Impaired GSH biosynthesis disrupts eye development, lens morphogenesis and PAX6 function.
Impaired GSH biosynthesis disrupts eye development, lens morphogenesis and PAX6 function.
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DOI：
10.1016/j.jtos.2021.08.010
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发表时间：
2021-10
期刊：
影响因子：
--
通讯作者：
Vasiliou V
中科院分区：
文献类型：
--
作者：
Thompson B;Chen Y;Davidson EA;Garcia-Milian R;Golla JP;Apostolopoulos N;Orlicky DJ;Schey K;Thompson DC;Vasiliou V
本研究的目的是阐明谷胱甘肽（GSH）生物合成受损对眼发育的作用和分子后果。通过将Gclcf/f小鼠与半合子Le-Cre转基因小鼠杂交以产生Gclcf/f/Le-CreTg/-（KO）小鼠，在表面外胚层来源的眼组织中GSH生物合成受损。对照小鼠包括Gclcf/f和Gclcwt/wt/Le-CreTg/-小鼠（CRE）。对来自所有小鼠（在眼睛发育的各个阶段）的眼睛进行组织学、免疫组织化学、蛋白质印迹、RT-qPCR、RNA-seq和随后的基因本体学、免疫途径分析和TRANSFAC分析。在使用丁硫氨酸亚砜亚胺（BSO）耗尽GSH的HEK 293 T细胞中使用荧光素酶报告基因测定研究PAX 6反式激活活性。Gclc的缺失降低了GSH水平，增加了活性氧（ROS），并引起了明显的小眼表型，其特征在于角膜、虹膜、透镜和视网膜的畸形，这与在CRE小鼠中观察到的小眼表型不同，并且比在CRE小鼠中观察到的小眼表型严重得多。此外，仅KO小鼠的晶状体显示晶状体蛋白（α，β）、PITX 3和Foxe 3表达降低。出生后第1天的RNA-seq分析显示，相对于Gclcf/f小鼠，KO小鼠晶状体中有1，552个差异表达基因（DEG），其中晶体蛋白和透镜纤维细胞身份基因下调，而透镜上皮细胞身份和免疫应答基因上调。DEG的生物信息学分析表明PAX 6是一个关键的上游调节因子。在BSO处理的HEK 293 T细胞中PAX 6反式激活活性受损。这些数据表明，受损的眼部GSH生物合成可能会破坏眼睛发育和PAX 6功能。
The purpose of this study was to elucidate the role and molecular consequences of impaired glutathione (GSH) biosynthesis on eye development. GSH biosynthesis was impaired in surface ectoderm-derived ocular tissues by crossing Gclcf/f mice with hemizygous Le-Cre transgenic mice to produce Gclcf/f/Le-CreTg/- (KO) mice. Control mice included Gclcf/f and Gclcwt/wt/Le-CreTg/- mice (CRE). Eyes from all mice (at various stages of eye development) were subjected to histological, immunohistochemical, Western blot, RT-qPCR, RNA-seq, and subsequent Gene Ontology, Ingenuity Pathway Analysis and TRANSFAC analyses. PAX6 transactivation activity was studied using a luciferase reporter assay in HEK293T cells depleted of GSH using buthionine sulfoximine (BSO). Deletion of Gclc diminished GSH levels, increased reactive oxygen species (ROS), and caused an overt microphthalmia phenotype characterized by malformation of the cornea, iris, lens, and retina that is distinct from and much more profound than the one observed in CRE mice. In addition, only the lenses of KO mice displayed reduced crystallin (α, β), PITX3 and Foxe3 expression. RNA-seq analyses at post-natal day 1 revealed 1,552 differentially expressed genes (DEGs) in the lenses of KO mice relative to those from Gclcf/f mice, with Crystallin and lens fiber cell identity genes being downregulated while lens epithelial cell identity and immune response genes were upregulated. Bioinformatic analysis of the DEGs implicated PAX6 as a key upstream regulator. PAX6 transactivation activity was impaired in BSO-treated HEK293T cells. These data suggest that impaired ocular GSH biosynthesis may disrupt eye development and PAX6 function.
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影响因子：
3.7
作者：
Dorà NJ;Collinson JM;Hill RE;West JD
通讯作者：
West JD
影响因子：
3.4
作者：
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影响因子：
2.3
作者：
Giblin, FJ
通讯作者：
Giblin, FJ
DOI：
10.1111/j.2517-6161.1995.tb02031.x
发表时间：
1995-01-01
影响因子：
5.8
作者：
BENJAMINI, Y;HOCHBERG, Y
通讯作者：
HOCHBERG, Y
影响因子：
15.9
作者：
Gregory-Evans, Cheryl Y.;Wang, Xia;Gregory-Evans, Kevin
通讯作者：
Gregory-Evans, Kevin