Exp Mol Med.  2010 Apr;42(4):270-279. 10.3858/emm.2010.42.4.026.

Co-treatment with hepatocyte growth factor and TGF-beta1 enhances migration of HaCaT cells through NADPH oxidase-dependent ROS generation

Affiliations
  • 1Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 443-721, Korea. jhlee64@ajou.ac.kr
  • 2Department of Molecular Science and Technology, Graduate School of Ajou University, Suwon 443-721, Korea.

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 hapatocyte 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 H2O2. 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.

Keyword

cell movement; hepatocyte growth factor; keratinocytes; NADPH oxidase; reactive oxygen species; transforming growth factor beta1
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