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S. Kenchesowda, Sr., F. R. Jackson, J. A. Johnson, C. Zhang, N. G. Bazan, H. E. P. Bazan; EGF-Mediated Lipoxin Synthesis Modulates Epithelial Wound Repair. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2955.
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Epidermal growth factor (EGF) is a strong inducer of the 12-lipoxygenase (12-LOX) expression in rabbit corneal epithelial (RCE) cells (Exp Eye Res 76:613,2003). This enzyme catalyzes the conversion of arachidonic acid to 12-(S) HETE, a mediator that increases after corneal injury (JBC 266:6726:1991).12-LOX is also involved in the synthesis of lipoxins (LxA4) using as substrate leukotriene A4 (LTA4). We tested the hypothesis that EGF stimulates the synthesis of LxA4 in RCE cells, and that in turn this mediator induces mitogen-activated protein kinase (MAPK) signaling, modulating cell migration and proliferation.
RCE cells were stimulated with EGF (10 ng/ml) with and without the 12-LOX inhibitor CDC (10mM) or with 12(S) HETE, LxA4 or aspirin triggered LxA4 (epi-LxA4) (100 nM) for different times. Phosporylation of Erk1/2 and p38 were analyzed by western blot. To assess wound closure, 7 mm epithelial debridement wounds were made in corneas in organ culture. Migration and proliferation of RCE cells were measured. Total homogenate of RCE cells stimulated with EGF were incubated for 24 h with LTA4 and the synthesis of 12(S) HETE and LxA4 was analyzed by LC-tandem mass spectrometry.
RCE cells stimulated with EGF activated the synthesis of 12(S) (HETE) 15-fold and the synthesis of LxA4 8-fold. EGF, LxA4 and epi-LxA4 activated ERK1/2 up to 30 min and CDC inhibited the EGF-stimulated phosphorylation. On the other hand, 12(S) HETE triggered short term phosphorylation (5- 10 min). Phosphorylation of p-38 was stimulated by EGF up to 4 h and CDC inhibited by 50 %. Activation of p38 up to 120 min was shown with 12(S) HETE, LxA4 and epi-LxA4. EGF promoted a 27% increase in wound closure at 24 h after de-epithelization in organ cultured corneas that was partially inhibited with CDC. 12(S) HETE produced a small but significant 5% increase in wound closure, while LxA4 and epi-LxA4 activated wound closure by 19 %. RCE cell migration was stimulated by EGF, 12(S) HETE and the lipoxins in a similar manner, and CDC inhibited the effect of EGF. The lipoxins were more active in proliferation than 12(S) HETE.
An increase in synthesis of LxA4 in RCE cells stimulated with EGF suggests that during corneal injury, LTA4 from infiltrated PMNs use the growth factor-induced 12-LOX for LxA4 synthesis. The potent action of lipoxins in wound closure, migration and proliferation and the similar kinetics to EGF in MAPK activation demonstrate that they are potent mediators of the action of EGF in epithelial repair.
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