Injury and nucleotides induce phosphorylation of epidermal growth factor receptor: MMP and HB-EGF dependent pathway

Abstract

The early events that occur rapidly after injury trigger signal cascades that are essential for proper wound closure of corneal epithelial cells. We hypothesize that injury releases ATP, which stimulates purinergic receptors and elicits the phosphorylation of epidermal growth factor receptor (EGFR) tyrosine residues and subsequent cell migration by a MMP and HB-EGF dependent pathway. We demonstrated that the inhibition of purinergic receptors with the antagonist, Reactive Blue 2, abrogated the phosphorylation of EGFR and ERK. Preincubation of cells with the EGFR kinase inhibitor, AG1478, and subsequent stimulation by injury or ATP resulted in a decrease in phosphorylation of EGFR and migration. Furthermore, downregulation of EGFR by siRNA, inhibited the EGF induced intracellular Ca 2+ wave. However, the response to injury and ATP was retained indicating the presence of 2 signaling pathways. Inhibition with either CRM197 or TIMP-3 decreased injury and nucleotide induced phosphorylation of both EGFR and ERK. Incubation in the presence of a functional blocking antibody to HB-EGF also resulted in a decrease in the phosphorylation of EGFR. In addition, cell migration was inhibited by CRM197 and rescued when cells were incubated with HB-EGF. We showed that injury induced phosphorylation of specific tyrosine residues and found that a similar pattern of phosphorylation was induced by trinucleotides. These studies indicate that injury induced purinergic receptor activation leads to phosphorylation of EGFR, ERK and migration.

Dual modes of motility at the leading edge of migrating epithelial cell sheets

“…Purse strings can nonetheless form at the leading edges of moving epithelial cell sheets because EGFR activation occurs only at straight and convex edges. Wounding sheets of corneal epithelial cells causes release of heparin-binding EGF-like growth factor and amphiregulin (19,20,22,32). Both of these ligands bind strongly to negatively charged glycans in the extracellular matrix and the cell surface (56)(57)(58).…”

“…The EGFR is activated upon wounding epithelial cell sheets (17)(18)(19)(20)(21)(22)(23). The finding that EGFR signaling disrupts purse strings suggested that activation occurs only at straight and convex edges.…”

“…Also, loss of cells results in disruption of cell-cell interactions, changes the mechanical forces in the cell layer, and allows receptors present at lateral edges of cells to interact with ligands present in apical regions. These stimuli contribute variously to EGF receptor (EGFR) activation, which is required for healing of large wounds in many types of epithelia (17)(18)(19)(20)(21)(22). Of this great number of stimuli, my colleagues and I determined previously that the presence of physically unconstrained edges in the cell sheet is sufficient to induce activation of the EGFR and increase cell motility and, thus, constitutes a minimal trigger that can induce the epithelium to move (23).…”

“…Signaling elicited by wounding has usually been studied by creating linear wounds by scraping cell layers with a needle or a pipette tip (17)(18)(19)(20)(21)(22). Although simple in execution, these procedures actually provide a complex set of signals: cells are broken and release intracellular components, some cells become transiently permeable to extracellular molecules, and extracellular matrix covered previously by cells becomes abruptly accessible.…”

Extracellular adenosine triphosphate and adenosine in cancer

“…During skin wound healing, the release of extracellular ATP has been shown to be critical for efficient epithelial repair, revealing the role of purinergic signaling in general cell functions. Indeed, activation of ATP receptors stimulates the proliferation of epidermal keratinocytes (Greig et al, 2003) and smooth muscle cells (Wilden et al, 1998), and initiates migration of epithelial cells (Boucher et al, 2007). Likewise, extracellular ATP released from hepatocytes and Kupffer cells immediately after hepatectomy has been shown to promote liver regeneration (Gonzales et al, 2010).…”