Distinct Sub-cellular Localization of 15-lipoxygenase-1 and -2 in Human Corneal Epithelial Cells

Abstract

Abstract: : Purpose: 15-Lipoxygenase (15-LOX) has been implicated in cellular proliferation and differentiation. There are two 15-LOX enzymes in humans, 15-LOX-1 and 15-LOX-2, and both are expressed in corneal epithelial cells. The two lipoxygenases share only 35-40% amino acid identity, yet both produce 15(S)-hydroxy-eicosatetraenoic acid (15(S)-HETE) from arachidonic acid. In this study we investigated how the expression of these two enzymes is coordinated within the cell to mediate their physiologic actions. Methods: Full-length cDNA for human 15-LOX-1 and -2 was subcloned into the pEGFP vector (Clontech) to express GFP-15-LOX-1 and -2 fusion proteins. The plasmids were transfected separately into SV40- immortalized human corneal epithelial cells using Lipofectamine 2000 (Invitrogen). After 18 to 24 hours, the transfected cells were viewed by confocal microscopy. Time-lapsed phase contrast microscopy was also performed over an 8 to 12 hour period. Results: The subcellular expression of GFP-15-LOX-1 is diffuse and occurs only in the cytoplasm, while GFP-15-LOX-2 is expressed both in the cytoplasm and in the nucleus. By fluorescence recovery after photo-bleaching (FRAP), the GFP-15-LOX-2 appears to be actively accumulated in the nucleus. By time-lapsed phase contrast microscopy, the cells expressing the GFP-LOX proteins appear to undergo blebbing followed by cell death. The specificity of this response to overexpression of lipoxygenase is unclear. Conclusions: These results demonstrate that 15-LOX-1 and 15-LOX-2 have different subcellular localization indicating compartmentalization of 15S-HETE produced by the two 15-lipoxygenases. This suggests distinct physiologic roles for the two enzymes in human corneal epithelium despite their similar enzymatic activity.

Keywords: cornea: epithelium • eicosanoids • cornea: basic science