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Min S. Chang, Claus Schneider, Richard L. Roberts, Scott B. Shappell, Fredrick R. Haselton, William E. Boeglin, Alan R. Brash; Detection and Subcellular Localization of Two 15S-Lipoxygenases in Human Cornea. Invest. Ophthalmol. Vis. Sci. 2005;46(3):849-856. doi: https://doi.org/10.1167/iovs.04-1166.
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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 [1-14C]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. [1-14C]Arachidonic acid incubations yielded 15S-HETE as the only LOX product. Treatment with 15S-HETE (5–10 μM) 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.
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