In this study, we demonstrate that EGF induces the synthesis of LXA4 and that this lipid mediator plays an important role in EGF-stimulated corneal epithelial wound healing (). The action of EGF on 12/15-LOX activation was demonstrated by, first, complete inhibition of wound closure by EGF in the 12/15-LOX knockout mice, whereas addition of LXA4 produced similar stimulation than in wild-type mice, and, second, inhibition of cell proliferation, migration, and wound healing when EGF stimulation was blocked with a selective 12/15-LOX inhibitor.
Figure 8. Diagram of EGF-stimulated wound healing through LXA4 synthesis. Injury increases the synthesis of EGF, thus stimulating the 12-LOX in rabbit corneas. Inflammatory cells that arrive to the cornea after injury, such as PMNs, release LTA4, which is converted (more ...)
Growth factors are key regulators of many of the processes essential for maintenance of the normal ocular surface and wound healing.31,35–37
EGF increases rapidly in tears after corneal injury19
and facilitates corneal epithelial wound repair by promoting migration and mitosis of epithelial cells. This growth factor increases cell replication as measured by an increase in DNA content in the regenerating epithelium,38
and inhibition of EGF receptor activation significantly decreases epithelial migration.35
A common theme in the arrangement of the pathways stimulated by growth factors is the integration and crosstalk between contiguous signaling cascades, which allows for fine tuning of biological outcomes such as cell proliferation, differentiation, and migration. In addition to stimulating MAPK signaling pathways, activation of the EGF receptor in corneal epithelium stimulates the phosphoinositide 3-kinase, adenylate cyclase, phospholipase C induced-Ca2+
signaling, and phospholipase D-mediated phosphatidic acid formation.39–43
EGFR activation also can occur through transactivation by other receptors and mediators.44–47
Our results indicate that during the inflammatory process, EGF-induced LXA4 (through ERK1/2 and p38 activation) is an additional mechanism by which EGF stimulates corneal wound healing.
We have previously shown that activation of ERK1/2 and p38 by growth factors is involved in proliferation and migration of corneal epithelial cells.24
When 12/15-LOX was chemically blocked, we observed downregulation in ERK1/2 and p38 activation, as well as a decrease in cell proliferation and migration. This clearly establishes that 12/15-LOX plays an important role in MAPK activation and epithelial wound healing.
We show here that the kinetics of MAPK activation is sustained when MAPK is stimulated with LXA4 and EGF. Cells can use transient or sustained activation of MAPK to produce different responses.48
In fibroblasts, sustained activation of ERK1/2 is associated with growth factor-induced proliferation.49
LXA4 seems to be more strongly involved in migration than proliferation since, in the organ culture system, blocking the p38 pathway completely abolishes LXA4-stimulated wound closure, while only partial inhibition occurs when the ERK1/2 pathway is inhibited. The action of LXA4 is probably through activation of a seven-membrane receptor named ALX (). This receptor is expressed in rabbit and mouse corneas.27,50
We also demonstrate that EGF stimulates LXA4 synthesis. LXA4 is synthesized by the action of two lipoxygenases during cell-cell interactions: the 15- and 5-LOX or the 5- and 12-LOX. Our present results suggest that in RCE cells the enzyme involved in LXA4 synthesis is the 12-LOX platelet type, which is the major lipoxygenase expressed in rabbit corneas.12,26
In our assay we added LTA4 (a product of 5-LOX) as an external substrate; however, in an “in vivo” situation, inflammatory cells that arrive to the cornea after injury (such as neutrophils that contain an active 5-LOX) may generate LTA4, which is released from inflammatory cells and converted to LXA4 ().
In the mouse, however, expression of a leukocyte type 12/15-LOX occurs that can form both 12- and 15-HETE.51
A possible secondary source of LXA4 synthesis, in addition to the corneal epithelium, could be platelets. It has been reported that platelets localize in the limbal vessels of mouse corneas after epithelial abrasion and are important regulators of wound healing.52
The arrival of PMNs after corneal injury could then produce transcellular biosynthesis of LXA4.
When 12/15-LOX was blocked with its inhibitor in our experiments, there was complete inhibition of EGF-stimulated LXA4 synthesis. Furthermore, when we blocked EGF-induced activation using the EGF receptor inhibitor, inhibition of LXA4 synthesis also occurred.
Lipid-derived mediators are very effective as signaling molecules in inflammation because they are small, rapidly generated molecules that can act and then be locally inactivated. In murine systems of acute inflammation, a return to homeostasis occurs after synthesis of lipid mediators that have anti-inflammatory or pro-resolving functions. One class of AA-derived mediators, the lipoxins, were the first mediators recognized as having both endogenous anti-inflammatory and pro-resolving actions.53
Previous studies from our laboratory show that lipid mediators such as platelet activating factor (PAF), cyclooxygenase-derived prostaglandins, and lipoxygenase derivatives are synthesized after injury and that many of them have important roles in inflammation and wound healing.5,7,12,54
While some lipid mediators (e.g., PAF) inhibit wound healing,55
the products of the 12/15-LOX promote epithelial repair instead.4,14
To our knowledge, there are no reports to date demonstrating that EGF induces LXA4 synthesis or that this lipid stimulates corneal wound healing through ERK1/2 and p38 stimulation. Our findings provide a better understanding of the wound-healing process and raise the possibility of the therapeutic use for LOX derivatives in wounds that are difficult to repair.