Abstract
ObjectiveNitric 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.MethodsThe 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.ResultsIt 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.ConclusionOur 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.
Highlights
Inflammatory mediators produced locally or systemically initiate and regulate cell migration and other processes during wound healing
It was found HaCaT cell migration was increased by sodium nitroprusside (SNP) in a dose-dependent manner, and the other two Nitric oxide (NO) donors either spermine NONOate or SNAP had almost the same effects on HaCat cell migrations
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 cyclic guanosine monophosphate (cGMP) or protein kinase G (PKG)
Summary
Inflammatory mediators produced locally or systemically initiate and regulate cell migration and other processes during wound healing. Many types of inflammatory factors are produced after wounds occur, such as IL-1, IL-3, TNF-α and NO. Studies have demonstrated that treatment with an NO donor enhances wound healing, but the underlying mechanism is unclear [1,2]. Residual keratinocytes in or around the wound are believed to be one of the crucial cell types responsible for reepithelialization via cell migration and proliferation during wound healing. The mechanism initiating the migration of keratinocytes has not yet been elucidated. The effect and the mechanism of NO action in keratinocyte migration are not well established [7]
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