Abstract
We have shown previously that wounding of human corneal epithelial (HCE) cells resulted in epidermal growth factor receptor (EGFR) transactivation through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF). However, the initial signal to trigger these signaling events in response to cell injury remains elusive. In the present study, we investigated the role of ATP released from the injured cells in EGFR transactivation in HCE cells as well as in BEAS 2B cells, a bronchial epithelial cell line. Wounding of epithelial monolayer resulted in the release of ATP into the culture medium. The wound-induced rapid activation of phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) pathways in HCE cells was attenuated by eliminating extracellular ATP, ADP and adenosine. The nonhydrolyzable ATP analog ATP-gamma-S induced rapid and sustained EGFR activation that depended on HB-EGF shedding and ADAM (a disintegrin and metalloproteinase). Targeting pathways leading to HB-EGF shedding and EGFR activation attenuated ATP-gamma-S-enhanced closure of small scratch wounds. The purinoceptor antagonist reactive blue 2 decreased wound closure and attenuated ATP-gamma-S induced HB-EGF shedding. Taken together, our data suggest that ATP, released upon epithelial injury, acts as an early signal to trigger cell responses including an increase in HB-EGF shedding, subsequent EGFR transactivation and its downstream signaling, resulting in wound healing.
Highlights
Epithelial cells form a protective barrier for all tissues and are subjected to constant physical, chemical and biological insults, often resulting in a wound and loss of functions
We and others have shown that epithelial wounding induces epidermal growth factor (EGF) receptor (EGFR) transactivation via ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF) in human corneal epithelial (HCE) cells, and this woundinduced activation of EGFR and its co-receptor erbB2 is required for epithelial migration and wound closure (Block et al, 2004; Mazie et al, 2006; Xu et al, 2004a; Xu et al, 2004b)
Wounding increases ATP concentration in the culture medium of epithelial cells To assess the release of ATP, THCE cells, an SV-40 immortalized HCE cell line, at confluence were extensively injured and ATP released into the medium was assessed by luciferase-luciferin ATP bioluminescent assay
Summary
Epithelial cells form a protective barrier for all tissues and are subjected to constant physical, chemical and biological insults, often resulting in a wound and loss of functions. Rapid healing of a wound is essential for an injured tissue to restore its homeostasis and function. The wound repair process involves cell adhesion, migration, proliferation, matrix deposition and tissue remodeling (Martin, 1997). Many of these biological processes are mediated by growth factors, cytokines and other mediators released in the injured tissues or cells (Werner and Grose, 2003). We and others have shown that epithelial wounding induces EGF receptor (EGFR) transactivation via ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF) in human corneal epithelial (HCE) cells, and this woundinduced activation of EGFR and its co-receptor erbB2 is required for epithelial migration and wound closure (Block et al, 2004; Mazie et al, 2006; Xu et al, 2004a; Xu et al, 2004b)
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