Zhang Ling, Wahlin Karl, Li Yuanyuan, Masuda Tomohiro, Yang Zhiyong, Zack Donald J, Esumi Noriko
The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Mol Vis. 2013 Jun 17;19:1371-86. Print 2013.
Ras-like without CAAX 2 (RIT2), a member of the Ras superfamily of small guanosine triphosphatases, is involved in regulating neuronal function. RIT2 is a unique member of the Ras family in that RIT2 is preferentially expressed in various neurons, including retinal neurons. The mechanisms that regulate RIT2 expression in neurons were studied.
Reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry, western blotting, bioinformatic prediction, electrophoretic mobility shift assay (EMSA), and cell transfection methods were used.
With immunohistochemistry of the mouse retina, RIT2 protein was detected in the ganglion cell layer (GCL), inner plexiform layer, inner nuclear layer, and outer plexiform layer, with the strongest staining in the GCL and the inner plexiform layer. RT-qPCR combined with laser capture microdissection detected Rit2 messenger RNA in the GCL and the inner nuclear layer. Western blot analysis showed a large increase in the RIT2 protein in the retina during maturation from newborn to adult. Transient transfection identified the 1.3 kb upstream region of human RIT2 as capable of driving expression in neuronal cell lines. Based on the known expression pattern and biological activity, we hypothesized that POU4 family factors might modulate RIT2 expression in retinal ganglion cells (RGCs). Bioinformatic analyses predicted six POU4 factor-binding sites within the 1.3 kb human RIT2 promoter region. EMSA analyses showed binding of POU4 proteins to three of the six predicted sites. Cotransfection with expression vectors demonstrated that POU4 proteins can indeed modulate the human RIT2 promoter, and that ISL1, a LIM homeodomain factor, can further modulate the activity of the POU4 factors.
These studies confirm the expression of RIT2 in retinal neuronal cells, including RGCs, begin to reveal the mechanisms responsible for neuronal expression of RIT2, and suggest a role for the POU4 family factors in modulating RIT2 expression in RGCs.
无CAAX结构的Ras样蛋白2（RIT2）是小GTP酶Ras超家族的成员，参与调节神经元功能。RIT2是Ras家族的独特成员，因为它在包括视网膜神经元在内的各种神经元中优先表达。本研究对调节神经元中RIT2表达的机制进行了探究。
采用逆转录定量PCR（RT-qPCR）、免疫组织化学、蛋白质免疫印迹、生物信息学预测、电泳迁移率变动分析（EMSA）和细胞转染方法。
通过对小鼠视网膜进行免疫组织化学检测，在神经节细胞层（GCL）、内网状层、内核层和外网状层检测到RIT2蛋白，其中在GCL和内网状层染色最强。RT-qPCR结合激光捕获显微切割技术在GCL和内核层检测到Rit2信使核糖核酸。蛋白质免疫印迹分析显示，从新生小鼠到成年小鼠，视网膜中RIT2蛋白大幅增加。瞬时转染实验表明，人RIT2基因上游1.3 kb区域能够驱动其在神经元细胞系中的表达。基于已知的表达模式和生物学活性，我们推测POU4家族因子可能调节视网膜神经节细胞（RGC）中RIT2的表达。生物信息学分析预测在人RIT2基因1.3 kb启动子区域内有6个POU4因子结合位点。EMSA分析显示POU4蛋白与6个预测位点中的3个结合。与表达载体共转染表明，POU4蛋白确实可以调节人RIT2启动子，而LIM同源结构域因子ISL1可以进一步调节POU4因子的活性。
这些研究证实了RIT2在包括RGC在内的视网膜神经元细胞中的表达，开始揭示RIT2在神经元中表达的机制，并提示POU4家族因子在调节RGC中RIT2表达方面发挥作用。
