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

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.

Figure 1

Co-treatment with HGF and TGF-β1 enhanced HaCaT cell migration. (A) Scratch wound assay. HaCaT cells were seeded in 24-well plates. After 24 h, cells were scratched using a micropipette tip, and growth factors were added as indicated. After incubation for 24 h, the indicated wells were stained with crystal violet. Representative pictures from three independent experiments are shown. (B) The width of the wound was measured by using ocular lens with ruler. Data are presented as mean ± SD of three independent experiments. W: No treatment, WT: TGF-β1 2 ng/ml, WH: HGF 30 units/ml, WTH: HGF 30 units/ml + TGF-β1 2 ng/ml (n = 25, ^* and ^**; P < 0.01 compared to control, #; P < 0.01 between WH and WTH; student's t-test). (C) Cell proliferation assay. HaCaT cells were seeded in 96-well plates before the respective growth factors were added. After 24 h of the treatment with growth factors as indicated, MTT assay was performed. (n = 6, ^*: P < 0.01 compared to 10% FBS group, ^**: P < 0.01 compared to 0.5% FBS group by student's t-test). Data are presented as mean ± SD of two independent experiments. (D) Cell migration assay. HaCaT cells were seeded at a density of 1.0 × 10⁴ cells/well in upper chamber prior to the treatment with cytokines indicated. After 24 h of the treatment with cytokines, cells were fixed with acetone: methanol (1:1), and stained with crystal violet. Representative pictures from two independent experiments are shown.

Figure 2

ROS induction at early and late time points following scratch wound was enhanced by combined growth factor treatment. (A) Enhanced ROS production by co-treatment with HGF and TGF-β1. HaCaT cells were scratched using a comb and cytokines were treated as indicated. After incubating for 30 min or 20 h, cells were incubated for 10 min with DCF-DA, and then harvested for the FACS analysis. Data are presented as mean ± SD of three independent experiments (^*; P < 0.01 compared to that without cytokine treatment by student's t-test). (B) N-acetylcyteine (NAC) could block the re-epithelialization of HaCaT cells induced by co-treatment with HGF and TGF-β1. HaCaT cells were pre-treated with or without indicated concentrations of NAC (2.5 µM, 5 µM, 10 µM) for 30 min before scratched and growth factors were added as indicated. Data are presented as mean ± SD of three independent experiments.

Figure 3

Diphenyliodonium (DPI) inhibited the increase of ROS significantly at early time point, but not at late time point, followed by inhibition of HaCaT cell migration. (A) and (B) diphenyliodonium inhibited significantly the increase of ROS at early time point. (A) HaCaT cells were pretreated with diphenyliodonium (5 µM) for 1 h before scratching, and growth factors for 30 min were added as indicated. Representative images of FACS analysis of three independent experiments are presented. Arrowheads denote FACS profiles of diphenyliodonium-treated samples. (B) Diphenyliodonium as well as growth factors were applied as described in (A). After incubation with DCF-DA, cells were observed under a confocal microscope. Representative images from two independent experiments are presented. (C) Migration of HaCaT cells was inhibited by diphenyliodonium treatment. HaCaT cells were pretreated for 1 h with indicated concentrations of diphenyliodonium before scratching and growth factors were applied as indicated. Data are presented as mean ± SD of two independent experiments (^*; P < 0.05 compared to that without diphenyliodonium by student's t-test).

Figure 4

Knock-down of Nox-1 or Nox-4 confirmed the involvement of NADPH oxidase in ROS generation after wound. (A) Depletion of either Nox-1 or Nox-4 significantly inhibited ROS production. After indicated plasmids were electroporated cells were scratched and growth factors were added as indicated. Thirty min later, cells were harvested and subjected to FACS analysis after DCF-DA staining. Representative data from two independent experiments are shown (right panel), and the extent of knock-down of Nox-1 and Nox-4 was measured by western blotting and RT-PCR, respectively (left lower panel). Representative FACS profiles are overlaid to reveal the differences between samples. Only the differences between samples with both wound and growth factor treatment were shown (left upper panel). Con shRNA: pSuper plasmid itselfplasmid transfected cells. (B) HaCaT cell migration was inhibited by depletion of either Nox-1 or Nox-4. Cells harboring indicated shRNAs were subjected to scratch wound assay. Data are presented as mean ± SD of two independent experiments (^**; P < 0.01 compared to pSuper-transfected cells by student's t-test).

Figure 5

PI3K pathway was involved in cell migration, but not in ROS generation. (A) and (B) HaCaT cell migration was inhibited by PI3K inhibitors. HaCaT cells were pretreated for 1 h with wortmannin (A) or LY294002 (B) at the indicated concentrations before scratching. Growth factors were added as indicated just after the scratch. Data are presented as mean ± SD of two independent experiments. (^**; P < 0.01 compared to that without inhibitors by student's t-test). (C) and (D) Wortmannin (1 µM) and LY294002 (30 µM) could not abolish the increase of ROS. HaCaT cells were prepared as indicated (A). Then, cells were incubated for 10 min with DCF-DA harvested for FACS analysis. Peak fluorescence intensity was measured for each sample, and a representative data from two independent experiments are shown. Vertical dashed line was drawn to help compare the location of two peaks.

Figure 6

HaCaT cell migration was not enhanced by H₂O₂ treatment. HaCaT cells were scratched and different concentrations of H₂O₂ were added as indicated. After incubation for 24 h, the respective wells were stained with crystal violet to measure the wound closure (A) or cells were harvested and stained by tryphan blue to measure the cell death (B). Data are presented as mean ± SD of two independent experiments (^**: P < 0.01 compared to that with cytokine treatment by Student's t-test).