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P Ottino, F Taheri, HE P Bazan; Stimulation of Platelet-Type 12-Lipoxygenase (12-LOX) Expression by Growth Factors in Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1641.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The growth factors EGF, HGF, and KGF are key elements in the regulation of corneal epithelial wound healing following an injury. Recent studies in our laboratory have shown that the mitogenic response of corneal epithelial cells to growth factor treatment is through stimulation of 12(S)-HETE synthesis, the product of 12-LOX. To characterize whether this effect is due to an increase in the expression of 12-LOX or only to stimulation of the enzyme activity, we investigated the effect of EGF, HGF, and KGF treatment on 12-LOX gene and protein expression in corneal epithelial cells. Methods: Primary cultures of rabbit corneal epithelial cells were stimulated with EGF, HGF or KGF (10 ng/ml) for 12, 24, 36, and 48 hours. mRNA was extracted and analyzed by real-time PCR using primers specific for the platelet-type 12-LOX gene. For protein expression, microsomal preparations from 24- and 48-hour growth factor stimulated cultures were subjected to Western blot using a platelet-type 12-LOX antibody. Proliferation assays were carried out in the presence of two selective 12-LOX inhibitors, biacalein (BC)and cinnamyl 3,4-dihydroxy-(alpha)-cyanocinnamate (CDC) (0.6 µM) followed by growth factor supplementation. To demonstrate the ability of 12(S)-HETE to reverse the effects of BC and CDC, 12-LOX inhibitors and 12(S)-HETE were added 45 minutes prior to adding the growth factor. The mitogenic response was determined with a CyQUANT cell proliferation assay kit. Results: Treatment with the growth factors for 12 hours induced 12-LOX mRNA expression in rabbit corneal epithelial cells, with EGF producing higher induction than did HGF and KGF. This increase in mRNA was followed by the expression of 12-LOX protein at 24 and 48 hours. Pretreatment of corneal epithelial cells with the 12-LOX inhibitors decreased the mitogenic response of the cells to growth factor treatment at 24, 48, and 72 hours. Furthermore, we found that the inhibitory effect of BC and CDC on EGF-stimulated proliferation of epithelial cells was reversed by exogenous 12(S)-HETE. Conclusion: These findings indicate that growth factors EGF, HGF, and KGF enhance the transcription of the 12-LOX gene in rabbit corneal epithelial cells, resulting in increased expression of platelet type 12-LOX. In addition, we provide further evidence that EGF, HGF and KGF regulate corneal epithelial cell proliferation through 12(S)-HETE, suggesting that this arachidonic acid metabolite, which increases after corneal injury, has an important role in epithelial proliferation and repair. [Supported by NIH NEI EY04298]
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