Abstract
Wound healing requires re-epithelialization from the wound margin through keratinocyte proliferation and migration, and some growth factors are known to influence this process. In the present study, we found that the co-treatment with hepatocyte growth factor (HGF) and TGF-beta1 resulted in enhanced migration of HaCaT cells compared with either growth factor alone, and that N-acetylcysteine, an antioxidant agent, was the most effective among several inhibitors tested, suggesting the involvement of reactive oxygen species (ROS). Fluorescence-activated cell sorter analysis using 2,7-dichlorofluorescein diacetate (DCF-DA) dye showed an early (30 min) as well as a late (24 h) increase of ROS after scratch, and the increase was more prominent with the growth factor treatment. Diphenyliodonium (DPI), a potent inhibitor of NADPH oxidase, abolished the increase of ROS at 30 min, followed by the inhibition of migration, but not the late time event. More precisely, gene knockdown by shRNA for either Nox-1 or Nox-4 isozyme of gp91phox subunit of NADPH oxidase abolished both the early time ROS production and migration. However, HaCaT cell migration was not enhanced by treatment with H((2))O((2)). Collectively, co-treatment with HGF and TGF-beta1 enhances keratinocyte migration, accompanied with ROS generation through NADPH oxidase, involving Nox-1 and Nox-4 isozymes.
Highlights
Wound healing after injury is a complex process encompassing many cellular and biochemical events to restore tissue integrity
We found that the co-treatment with Hepatocyte growth factor (HGF) and TGF-β1 resulted in enhanced migration of human keratinocyte HaCaT cells, compared with either growth factor alone
To gain insight into the function of HGF and/or TGF-β1 in keratinocyte migration, we performed scratch wound assay using HaCaT human keratinocytes with a micropipette tip in the presence or absence of growth factors
Summary
Wound healing after injury is a complex process encompassing many cellular and biochemical events to restore tissue integrity. It is well known that released growth factors and cytokines control tissue repair process: Exogenous addition of growth factors such as PDGF, fibroblast growth factor (FGF), epidermal growth factor (EGF), TGF-α and TGF-β1 to animal wounds usually accelerates tissue repair rate by enhancing granulation tissue formation or keratinocyte migration (Robinson, 1993; Steed, 1998; Carter, 2003). Among many molecules known to influence wound healing, TGF-β1 has the broadest spectrum of actions, affecting all cell types that are involved in all stages of wound healing. Both positive and negative effects of TGF-β1 on wound healing have been reported (Wang et al, 2006). HGF and TGF-β1 usually acts antagonistically in many systems, the reports on the positive effect of TGF-β1 in skin wound healing led us to assume that HGF and TGF-β1 might not act antagonistically on keratinocyte migration, and to compare the effect of co-treatment with HGF and TGF-β1 with either growth factor alone in keratinocyte migration
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