Chang Min S, Schneider Claus, Roberts Richard L, Shappell Scott B, Haselton Fredrick R, Boeglin William E, Brash Alan R
Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, 8000 MCE, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Invest Ophthalmol Vis Sci. 2005 Mar;46(3):849-56. doi: 10.1167/iovs.04-1166.
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.
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.
[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.
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.
人类有两种15 - 脂氧合酶（LOX），即15 - LOX - 1和15 - LOX - 2，它们可将花生四烯酸转化为15S - 羟基二十碳四烯酸（15S - HETE）。人角膜中同时存在这两种15 - LOX，促使本研究来阐明它们在人角膜上皮中的作用。
使用来自供体角膜的人角膜上皮和一种人角膜上皮（HCE）细胞系进行[1 - （14）C]花生四烯酸孵育、蛋白质印迹分析和实时定量逆转录 - 聚合酶链反应（RT - PCR）。用15S - HETE处理HCE细胞培养物以测量其对细胞生长的影响。用质粒转染HCE细胞以表达15 - LOX - 1和15 - LOX - 2的绿色荧光（GFP）融合蛋白，并进行体内激光共聚焦显微镜检查以确定15 - LOX融合蛋白的亚细胞定位。
[1 - （14）C]花生四烯酸孵育产生的唯一LOX产物是15S - HETE。用15S - HETE（5 - 10 microM）处理以剂量依赖方式降低了培养的HCE细胞的生长速率并诱导其凋亡。蛋白质印迹分析检测到15 - LOX - 2而非15 - LOX - 1，尽管我们通过实时定量RT - PCR能够检测到两种15 - LOX mRNA的相似水平。15 - LOX - 1和15 - LOX - 2蛋白表现出不同的亚细胞表达模式。15 - LOX - 2 GFP在细胞质和细胞核中表达（可主动进入细胞核）。15 - LOX - 1 GFP融合蛋白的表达局限于细胞质。
这些发现表明15 - LOX - 2是人类角膜中主要的15 - LOX蛋白，其产物15S - HETE在细胞增殖中起作用。由于两种15 - LOX具有不同的亚细胞分隔，作者推测它们的产物也存在分隔，因此在人角膜上皮中发挥不同的分子效应。
