Nitric oxide enhances keratinocyte cell migration by regulating Rho GTPase via cGMP-PKG signalling

PLoS One. 2015 Mar 23;10(3):e0121551. doi: 10.1371/journal.pone.0121551. eCollection 2015.

Abstract

Objective: Nitric oxide (NO) has been shown to improve wound healing, but the mechanism underlying this function is not well defined. Here, we explored the effect of NO on the migration of a human keratinocyte cell line (HaCaT) and its possible mechanism.

Methods: The effects of NO on HaCaT cells in the presence of different concentrations of the NO donor sodium nitroprusside (SNP) were evaluated in a cell migration assay. Subsequently, the cytoskeleton reorganization of cultured HaCaT cells stained with rhodamine-phalloidin was observed with a confocal laser scanning microscope. The mRNA expression and active proteins of CDC42, Rac1 and RhoA in the cultured cells were determined via RT-PCR and pull-down assays, respectively. Furthermore, the roles of various inhibitors or agonists specific to cGMP, PKG and CDC42, Rac1, RhoA in the effects of NO on HaCaT cell migration, F-actin stress fibre formation, and Rho GTPase expression were observed.

Results: It was also found HaCaT cell migration was increased by SNP in a dose-dependent manner, and the other two NO donors either spermine NONOate or SNAP had almost the same effects on HaCat cell migrations. The formation of F-actin stress fibres in SNP-treated HaCaT cells was increased. The mRNA expression and the active proteins of CDC42, Rac1 and RhoA were found to be upregulated after SNP treatment. Similar effects were observed after the cells were treated with a cGMP or PKG agonist. Additionally, the SNP-mediated upregulation of the mRNA expression and the active proteins of CDC42, Rac1 and RhoA were inhibited by the addition of an inhibitor of cGMP or PKG. Moreover, the SNP-mediated promoting effects of migration and cytoskeleton reorganization were inhibited by treatment with inhibitors of cGMP, PKG, CDC42, Rac1 and RhoA respectively.

Conclusion: Our data indicated that the stimulatory effects of NO on cell migration of HaCaT cells are mediated by the cGMP signalling pathway via the upregulation of Rho-GTPase expression, which might promote cytoskeleton reorganization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cell Movement*
  • Cyclic GMP / metabolism
  • Cyclic GMP-Dependent Protein Kinase Type I / metabolism*
  • Humans
  • Keratinocytes / drug effects
  • Keratinocytes / metabolism*
  • Keratinocytes / physiology
  • Nitric Oxide / metabolism*
  • Nitroprusside / pharmacology
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Signal Transduction*
  • rho GTP-Binding Proteins / genetics
  • rho GTP-Binding Proteins / metabolism*

Substances

  • RNA, Messenger
  • Nitroprusside
  • Nitric Oxide
  • Cyclic GMP-Dependent Protein Kinase Type I
  • rho GTP-Binding Proteins
  • Cyclic GMP