An in vitro analysis of mechanical wounding-induced ligand-independent KGFR activation.

Abstract

BACKGROUND:

KGFR (keratinocyte growth factor receptor), exclusively expressed in epithelial cells, plays an important role in wound healing. However, mechanisms of KGFR activation and signaling in wound healing are not clearly understood.

OBJECTIVES:

We utilized an in vitro mechanical wounding model to examine ligand-independent KGFR activation, its regulation by reactive oxygen species (ROS) and the functional significance of this activation mechanism.

METHODS:

Confluent HaCaT cell line cultures were mechanically wounded and KGFR internalization and phosphorylation were examined using immunostaining with confocal microscopy and immunoprecipitation with Western blotting. Wounding-induced generation of reactive oxygen species and ligand-independent activation of KGFR were examined. In addition, phosphorylation of its associated molecules FRS2 and c-Src were examined in the presence and absence of the ROS and pathway specific inhibitors. The importance of this activation process on cell migration was also examined in the presence and absence of these inhibitors.

RESULTS:

Mechanical wounding induced ligand-independent KGFR activation and internalization. KGFR internalization and phosphorylation was associated with ROS generation along the wound edge and scavenging of ROS with NAC inhibited KGFR phosphorylation. Intracellularly, c-Src was phosphorylated by wounding but its inhibitor, PP1, significantly inhibited KGFR activation and associated FRS2 phosphorylation. Mechanical wounding induced wound edge migration, which was significantly reduced by the selective receptor and pathway inhibitors PP1 (82.7%), KGFR inhibitor SU5402 (70%) and MAPK inhibitor PD98059 (57%).

CONCLUSION:

Mechanical wounding induces significant ROS generation at the wound edge which, in turn, induced ligand-independent KGFR and FRS2 activation via c-Src kinase signaling. Functionally, downstream MAPK signaling induced wound edge cell migration.

• Authors: Min Li, James D Firth, Edward E Putnins
• Journal: Journal of dermatological science (J Dermatol Sci) Vol. 53 Issue 3 Pg. 182-91 (Mar 2009) ISSN: 0923-1811 [Print] Netherlands
• PMID: 19111446 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Adaptor Proteins, Signal Transducing
• Enzyme Inhibitors
• FRS2 protein, human
• Flavonoids
• Ligands
• Membrane Proteins
• Pyrroles
• Reactive Oxygen Species
• SU 5402
• Hydrogen Peroxide
• Protein-Tyrosine Kinases
• Receptor, Fibroblast Growth Factor, Type 2
• keratinocyte growth factor receptor
• CSK Tyrosine-Protein Kinase
• src-Family Kinases
• CSK protein, human
• Mitogen-Activated Protein Kinase 3
• 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one

Topics

• Adaptor Proteins, Signal Transducing (metabolism)
• CSK Tyrosine-Protein Kinase
• Cell Line
• Cell Movement (physiology)
• Cells, Cultured
• Enzyme Inhibitors (pharmacology)
• Flavonoids (pharmacology)
• Humans
• Hydrogen Peroxide (metabolism)
• Keratinocytes (cytology, metabolism)
• Ligands
• Membrane Proteins (metabolism)
• Mitogen-Activated Protein Kinase 3 (metabolism)
• Phosphorylation (physiology)
• Protein-Tyrosine Kinases (metabolism)
• Pyrroles (pharmacology)
• Reactive Oxygen Species (metabolism)
• Receptor, Fibroblast Growth Factor, Type 2 (metabolism)
• Signal Transduction (drug effects, physiology)
• Wound Healing (physiology)
• src-Family Kinases