Summary
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.
Keywords
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References
- Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth.Science. 1989; 245: 752-755
- Keratinocyte growth factor.Cell Biol Int. 1995; 19: 399-411
- Large induction of keratinocyte growth factor expression in the dermis during wound healing.Proc Natl Acad Sci USA. 1992; 89: 6896-6900
- Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin.J Exp Med. 1995; 182: 1369-1376
- Altered expression of keratinocyte growth factor and its receptor in psoriasis.Am J Pathol. 1997; 151: 1619-1628
- Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse.J Invest Dermatol. 1994; 103: 469-473
- Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis.Biochem Biophys Res Commun. 2005; 327: 523-532
- Reactive oxygen species and human inflammatory periodontal diseases.Biochemistry (Mosc). 2005; 70: 619-628
- Reactive oxygen species and antioxidants in inflammatory diseases.J Clin Periodontol. 1997; 24: 287-296
- Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells.Biochem J. 1988; 250: 95-101
- Ligand-independent trans-activation of the platelet-derived growth factor receptor by reactive oxygen species requires protein kinase C-delta and c-Src.J Biol Chem. 2002; 277: 44695-44700
- H2O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes.J Invest Dermatol. 1998; 110: 966-971
- H2O2 is required for UVB-induced EGF receptor and downstream signaling pathway activation.Free Radic Biol Med. 1999; 27: 1197-1202
- Redox modulation of tyrosine phosphorylation-dependent signal transduction pathways.Free Radic Biol Med. 1996; 21: 323-333
- Oxidative stress induces tyrosine phosphorylation of PDGF alpha-and beta-receptors and pp60c-src in mesangial cells.Kidney Int. 1996; 50: 164-173
- Cellular functions regulated by Src family kinases.Annu Rev Cell Dev Biol. 1997; 13: 513-609
- The interplay between Src family kinases and receptor tyrosine kinases.Oncogene. 2004; 23: 7957-7968
- UVB-induced activation and internalization of keratinocyte growth factor receptor.Oncogene. 2003; 22: 2422-2431
- Endocytic pathways and biological effects induced by UVB-dependent or ligand-dependent activation of the keratinocyte growth factor receptor.FASEB J. 2006; 20: 395-397
- Nickel-induced keratinocyte proliferation and up-modulation of the keratinocyte growth factor receptor expression.Exp Dermatol. 2003; 12: 497-505
- Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers: involvement of mitogen-activated protein kinase cascades.Biochem J. 2001; 354: 99-106
- Keratinocyte growth factor 1 inhibits wound edge epithelial cell apoptosis in vitro.J Invest Dermatol. 2004; 122: 222-231
- The docking protein FRS2alpha controls a MAP kinase-mediated negative feedback mechanism for signaling by FGF receptors.Mol Cell. 2002; 10: 709-719
- Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor.J Exp Med. 1994; 179: 831-840
- The function of KGF in morphogenesis of epithelium and reepithelialization of wounds.Science. 1994; 266: 819-822
- Wound repair: overview and general considerations.The molecular and cellular biology of wound repair. Plenum, New York1996 (pp. 3–50)
- Dynamic characterization of the molecular events during in vitro epidermal wound healing.J Invest Dermatol. 2002; 119: 56-63
- Scraped-wounding causes activation and association of C-Src tyrosine kinase with microtubules in cultured keratinocytes.Cell Struct Funct. 2000; 25: 351-359
- Metalloprotease disintegrin-mediated ectodomain shedding of EGFR ligands promotes intestinal epithelial restitution.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G1213-1219
- Cellular signaling by fibroblast growth factor receptors.Cytokine Growth Factor Rev. 2005; 16: 139-149
- Protection of epithelial cells by keratinocyte growth factor signaling.Proc Am Thorac Soc. 2005; 2: 221-225
- Dermal wound healing is subject to redox control.Mol Ther. 2006; 13: 211-220
- Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.Science. 1995; 270: 296-299
- Src-dependent phosphorylation of the EGF receptor Tyr-845 mediates Stat-p21waf1 pathway in A431 cells.Genes Cells. 2003; 8: 995-1003
- Redox signaling: hydrogen peroxide as intracellular messenger.Exp Mol Med. 1999; 31: 53-59
- Interactions between non-immune host cells and the immune system during periodontal disease: role of the gingival keratinocyte.Crit Rev Oral Biol Med. 1998; 9: 292-305
- Gbetagamma subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation.J Biol Chem. 1997; 272: 4637-4644
- Epidermal growth factor receptor transactivation by angiotensin II requires reactive oxygen species in vascular smooth muscle cells.Arterioscler Thromb Vasc Biol. 2001; 21: 489-495
- Integrin-induced epidermal growth factor (EGF) receptor activation requires c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosines.J Biol Chem. 2002; 277: 9405-9414
- Keratinocyte growth factor (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes.J Invest Dermatol. 1993; 101: 49-53
- Purification and characterization of a newly identified growth factor specific for epithelial cells.Proc Natl Acad Sci USA. 1989; 86: 802-806
- Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family.J Cell Physiol. 1990; 144: 326-332
- Localizing NADPH oxidase-derived ROS.Sci STKE. 2006; (2006:re8)
- Role of hydrogen peroxide and oxidative stress in healing responses.Cell Mol Life Sci. 2002; 59: 1872-1891
- Protein kinase B activation by reactive oxygen species is independent of tyrosine kinase receptor phosphorylation and requires SRC activity.J Biol Chem. 2003; 278: 20828-20834
- Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation.J Immunol. 1983; 130: 1910-1917
- Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors.Science. 1997; 276: 955-960
- The pp60c-Src inhibitor PP1 is non-competitive against ATP.FEBS Lett. 2003; 537: 47-52
- FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern.EMBO J. 1991; 10: 1347-1354
- Zinc, copper and manganese enhanced keratinocyte migration through a functional modulation of keratinocyte integrins.Exp Dermatol. 2000; 9: 407-416
- Vulnerability of central neurons to secondary insults after in vitro mechanical stretch.J. Neurosci. 2004; 24: 8106-8123
- Shear-induced reactive nitrogen species inhibit mitochondrial respiratory complex activities in cultured vascular endothelial cells.Am J Physiol Cell Physiol. 2007; 292: C1103-C1112
- Src autophosphorylation is an early event in pressure-mediated signaling pathways in isolated resistance arteries.Hypertension. 2002; 39: 502-507
- Stretch enhances contraction of bovine coronary arteries via an NAD(P)H oxidase-mediated activation of the extracellular signal-regulated kinase mitogen-activated protein kinase cascade.Circ Res. 2003; 92: 23-31
Article info
Publication history
Published online: July 12, 2010
Accepted:
October 14,
2008
Received in revised form:
October 1,
2008
Received:
March 2,
2008
Footnotes
☆Supported by The Canadian Institutes of Health Research.
Identification
Copyright
© 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Inc. All rights reserved.
