, 01.04.2003.

Research output: Contribution to journalArticle

Rao, Jaladanki N. ; Guo, Xin ; Liu, Lan ; Zou, Tongtong ; Murthy, Karnam S. ; Yuan, Jason ; Wang, Jian Ying. / Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution. In: American Journal of Physiology - Cell Physiology. 2003 ; Vol. 284, No. 4 53-4.
@article{10ff49337aa64a88b200532d1364deb7,
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 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.",
keywords = "Cdx2 gene, Cytoskeleton, Dominant negative mutant RhoA, Intracellular calcium, Mucosal injury",
author = "Rao, {Jaladanki N.} and Xin Guo and Lan Liu and Tongtong Zou and Murthy, {Karnam S.} and Jason Yuan and Wang, {Jian Ying}",
year = "2003",
month = "4",
day = "1",
language = "English (US)",
volume = "284",
journal = "American Journal of Physiology",
issn = "0363-6143",
publisher = "American Physiological Society",
number = "4 53-4",

}

TY - JOUR

T1 - Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution

AU - Rao, Jaladanki N.

AU - Guo, Xin

AU - Liu, Lan

AU - Zou, Tongtong

AU - Murthy, Karnam S.

AU - Yuan, Jason

AU - Wang, Jian Ying

PY - 2003/4/1

Y1 - 2003/4/1

N2 - 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.

AB - 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.

KW - Cdx2 gene

KW - Cytoskeleton

KW - Dominant negative mutant RhoA

KW - Intracellular calcium

KW - Mucosal injury

UR - http://www.scopus.com/inward/record.url?scp=0037377147&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037377147&partnerID=8YFLogxK

M3 - Article

VL - 284

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6143

IS - 4 53-4

ER -