Cell Cycle Arrest by Kynurenine in Lens Epithelial Cells
Cell Cycle Arrest by Kynurenine in Lens Epithelial Cells
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DOI：
10.1167/iovs.08-2374
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发表时间：
2008-12-01
影响因子：
4.4
通讯作者：
Nagaraj, Ram H.
中科院分区：
文献类型：
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作者：
Mailankot, Maneesh;Smith, Dawn;Nagaraj, Ram H.
目的.吲哚胺2，3-双加氧酶（IDO）介导的色氨酸氧化产生犬尿氨酸（KYN），其可能在白内障形成中起作用。KYN引起细胞变化的分子机制知之甚少。研究KYN对过表达人IDO的小鼠透镜上皮细胞的影响。使用来源于人IDO过表达半合子转基因（hemTg）和野生型（Wt）小鼠的透镜上皮细胞（mLEC）。通过HPLC定量犬尿氨酸（KYN）来测量IDO活性。KYN介导的蛋白质修饰通过免疫细胞化学检测并通过ELISA测量。用市售试剂盒测量细胞增殖和凋亡。用流式细胞术分析细胞周期时相之间的细胞分布。免疫沉淀法，然后LC/MS用于鉴定犬尿氨酸修饰的蛋白质。来自hemTg动物的mLECs表现出相当大的IDO免疫反应性和酶活性，这在Wt mLECs中几乎检测不到。KYN和KYN介导的蛋白质修饰在hemTg中检测到，但在Wt mLEC中未检测到;修饰的蛋白质是肌球蛋白II和α/γ-肌动蛋白。HemTg mLEC表现出降低的活力和增殖。hemTg mLEC培养物的细胞周期分析显示，相对于Wt mLEC，处于G（2）/M或两个阶段的细胞增加约两倍。用1-甲基-D，L-色氨酸阻断hemTg mLEC中IDO活性可防止KYN形成、KYN介导的蛋白修饰和G（2）/M阻滞。mLEC中过量的IDO活性导致KYN产生、KYN介导的肌球蛋白II和α/γ-肌动蛋白的修饰以及细胞周期扰动。KYN对肌球蛋白II和γ-肌动蛋白的修饰可能会干扰胞质分裂，导致上皮细胞分裂缺陷，从而减少纤维细胞的数量。（Invest Ophthalmol维斯科学。2008;49：5466-5475）DOI：10.1167/iovs.08-2374
PURPOSE. Indolemine 2,3-dioxygenase (IDO)-mediated oxidation of tryptophan produces kynurenines (KYNs), which may play a role in cataract formation. The molecular mechanisms by which KYNs cause cellular changes are poorly understood. The effects of KYNs on mouse lens epithelial cells by overexpression of human IDO were investigated.METHODS. Lens epithelial cells (mLECs) derived from human IDO-overexpressing hemizygous transgenic (hemTg) and wildtype (Wt) mice were used. IDO activity was measured by quantifying kynurenine (KYN) by HPLC. KYN-mediated protein modifications were detected by immunocytochemistry and measured by ELISA. Cell proliferation and apoptosis were measured with commercially available kits. Cell distribution between cell cycle phases was examined with flow cytometric analysis. Immunoprecipitation followed by LC/MS was used to identify kynurenine-modified proteins.RESULTS. mLECs derived from hemTg animals exhibited considerable IDO immunoreactivity and enzyme activity, which were barely detectable in Wt mLECs. KYN and KYN-mediated protein modification were detected in hemTg but not in Wt mLECs; the modified proteins were myosin II and alpha/gamma-actin. HemTg mLECs displayed reduced viability and proliferation. Cell cycle analysis of hemTg mLEC cultures showed approximately a twofold increase in cells at G(2)/M or in both phases, relative to Wt mLECs. Blocking IDO activity with 1-methyl-D,L-tryptophan in hemTg mLECs prevented KYN formation, KYN-mediated protein modification, and G(2)/M arrest.CONCLUSIONS. Excess IDO activity in mLECs results in KYN production, KYN-mediated modification of myosin II and alpha/gamma-actin, and cell cycle perturbation. Modification of myosin II and gamma-actin by KYN may interfere with cytokinesis, leading to defective epithelial cell division and thus a decreased number of fiber cells. (Invest Ophthalmol Vis Sci. 2008;49:5466-5475) DOI: 10.1167/iovs.08-2374