J Korean Med Sci.  2014 Nov;29(Suppl 3):S210-S216. 10.3346/jkms.2014.29.S3.S210.

Synergistic Effect of Interleukin-6 and Hyaluronic Acid on Cell Migration and ERK Activation in Human Keratinocytes

• Choi JH
• Jun JH
• Kim JH
• Sung HJ
• Lee JH

• KMID: 2151415
• DOI: http://doi.org/10.3346/jkms.2014.29.S3.S210

Abstract

Wound healing is initiated and progressed by complex integrated process of cellular, physiologic, and biochemical events, such as inflammation, cell migration and proliferation. Interleukin 6 (IL-6) is a multifunctional cytokine, and it could regulate the inflammatory response of wound healing process in a timely manner. Hyaluronic acid (HA) is an essential component of the extracellular matrix, and contributes significantly to cell proliferation and migration. The purpose of this study was to investigate the effects of IL-6 or/and HA on the cell migration process in human keratinocytes. Combining IL-6 and HA significantly increased the cell migration in scratch based wound healing assay. The phosphorylation of extracellular-signal-regulated kinase (ERK) was significantly increased after 1 hr of IL-6 and HA treatment, but the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was not. We also found that significant increase of the NF-kappaB translocation from cytoplasm into nucleus after 1 hr of IL-6 or/and HA treatments. This study firstly showed that synergistic effects of combining IL-6 and HA on the cell migration of wound healing by activation of ERK and NF-kappaB signaling. Further studies might be required to confirm the synergistic effects of HA and IL-6 in the animal model for the development of a novel therapeutic mixture for stimulation of wound healing process.

Keyword

Interleukin-6; Hyaluronic Acid; Cell Movement; ERK; Phosphorylation; Wound Healing; Keratinocytes

MeSH Terms

Active Transport, Cell Nucleus/drug effects
Cell Line
Cell Movement/*drug effects
Cell Proliferation/drug effects
Cell Survival/drug effects
Enzyme Activation/drug effects
Extracellular Signal-Regulated MAP Kinases/*metabolism
Humans
Hyaluronic Acid/*pharmacology
Interleukin-6/*pharmacology
Keratinocytes/*metabolism
MAP Kinase Signaling System/drug effects
NF-kappa B/metabolism
Phosphorylation/drug effects
Protein Transport/drug effects
Wound Healing
p38 Mitogen-Activated Protein Kinases/metabolism
Extracellular Signal-Regulated MAP Kinases
Interleukin-6
NF-kappa B
Hyaluronic Acid
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 The viability analysis of IL-6 and HA in HaCaT cells. There was no significantly difference in all treated groups. HaCaT cells were incubated with indicated concentrations of IL-6 (A; 0.1-1.0 ng/mL) and HA (B; 0.05-0.2 mg/mL) in 1% FBS containing DMEM for 24 hr. Absorbances were measured at 530 nm using ELISA reader. Data are presented as the means±SEM of three experiments.

• 

  Fig. 2 Effects of IL-6 and HA on the migration of HaCaT cells. In scratch based wound healing assay, IL-6 treated group and HA treated group significantly increased cell migration compared to control group at 24 and 48 hr, respectively. Additional, combining IL-6 and HA treated group significantly increased cell migration compared to control and IL-6 treated group at 24 and 48 hr, respectively. Cultured HaCaT cells on culture dished were scratched with a pipette tip. Data are presented as the means±SEM of three experiments. P-values compared to the control group (*P < 0.001; †P = 0.008) and the IL-6 group (‡P = 0.042; §P = 0.004).

• 

  Fig. 3 Phosphorylation of ERK by treatments of IL-6 and HA in HaCaT cells. (A) HaCaT cells were treated with IL-6 and HA for 1 hr. Phosphorylation of ERK was significantly increased by combining IL-6 and HA treated group. (B) HaCaT cell lines were treated with IL-6 and HA for 24 hr. Phosphorylated ERK was no significant difference for 24 hr all treated groups. Phosphorylation and total protein expression of ERK were detected by Western blot analysis. Densitometry analysis of ERK phosphorylation is showed as fold change versus control at 1 hr (C) and 24 hr (D). Data are presented as the means±SEM of three experiments. *P = 0.009 compared to the control group.

• 

  Fig. 4 Phosphorylation of p38 by treatments of IL-6 and HA in HaCaT cells. HaCaT cells were treated with IL-6 and HA for 1 hr (A) and 24 hr (B). There was no significant difference of phosphorylation of p38 in all treated groups. Phosphorylated and total p38 protein was detected by Western blot analysis. Densitometry analysis of p38 phosphorylation is showed as fold change versus control at 1 hr (C) and 24 hr (D). Data are presented as the means±SEM of three experiments.

• 

  Fig. 5 Immunocytochemistry of NF-κB in HaCaT cells after treatments of IL-6 and HA. (A) HaCaT cells were labeled to visualize NF-κB (Alexa 594; red) and nucleus (Hoechst; blue) by Immunocytochemistry. (B) Fluorescent microscopic images were captured and analyzed with i-solution software. Fluorescent intensities of NF-κB were calculated on a ratio of NF-κB intensity of nucleus to whole cell boundary. Fluorescent intensities are expressed as fold of control group. Data were expressed on the mean±SEM of to the individual cells. P values compared to the control group (*P < 0.001; †P = 0.002).

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