Claus Schneider
Last active: 4/5/2016

Detection and subcellular localization of two 15S-lipoxygenases in human cornea.

Chang MS, Schneider C, Roberts RL, Shappell SB, Haselton FR, Boeglin WE, Brash AR
Invest Ophthalmol Vis Sci. 2005 46 (3): 849-56

PMID: 15728540 · DOI:10.1167/iovs.04-1166

PURPOSE - There are two human 15-lipoxygenases (LOX), 15-LOX-1 and -2, which convert arachidonic acid to 15S-hydroxyeicosatetraenoic acid (15S-HETE). The presence of both 15-LOXs in the human cornea prompted this study to delineate their roles in the human corneal epithelium.

METHODS - Human corneal epithelia from donor corneas and a human corneal epithelial (HCE) cell line were used in [1-(14)C]arachidonic acid incubations, Western blot analysis, and quantitative real-time RT-PCR. Cell cultures of HCE were treated with 15S-HETE to measure its effect on cell growth. HCE cells were transfected with plasmids to express green fluorescent (GFP) fusion proteins of 15-LOX-1 and -2, and in vivo laser confocal microscopy was performed to determine the subcellular localization of the 15-LOX fusion proteins.

RESULTS - [1-(14)C]Arachidonic acid incubations yielded 15S-HETE as the only LOX product. Treatment with 15S-HETE (5-10 microM) reduced growth rate and induced apoptosis in cultured HCE cells in a dose-dependent manner. 15-LOX-2 but not 15-LOX-1 was detected by Western blot analysis, although we were able to detect similar levels of both 15-LOX mRNAs by real-time quantitative RT-PCR. 15-LOX-1 and -2 proteins showed different subcellular expression patterns. 15-LOX-2 GFP was expressed in the cytoplasm and nucleus (actively taken up into the nucleus). 15-LOX-1 GFP fusion protein expression was restricted to the cytoplasm.

CONCLUSIONS - These findings indicate that 15-LOX-2 is the predominant 15-LOX protein in human cornea, and its product, 15S-HETE, plays a role in cellular proliferation. Because the two 15-LOXs have different subcellular compartmentalization, the authors hypothesize that their products are also compartmentalized and therefore exert different molecular effects in the human corneal epithelium.

MeSH Terms (14)

Arachidonate 15-Lipoxygenase Arachidonic Acids Blotting, Western Cell Line Cell Proliferation Cornea Dose-Response Relationship, Drug Epithelium, Corneal Humans Hydroxyeicosatetraenoic Acids Microscopy, Confocal Reverse Transcriptase Polymerase Chain Reaction RNA, Messenger Subcellular Fractions

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